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CHEMISTRY RESEARCH AND APPLICATIONS

CHEMICAL REACTIONS IN GAS,


LIQUID AND SOLID PHASES:
SYNTHESIS, PROPERTIES
AND APPLICATION

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CHEMISTRY RESEARCH AND APPLICATIONS

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CHEMISTRY RESEARCH AND APPLICATIONS

CHEMICAL REACTIONS IN GAS,


LIQUID AND SOLID PHASES:
SYNTHESIS, PROPERTIES
AND APPLICATION

G. E. ZAIKOV
AND
R. M. KOZLOWSKI
EDITORS

Nova Science Publishers, Inc.


New York
Copyright © 2010 by Nova Science Publishers, Inc.

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LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA

Chemical reactions in gas, liquid, and solid phases : synthesis, properties,


and application / editors, G.E. Zaikov, R.M. Kozlowski.
p. cm.
Includes index.
ISBN 978-1-61668-906-3 (eBook)
1. Polymers--Biodegradation. 2. Composite materials--Biodegradation. I.
Zaikov, Gennadii Efremovich. II. Kozlowski, R. (Ryszard)
QD381.9.D47C54 2009
541'.39--dc22
2010015588

Published by Nova Science Publishers, Inc. Ô New York


CONTENTS

Preface ix 
Chapter 1 Classification of Polymers in Reactivity Toward Nitrogen Oxides 1 
E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii, T. V. Pokholok
and G. E. Zaikov 
Chapter 2 Influence of the Initiation Rate of Radicals on the Kinetic
Characteristics of Quercetin and Dihydroquercetin in the Methyl
Oleate Oxidation 11 
L. I. Mazaletskaya, N. I. Sheludchenko and L. N. Shishkina 
Chapter 3 An Antioxidant from Hindered Phenols Group Activates Cellulose
Hydrolysis By Celloviridin in a Wide Concentration Range,
Including Ultralow Doses 21 
E. M. Molochkina, Yu. A. Treschenkova, I. A. Krylov
and E. B. Burlakova 
Chapter 4 a-Tocopherol as Modifier of the Lipid Structure of Plasma
Membranes In Vitro in a Wide Range of Concentrations
Studied by Spin-Probes 29 
V. V. Belov, E. L. Maltseva and N. P. Palmina 
Chapter 5 Supercritical Carbon Dioxide Swelling of Polyheteroarylenes
Synthesized in N-Methylpyrrolidone 45 
Inga A. Ronova, Lev N. Nikitin, Gennadii F. Tereschenko
and Maria Bruma 
Chapter 6 Inhibition of 2-Hexenal Oxidation By Essential Oils of Ginger,
Marjoram, Juniper Berry, Black and White Pepper 65 
T. A. Misharina, M. B. Terenina, N. I. Krikunova
and I. B. Medvedeva 
Chapter 7 The Organophosphorus Plant Growth Regulator Melaphen
as Adaptogen to Low Moisher 75 
I. V. Zhigacheva, E. B. Burlakova, T. A. Misharina, M. B.Terenina,
N. I. Krikunova, I. P. Generozova and A. G. Shugaev 
vi Contents

Chapter 8 Antioxidant Properties of Essential Oils from Clove Bud,


Laurel, Cardamom, Nutmeg and Mace 83 
T. A. Misharina, M. B. Terenina, and N. I. Krikunova 
Chapter 9 Specific Properties of Some Biological Composite Materials 91 
N. Barbakadze, E. Gorb and S. Gorb 
Chapter 10 Properties and Applications of Aminoxyl Radicals
in Polymer Chemistry 123 
E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii, T. V. Pokholok,
and G. E. Zaikov  
Chapter 11 Synthesis of Flexible Manufacturings for Phosphoric Industry
Waste Utilization Based on the Cals-Concept 155 
A. M. Bessarabov, A. V. Kvasyuk and G. E. Zaikov 
Chapter 12 Practical Hints on the Application of Nanosilvers in Antibacterial
Coating of Textiles 165 
S. Dadvar, A. Oroume and A. K. Haghi 
Chapter 13 The Nanostructure and Yield Process of Cross-Linked Epoxy
Polymers 191 
Z. M. Amirshikhova, G. V. Kozlov, G. M. Magomedov
and G. E. Zaikov 
Chapter 14 Nanostructures in Cross-Linking Epoxy Polymers and Their
Influence on Mechanical Properties 197 
Z. M. Amirshikhova, G. V. Kozlov, G. M. Magomedov
and G. E. Zaikov  
Chapter 15 The Degradation Heterochain Polymers in The Presence of
Phosphorus Stаbilizers 205 
E. V. Kalugina, N. V. Gaevoy,  
K. Z. Gumargalieva and G. E. Zaikov 
Chapter 16 Quantum-Chemical Calculation of Olefins of Cationic
Polymerization Branching in -, - and  Position on Relations to
Double Connection By Method MNDO 221 
V. A. Babkin, D. S. Andreev, T. V. Peresypkina and G. E. Zaikov  
Chapter 17 Thermodynamics for Catalase and Hydrogen Peroxide Interaction 227 
A. A. Turovsky, A. R. Kytsya, L. I. Bazylyak and G. E. Zaikov
Chapter 18 Dr. Rer. Nat. Wolfgang Fritsche – Scientist and Organizer of
International Science (Secretary General Rtd. of Gesellschaft
Deutscher Chemiker, Honorary President of Federation
of European Chemical Societies) 245 
G. E. Zaikov  
Chapter 19 Professor Victor Manuel De Matos Lobo on His 70th Anniversary 249 
Gennady E. Zaikov and Artur J. M. Valente  
Contents vii

Chapter 20 The Scientist Who Outstripped His Time 251 


Revaz Skhiladze and Tengiz Tsivtsivadze  
Chapter 21 Prof. Dr. Ryszard Michal Kozlowski: Half a Century in Science
and Technology 263 
Gennady Zaikov 
Chapter 22 The Second International Conference on Biodegradable Polymers
and Sustainable Composites (BIOPOL-2009) 267 
G. E. Zaikov, L. L. Madyuskina and M. I. Artsis  
Index 271 
“If you are sixty (or more)
and you are not feeling any pain in your body
when you are getting up in the morning,
it means that you have passed away (already)”
Russian proverb

PREFACE

This epigraph is very correct for the Russian Federation today because the average
lifespan of Russian men in this country is 57 years (Russian women are living on ten years
more). It is statistical data. Russian scientists are an exception. Particularly, the majority of
contributors of this volume are older then 60 and the editor of volume is older then 75.
We can explain this exception (phenomena) if we take in account the next Russian
proverb: “All illnesses are from nerves and only a small amount from pleasure”. We expect
that readers immediately are thinking about sex as a part of pleasure. It is only partly right.
Alcohol, tobacco and narcotics should also be included in the “pleasure” part. Unfortunately,
the Russian people are world champions for drinking. The average Russian man (including
ladies, children and even babies) drinks 18 liters of pure ethyl alcohol (calculation done in
pure alcohol) per year. It is twice more than twice the critical amount (9 liters).
Russian scientists are again an exception because the majority of them have pleasure only
in the case of communication with SCIENCE!
Now we should remember the Kazakh (people living in the Asian part of former USSR)
proverb: “If sixty years are coming the mind (brain, memory) will go back (to childhood
conditions)”. We expect that this proverb is also not correct for Russian scientists. As
evidence of this opinion you can read the chapters of this volume where the most part of
chapters were prepared by scientists from Russian Research Centers and from Research
Centers of former Republics (now independent states) of the USSR.
It is now the right time to remember English proverb: “Please eat one apple every day
and you will not need a physician” (it is a reverse translation from Russian to English). We do
not know exactly if it is enough to eat one apple a day to be permanently healthy or not. We
do know that positive emotions are in favor for good health. We expect that this book can
(should) give only positive emotions to readers and we are waiting for the opinions of readers
in this case.
So, we should stop about proverbs and start about chemical science and application.
This volume includes information about kinetics and mechanism of chemical reactions in
different phases: classification of polymers in reactivity toward nitrogen oxides (polluted
atmosphere), influence of the initiation rate of radicals on the kinetic characteristics of
quercetin and dihydroquercetin in the methyl oleate oxidation, an antioxidant from hindered
x Gennady E. Zaikov and R. M. Kozlowski

phenols group activates cellulose hydrolysis by celloviridin in a wide concentration range,


including ultralow doses, α-tocopherol as modifier of the lipid structure of plasma membranes
in vitro in a wide range of concentrations studied by spin-probes, supercritical carbon dioxide
swelling of polyheteroarylenes synthesized in N-methylpyrrolidone, inhibition of 2-hexenal
oxidation by essential oils of ginger, marjoram, juniper berry, black and white pepper,
specific properties of some biological composite materials, the organophosphorus plant
growth regulator melaphen as adaptogen to low moisher, properties and applications of
aminoxyl radicals in polymer chemistry, synthesis of flexible manufacturings for phosphoric
industry waste utilization based on the cals-concept, practical hints on application of
nanosilvers in antibacterial coating of textiles, the degradation heterochain polymers in the
presence оf phosphorus stаbilizers, quantum-chemical calculation of olefins of cationic
polymerization and antioxidant properties of essential oils.
The nanostructure and yield process of cross-linked epoxy polymers as well as
nanostructures in cross-linking epoxy polymers and their influence on mechanical properties
are discussed in this volume.
Somebody asked Henry Ford: “Which car is better?” Ford answered: “A new one”. No
doubt a new car as well as new scientific information is better than old ones (only old cognac
is better than the new one). We took this idea into account in case preparation of our volume.
So, this volume is a complete guide to the subject of kinetic and mechanisms of chemical
reactions in gas, liquid and solid states. The editors and contributors will be happy to receive
from the readers some comments which we can use in our research in the future.
Once, Agatha Christie was filling out a form. There was a question: “What is your
occupation” and she wrote “A married lady”.
Of course Agatha Christie could write on any form whatever she wanted because
everybody knew her.
What is important for us is that in filling out any form we contributors could write with a
clear conscience – “a scientist.”

Gennady E. Zaikov
N.M. Emanuel Institute of Biochemical Physics
Russian Academy of Sciences
4 Kosygin Str., Moscow 119334, Russia

Ryszard M. Kozlowski
Institute for Engineering of Polymer Materials and Dyes
Torun, Poland
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.1-10 ©2010 Nova Science Publishers, Inc.

Chapter 1

CLASSIFICATION OF POLYMERS IN REACTIVITY


TOWARD NITROGEN OXIDES

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii,


T. V. Pokholok, and G. E. Zaikov*
N.M. Emanuel Institute of Biochemical Physics,
Russian Academy of Sciences, Moscow, Russia

ABSTRACT
The review is includies information about the kinetics and mechanism of interaction
of nitrogen oxides with carbon-chain polymers, rubbers, aliphatic polyamides and
polyuretanes.

Keywords: nitrogen oxides, reactivity, polymers, classification, kinetics, mechanism.

The general review of influence of the pollutants on polymers has been presented by
Jellinek et al. [1]. Therein the characterization of reactivity of polymeric materials toward
aggressive gases is given. The various polymers were used as films of 20 μ thickness. The
thickness is enough small to exclude in most cases the diffusion as the determining factor of
the pollutant action. The films were investigated under different conditions: 1) the pollutant
action; 2) the oxygen action; 3) UV light action; 4) UV light and oxygen; 5) UV light, oxygen
and pollutants. For NO2, the exposure of samples was usually realized under the pressure of
15 mm Hg during 30 hours at 308 K. However, in a case of nylon 66 and butyl rubber, the
NO2 pressure was lowered up to 1 mm Hg at during 30 min. Polyisoprene and polybutadiene
were exposed to NO2 during 5 min under a pressure of 1 mm Hg. As a light source (λ > 290
nm), a mercury lamp was used. The intrinsic viscosity of polymer solutions was measured
before and after exposure of samples in the chosen conditions. The rather high concentration

* N.M. Emanuel Institute of Biochemical physics, Russian Academy of Sciences, 4 Kosygin Street, 119334
Moscow, Russia, Fax: (7-495)1374101, E-mail: pgb@sky.chph.ras.ru , chembio@sky.chph.ras.ru
2 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

of nitrogen dioxide in these experiences was used to be convinced that the certain effects can
be observed for a reasonable time.
The polymers on the basis of their reactivity with respect to NO2 can be divided into two
main classes [1]. The saturated polymers, for instance, polyethylene (PE) and polypropylene
(PP) belong to the first group, but nylon 66 is not included into this series. The second group
covers elastomers. Butyl rubber undergoes scissions of the main chain, and polybutadiene is
restrictedly cross-linked under the action of NO2. These elastomers have approximately the
same reactivity with respect to NO2 as to ozone. All films exposed to NO2 become yellow,
and their IR spectra show that nitro groups enter into macromolecules. In polyvinylchloride in
the presence of NO2, some decreasing the amount of chlorine along with the appearance of
nitro and nitrite groups are observed from IR spectra.
It is the author’s opinion [1] that some estimations concerning influences of so low
concentration of nitrogen dioxides in an atmosphere (2⋅10−9 − 2⋅10−8 mol⋅l−1) on polymeric
materials can be obtained from the experiences with using concentrations of the gas of several
orders of magnitude higher. The formulated assumption says that there is linear dependence
of the concentration effect of aggressive pollutants. This means that the effect of aggressive
gases at low concentrations can be determined by the linear extrapolation of results obtained
under the influence of high concentrations. The author pointed out that this procedure
contains an element of risk because scissions of macromolecules in some cases are not always
linearly decreased with the pressure reduction of the aggressive gas, but the rate of breaks can
change drastically at very low concentrations.
The procedure of extrapolation was used for an estimation of the scission average number
S under the action of aggressive gases at concentrations of 1 − 5ppm within 1 hour [1]. This
value is given by the equation:

DPn ,0
S= −1 (I.1)
DPn ,t

where DPn,0 and DPn ,t are lengths of macrochains at t =0 and t correspondingly. On the
basis of these estimations, it was concluded that aggressive gases, for instance NO2 and SO2,
slightly effect on vinyl polymers in concentrations really available in polluted air. Even in a
combination with UV light, the deterioration of these polymers is hardly noticeable.
However, nylon 66 is quite subjected to the action of small concentrations of NO2 with
essential degradation.

I.1. INTERACTION OF CARBON-CHAIN POLYMERS WITH NO2

Pioneering studies of the reaction of nitrogen dioxide with polyethylene (PE) and
polypropylene (PP) have been carried out by Ogihara et al. [2, 3]. Using IR spectroscopy,
they have found that nitrogen dioxide cannot abstract secondary and tertiary hydrogen atoms
from PE and PP at 298 K. It can only add to the vinylene and vinylidene units that are formed
Classification of Polymers in Reactivity Toward Nitrogen Oxides 3

in the synthesis of the polymers. These reactions resulted in dinitro compounds and nitro
nitrites:

C=C + NO2 C C
NO2 (I.2)
H
C C
H (I.3)
O2N NO2
C C + NO2
H
NO2
C C
ONO NO2 (I.4)

At T > 373 K, nitro, nitrite, nitrate, carbonyl and hydroxy groups are formed in these
polymers. The following reaction mechanism at high temperatures was proposed:

RH + NO2 → R• + HNO2 (I.5)

R• + NO2 → RNO2 (I.6)

R• + ONO → RONO (I.7)

RONO → RO• + NO (I.8)

RO• + NO2 → RONO2 (I.9)

RH + RO• → R• + ROH (I.10)

~CH2-CH2-CH2-O• → ~CH2-CH2-CHO + H (I.11)

This scheme allows rationalization of the accumulation of the nitro groups, which
proceeds at a constant rate and autoaccelerated formation of nitrates, alcohols and carbonyl
compounds.
However, it provides no explanation for S-shaped dependence of the accumulation of
nitrites.
The activation energies for the NO2 addition to the double bonds of PE are 8-16 kJ⋅mol−1.
The activation energy for hydrogen abstraction is within of 56 and 68 kJ⋅mol−1 for PE and 60
kJ⋅mol−1 for PP.
At room temperature and at NO2 concentrations of 5.4⋅10−4 – 5.4⋅10−3 mol⋅l−1, the
characteristics of PE, PP, polyacrylonitrile and polymethylmethacrylate (PMMA) are changed
only slightly even if they simultaneously undergo to a combined action of NO2, O2 and UV
radiation [4]. Reactions of NO2 with polyvinylchloride and polyvinyl fluoride resulted in a
slight decrease in the content of chlorine and fluorine atoms, respectively [1, 4].
4 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

In the temperature range of 298−328 K nitrogen dioxide (7.8⋅10−3−3.4⋅10−2 mol⋅l−1) can


abstract tertiary hydrogen atoms from polystyrene (PS) molecules to introduce nitro and
nitrite groups into macromolecules in result of subsequent reactions [1]. This process
proceeds at low rates and is accompanied by chain scissions [1, 5, 6]. The number of chain
scissions on time α(t) was determined from intrinsic viscosity using the equation (I.1). The
experiments have been carried out at the temperature range of 298-328 K. According to
Jellinek, the dependence of the decrease in the degree of polymerization of PS on the
exposure time in NO2 has three linear regions: initial, middle and final. A decrease in the
apparent degradation rate was observed in the middle region of the dependence. Presumably
this was related to the association of the macromolecules in solution, which is due to the
effect of polar groups and can affect the results of viscosimetric measurements. Subsequent
increase in the apparent degradation rate was attributed to the consumption of these nitrogen-
containing groups and to a decrease in the degree of association of the macromolecules. PS
films were also simultaneously exposed to NO2 (1.1·10−4 mol·l−1) and light (λ > 280 nm) [6].
No polymer degradation was observed in the initial stage during 10 h. Then chain scission
occurred at a constant rate.
An attempt to determine quantitative characteristics of the ageing of PS and poly-t-
butylmethacrylate (PTBMA) under the action of NO2 has been undertaken by Huber [7]. The
samples were exposed to a stream of air containing NO2 (2.5⋅10−6 – 3.7⋅10−5 mol⋅l−1) at 300 K
and simultaneously irradiated with light (λ>290 nm). The number of chain scission per 10000
monomer units α(t) can be described by the empirical equation:

P
α= (exp Qt − 1) (I.12)
Q

where P and Q are constants. This equation describes an autocatalytic process. At Q → 0,


degradation occurs at a constant rate. Autocatalytic process is more pronounced for thin films.
Degradation of thin PS films under the same conditions occurs slower than that of the
PTBMA films and its autocatalytic nature is more pronounced.
The autocatalytic path of degradation of PTBMA was associated [7] with the photo-
induced formation of isobutylene, which reacts with NO2, thus initiating free-radical
degradation processes of macromolecules. The IR spectrum of PS exposed toNO2 and light
exhibits two bands at 1686 and 3400 cm−1 corresponding to the carbonyl and hydroxyl
groups, respectively. The formation of nitrogen-containing products has not been observed in
both PTBMA and PS. The following reactions have been proposed [7] in PS:

~ CH2−C(Ph)H ~ + NO2 → HNO2 + ~ CH2−C•(Ph) ~ (R1•) (I.13)

R1• + O2 → R1O2• (I.14)

R1O2• + RH → ROOH + R1• (I.15)


Classification of Polymers in Reactivity Toward Nitrogen Oxides 5

R1NO2 (I.16)
R1 + NO2
R1ONO (I.17)


R1ONO ⎯⎯→ R1O• + NO (I.18)

R1OOH + NO → R1O• + •OH + NO (I.19)


R1OOH ⎯⎯→ R2• + •OH (I.20)

R1O• → R2• + degradation products (I.21)

It is believed that the decomposition of hydroperoxides exposed to NO2 and light leads to
autocatalytic degradation of PS.

I.2. INTERACTION OF RUBBERS WITH NO2


Rubbers are much more susceptible to NO2 than the polymers containing no double
bonds. First, this is due to the ability of NO2 to add reversibly to carbon-carbon double bonds
to give nitroalkyl radicals (reaction (I.2)), thus initiating free radical conversions of
elastomers. Second, nitrogen dioxide is able of abstracting hydrogen atoms in β−position to
the double bond to give allyl radicals, which then recombine with NO2 [8]. Depending on the
structure of the alkene, the reaction resulting in the formation of the allyl radical can be either
weakly exothermic or weakly endothermic. For instance, the strength of the weakest C−H
bond in the structure CH2=C(CH3)CH2−H is only 314 kJ·mol−1 [9].
The exposure of polyisoprene and polubutadiene to nitrogen dioxide leads to both
degradation and cross-linking of macromolecules, whereas butyl rubber (BR) (a copolymer of
36% isobutylene and 54% isoprene units) only undergoes degradation [10]. The detailed
study of the ageing BR exposed to NO2 (5.2⋅10−7 – 5.2⋅10−5 mol⋅l−1) alone, an NO2−O2
mixture and an NO2−O2 mixture plus UV light (λ > 280 nm) at 298−358 K has been
performed by Jellinek et al.[11, 12]. IR spectra before and after the exposure of samples show
that the band at 1540 cm−1 of ~ C=C ~ bonds disappears, and the new band at 1550 cm−1
arises. The latter belongs to nitro groups appearing as a result of addition to double bonds by
the reaction (I.2).
The chain scission process in BR causes by the following scheme:

NO2
k1 k3
~C(CH3)=CH~ + NO2 ~C CH~ + NO2 chain scission + NO2 (I.22)
k2 CH3
R

Then the rate of scissions is:


6 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

d [n ' ]
− = k 3 [ R ⋅ ][NO 2 ] (I.23)
dt

where n ' is a number of isoprene units in BR. After integration of (I.23) taking into account
a stationary concentration of R•, the following equation for the degradation degree is derived:

k1k3[n' ]0 [ NO 2 ]t
α= (I.24)
[n ]0 ( k 2 + k3[ NO 2 ]

where [n' ]0 and [n ]0 are the initial concentrations of isoprene units and all units. The
amount of double bonds remains practically constant because only a small number of those
are destroyed. Really, only 1/50 of macromolecules of BR are subjected to scissions. Taking
into account low concentrations of NO2, the linear dependence on time is obtained:

α = k exp t (I.25)

where k exp is the experimentally determined constant. This constant is represented by the
following Arrhenius equation: kexp = 3.8 ⋅ 10 − 2 e − 7450 / RT , h−1.
The degradation of BR in a polluted atmosphere runs in three directions: 1) the action of
NO2 alone, 2) the action of O2, 3) the combined (synergetic) action of these gases. The
general scheme of the process can be represented as follows:

k
RH + O2 ⎯⎯→
4
R• + HO2 (I.26)

k
R • + O2 ⎯⎯→
5
RO2• (I.27)

k
RO2• + RH ⎯⎯→
6
ROOH + R• (I.28)

k
ROOH ⎯⎯→
7
stable products (I.29)

k8 k10
ROOH [cage1] + O2 chain scission products + O2 (I.30)
k9

The effect of NO2 + O2:

k
ROOH + NO2 ⎯⎯→

11
NO2−ROOH (I.31)
Classification of Polymers in Reactivity Toward Nitrogen Oxides 7

k12 k14
NO2-ROOH [cage2] chain scission products (I.32)
k13
k
2 R• ⎯⎯→

15
[cage 3] (I.33)

k
[cage 3] + O2 ⎯⎯

16
→ 2 R• (I.34)

k
[cage 3] ⎯⎯→

17
R−R (I.35)

The synergetic action of NO2 and O2 can be seen from the scheme:

~CH2C(CH3)=CHCH2CH2~ + O2 → ~CH2C(CH3)=CHCH(OO•)CH2~ + HO2• (I.36)

~CH2C(CH3)=CHCH(OO•)CH2~ + RH → ~CH2C(CH3)=CHCH(OOH)CH2~ + R (I.37)

~CH2C(CH3)=CHCH(OOH)CH2~ + NO2 → ~CH2C(CH3)=C(NO2)CH(OOH)CH2~ (I.38)

BR is not sensitive to UV light (λ > 290 nm) alone. Probably, UV light in the presence of
NO2 effects on nitro groups of macromolecules.

I. 3. INTERACTION OF NITROGEN DIOXIDE WITH ALIPHATIC


POLYAMIDES AND POLYURETHANES
Polymers containing amide and urethane groups form a particular class of materials
sensitive to NO2. Jellinek et al. [13, 14] showed that exposure of nylon-66 films of different
morphology to NO2 (10−5 − 2.6⋅10−1 mol⋅l−1) causes main-chain scission in the polymers. The
degradation of nylon is a diffusion-controlled reaction. Its rate and depth depend essentially
on the degree of crystalline of samples and on the size of crystallites. The degradation is
accelerated in the presence of air and UV light in addition to NO2. The following mechanism
for the polymer degradation under the action of NO2 was proposed:

O H O
C N CH2 CH2 + NO2 C N CH2 CH2 + HNO2
O NO2 O
NO2
C N CH2 CH2 C N CH2 + CH2 (I.39)

The degradation process is inhibited by small amounts of benzaldehyde or benzoic acid.


It is believed that these compounds block the amide groups and that only a few of them, not
involved in hydrogen bonding, enter into the reaction:
8 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

O O
C NH CH2 + Ph C OH C N (I.40)
O H
H O
O C Ph

Jellinek et al. [14, 15] studied the effect of NO2 on films of linear polyurethane
synthesized from tetramethylene glycol and hexamethylene diisocyanate. It was found that
the degradation of polyurethanes is accompanied by cross-linking of macromolecules and that
the degree of degradation and the yield (the weight percentage) of the gel fraction are
complex functions of the exposure time. For instance, the yield of the gel fraction initially
increases up to 20% and then decreases down to nearly zero at 330 K and NO2 concentration
of 10−3 mol⋅l−1. The number of chain scissions in the sol fraction (the degree of degradation)
increases initially, then decreases and eventually increases again; however, the final
degradation rate is lower than the initial one. Exposure of the polyurethane films to NO2 is
accompanied by release of CO2. The IR spectra of the films allow assessment of the
consumption of NH bonds (ν = 3300 cm−1).
The reaction mechanism proposed [15, 16] involves the abstraction of hydrogen atoms
from two types of structures, namely, a carbamate structure (A) and a tertiary amide structure
(B):

O O
O C NH CH2 O C N CH2

B ZH
A

where Z is a side alkyl group. The next stages are represented as follows:
O
NO2
A O C N CH2 + HNO2 (I.41)
R1
O
NO2
B O C N CH2 + HNO2 (I.42)
Z R2
(I.43)
R1 + NO2 R1NO2
O

R1 H2C N + C O CH2 (I.44)


R3

R3 (I.45)
CO2 + H2C

R1 + R 2 cross-linking product (I.46)


Classification of Polymers in Reactivity Toward Nitrogen Oxides 9

According to the Jellinek, recombination of R1• and R2• radicals leads to cross-linking of
the polymer chains, while decomposition of R1• radicals results in the degradation of
macromolecules and the CO2 release. Energetically, the decomposition of the R1• radicals
seems to be improbable since this reaction results in the formation of terminal macroradical
R3•and a nitrene, which is a very reactive species. On the other hand, the R1• decomposition
reaction involving cleavage of C−C or C−O is more bonds produce no alkoxycarbonyl
macroradicals R3•, which can undergo decarboxylation [16]. Therefore, the ageing of
polyurethanes in an NO2 atmosphere can be represented as follows [17]:

Reaction (I.41)

Reaction (I.42)

O
NO2
B O C N CH-CH2 + HNO2 (I.47)
ZH R
4

R4 R3 + HZ- N=CH (I.48)

R3 CO2 + CH 2 (I.49)

2Ri + NO2 nitration products (I.50)

2Ri cross-linking products (I.51)

where i = 1−4.
This scheme expresses the degradation accompanied by cross-linking of macromolecules,
the consumption of NH groups of the polymer as well as the release of carbon dioxide upon
degradation.
The investigations performed earlier characterize in general the reactivity of polymers of
different classes in their reactions with nitrogen dioxide. However, mechanisms of free
radical processes proposed on basis of the results considered are enough formal. As a rule
they take account of changing molecular weights and the composition of final molecular
products of the nitration. In connection with this, the study of structures of free radicals
forming in primary and intermediate stages of polymer conversions attracts an especial
interest. Such researches allow drawing conclusions on the mechanism of initiation of free
radical conversions dependent on nature of functional groups of macromolecules. As is
shown by ESR measurements, different stable nitrogen containing macroradicals are formed
on exposure of polymers to NO2 [17]. The analysis of the radical composition from ESR
spectra gives an opportunity for estimation of the polymer stability by the quite simple
method [18].
10 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

REFERENCES

[1] Jellinek H. H. D. Degradation and Stabilization of Polymers. New York: Elsevier,


1978.
[2] Ogihara T.. Bull. Chem. Soc. Jap. 1963, 31, 58-
[3] Ogihara T., Tsuchiya S., Kuratani K. Bull. Chem. Soc. Jap. 1965, 38, 978-
[4] Jellinek H. H. D. The 2nd International Symposium on Degradation and Stabilization of
Polymers (Abstracts of Reports), Dubrovnik, 1978.
[5] Jellinek H. H. D., Toyoshima Y. J. Polym. Sci. A-1. 1967, 5, 3214-
[6] Jellinek H. H. D., Flajsman F. J. Polym. Sci. A-1. 1969, 7, 1153-
[7] Huber A. Diss. Doktor Naturwiss. Stutgart: Fakultät Chemie der Universität Stutgart,
1988.
[8] Giamalva D. H., Kenion G. B., Church D. F., Pryor W. A. J. Am. Chem. Soc. 1987,
108, 7059-7063.
[9] Rånby B., Rabek J. F. Photodegradation, Photo-oxidation and Photostabilization of
Polymers. London: Wiley, 1975.
[10] Jellinek H. H. D., Flajsman F., Kryman F. J. J. Appl. Polym. Sci. 1969, 13, 107-
[11] Jellinek H. H. D., Flajsman F. J. Polym. Sci. A-1. 1970, 8, 711-
[12] Jellinek H. H. D., Hrdlovič P. J. Polym. Sci. A-1. 1971, 9, 1219-
[13] Jellinek H. H. D., Chandhuri A. J. Polym. Sci. A-1. 1972, 10, 1773-
[14] Jellinek H. H. D., Yokata A. R., Itoh Y. Polym. J. 1973, 4, 601.
[15] Jellinek H. H. D., Wang A. T. J. W. J. Polym. Sci., Polym. Chem. Ad. 1973, 11, 3227-.
[16] [Jellinek H. H. D., Martin F. H., Wegener H. J. Appl. Polym. Sci. 1974, 18, 1773-
[17] Pariiskii G. B., Gaponova I. S., Davydov E. Ya. Russ. Chem. Rev. 2000, 69, 985-999.
[18] Zaikov G.E. Success in chemistry and biochemistry, New York, Nova Science
Publishers, 2009
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.11-20 ©2010 Nova Science Publishers, Inc.

Chapter 2

INFLUENCE OF THE INITIATION RATE OF


RADICALS ON THE KINETIC CHARACTERISTICS OF
QUERCETIN AND DIHYDROQUERCETIN IN THE
METHYL OLEATE OXIDATION

L. I. Mazaletskaya*, N. I. Sheludchenko and L. N. Shishkina


Emanuel Institute of Biochemical Physics of Russian Academy of Sciences,
Moscow, Russia

ABSTRACT
The antioxidant activity of quercetin (Q) and dihydroquercetin (QH2) is studied by
the methyl oleate autooxidation model at 323 K in thin layers, and also the antiradical
activity of these substances is investigated by the initiated oxidation of methyl oleate at
333 K. It is shown that the rate constant of the inhibition for Q is equal 1.7×105 dm-3 mol-1
s-1, that is 2.2 times greater than king for QH2 (7.9×104 dm3 mol-1 s-1). Under the
autooxidation condition the inhibitory effectiveness of Q is also 1.6 times greater than
that for QH2. The existence of the direct correlation between the induction period of the
methyl oleate autooxidation in the presence of flavonoids and the Q and QH2
concentrations indicates their primary interaction with peroxyl radicals.

Keywords: autooxidation, initiated oxidation, kinetics, flavonoids, methyl oleate.

AIMS AND BACKGROUND


Flavonoids are the compounds within the group of the vitamin P and of interest as the
biologically active substances with a wide spectrum of action. Biological activity of
flavonoids is associated with their ability to inhibit of oxidation processes by the reaction

* Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, 4 Kosygin st., 119334, Moscow,
Russia e-mail: lim@sky.chph.ras.ru
12 L. I. Mazaletskaya, N. I. Sheludchenko and L. N. Shishkina

with reactive oxygen species, as well as free radicals [1-6]. In addition to the use of
flavonoids as drugs and the component of the biologically active additives for the
stabilization of the food products, cosmetics, preparations, etc, from the air oxygen oxidation
is of the great practical interest, because the number permitted to be used for this purpose
synthetic antioxidants is extremely limited.
Many investigations are made to examine the antioxidant activity of flavonoids and their
content in food product consistent by using the different methods. Advantages and
disadvantages of these methods are discussed in detail in review [7].
The analysis of the available literature data shows that the parameters characterizing the
antioxidant activity as well as the rate constant of the antiradical activity of flavonoids
obtained under different conditions substantially differ and depend on many factors, including
the chemical structure of flavonoids and the oxidation model. Thus, one would expect that the
antioxidant activity of the two closest to the structure of flavonoids - quercetin (Q) and
dihydroquercetin (QH2), the molecule structure of which is the presence or absence of the C2-
C3 double bond, will be little different, because their rate constants with oxygen anion-
radicals are close and equal 1.7×105 dm3 mol-1 s-1 and 1.5×105 dm3 mol-1 s-1, correspondingly
[8]. However, the effectiveness of Q is more 2 times greater than that for QH2, when it is
determined by the NADPH- and CCl4-dependent lipid peroxidation in microsomal
membranes of the rat liver8. On the contrary, the greatest antioxidant activity has QH2 that
follows from the results of the comparative tests presented in the paper [9].
As shown, the antiradical activity of flavonoids significantly changes depending on the
oxidizing substrate and the oxidation conditions. So, the rate constants of their interaction
with peroxyl radicals obtained in the initiated oxidation reaction of diphenylmethane10
significantly higher than that obtained by methyl linoleate oxidation in the homogenous and
micellar solutions [11]. Besides, as noted in Ref. 10 - 11, flavonoids did not behave as the
classical antioxidants and the constant of their antiradical activity depends on the initial
concentrations of both flavonoid and the oxidation substrate.
To use flavonoids as stabilizers from the spontaneous oxidation of foods, cosmetics and
medicinal agents, it is necessary to establish the regularities of their antioxidant action under
the autooxidation conditions, which simulates the natural aging process taking place under the
air oxygen action. As known, it is the side-reactions of antioxidants (InH) play the most
substantial role under the degenerate branching chain in the autooxidation process, they
proceed with the participation of the initial molecules of antioxidants and products of their
oxidative conversion and lead to loss of the effectiveness of the antioxidant action. For this
reason, there may exist substantial differences in regularities of the antioxidant action of these
substances between the autooxidation reactions and reactions with the constant rate of the free
radical initiation. So, for example, the deviation from linearity is observed for the dependence
of the induction period on the initial concentration for the natural antioxidant α-tocopherol
(TP) under the autooxidation conditions, and, in some cases, this dependence can be
extremal12. On the contrary, the linear relation between these parameters was detected by both
the initial oxidation and autooxidation for the hindered phenols, radicals of which are
practically not consumed in side-reactions [13,14].
The aim of our research was to study the influence of the quercetin and dihydroquercetin
concentrations on the effectiveness of their inhibitory action and the antiradical activity
depending on the oxidation conditions of the same substrate – methyl oleate.
Influence of the Initiation Rate of Radicals on the Kinetic Characteristics… 13

EXPERIMENTAL
Autooxidation of methyl oleate is carried out by the atmospheric oxygen in a thin layer at
323 K. The course of oxidation was followed by the accumulation of hydroperoxides
(ROOH), the concentration of which was determined by iodometric titration. The error of the
ROOH concentration determination did not exceed 1.5%. The antioxidant effectiveness was
evaluated by τ values, which were graphically determined from the kinetic curves of the
ROOH accumulation during the methyl oleate autooxidation in the absence and presence of
InH. As duration of the oxidation induction period (τ), we took the interval from zero to the
perpendicular dropped on the X axis from point of intersection of the linear plots of kinetic
curve of the ROOH accumulation: the initial oxidation rate within the induction period and
the maximal rate of the ROOH accumulation.
The initiated oxidation of methyl oleate in a mixture (1:1) with an inert solvent
(chlorobenzene) was performed by the air oxygen at 333 K. The reaction mixture containing
methyl oleate, chlorobenzene and the initiator – dinitrile of azoizobutyric acid, placed in
oxidative cell equipped with a magnetic mixer. The reaction mixture was thermostated at 333
K, and then InH was injected and measured the kinetics of oxygen uptake by using the
volumetric method. The initiation rate and the InH concentration were varied. The initial rate
of oxidation, as well as the value of the induction period τ, were determined from the kinetic
curves of oxygen absorption by method described in Ref. 15. The interval from the beginning
of the experience to the point of intersection of two straight lines for which tg α1 = 2 tg α2 was
taken as τ in the presence of InH. The first line is an extension to a straight of the oxygen
uptake, when the reaction rate is constant after the complete consumption of InH. The second
line is tangent to the kinetic curve of the oxygen uptake in the point, the reaction rate of
which is twice less than that in the absence of inhibitor.
The concentration of Q was determined spectrophotometrically at the wavelength λmax =
368 nm. The formation of the intermediate product is recorded by the absorption spectrum at
the wavelength λmax = 526 nm. To prepare the InH solutions, their sample is dissolved in
ethanol and then diluted by chlorobenzene. The proportion of ethanol in the oxidative cell is
not exceeded about 1.3 % (v/v). Methyl oleate was purified by vacuum distillation.
Dihydroquercetin and Quercetin (Sigma) were used without additional purification.
The kinetic data were processed by KINS program given in Ref. 16.

RESULTS AND DISCUSSION


Methyl oleate is one of the most used model system for the analyzing the antioxidant
properties of different individual InH and their mixtures, but the employment of the methyl
oleate autooxidation in the thin layer is rare. For this reason, in our research it was used the
methyl oleate autooxidation in thin layer. It is obtained that Q and QH2 were effectively
inhibited this process (Fig. 1). The dependence of τ on the initial concentration of these
antioxidants is closely to linear in the studied range of concentrations. The slope of
dependences of τ on the [InH]0 concentrations for Q and QH2 is significantly different.
Besides, there is a need to note that the antioxidant activity of Q is 1.6 times greater than for
QH2 (Fig. 1). The similar results were obtained during the oxidation of lard17: it was shown
14 L. I. Mazaletskaya, N. I. Sheludchenko and L. N. Shishkina

that τQ is 1.4 times greater then τQH2. Besides, our results are also consistent of the data
presented in Ref. 18 about the antioxidant activity of the two flavonoids – fisetin and fustin,
the molecular structure of which is also characterized by presence or absence of the C2 – C3
double bond. According to Ref.18, the IC50 values for fisetin and fustin during the NADPH-
dependent microsomal lipid peroxidation are equal 20.8 and 66 μmol, correspondingly, e. i.
their antioxidant activities differ about 3.2 times.

300 τ, h

3
250

1
200

150 2

100

50

0
0 0,5 1 1,5 2 2,5 3
4 -3
[InH]0×10 , mol dm

Figure 1. Dependence of the induction period (τ) on the antioxidant concentrations during
autooxidation of methyl oleate 1 – Q; 2 – QH2; 3 – TP

Hence, the obtained results and the literature data point to the fact that the presence of the
C2 – C3 double bond in the C-ring caused the conjugation at all rings in the flavonoid
molecule results in the increase of their antioxidant activity.
In order to clear the behavior of flavonoids during the oxidation, a detail computer
analysis of the kinetics of the methyl oleate autooxidation in the absence and presence of
flavonoids was performed. It was showed that, in all cases, the peroxide accumulation is well
described by the exponential law: [ROOH] = a×exp(kt), the correlation coefficients of which
are equal 0.99 – 1.0. Earlier the similar relationship was revealed for the methyl oleate
autooxidation when the oxygen concentration provides the oxidation in the kinetic range14.
The exponential index k, the value of which is proportional to the total oxidation rate14, is not
reliable differ for Q and QH2 and is not depend on their concentrations: k = (5.3 ± 0.6)×10-2 h-
1
and k = (4.94 ± 0.04)×10-2 h-1 for Q and QH2, correspondingly. Thus, the results of
computations indicate the complete consumption of flavonoids within the induction period.
The preexponential factor a of the kinetic curves of the peroxide accumulation, the values of
which are due to the rates of radical initiation and the chain propagation [14], is substantially
less in the presence of flavonoids than the same during the methyl oleate autooxidation.
Influence of the Initiation Rate of Radicals on the Kinetic Characteristics… 15

Furthermore, this parameter decreases with a growth of the flavonoid concentration (Fig. 2).
As can be seen from Fig. 2, the value a more significantly reduces during the methyl oleate
autooxidation inhibited by Q than that in the QH2 presence. These data corroborate the higher
antioxidant activity of Q as compared with that for QH2.

3
6 а ×10

4
2
3

2
1
1

0
0 0,5 1 1,5 2 2,5 3
4 -3
[InH]0 ×10 , mol dm

Figure 2. Dependence of the preexponential factor a on the quercetin (1) and dihydroquercetin (2)
initial concentrations.

Dependences of τ on the initial concentrations of flavonoids [Q]0 and [QH2]0 differ from
those for the natural antioxidant TP obtained under the same conditions. It can be seen from
the comparison of data, which are presented in Fig. 1. Unlike Q and QH2, the dependence of τ
on [TP]0 is nonlinear (Fig. 1, curve 3) in the range of the studied concentrations. TP inhibits
better the methyl oleate autooxidation compared with Q and QH2 at the low concentrations,
where the contribution of side-reactions was negligible. However, the most active of the
investigated flavonoids Q provides the some longer induction period compared with TP
already at [InH]0 = 2.5×10-4 mol dm-3 (Fig. 1).
The inhibitory action effectiveness is above noted to depend on side-reactions involving
the antioxidant. One of these reactions is the interaction of InH with the molecular oxidation
product – ROOH. As known, TP has the antiperoxide activity decomposing the methyl oleate
hydroperoxide already at room temperature [19]. In this work the reaction of Q and ROOH
was studied by the Q consumption under anaerobic conditions at 333 K. It is established that
the consumption rate of Q (WQ) is independent on [Q]0 at [ROOH]0 = const. in the range of Q
concentrations providing the linear chain termination. However, WQ increases with the
growth in the [ROOH]0 concentration at the fixed initial concentration of Q, as can be seen in
Fig. 3, The results obtained indicate that Q does not practically interact with ROOH under
experimental conditions. The observed consumption of Q is due to its interaction with free
16 L. I. Mazaletskaya, N. I. Sheludchenko and L. N. Shishkina

radicals forming during the thermal decay of ROOH. From the dependence of WQ on
[ROOH]0 presented in Fig. 3 the rate constant of the ROOH decay with the free radical
formation (k3΄) was calculated. It’s value is equal k3΄ = WQ /[ROOH]0 = 2.8×10-7 s-1.

8 -3 -1
4 W Q x10 , mol dm s

0
0 0,02 0,04 0,06 0,08 0,1 0,12
-3
[ROOH]0, mol dm

Figure 3. Effect of [hydroperoxide]0 on the initial rate of the quercetin consumption.

To compare the antioxidant action effectiveness of studied flavonoids with their


antiradical activity, the stoichiometric inhibition coefficient f and the fk7/k60.5 parameter,
where k7 and k6 are the rate constants of the antioxidant interaction with peroxyl radicals and
the square recombination of peroxyl radicals, correspondingly, were measured by the model
reaction of the methyl oleate initiated oxidation. The kinetic curves of the oxygen uptake
during the methyl oleate oxidation in presence of Q and QH2 have the particularly pronounced
induction period. It can be seen in Fig.4, curve 1 for Q. To calculate f, the τ values were
measured from the kinetic curves of the oxygen uptake at the different initiated rates (Wi) and
initial concentrations of InH. These data are given in Table. Then we made plot on the τWi –
f[InH]0 coordinates presented in Fig. 5. It is seen that these dependences are linear (Fig. 5).
From the slope of these straight lines the f values for flavonoids were calculated which are
equal fQ = 2 ± 0.2 and f QH2 = 2,6 ± 0.3. Thus, despite the fact that the molecules of these
flavonoids have the same number of OH groups there is a some difference among their
stoichiometric inhibition coefficients. The f value for Q obtained from kinetic curves of the
oxygen uptake is in good agreement with fQ = Wi/WQ = 2 calculated from the rate of Q
consumption (WQ) (Fig. 4, curve 1΄). As observed, there is the formation of coloured
intermediate product with the maximal absorption at the wavelength λ = 526 nm during the
initiated oxidation of the methyl oleate inhibited by Q. However, the practically complete
discolouration of the reaction mixture is observed by the induction period end (Fig. 4, curve
1΄΄). This result may indicate that the coloured intermediate product formed in the inhibition
reaction also possesses the antioxidant properties.
Influence of the Initiation Rate of Radicals on the Kinetic Characteristics… 17

0,8 70

0,7 1
60

0,6
50
'1
0,5
40
Absorption

V, a.u.
0,4
30
0,3

1'' 20
0,2

0,1 10

0 0
0 10 20 30 40 50 60 70 80 90
min

Figure 4. Kinetic curves of the oxygen uptake in the absence (the dotted line) and presence (1) of Q
([Q]0 = 1.25×10-4 mol dm-3) during the methyl oleate initiated oxidation at 333 K and Wi = 10-7 mol
dm-3 s-1.The Q consumption (1΄) and the formation of their conversion coloured product (1΄΄) under
the methyl oleate initiated oxidation

4 -3
τ W i × 10 , mol dm
6
2
5

1
4

0
0 0,5 1 1,5 2 2,5
4 -3
[InH]0 × 10 , mol dm

Figure 5. Plot of τWi versus [InH]0: 1 – Q, 2 – QH2; the methyl oleate initiated oxidation at 333 K.

From the kinetic curves of the oxygen uptake at the initial step the rates of the inhibited
oxidation (Wing) were determined. To calculate the rate constant of the Q and QH2 interaction
with peroxyl radicals of methyl oleate (k7), the following equation was used:
18 L. I. Mazaletskaya, N. I. Sheludchenko and L. N. Shishkina

ω = W0/Wing – Wing/W0 = fk7[InH]/(Wik6)0.5 = king[InH]/(Wik6)0.5,

where W0 is the oxidation rate of methyl oleate without additivies InH, Wi – the initiation rate.
Since Wing is determined at t > 0, the concentration of InH is calculated by equation [InH] =
[InH]0 – tWi/f. From the slope of straight lines presented in Fig. 6 the values fk7k6-0.5 were
calculated which are equal 55 and 25 (dm3 mol-1 s-1)0.5 for Q and QH2, respectively. For
comparison, the fk7k6-0,5 parameter for the well-known synthetic antioxidant 2,6-di-tert-butyl-
4-methylphenol (BHT) obtained under the same conditions was calculated as fk7k6-0,5 = 4,5
(dm3 mol-1 s-1)0,5 which was a good agreement with published data about its smaller reactivity
compared with flavonoids [10].

0.5 4 -3
ωW i x10 , mol dm
70
1
60

50

40
2
30

20

10

0
0 0,2 0,4 0,6 0,8 1 1,2 1,4
4 -3
[InH]0×10 , mol dm

Figure 6. Plot of ωWi versus [InH]0: 1 – Q, 2 – QH2; the methyl oleate initiated oxidation at 333 K.

Assuming k6 = 1×107 dm3 mol-1 s-1, king = f k7 were calculated as 1.7×105 and 7.9×104 dm3
-1 -1
mol s for Q and QH2, respectively. The obtained value king for Q is satisfactory agreement
with king = 4.3×105 dm3 mol-1 s-1 calculated from the kinetic curves for oxygen uptake during
the methyl liloleate oxidation (0,242 M in chlorobenzene), inhibited by quercetin [20]. Some
difference in values king can be associated with greater reactivity of peroxyl radicals from the
methyl liloleate compared with that from methyl oleate. From the values king the rate
constants of flavonoids with peroxyl radicals was calculated, which have values k7 = 8.5×104
and 3.0×104 dm3 mol-1 s-1 for Q and QH2, respectively. The ratio of parameters characterizing
the antiradical activity of Q and QH2 also shows that the presence of the C2 – C3 double bond
in the C-ring leads to a 2-fold increase in the inhibition constant. It is a good agreement with
the ratio = 2,6 which are calculated by the concentrations of flavonoids for 50% inhibition of
lipid peroxidation from the half-wave potential of the first oxidation wave measured by flow-
Influence of the Initiation Rate of Radicals on the Kinetic Characteristics… 19

through column electrolysis and the octanol/water partition coefficient for fisetin and fustin
presented in Ref. 18.
Thus, the data obtained allow us to conclude that Q has the highest rate constant
interaction with the peroxyl radicals (k7) which is 2,8 and more 10 times greater than that for
QH2 and BHT, respectively. The antioxidant activity of flavonoids and also their antiradical
properties is above note to depend substantially on the oxidation system [10,18,20]. It is
associated with the occurrence of the different side-reactions, the lipophilicity of flavonoids
or the hydrogen-bonding ability of solvents [10,11,18,20]. Our results showed that the
antioxidant activity of Q and QH2 correlates with the parameter fk7k6-0,5, characterizing their
antiradical activity under the different conditions of the methyl oleate oxidation. From the
data obtained, the magnitudes fk7k6-0,5 for Q and QH2 differ about 2 times while the inhibition
action effectiveness for Q is 1.6 times greater than that for QH2 calculated from the
dependence of τ on [InH]0 under the autooxidation condition. The existence of the direct
correlation between the induction period of the methyl oleate autooxidation in the presence of
flavonoids and the Q and QH2 concentrations indicates their primary interaction with peroxyl
radicals.

Table. Effect of initial rate of radicals on induction periods ( ) of the methyl oleate
oxidation in presence of antioxidants, temperature 333 .

InH [InH]0×104 Wi×107 τ


(mol dm-3) (mol dm-3 s-1) (min)
Q 0.2 0.3 22
Q 0.3 0.5 22.5
Q 0.6 1.0 18
Q 0.75 0.5 54
Q 1.25 1.0 42
Q 2.0 1.0 67
QН2 0.3 0.5 25
QН2 0.45 1.0 22
QН2 0.6 0.5 50
QН2 1.0 1.0 43
QН2 2.0 1.0 82

REFERENCES
[1] U. Takahama: Photochem. Photobiol. 38, 363 (1983).
[2] U. Takahama: Plant Physiol. 71, 598 (1983).
[3] V.A. Kostyuk, A.I. Potapovich, S.M. Tereshchenko, I.B. Afanas΄Yev: Biokhimiya, 53
(8), 1365 (1988) (in Russian).
[4] Yu.O. Teselkin, B.A. Zhambalova, I.V. Babenkova, G.I. Klebanov, N.A. Tyukavkina:
Biofizika, 41 (3), 620 (1996) (in Russian).
[5] M.R. Cholbi, M. Paya, M.J. Alcaraz: Experientia, 47 (2), 195 (1991).
[6] W. Bors, W. Heller, C. Michel, M. Saran: Methods Enzymol., 186. 343 (1990).
[7] V. Roginsky, L.A. Eduadro: Food Chem., 92 (2), 235 (2005).
[8] A.I. Potapovich, V.A. Kostyuk: Biokhimiya, 68 (5), 632 (2003) (in Russian).
20 L. I. Mazaletskaya, N. I. Sheludchenko and L. N. Shishkina

[9] S. Ya. Sokolov, N.A. Tyukavkina, V.K. Kolkhir, Yu.A. Kolesnik, A.P. Arzamastsev,
N.G. Glazova, V.A. Zyuzin, A.I. Baginskaya, V.A. Babkin, L.A. Ostroukhova: Patent
№ 2014841 (Russian Federation).
[10] V.A. Belyakov, V.A. Roginsky, W. Bors: J. Chem. Soc., Perkin Trans. II, (12), 2319
(1995).
[11] V.A. Roginsky, T.K. Barsukova, A.A. Remorova, W. Bors: J. Am. Oil Soc. 73 (6), 777
(1996).
[12] V.V. Naumov, R.F. Vasil΄Yev: Kitetika i kataliz, 44 (1), 111 (2003) (in Russian).
[13] L.I. Mazaletskaya, G.V. Karpukhina: Neftekhimiya, 24 (2), 502 (1984) (in Russian).
[14] N. V. Khrustova, L.N. Shishkina: Kinet. Catal., 45 (6), 799 (2004).
[15] N.M. Emanuel, G.P. Gladyshev, E.T. Denisov, V.F. Tsepalov, V.V. Kharitonov: The
testing procedure of chemical compounds as stabilizers of polymer materials. Preprint.
Chernogolovka, 1976, 35 p. (in Russian).
[16] E.F. Brin, S.O. Travin: Chem. Phys. Reports 10 (6), 830 (1990).
[17] S. V. Antoshina, А.А. Selishcheva, G.M. Sorokoumova, E.A. Utkina, P.S. Degtyarev,
V.I. Shvets: Prikladnaya biokhimiya i mikrobiologiya, 41 (1), 23 (2005) (in Russian).
[18] B. Yang, A. Konani, K. Arai, F. Kusu: Analytical Sciences, 17, 599 (2001).
[19] L.I. Mazaletskaya, N.I. Sheludchenko, L.N. Shishkina: Petroleum chemistry, 48, 105
(2008).
[20] P. Pedrielli, G.F. Pedulli, L.H. Skibsted: J. Agric. Food Chem., 49, 3034, (2001).
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.21-28 ©2010 Nova Science Publishers, Inc.

Chapter 3

AN ANTIOXIDANT FROM HINDERED PHENOLS


GROUP ACTIVATES CELLULOSE HYDROLYSIS BY
CELLOVIRIDIN IN A WIDE CONCENTRATION RANGE,
INCLUDING ULTRALOW DOSES

E. M. Molochkina1, Yu. A. Treschenkova1,


I. A. Krylov2 and E. B. Burlakova1
1
N.M. Emanuel Institute of Biochemical Physics,
Russian Academy of Sciences, Moscow, Russia
2
D.I. Mendeleev Russian Chemical-Technological University,
Moscow, Russia

ABSTRACT
For the first time, the effect of a synthetic antioxidant – an inhibitor of free-radical
processes, phenosan, on the cellulase activity was studied.
A considerable increase of product yield of microcrystalline cellulose hydrolysis by
celloviridin (a complex of cellulases from Trichoderma Viride) under the effect of a wide
range of concentrations of phenosan, including ultralow doses, not associated with its
antioxidant properties was observed.
The concentration dependence of the effect is complex and non-monotonic.
Ultralow concentrations of phenosan increase the product yield similarly or greater
than “usual” ones.
When increasing the process efficiency by phenosan at different concentrations of
celloviridin protein, it is shown that phenosan action (including ultralow concentrations)
allows a considerable decrease of the amount of an expensive enzymic complex
necessary for obtaining the target product.

Keywords: antioxidants, hindered phenols, hydrolysis, cellulose, ultralow doses, wide


concentrations.
22 E. M. Molochkina, Yu. A. Treschenkova, I. A. Krylovet al.

INTRODUCTION
While the population of the Earth increases and food resources based on non-food raw
materials and, which is the most important alternative sources of energy are actively searched
for, the interest to cellulose-containing substrates (CCS), which renewable resources are
almost unlimited, increased. Annually, about 1011 tons of plant biomass on the Earth is
hydrolyzed by microorganisms’ enzymes, and released energy is equivalent to 640 billion
barrels of oil. Therefore, it is clear why CCS enzymic hydrolysis using cellulase complexes
produced by microorganisms is considered to be the most perspective way for obtaining
alternative fuel [1].
Beside searching for and cultivation of new microorganisms – cellulase complex
producers, new approaches to increase the yield of end products under the effect of currently
used enzymic preparations shall be developed.
Celloviridin produced from Trichoderma Viride fungus in two variants – celloviridin
G20x and celloviridin G3x, is one of cellulase complexes used for CCS hydrolysis in Russia.
The preparations catalyze degradation of cellulose from a plant cell to oligosaccharides,
mono- and disaccharides by enzymes of three types: endoglucanase (Е.С. 3.2.1.4),
cellobiohydrolase (Е.С. 3.2.1.91) and beta-glucosidase (Е.С. 3.2.1.21). Cellulolytic activity of
celloviridin-V G20x equals 2000 ± 200 units/g. The content of enzymic system components
in celloviridin G3x is not constant, and its activity equals 100-200, 300-370 or 500-600
units/g [2].
Celloviridin production from microbiological material is rather labor consuming and
expensive. It is of importance to find possibilities of increasing the action efficiency of
cellulases of celloviridin complex and, thus, to reduce the amount of required celloviridin.
Celloviridin represents a complex set of components containing a biomass, in particular,
having lipids subjected to peroxidation (LPO), which products may affect the enzyme
activity.
To regulate LPO intensity in biological and chemical systems, antioxidants (AO),
including free-radical reaction inhibitors from the group of hindered phenols, are used.
Recently, it has been found that AO of this type relate to substances, which ultralow doses
(ULD) affect biological systems of different complexity [3].
In view of possible increase of process efficiency, we consider perspective to study the
effect of a wide range of AO concentrations from the class of hindered phenols – phenosan K
(potassium salt of beta-(3’,5’-ditert.butyl-4’-hydroxyphenyl)propane acid), on cellulose
hydrolysis by celloviridin complex.
Phenosan in the form of industrially produced acid is used in cattle breeding by inducing
the growth-stimulating action in the forage composition for the farm livestock (chicken
broilers, veals). It is assumed that phenosan acts as a bioantioxidant, preventing oxidation of
fat-soluble vitamins A and E in the combined fodder composition [4 - 6].
Being the inhibitor of free-radical oxidation, phenosan may stabilize celloviridin
preparation, because the latter may contain biomass components, lipids, in particular, capable
of easy oxidation, giving products capable of damaging enzymic proteins.
However, for us the main precondition of phenosan use is the fact that this substance is
one of the agents in ULD affecting various biological systems [3]. In particular, it is known as
a superactivator of enzymic activity, this effect being not associated with antioxidant
An Antioxidant from Hindered Phenols Group Activates Cellulose Hydrolysis… 23

properties. This fact is described in ref. [7], which shows that injection of this preparation to
smooth muscle cells of mouse aorta in the cellular culture in 10-18 M concentration induces
protein kinase C activation by 400%.
The aim of this work was to determine the presence and to estimate the level of phenosan
K effect on cellulose hydrolysis by celloviridin complex, using a wide range of AO
concentrations, including ultralow doses.
The tasks included:

1. Determination of microcrystalline cellulose (MCC) hydrolysis products yield - total


sugars and glucose - in different periods after initiation of the process, carrying out
the reaction in the presence of various phenosan concentrations, including ultralow
doses.
2. Investigation of phenosan effect on the target product yield at different
concentrations of celloviridin.

EXPERIMENTAL
In the work microcrystalline cellulose (MCC) by Merck Company (Microcrystalline
Cellulose, Charge Nr.: 5010160202) was used; enzymic preparation celloviridin G3x by OAO
(BK) “Vostok” was presented by Biotechnology Department, D.I. Mendeleev RCTU. The
studies were performed on two celloviridin G3x samples taken from different batches of
preparation (CV-1 and CV-2 samples), which demonstrated different cellulolytic activity
estimated by the rate of total sugars accumulation with the help of phenol-sulfuric method [8,
9]. Activity of CV-1 sample estimated by this method was by 65% higher than activity of
CV-2 sample.
To purify the “grout” of the initial celloviridin powder in the acetate buffer (pH) from
components insoluble in the aqueous medium (filler, biomass residues) and to obtain samples
containing cellulases soluble in the aqueous medium, the “grout” was centrifuged, and
supernatant fluid was used in the work. Protein concentration in supernatant fluid was
determined by the Lowry method [10].
Synthetic AO phenosan K was synthesized in N.M. Emanuel Institute of Biochemical
Physics, Russian Academy of Sciences.
The rate of cellulose enzymic hydrolysis was estimated by accumulation of total sugars,
which quantity was determined by the phenol-sulfuric method [8, 9], and glucose, which
concentration was determined by glucose oxidase test [11] using the assay kit “Novogluk-K,
M”.
The reaction was performed in round-bottomed flasks in the acetate buffer solution with
pH 4.5, containing 50 mg/ml of cellulose. CV “solution” was added to the reaction vessels so
that the final concentration of protein containing the enzymic complex equaled 0.15-0.62
mg/ml. Flasks were suspended on a rubber hose strained over the thermostat bath so that their
bottoms were dipped into the water. The incubation temperature was 40°C. Rather intensive
and uniform mixing of the reaction mixture was provided by flasks swinging due to water
motion in the thermostat. To each flask solution of the substance under test in corresponding
concentration or acetate buffer solution (in the control) was added. The stock phenosan K
24 E. M. Molochkina, Yu. A. Treschenkova, I. A. Krylovet al.

solution (10-3 М) was prepared by dissolving a weighed portion of the preparation in the
acetate buffer solution with pH 4.5. The effect of phenosan was studied at its concentration in
the reaction mixture ranged within 5⋅10-17 − 10-5 M, every time adding solutions of
corresponding concentrations obtained by subsequent dilutions of the primary solutions by
100 times into reaction vessels.
From celloviridin lipids were extracted and concentrations of total lipids and
phospholipids were determined, using the guidelines [12].
The results were treated and graphically designed using the software Sigma Plot for
Windows v. 8 (demo).

RESULTS AND DISCUSSION


Celloviridin analysis performed demonstrated that the concentration of lipids in the solid
preparation is 0.001±0.0001 g/g, and concentration of phospholipids (the main LPO
substrates) is 0.1±0.01 mg/g (of about 1.2·10-4 М). For possible effect of their oxidation
products on enzymic proteins in the solid celloviridin, this is rather high concentration, and
addition of an antioxidant may stabilize potential cellulase activity of the powder.
However, the aim of this work was to estimate the possibility of direct phenosan
participation in the enzymic process. Therefore, it was added to the reaction mixture together
with the homogeneous solution containing enzymic proteins, obtained from celloviridin
powder (see Experimental).
Figure 1 illustrates the phenosan effect on the yield of products of MCC hydrolysis by
celloviridin. It shows the dependence of the amount of sugars formed on the antioxidant
concentration in the incubation medium 30, 60 and 120 min after the process beginning.
Every column represents the average value obtained from three-four parallel measurements ±
SE.
It is obvious the intense stimulating effect of phenosan on the yield of total sugars and the
target product (glucose).
The dependence of sugars yield on the phenosan concentration is complex and
nonmonotonous. Such dose dependence resembles the “dose-effect” curve characteristic of
substances (of various classes) capable of acting in ultralow concentrations [3]. As mentioned
above, phenosan also studied on some biological systems, manifested itself as the agent of
this kind. Enzymes also are the targets affected by its ultralow doses. These are, for example,
soluble and membrane-bound acetylcholinesterase, protein kinase C and lactate
dehydrogenase [3, 7, 13, 14]. Now cellulases can be added to this list.
As shown in the figure, this effect of phenosan in ULD is either comparable with its
action in “usual” concentrations (10-7 - 10-5 М), or is much greater expressed. (For “usual”
AO concentrations the concentrations are taken, in which AO from the group of hindered
phenols are able to inhibit free-radical oxidation processes).
The glucose percentage in total sugars in the presence of phenosan either increased
insignificantly compared with the control (except of a considerable increase from 13 to 23%
in the case of 30-minute incubation with 10-4 М) or even demonstrated a tendency to
decrease. This indicates that, apparently, the effect of phenosan mostly stimulates the stages
associated with the pool of enzymes catalyzing formation of oligo- and disaccharides.
An Antioxidant from Hindered Phenols Group Activates Cellulose Hydrolysis… 25

Figure 1. The yield of total sugars and glucose in different times from the incubation beginning in the
presence of different phenosan concentrations. CV-1 protein content in the incubation medium is 0.62
mg/ml. Abscissa axis - common logarithm of phenosan concentration; ordinate axis – concentrations of
total sugars (at the top) and glucose (at the bottom) in mg · ml-1.
26 E. M. Molochkina, Yu. A. Treschenkova, I. A. Krylovet al.

Figure 2.Phenosan effect on the initial accumulation rate and yield of glucose 2 h after the process
beginning at different celloviridin (CV-2) protein concentrations in the reaction mixture. At the right –
phenosan concentrations used. Arbitrary units – mg · ml-1· min-1· 104

Thus the expressed increase of the product yield of MCC hydrolysis by celloviridin
complex under the effect of a wide spectrum of phenosan concentrations, including ultralow
doses was observed. Ultralow concentrations of phenosan increase the product yield similarly
or greater than “usual” ones.
An Antioxidant from Hindered Phenols Group Activates Cellulose Hydrolysis… 27

Apparently, the observed action of the preparation, even in “usual” concentrations, is


associated not with its antioxidant (antiradical) properties, but with direct effect on the
enzymic system components.
The results obtained allow a suggestion that the phenosan effect on productivity and
weight gain of livestock (associated with its antioxidant activity) used in the agriculture may
be contributed by its activating effect on cellulases. Apparently, ultralow dose may have more
expressed effect than those currently used.
At the next stage we studied the phenosan effect on the target product (glucose)
formation in cellulose hydrolysis with different celloviridin concentrations. In this part of the
work, CV-2 was used. Figure 2 shows results on the phenosan influence on glucose formation
at different CV-2 concentrations.
As CV-2 protein concentration in the reaction mixture increased from 0.15 to 0.62
mg/ml, the initial reaction rate and glucose yield 2 h after beginning also increased. All
studied phenosan concentrations caused similar stimulating effect on these parameters. From
the Figure 2 one may calculate that in the presence of phenosan reaching of the initial rate of
glucose formation equal to control requires almost twice less celloviridin; obtaining of the
yield of glucose 2 h after the reaction beginning equal to control requires 1.5 times lower
amount of the enzyme preparation. Thus, the possibility to economize the expensive enzyme
complex applied in the industry for CCS hydrolysis using phenosan to stimulate the process,
including in ULD, is obvious.

CONCLUSIONS
At this stage of investigation, we may not indicate what namely components of the
studied enzymic system and stages are touched by the phenosan action, and what the
mechanisms of its influence are. Nevertheless, the fact of stimulating effect of this antioxidant
on the action of the complex cellulase system may be considered established. As a prospect,
this may be used for production of fuel and foodstuff from CCS. Since cellulase complexes
obtained from different microorganisms act by similar mechanisms, apparently, phenosan will
also activate other cellulase complexes produced from microbiological materials more
effective than Trichoderma Viride.

ACKNOWLEDGMENTS
The authors are thankful to T. Onishchenko (Murashova) and M. Zakharov for great help
in the experimental work.

REFERENCES
[1] Rabinovich M.L.// Prikl Biokhim Mikrobiol. 2006. V.42(1).P.5-32.
[2] http://www.sibbio.ru/products/bird/celo_bird.php
28 E. M. Molochkina, Yu. A. Treschenkova, I. A. Krylovet al.

[3] Burlakova E.B., Konradov A.A., Maltseva E.L. // Khimicheskaya Fizika. 2003. V.
22(2). P. 21-40. (in Russian)
[4] http://www.barva.com.ua/page.php?14
[5] http://www.alvi.tamb.ru/production/fenozan_kisl1.htm
[6] http://chemindustry.ru/rus/chemicals/DiButylOxyphenylPropionicAcid.php
[7] Maltseva E. L., Palmina N. P., Burlakova E. B. // Membr Cell Biol. 1998. V.12(2).
P.251-268.
[8] Klesov A. A., Chernoglazov V. M., Rabinovich M. L., Glazov M. V., Adamenkova M.
D. // Biokhimia. 1983. V.48(9). P.1411-1420 (in Russian).
[9] Pustovalova L. M. Laboratory Manual on Biochemistry. Rostov-on-Don. Phoenix.
1999. 542 P. (Russian)
[10] Lowry O. H., Rosebrough N. J., Fare A. L., Randall R. J.// J Biol Chem. 1951.V.193.
N1. р. 265-275
[11] Klesov A. A., Grigorashch S. Iu. // Biokhimia. 1980. V. 45(2). P. 228-241. (in Russian)
[12] Kates M. Techniques of Lipidology: Analysis, Isolation and Identification of Lipids.
American Elsevier Pub. Co. Inc. New York. N.Y. 10017. 1973
[13] Molochkina E. M., Ozerova I. B. // Radiats Biol Radioecol. 2003. V. 43(3). P. 294-300
(in Russian)
[14] Treschenkova Yu. A., Burlakova E. B., Goloschapov A. N. // Radiats Biol Radioecol.
2003. V. 43(3) P.320-323 (in Russian)
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.29-43 ©2010 Nova Science Publishers, Inc.

Chapter 4

A-TOCOPHEROL AS MODIFIER OF THE LIPID


STRUCTURE OF PLASMA MEMBRANES IN VITRO IN A
WIDE RANGE OF CONCENTRATIONS
STUDIED BY SPIN-PROBES

V. V. Belov, E. L. Maltseva and N. P. Palmina*


N.M. Emanuel Institute of Biochemical Physics RAS, Moscow, Russia

ABSTRACT
α-Tocopherol (α-TP) is an effective natural antioxidant and important component of
biological membranes localized in the lipid bilayer and capable of changing their
structural dynamic state. . For this reason, it was of interest to study α-TP effect in a wide
range of concentrations (10-25 М - 10-4 М) on viscosity parameters and thermally-induced
structural transition of the lipid bilayer of plasmatic membranes of liver cells of mice in
vitro. The changes of structural parameters, namely, the order parameter of surface and
micro-viscosity of hydrophobic regions of the lipid bilayer in membranes, were measured
on Bruker EMX ESR-spectrometer (Germany) by the spin probe method by use two
stable nitroxyl radicals - 5- and 16-doxylstearic acids localized at the different depths in
the membrane ~8 A0 and ~20 A0 correspondingly. The “dose - effect” dependencies were
nonlinear and polymodal, with statistically reliable increases of viscous parameters of the
membrane in three ranges of α-TP concentrations: (1) in the range of traditional
“physiological” concentrations of 10-9 – 10-4 M; (2) in the range of ultra-low doses of 10-
17
– 10-9 М, and even (3) in the range of apparent concentrations or dilutions of 10-25 – 10-
17
М. The mechanisms of the effect of α-TP in each of these ranges localized in the lipid
bilayer are discussed. By study the temperature dependencies of micro-viscosity value a
new thermally induced structural transition has been found at “physiological”
temperatures of 309 – 3130K for α-TP concentrations, including ultra-low doses, to which
maxima on dose dependencies.

* Corresponding Address to: N.P. Palmina (Ph.D., D.Sci.), IBCP RAS, Kosygin str.4, 119334 Moscow, Russia
30 V. V. Belov, E. L. Maltseva and N. P. Palmina

Keywords: α-tocopherol (α-TP), ultra-low doses (ULD), spin probes, microviscosity and
rigidity of lipids, thermo- induced structural transitions in lipids (TIST)

AIMS AND BACKGROUND


In biological membranes α-tocopherol (α-TP) performs an important function of
preserving its wholeness by participating in regulation of peroxide oxidation of lipids (POL)
as one of the most effective natural antioxidants [1, 2]. Due to its lipophily and localization in
all cell membranes α-TP affects their structural state which is a central unit in the POL cycle
and on which, in turn, activity of many membrane-bound enzymes participating in the most
important biochemical processes in the cell depends [3].
At present, the function of α-TP that modifies the membrane structure is studied only in a
rather limited range of physiological concentrations; meanwhile, the problem of biologically
active substance (BAS) action in ultra-low doses (ULD) becomes more and more urgent [4 -
6]. On this subject a broad experimental material (references of many works are present in the
review [4]) is accumulated; however, the mechanisms of phenomenon are not known
completely. Hence, it is suggested that cellular and subcellular membranes may be one of
critical targets. Previously, in a series of researches [7, 8] performed in our laboratory as well
[9 – 13] it has been found that ULD of BAS injected to the laboratory animals, cellular
culture or suspension of biological membranes changed the structural characteristics of the
membrane lipids [13]. The plasma membranes are of special interest as a localization of some
vitally important regulatory systems, which define general metabolic status of the organism,
secondary messenger systems, in particular [14, 15]. This was the reason for performance of
this work, aimed at the study of α-TP effect on the plasma membrane structure isolated from
mice liver cells in a wide concentration range (10-25 - 10-4 М) in vitro.

EXPERIMENTAL
Plasma membranes were extracted from liver cells of 40-50 mice of F1(C57xDBA2) line
by consequent centrifuging, using the method described in [16]. Protein concentration was
determined by Lowry method [17]. The membranes obtained were resuspended in the
extraction medium; the protein concentration was increased to 2.5 mg/ml of suspension.
Thereafter, this suspension was poured into 1 ml Eppendorf tubes and stored in a freezer at -
50°C. In the day of experiment the required quantity of membranes is defrosted, the
suspension was shaken and used in the work. A specimen for measurements on ESR
spectrometer represented a non-viscous liquid suspension of membranes in the SET
extraction medium (pH 7.3) containing saccharose (0.25 M), tris-HCl (10 mM) and EDTA (1
mM). This solution (150 μl) was placed to a long, narrow quartz trough with high Q-factor.
This provided homogeneous temperature by the trough volume. Thermostatics accuracy was
controlled by a thermocouple placed to the resonator cavity. By thermal accessory ER 4131
VT, this accuracy equaled ”0.05°C. Record of ESR spectra performed every 3-5 minutes.
After multiple recording of spectra in the control the membranes were moved to a flask and
a-Tocopherol as Modifier of the Lipid Structure… 31

added by α-TP at any concentration. The suspension was thoroughly blended and placed back
to the trough and then to the ESR resonator cavity. Thus, control and test measurements were
performed for the same membranes. α-TP alcohol-aqueous solutions were obtained by
consecutive dissolution in a quartz dish next by one order of magnitude from its initial 10-1 M
solution in alcohol of advanced purification by MERCK Company (Germany) down to 10-3
M, and then by distilled water. Liquid was taken from the volume by semiautomated pipette
with one-off nozzles. All solutions were blended in an electric shaker with controlled speed
and time of 1 min. Dynamic state of the membrane lipids was studied by the spin probe
method [18, 19] on Bruker EMX ESR-spectrometer and was described by structural
parameters at constant temperature of 293 K – order parameter (S) which means rigidity of
surface areas of lipids and micro-viscosity of deep hydrophobic regions, as well as thermally
induced structural transitions in the lipid bilayer. The required parameters were calculated
from ESR spectra obtained in three accumulations, in semiautomatic mode with the help of
the original software in Origin 6.1 environment. The spin probe - stable nitroxyl radical of 5-
doxylstearic acid (probe C5) – was used to study the rigidity of the surface lipid slices of the
membrane (Fig.1a). Rigidity of the membrane in the probe localization zone (~8A0) was
described by the order parameter - S [19], which depends on the variation amplitude of probe
rotation transversal line from the middle orientation of surrounding lipid molecules:

TII − T⊥ 1
S= ⋅ ,
1 k
T zzc− (Txxc + Tyyc )
2

TII + 2T⊥
where k=
Txxc + Tyyc + Tzzc

is a correction factor for difference in monocrystal and experimental specimen polarities.


Txxc , T yyc , Tzzc are principal values of hyperfine interaction (HFI) tensor obtained for
monocrystal. In the case of fast rotation of spin probes about their molecular axis (S< 0.8), the
HFI tensor becomes axially symmetrical, and its principal values TII and T⊥ averaged by
motion may be obtained from maximal and minimal splits 2Tmax and 2Tmin at the ESR
spectrum (Fig. 1a):

TII = Tmax ,

T⊥ = Tmin + 1.32 + 1.86 lg(1 − S пр ) ,

Tmax − Tmin
where S пр =
1
Tzzc − (Txxc + T yyc )
2
32 V. V. Belov, E. L. Maltseva and N. P. Palmina

is the approximate orderliness parameter without regard to corrections for polarity.

5-doxylstearic acid 16-doxylstearic acid


(probe С5) (probe С16)
OH
O N O N O
O O
OH

a b

Figure 1. The ESR spectra and structural formulae of С5 (a) и С16 (b) spin probes localized in plasma
membranes of liver cells in mice. The probe concentration is 6×10-5 M. Protein concentration is 2.5
mg/ml. Temperature is 293 K.

To study the micro-viscosity of the deep hydrophobic regions of the lipid bilayer the
stable nitroxyl radical – 16-doxylstearic acid (probe C16) was used as spin probe (Fig.1b). In
the localization zone of this probe (~20A0) the micro-viscosity value was characterized by its
rotational correlation time, τс which is numerically equal to time required for C16 probe to
turn around its longitudinal axis by π/2 angle. It was calculated by the formula used for fast
anisotropic rotation of the radical [18]:

I0
τ с = 6,5 ⋅ ΔН 0 ( − 1) ⋅ 10−10 , s
I−

Spectrum parameters in the formulae for S and τс were measured automatically, using
original software in the Origin 6.1 environment with standard extreme search algorithms at
the given sampling interval of 0.06 Gs. For instance, measuring error for the distance between
internal and external extremes in the well resolved ESR spectrum of the probe C5 did not
exceed 0.2 Gs. The main contribution to the error of mean S and τс determination was made
by the random component determined, first of all, by statistic character of the values under

( )
study: Sϕ
random
=
t (95%, f ) * σ
n
(where ϕ is τс or S parameter calculated from the

experimental data). As a result, for τс, relative random error of the mean value determination
a-Tocopherol as Modifier of the Lipid Structure… 33

did not exceed 1%, and for S – 0.15%. In each particular experiment, calculation error for
separate τс and S values did not exceed 0.04 ns and 0.002, respectively. Since we operated
with a homogeneous material, deviations from the mean values obtained with all controls
considered for membranes of a single extraction coincided with the mentioned values. The
analysis of literature data shows the precision of our results to deviations of τс and S values
obtained for the different membranes [20 – 24].
To obtain temperature dependencies of dynamic characteristics of the membrane lipids,
the sample was thermostatically controlled with the accuracy ±0.050K on a thermal accessory
ER 4131 VT by Bruker Company. The measurements were performed in the temperature
range of 285 – 320 K with 2 K step. Thermo-induced structural transitions in the lipid bilayer
were demonstrated by salient points between linear sections of temperature dependencies
plotted in Arrhenius coordinates: Lg (dynamic parameter) vs 1/T. Salient points were the
points added to straight line put the correlation coefficient out of the norm defined by the
number of degrees of freedom and 95% statistical reliability.
For each α-TP concentration, on each membrane extraction 3-5 parallel determinations of
the effect were performed; totally, 2 series of experiments on membranes isolated in different
times of year were performed. The average effects of α-TP are shown on graphs, expressed in
percents in relation to control, obtained after statistical treatment of all these results by
parametric and nonparametric statistics methods using Statistica and Origin 6.1 software
packages with 95% statistical reliability.

RESULTS AND DISCUSSIONS


Structural parameters of the membrane lipids are important to describe its structural
dynamics state at the given temperature. Therefore, it was studied the effect of α-TP on the
rigidity of surface areas and microviscosity of deep lipid regions of plasma membranes at 293
K. These data are shown in Figs. 2a and 2b, and are expressed in percents in relation to the
control. Hence, each point is the result of averaging of 6 to 10 independent measurements (for
every concentration). The dose dependencies shown are polymodal, which is typical of BAS
action in a wide range of concentrations, including ULD, where maxima corresponding to
increase of a S and τс observed in definite dose ranges, separated from one another by the so-
called “dead zones”, in which effect is not displayed. In the range of traditional
“physiological” concentrations (10-9 – 10-4 М), in which α-TP usually acts in the organism,
and in the ULD range (10-17 – 10-9 М) the effects are observed in both surface and deep
hydrophobic regions of the membrane lipids. However, in the range of the so-called apparent
concentrations or dilutions (<10-17 М), when the probability of containing even one molecule
in the membranes is close to zero, the effect is observed exclusively in the depth the
membrane. It should be noted that the values of α-TP effects in all three dose areas are
similar. Hence, the effect in the surface areas of plasma membrane lipids is markedly higher
than in microsomal membranes obtained by us [13].
34 V. V. Belov, E. L. Maltseva and N. P. Palmina

3.5
a
3.0

2.5

2.0
EFFECT, %

1.5

1.0

0.5

0.0

-0.5
4 6 8 10 12 14 16 18 20 22 24 26
-Lg[α-tocopherol]

8
b
7

5
EFFECT, %

-1
4 6 8 10 12 14 16 18 20 22 24 26
-Lg[α-tocopherol]
Figure 2. The effect of α-TP on the rigidity of surface (a) and microviscosity of deep hydrophobic (b)
regions of plasma membranes at 293 K. Protein concentration in the membrane suspension is 2.5
mg/ml. The control values: S = 0.644±0.004, τс = (2.10±0.04)×10-9 s.

Beside changes of the main parameters (τс and S) of the lipid bilayer at constant
temperature, very important characteristics described the dynamic state of the membrane
lipids are the quantity and quality of thermally induced structural transitions. They represent
a-Tocopherol as Modifier of the Lipid Structure… 35

cooperative structural transformations of groups of lipids with temperature increase, which


are accompanied by a discontinuous change of the main parameters (τс and S). As described
in “Materials and Methods”, for the purpose of detecting transitions the temperature
dependencies of τс and S for the control and α-TP concentrations, to which maxima or the
absence of effects on dose dependencies (Fig. 2) correspond, were presented in the Arrhenius
coordinates – Lg (dynamic parameter) on 1/T (Figs. 3 and 4). The main data on location of
transitions in the hydrophobic regions of lipid bilayer are summarized in Table 1, and on
surface lipid areas - in Table 2. Despite the fact that thermo- induced structural transitions
occur in different depth areas of the membrane, some general regularities are observed. For
instance, compared with the control characterized by two transitions, all α-TP concentrations,
to which maxima on dose dependencies (Figs. 2a and 2b), including ULD, induced the third
additional transition. And ULD - 10-15 М of α-TP in the surface areas of the membrane lipids
also induced the fourth transition in the range of “physiological” temperatures at 309 – 313 K
(dashed). The total number of transitions does not change for concentrations corresponding to
“dead zones” on the dose dependencies in Figs. 2a and 2b. However, compared with the
control, they shift towards higher temperatures, including “physiological” ones. Taking into
consideration an important role of phase transitions in lipids of biological membranes in the
vital activity of the cell (in the change of membrane permeability, pore formation, fusion of
membranes, excitability of nerve tissues and nervous pulse conduction by axon,
thermoregulation, synaptic exocytosis, etc. [25, 26], it was suggested that thermo-induced
transitions observed by α-TP may be regulatory for the cells and their membranes.

Figure 3. The temperature dependencies of rotation correlation time - τс of C16 presented in Arrhenius
coordinates in control and at α-TP effect in 10-4, 10-7, 10-10, 10-14, 10-18 and 10-22 M concentrations in
vitro. Maximal relative error of values Lg [τс] obtained in the series of three measurements for each
temperature did not exceed 0.2%.
36 V. V. Belov, E. L. Maltseva and N. P. Palmina

Figure 4. The temperature dependencies of order parameter - S of C5 presented in Arrhenius


coordinates in control and at α-TP effect in 10-4, 10-9, 10-15 and 10-21 M concentrations in vitro.
Maximal relative error of values Lg[S] obtained in the series of three measurements for each
temperature did not exceed 0.2%.
a-Tocopherol as Modifier of the Lipid Structure… 37

Table 1. The thermo-induced structural transitions in hydrophobic lipid regions (~20


A0) of liver plasma membranes in control and under the effect of -TP at different
concentrations in vitro.

Т,К control 10-4М 10-7М 10-10М 10-14М 10-18М 10-22М


285
287
289
291
293
295
297
299
301
303
305
307
309
311
313
315
317
319
Note: The structural transitions are marked grey.

Table 2. The thermo-induced structural transitions in surface (~8 A0) of the lipid bilayer
of liver plasma membranes in control and under the effect of -TP at different
concentrations in vitro.

Т,К control 10-4М 10-9М 10-15М 10-21М


285
287
289
291
293
295
297
299
301
303
305
307
309
311
312
313
315
317
319
Note: The structural transitions are marked grey.
38 V. V. Belov, E. L. Maltseva and N. P. Palmina

As mentioned above, polymodal type of dose dependencies shown in Figs. 2a and 2b is


promoted by α-TP effects in three dose ranges. However, the main contribution in mechanism
of α-TP action in each range may be made by different processes.
Primarily, ordering of the surface lipids and increase of micro-viscosity of hydrophobic
regions of the lipid bilayer under the effect of α-TP at “physiological” concentrations (10-9-–
10-4 М) may be associated with restrictions for packing of hydrocarbon chains of lipids near
α-TP molecule reduced their conformational mobility [27]. A significant contribution to these
processes can be made by α-TP interaction with phospholipids molecules. The different
biophysical methods give indirect evidences of not random distribution of α-TP in the
membrane, but formation of complexes with definite stoichiometry [28 - 30]. Moreover, an
evidence of hydrogen bond formation between hydroxyl group of α-TP molecule and oxygen
either carbonyl or phosphate group of phospholipid molecules is present in [31]. However,
lateral distribution of the complexes in the membrane plane may be random, and they may
also form domains and induce phase separation [32]. Thus, being well mixed with
phospholipids, α-TP may cause a significant effect on their structural transformations. The
evidence for this opinion is occurrence of additional third thermo-induced structural transition
in the “physiological” temperature range in the depth of lipid bilayer under the action of α-TP
in 10-4 and 10-7 М concentrations (Table 1) and in surface membrane lipids for 10-4 М
concentration (Table 2). To our point of view, this is associated with formation of such
domains with denser packing of lipids. The increase of viscosity or decrease of fluidity of
membrane lipids in the processes of penetration of α-TP at “physiological” concentrations
have been demonstrated for many times in experiments on biological (see [33, 34], for
example) and artificial membranes by different physical methods, including ESR [27, 35, 36].
Our data correlate with these results.
In the ULD range (10-17 – 10-9 M) the effect in the surface lipids of plasma membranes is
markedly higher than in microsomal membranes studied by us before [13]. One of the
possible mechanisms may be specific interaction between α-TP and binding sites of the
membrane that, judging by data from the literature [7] and our results [9], can lead to a
change of viscosity properties of the membrane. We have suggested that in this case the role
of such binding site may be played by protein kinase C (PKC), the membrane-bound and
lipid-dependent enzyme, which content in plasma membranes is considerably higher than in
microsomal membranes. It was obtained the high correlation degree (ρ = 0.0047, r = 0.87)
between order parameter (S) of surface lipids of plasma membranes and inhibition of PKC
activity by α-TP in ULD [37]. This fact may be an indirect evidence of such kind mechanism
(Fig. 5).
To our point of view, another probable explanation of increase of the lipid micro-
viscosity under α-TP action in ULD may be given by α-TP induced initiation of new highly
ordered microdomain complex formation or modification of already existing ones. In this
case, the role of such complexes may be played by rafts representing special areas in the
plasma membrane, which demonstrate much higher ordering compared with their
microsurrounding due to increased content of cholesterol, sphingomyelin and gangliosides,
and mostly saturated fatty-acid chains of phospholipids [38, 39]. It is known that a lot of
proteins participating in signal transduction in the cell are capable of being localized in rafts
including PKC as well [39, 40]. The lifetime of such complexes is short (less than 1 ms);
however, the action of external signals (which role may be played by ligands) inducing
a-Tocopherol as Modifier of the Lipid Structure… 39

conformational changes in the protein molecules, can extend it significantly (up to several
minutes) and, moreover, lead to association of several rafts into a larger domain [40, 41].
Therefore, we suggest that such processes may be accompanied by the interaction of α-TP in
ULD with PKC during inhibition of enzyme activity.

Figure 5. The change of rigidity of the surface membrane lipids and inhibition of protein kinase-C
activity in vitro depending on α-TP concentration (a) and correlation between these parameters (b), ρ =
0.0047, r = 0.87.
40 V. V. Belov, E. L. Maltseva and N. P. Palmina

To our point of view, the presence of α-TP in the membrane in both “physiological”
concentration and ULD may also affect formation of rafts. For instance, as one of the
mechanisms of their formation, spatial incompatibility between solid styrene structure of
cholesterol molecule and rigid bending of unsaturated hydrocarbon chain of the neighboring
phospholipid molecule, having cis-double bond in С9-С10 position, in the liquid-crystal
phase is considered [41]. Such mutual packing of molecules leads to pushing cholesterol out
of the areas with unsaturated phospholipids, with its consequent concentration and
stabilization in the areas with mostly saturated fatty-acid chains of phospholipids. It should be
noted in this connection that α-TP, in turn, more preferably interacts with poly-unsaturated
phospholipids [29, 42], thus promoting cholesterol pushing out and simplifying formation of
rafts at both “physiological” concentrations and ULD.
However, in the range of so-called “apparent” concentrations (<10-17 М), where the
probability of containing even one α-TP molecule in the membrane is close to zero, the effect
was observed exclusively in the deep hydrophobic regions of the membrane lipids. As shown
in our previous work [9] the properties of polar solvent (water) of α-TP make a considerable
contribution into the mechanism of transmission of “information” about the substance, in one
form or another, during preparation of solutions and further interactions on the way of signal
transmission to the membrane. This, finally, provides the effects obtained. Unfortunately,
today we fail to know the precise molecular mechanism of the effects in the range of
“apparent” concentrations. However, some experimental data [43, 44] draw attention to
dynamic characteristics of the polar medium in these processes (for instance, frequency of
formation and degradation of short-living aqueous associates, and interaction between them),
which are rather sensitive to external impacts and variation will affect its dynamic structure.
To conclude the consideration, our work is the first presenting the study of α-TP effect in
a wide range of real and “apparent” concentrations (10-25 – 10-4 M), on lipid structure
characteristics of liver plasma membranes in vitro. Dose dependencies are polymodal with
statistically reliable increase of rigidity of surface (~8 A) and microviscosity of deep
hydrophobic areas (~20 A) of membrane lipids caused by different mechanisms of α-TP
action into three dose ranges: (1) 10-9 – 10-4 M – nonspecific effect on the lipid membrane
structure at incorporation into it; (2) 10-17 – 10-9 M – specific interaction with the binding sites
on the membrane surface; (3) 10-25 – 10-17 M – polar properties of α-TP solvent (in the surface
areas of the plasma membrane the effect is not observed). The temperature dependencies of τс
and S demonstrated occurrence of new thermo-induced structural transition compared with
the control in the range of “physiological” temperatures of 309 – 3130K at α-TP
concentrations (included ULD) giving the maximal effects at 2930K.
The authors are greatly thankful to Professor E.B. Burlakova for scientific discussion.
This work is supported by Grant No. 01-RAS-15 of the Federal Program of Fundamental
Research for RAS Department of Chemistry and Material Sciences “Biomolecular and
Medical Chemistry”

REFERENCES
[1] E.B. Burlakova., S.A. Krashakov, N.G. Krapova: The Role of Tocopherols in
Biomembrane Lipid Peroxidation. Membr. Cell Biol., 12, 173 (1998).
a-Tocopherol as Modifier of the Lipid Structure… 41

[2] P.J. Quinn: Molecular Associations of Vitamin E. Vitam. Horm., 76, 67 (2007).
[3] E.B. Burlakova, N.P. Pal’mina N.P., E.L. Mal’tseva: In: Membrane Lipid Oxidation.
Vol. 3. (Ed. Carmen Vigo-Pelfrey). CRC Press. Boca Rato-Ann Arbor- Boston., 1991,
209-237.
[4] E.B. Burlakova., A.A. Konradov, E.L. Mal’tseva: Effect of Super-Small Doses of
Biologically Active Substances and Low-Intensity Physical Factors. J. Adv. Chem.
Phys. 2, 140 (2003).
[5] I.P. Ashmarin, E.P. Karaseeva, T.V. Lelekova: To the Question about Development of
the Problem of Efficacy of Supersmall Doses of Biologically Active Substances. Russ.
Chem. J. 18, 21 (1999).
[6] S.V. Zaitsev, A.M. Efanov, L.A. Sazanov: The Common Phenomena and Possible
Mechanisms of Action of Biologically Active Substances in Super-Small Doses. Russ.
Chem. J. 18, 28 (1999).
[7] A.S. Polezina, K.A. Anikienko, V.K. Kurochkin: Elaboration of Methodological Basis
to Detect Damage Effect of Super-Small Concentrations of Physiologically Active
Substances on the Blood Human Formed Elements. Russ. Chem. J. 18, 72 (1999).
[8] G.T. Rikhereva, I.N. Golubev, S.A. Kopylovskii, I.A. Prudenko, I.I. Mihkaleva: The in
vitro Interaction of Delta-Sleep-Inducing Peptide with Cell Membranes. Bio-Org.
Chem., 25, 334 (1999).
[9] N.P. Pal’mina, N.G. Bogdanova, E.L. Mal’tseva, E.I. Pinzar: Phorbol Esters as
Modifiers of Structure of Biological Membranes. Biol. Membr. 6, 1077, (1993).
[10] E.L. Mal’tseva, N.P. Pal’mina: The Effect of Phorbol Ester in a Wide Range of
Concentrations on the Lipid Structure of Microsome Membranes of Tumor Cells in
vitro. Radiat. Biol. Radio-Ecol., 43, 301 (2003).
[11] V.E. Zhernovkov, N.G. Bogdanova, N.P. Pal’mina: Effect of Ultra Low Concentrations
of Thyroliberin on Structural Parameters of Membrane Lipids of Endoplasmic
Reticulum in Vitro. Biol. Membr. (Moscow), 22, 388 (2005).
[12] V.V. Belov, E.L. Mal’tseva, N.P. Pal’mina: A study of the Effect of Low D-Tocopherol
Concentrations on the Dynamical parameters of Microsomal Membranes by the Method
of Spin Probes. Biofizika, 52, 75 (2007).
[13] V.V. Belov, E.L. Mal’tseva, N.P. Pal’mina, E.B. Burlakova: The Role of Solvent
Polarity in the Mechanism of Action of Biologically Active Compounds at Ultra-Low
Concentrations. Dokl. Biochem. Biophys. (Moscow), 399, 362 (2004).
[14] Y. Nishizuka: The Protein Kinase C Family and Lipid Mediators for Trans-Membrane
Signaling and Cell Regulation. Alcohol. Clin. Exp. Res., 25, 3S (2001).
[15] M. Liscovich, L.C. Cantley: Lipid Second Messengers. Cell, 77, 329 (1994).
[16] LOTEN E.G., REDSHAW-LOTEN J.C.: Preparation of Rat Liver Membranes in a
High Yield. Analit. Biochem., 154, 183 (1986).
[17] Lowry O H.., Rosebrough N. J., Barr A.L., Randall R.J.: Protein Measurement with the
Folin Phenol Reagent. J. Biol. Chem., 193, 265 (1951).
[18] A.N. Kuznezov: The Spin-Probe Method. Nauka, Moscow, 1976.
[19] O.Griffit, P. Jost: In: Spin Labeling. Theory and Applications. (Ed. L.J. Berliner). Acad.
Press, New York-San Fransisco-London, 1979, 489-569.
[20] E.K. Ruuge, E.N. Gerasimova: In: Method of Spin Labels and Probes (Eds. N.M.
Emanuel, R.I. Zhdanov). Nauka, Moscow, 1986, 225-239.
42 V. V. Belov, E. L. Maltseva and N. P. Palmina

[21] O.A. Azizova, T.I. Torkhovskaya, Yu.M. Lopukhin: In: Method of Spin Labels and
Probes (Eds. N.M. Emanuel, R.I. Zhdanov). Nauka, Moscow, 1986, 239-250.
[22] M.T. Curtis, D. Gilfor, J.L. Farber: Lipid Peroxidation Increase the Molecular Order of
Microsomal Membranes. Arch. Biochem. Biophys., 235, 644 (1984).
[23] Parshina E.L, Gendel L.Ia., Rubin A.B. The Influence of New Hybrid Antioxidants
Ichphans on the Kinetics of Ascorbate-Induced Reduction of Spin Probe Radical
Centers in Liposomes. Biofizika, 50, 676 (2005).
[24] K.Tsuda: Electron Paramagnetic Resonance Iinvestigation of Modulatory Effect of
Benidipine on Membrane Fluidity of Erythrocytes in Essential Hypertension. Heart
Vessels, 14, 1505 (2008).
[25] V. F. Antonov, E.Yu. Smirnova, E.V. Shevshenko: Lipid Membranes under the Phase
ransitions. Nauka, Moscow, 1992.
[26] D. P. Kharakoz: Phase Transition in Lipids and the Problem of Homoiothermia.
Biofizika, 45, 569 (2000).
[27] S. Wassal, L.Wang, R.C. Mc-Cabe, W.D. Ehringer, W. Stillwell: Electron
Paramagnetic Resonance of the Interaction of D-Tocopherol with Phospholipid Model
Membranes. Chem. Phys. Lipids, 60, 29 (1991).
[28] V.E. Kagan, P.J. Quinn: The Interaction of Alpha-Tocopherol and Homologes with
Shorter Hydrocarbon Chains with Phospholipid Bilayer Dispersions. A Fluorescence
Probe Study. Eur. J. Biochem., 171, 661 (1988).
[29] W. Stillwell, T.Dallman, A.C. Dumanuel, F.T. Crump, L.J. Jenski: Cholesterol versus
D-Tocopherol Effects on Properties Bilayer Model from Heteroacid
Phosphatidylcholines. Biochemistry. 35, 13353 (1996).
[30] P.J. Quinn: Characterisation of Clusters of Alfa-Tocopherol in Gel and Fluid Phase of
Dipalmitoylglycerophosphocholine. Eur. J. Biochem.,233, 916 (1995).
[31] J.C. Gomez-Fernandez, J. Villalain, F.J. Aranda, A. Ortiz, V. Micol, A. Coutinho, M.N.
Berberan-Santos, M.J. Prieto: Location of D-Tocopherol in Membranes. Ann.N.Y.
Acad. Sci., 570, 109 (1989).
[32] X. Wang, H. Takahashi, I.Hatta, P.J.Quinn: An X-ray Diffraction Study of the Effect of
D-Tocopherol on the Structure and Phase Behavior of Bilayer of
Dimytristoylphosphatidyl-ethanolamine. Biochim. Biophys. Acta, 1418, 335 (1999).
[33] M. Steiner: Vitamin E Changes the Membrane Fluidity of Human Platelets. Biophys.
Biochim. Acta, 640, 100 (1981).
[34] S. Urano, Y. Inomori, T. Sugawara, Y.Kato, M. Kitahara, Y. Hasegawa, M. Matsuo, K.
Murai: Vitamin E: Inhibition of Retinol-Induced Hemolysis and Membrane-Stabilizing
Behavior. J. Biol. Chem., 267, 18365 (1992).
[35] Koyama T., Araiso T.: Effect of alpha-tocopherol-nicotinate administration on the
microdynamics of phospholipids of erythrocyte membranes in human subjects. J.
Nutr.Sci. Vitaminol., 34, 449 (1988).
[36] D. Scmidt, H. Steffen, C. Planta: Lateral Diffusion Order Parameter and Phase
Transition in Phospholipids Bilayer Membranes Containing Tocopheryl Acetate.
Biochim. Biophys. Acta, 443, 1 (1976).
[37] N.P. Pal’mina, Е.L. Mal’tseva, N.V. Кurnakova, Е.B. Burlakova: The D-Tocopherol
Effect in a Wide Concentrations Range (10-2 -10-17 M) on the Activity of Protein Kinase
a-Tocopherol as Modifier of the Lipid Structure… 43

C. Relationship to Cell Proliferation and Tumour Growth. Biokhimia (Moscow), 52,


193 (1994).
[38] P.H.M. Lommerse, H.P. Spaink, T. Schmidt: In vivo Plasma Membrane Organization:
Results of Biophysical Approaches. Boichim. Biophys. Acta., 1664, 119 (2004).
[39] L. J. Pike: Lipid Rafts: Bringing Order to Chaos. J. Lipid Res., 44, 655 (2003).
[40] K. Monastyrskaya, A. Hostettler, S. Buergi, A. Draeger: The NK1 Receptor Localizes
to the Plasma Membrane Microdomains and its Activation is Depended on Lipid Raft
Integrity. J. Biol. Chem., 280, 7135 (2005).
[41] W. Subczynski, A. Kusumi: Dynamics of Raft Molecules in the Cell and Artificial
Membranes: Approaches by Pulse EPR Spin Labeling and Signals Molecule Optical
Microscopy. Boichim. Biophys. Acta., 610, 231 (2003).
[42] A.N. Erin, M.M. Spirin, L.V. Tabidze, V.E. Kagan: Formation of Alfa-Tocopherol
Complexes with Fatty Acids. A Hypothetical Mechanism of Stabilization of
Biomembranes by Vitamin E. Biochim. Biophys. Acta., 774, 96 (1984).
[43] G.M. Zubareva, A.V. Kargapolov, L.S. Jaguzhinskii: Effect of Superlow Quantities of
Hydrogen Peroxide on Water Base of Solutions. Biofizika, 48, 581 (2003).
[44] A.N. Smirnov, A.V. Siroezhkin: Supramolecular Water Complexes. Russ. Chem. J., 28,
125 (2004).
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.45-63 ©2010 Nova Science Publishers, Inc.

Chapter 5

SUPERCRITICAL CARBON DIOXIDE


SWELLING OF POLYHETEROARYLENES
SYNTHESIZED IN N-METHYLPYRROLIDONE

Inga A. Ronova*1, Lev N. Nikitin1,


Gennadii F. Tereschenko1 and Maria Bruma2
1
A.N. Nesmeyanov Institute of Organoelement Compounds, Moscow, Russia
2
“Petru Poni” Institute of Macromolecular Chemistry, Iasi, Romania

ABSTRACT
Five series of polyheteroarylenes have been investigated with regard to their physical
properties before and after swelling with supercritical carbon dioxide. The study of the
dependence of glass transition temperature and free volume of polymers on their
conformational rigidity showed that the process of swelling in supercritical carbon
dioxide is influenced by the voluminous side groups and by the high boiling solvent N-
methylpyrolidinone used for the preparation of the polymers which facilitates the
formation of crosslinks or complexes with the macromolecular chains.

Keywords: polyheteroarylenes, supercritical carbon dioxide, swelling, conformational


rigidity, free volume, crosslinks

1. INTRODUCTION
It is known that polyheteroarylenes exhibit high thermal stability and excellent
mechanical properties determined by their conformational rigidity which differs significantly
from that of analogous aliphatic polymers, from 10-15Å to thousands of Angstroms [1]. Most
of these polyheteroarylenes are amorphous glassy polymers and most of their physical

* A.N. Nesmeyanov Institute of Organoelement Compounds, 28 Vavilova, 119991 Moscow, Russia, E-mail
ron@ineos.ac.ru
46 Inga A. Ronova, Lev N. Nikitin, Gennadii F. Tereschenko et al.

properties correlate well with their conformational rigidity [2]. At the same time, it is known
that physical properties of amorphous glassy polymers depend not only on their chemical
structure, but also on the history of their preparation, on the physical aging processes and
other. In recent years a great interest was given to the modification of amorphous glassy
polymers by treatment with supercritical carbon dioxide (sc CO2) with the aim to manipulate
their physico-chemical properties [3]. It is believed that the swelling process with sc CO2 can
change some of these properties. If the swelling process is directly connected with the
conformational rigidity of the polymers, this gives a good possibility to change their
properties. Therefore, the study of the influence of the swelling process on glassy polymers of
different chemical structure is very important.
Previously we have studied a polyetherimide containing hexafluoroisopropylidene groups
which was subjected to the treatment with supercritical CO2 under a temperature of 40ºC and
65ºC, and a pressure of 150 bar; we have found that under the temperature of 40ºC
microcavities were formed and an increase of the free volume of 23.1% took place. In the
case of treatment under 60ºC we have obtained nanocavities and an increase of the free
volume of 56.8% [4].
Here we present a study of the dependence of glass transition temperature on
conformational parameters and free volume for five groups of polyheteroarylenes and the
swelling process of polymer films in supercritical carbon dioxide.

2. EXPERIMENTAL
2.1. Preparation of Polymer Films

Five series of polymers have been studied whose synthesis was reported previously [5-
13]. Thus, the series having the code AMIC contains one polyimide (1) and two polyamidic
acids (2 and 3); the series having the code OXAD contains two polyoxadiazole-amidic acids
(4 and 6) and one polyoxadiazole-imide (5); the series having the code PEAM contains six
polyester-amides (7, 8, 9, 10, 11 and 12); the series having the code ACET contains three
poly(oxadiazole-amide)s with pendant acetoxybenzamide groups (13, 14, 15); the series
having the code CYAN contains four polyamidic acids (16, 18, 20, 22) and the corresponding
four polyimides (17, 19, 21, 23) based on a diamine having cyano substituents and four
dianhydrides containing pyromellitic, hexafluoroisopropylidene diphthalic, biphenylene or
diphenylketone units, respectively (Table 1). These polymers show good solubility in N-
methylpyrrolidinone (NMP) and other polar amidic solvents having high boiling temperature.
The films, having the thickness usually in the range of 30-40 mμ, were prepared by using
solutions of polymers in N-methylpyrrolidinone, having the concentration of 15%, which
were cast onto glass plates and heated gradually up to 210ºC. The films were carefully taken
out of the substrate and were used afterwards for various measurements.
Supercritical Carbon Dioxide Swelling of Polyheteroarylenes Synthesized… 47

Tabel 1. Repeating units of the studied polymers: AMIC, OXAD, PEAM, ACET and
O
R= NH-CO O C CH3
CYAN.

Polymer Series Repeating unit


code
O
1 AMIC O
O

C
O CH3
O O
C
O N N O C O N N
CH3
O O O O
O
2 AMIC H O
O
O H CH 3 H O
C
O H
C N O N C N
CH2 N C C O C C

C C CH3 C C
HO OH HO OH
O O O O
O
3 AMIC O
H O
O
O H CH3 H O
C
O H
C O
S N C C N O C N C C N

O C C CH 3 C C
HO OH HO OH
O O O O

O
4 OXAD H O O H
N N
C
O O N C C N
O
C C
HO OH
O O
O
5 OXAD O O
N N C
O O N N
O
O O
O
6 OXAD H O O H
N N
C
O O N C C N
O
C C
HO OH
O O

7 PEAM CO-NH CH2


NH-CO O-CO CO-O

8 PEAM CH2
NH-CO O-CO CO-O CO-NH

CH3 CH3
CN
9 PEAM
NH-CO O-CO CO-O CO-NH O O

CN
10 PEAM
NH-CO O-CO CO-O CO-NH O O

11 PEAM NH-CO O-CO CO-O CO-NH O O

CH3
12 PEAM
NH-CO O-CO CO-O CO-NH O C O
CH3
48 Inga A. Ronova, Lev N. Nikitin, Gennadii F. Tereschenko et al.

Table 1. (continued)

Polymer Series Repeating unit


code
13 ACET H H
O O N N
N C C N
O

R
H
14 ACET H O O N N
O N C C N O
O

R
H
15 ACET H O O N N
O N C C N O
O

R
H
16 CYAN CN H O O O

O O N C C C N
HOOC COOH
O
17 CYAN CN O O

C
O O N N

O O
H
18 CYAN CN H O O

O O N C C N
HOOC COOH
O
19 CYAN CN O

O O N N

O O
H
20 CYAN CN H O CF3 O

O O N C C C N
HOOC COOH
CF3
O
21 CYAN CN O CF3

C
O O N N
CF3
O O
CN
22 CYAN H O O H

O O N C C N

HOOC COOH

23 CYAN CN O O

O O N N

O O
Supercritical Carbon Dioxide Swelling of Polyheteroarylenes Synthesized… 49

2.2. Measurement of Density

To measure the density of polyheteroarylene films we used the hydrostatic weighing


method. The study was performed with an equipment for density measurement and an
electronic analytic balance Ohaus AP 250D, precision of 10-5 g, from Ohaus Corp US which
was connected to a computer. With this equipment we measured the change of sample weight
during the experiment, with a precision of 0.001 g/cm3 in the value of density. Ethanol was
taken as a liquid with known density. The studied polyheteroarylenes did not absorb and did
not disolve in ethanol, which for these polymers had a low diffusion coefficient. Since the
density of ethanol depends on temperature, every time it was measured using pycnometer.
The characteristic diffusion times were in the domain of 104 – 105 s, which are 1-2 order of
magnitude higher than the time of density measurement. This is why the sorption of solvent
and the swelling of the film must have only insignificant influence on the value of the
measured density. All measurements of the density were performed at 23ºC. The density was
calculated with the equation (1):

ρs = Wa / (Wa – Wl ) ρl , (1)

where ρs is density of the sample, Wa is the weight of the sample in air, Wl is the weight of
the sample in liquid, ρl is the density of liquid. The error of the density measurements was
0.1–0.3%.

2.3. Measurement of Glass Transition Temperature

The glass transition temperature (Tg) of the polymers was measured by differential
scanning calorimetry, using a Mettler DSC 12E apparatus. The samples were heated at a rate
of 15ºC/min under nitrogen to above 300ºC. Heat flow versus temperature scans from the
second heating run were plotted and used for reporting the Tg. The mid point of the inflection
curve resulting from the second heating run was assigned as the Tg of the respective
polymers. The precision of this method is ±7 – 10оС.

2.4. Calculation of Conformational Parameter and Free Volume

As conformational parameter we have taken the statistical Kuhn segment Afr, which was
calculated with the equation (2) [1]:

⎛ <R2 >⎞
A fr = lim ⎜ ⎟, (2)
n→∞ ⎝ nl o ⎠

where R2 is mean square distance between the ends of the chain calculated for all possible
conformations; L = nlo is the contour length of the chain, a parameter which does not depend
on the chain conformation; lo is the contour length of a repeating unit. All the values of Kuhn
50 Inga A. Ronova, Lev N. Nikitin, Gennadii F. Tereschenko et al.

segment were calculated with Monte Carlo method, geometry of the repeating unit was
evaluated by quantum chemical method AM1 [14].
To calculate the free volume we used the method previously described [15]. We built a
model of the repeating unit and its geometry was also evaluated by quantum chemical method
AM1. The atoms are described by spheres having the Van der Waals radius equal to the
corresponding radius of each type of atoms (Chart 1) [16].

Ly
Lx

Chart 1. The monomer introducing in the box.

This model was situated in a 3D rectangular box having the axes Lx, Ly, Lz given the
equation (3):

Lx = xmax + Rmax – (xmin - Rmax) = xmax – xmin + 2Rmax , (3)

where xmax and xmin are the maximum and the minimum values of the coordinates of atom
corresponding to the repeating unit, Rmax is the maximum value of the radius of atom
corresponding to the repeating unit. Ly and Lz were determined in the same way. The volume
of this model was calculated with Monte Carlo method. For that, in the volume corresponding
to the parameters of the box, random points were generated. The number of random points,
landing in the repeating unit, is m. In the beginning of calculation m is equal 0. For each
random point the following conditions were verified:

| rd – ri | ≤ Ri , i = 1…N,

where N is the number of atoms in the repeating unit, ⎥ rd – ri⎥ is the distance between a given
point and any other point in the repeating unit. In case of achievement of this conditions for at
least one atom, procedure of verification stopped, number of successful events began with
m+1, and next random point was generated.
Van der Waals volume (Vw) was calculated with the formula (4):
Supercritical Carbon Dioxide Swelling of Polyheteroarylenes Synthesized… 51

Vw = (m / M) Vbox (4)

where M is the total number of all points, Vbox is the volume of the box.
The free volume (Vf) was calculated with the formula (5):

1 N A • Vw
Vf = − (5)
ρ Mo

where NA is the number of Avogadro, ρ is the density of polymer, Mo is the molecular weight
of the repeating unit. The value Vf , thus calculated, shows the volume which is not occupied
by the macromolecules in one cm3 of polymer film. From this point on, we will call it “free
volume”.

2.5. Method of Treatment with Supercritical Carbon Dioxide

The experimental set-up and the method of impregnation with supercritical carbon
dioxide (sc-CO2) were described in previous papers [17-19]. This experimental set-up is
composed of a generator which can provide CO2 up to 35 MPa pressure (High Pressure
Equipment Company, USA). A system of valves ensures the CO2 access to the reaction cell
with the volume of 30 cm3. The pressure generator and the reaction cell are provided with
manometers to allow a control of the pressure and the letting-in and letting-out of gas. The
temperature control allows a precision better than ±0.2°С. The cell is designed for
experiments at pressures up to 50 MPa and temperatures up to 120°С.
CO2 desorption curves were obtained using the gravimetric technique [20]. Sample
weight was measured with an Ohaus AP 250 D electronic balance interfaced with a computer.
The following experimental technique was applied: the polymer sample was weighed and
placed into the cell. The sample had the form of a film (typically, a disk with 15 mm diameter
and thickness in the range from several to tens microns). After purging the cell with CO2 it
was sealed. The pressure was increased up to the necessary value and the sample was exposed
during a given time. The cell was decompressed, the polymer sample was placed on the
electronic balance and the weight decrease during CO2 desorption was recorded using a
computer. Then the weight swelling degree at zero time (the moment of decompression) and
the CO2 diffusion coefficient were calculated. All experiments were done at 150 bar and
40ºC. The decompression speed of CO2 was near 5 mL/s.
Approximate (asymptotic) formulas are typically used to analyze the desorption
dynamics using gravimetric technique [21, 22]. Such formulas are only valid either for the
initial or the final stage of the process. This approach can hardly give an answer to the
question about the diffusion type: whether the diffusion is subjected to the Fick law or not.
On the other hand, the progress of the computer technology during the recent years allows to
realize numerically the analysis of the experimental data even when the complex exact
solutions are used. We suppose that this makes the use of the approximate solutions
superfluous. Therefore, we used the exact solutions of the diffusion problem with the uniform
initial and zero boundary conditions valid in the Fick approximations. The equations
52 Inga A. Ronova, Lev N. Nikitin, Gennadii F. Tereschenko et al.

describing the dependence of the sorbate weight on time Z(t) are known from the diffusion
theory [23]. For a film-like sample this equation has the form (6):

Z( t ) 8 ∞
1 ⎛ − (2 n + 1)2 π 2 Dt ⎞
Z0
= 2
π

n = 0 (2 n + 1 )
2
exp⎜⎜
l 2


⎝ ⎠ (6),

where l is the film thickness; Z0 is equilibrium coefficient of swelling for the “0” time; D is
the diffusion coefficient.
A numerical algorithm to find the best fit for the experimental data was realized using the
least squares method. Theoretical dependencies (6) were used as the fit functions and the
parameters Z0 and D were varied. Thus, the best fit allows to determine both the values of the
diffusion coefficient and the initial sorbate weight (and therefore the equilibrium degree of
the polymer swelling in sc CO2). In case when the exact solution (6), valid for D = const,
gives a good fit for the experimental data, the diffusion is of normal type and it obeys the Fick
law. Figure 1 shows the typical desorption curve for the films of studied polyheteroarylene 4
and 5 and their fit with the theoretical dependence (6). As can be seen in figure 1 our case
corresponds to the normal diffusion (the experimental and theoretical curves coincide very
well). Therefore, we can calculate the values of Z0 and D. These values for the studied
polyheteroarylenes are given in table 2.

Figure 1. Desorption curve of CO2 for the polymer AMIC-2.


Supercritical Carbon Dioxide Swelling of Polyheteroarylenes Synthesized… 53

ble 2. Diffusion coefficients ( D ) of CO2 and equilibrium coefficient of swelling


( Zo )of the studied polymers.

Polymer D, Zo , Polymer D, Zo ,
10-10 cm2/s weight % 10-10 cm2/s weight %
1 40 6.9 13 4.3 0.968
2 14 8.7 14 5.8 2.98
3 29 12.5 15 3.6 2.93
4 19 - 16 15 4.49
5 0.8 6.9 17 8.7 4.78
6 22 - 18 25.9 3.86
7 - - 19 4.4 4.16
8 3.4 6.6 20 48 7.81
9 3.0 3.7 21 80.3 4.45
10 0.16 2.6 22 349 9.30
11 2.3 - 23 - -
12 0.8 6.9 - - -

3. RESULTS AND DISCUSSION


In order to understand how the physico-chemical properties of polyheteroarylenes change
after treatment with supercritical carbon dioxide (sc CO2), we analyzed such properties before
and after treatment with sc CO2. Table 3 shows the values of glass transition temperature
(Tg), Kuhn segment (Afr) and Van der Waals volume (Vw) of all ivestigated polymers. The
glass transition temperature of these polymers was in the range of 185ºC – 280ºC and the
conformational rigidity was in the domain of 12 Å – 72.5 Å. The dependence of Tg of these
polymers on Kuhn segment is described by three straight lines having high correlation
coefficients (Figure 2 a, b and c). For two samples OXAD ( polymers 5 and 6) the values of
Tg were identical. By using the equation Y = 255.369 + 0.629 X, having such a high
correlation coefficient R = 99.57 % , these Tg values can be calculated with high precision
and, indeed, the difference was only 0.74oC, that situated beyond the accuracy limits of
method of measuring Tg .
In the case of PEAM (fig. 2a), the samples 8 and 12 are out of the straight line which is
due to the low molecular weight of these two polymers. In such cases Gaussian coil has not
formed yet, the packing of the chains is loose, and the conformational transitions take place
very easy in glass transition state.
The dependence of glass transition temperature on Kuhn segment in the case of ACET
polymers is linear, with not a very high correlation coefficient (figure 2b). When measuring
the glass transition temperature by using the differential scanning calorimetry (DSC) method,
the error can be ±10%. From the dependence of Tg on Kuhn segment, y= 260.35 + 0.391x,
we calculated the Tg of ACET polymers 14 and 15. They are 273.8 ºC and 268.4оС,
respectively. In one case it is 3.8ºC higher and in the other case it is 1.6ºC lower than the
experimental values (270ºC for both polymers). These differences are in the domain of
experimental error of DSC method.
54 Inga A. Ronova, Lev N. Nikitin, Gennadii F. Tereschenko et al.

ACET

b
CYAN

Figure 2. Dependence of glass transition temperature (Tg) on Kuhn segment (Afr) for the studied
polymers: AMIC, OXAD, PEAM (fig. 2a), ACET (fig. 2b) and CYAN (fig. 2c)
Supercritical Carbon Dioxide Swelling of Polyheteroarylenes Synthesized… 55

The dependence of glass transition temperature on Kuhn segment for the CYAN
polymers is described by three lines (fig. 2c). The first line is for polymers 22 and 23 based
on pyromellitic dianhydride, the second is for polymers 20 and 21 based on
hexafluoroisopropylidene diphthalic dianhydride and the third line is for polymers 16, 17, 18
and 19 based on the dianhydride containing diphenylketone or biphenylene segment. Figures
2 show that the polymers AMIC, OXAD, PEAM, ACET and CYAN behave normally,
which means that the glass transition temperature increases with increasing the rigidity [2].
Now we examine the modification of density of the polymers after swelling and
desorption of sc CO2. We can presume that after treatment with sc CO2 the density of
polymers should decrease due to possible formation of nano- and micro-cavities [13, 19, 24].
Table 3 shows the density values before (ρ1) and after (ρ2) treatment with sc CO2.

Table 3. Glass transition temperature, density, conformational parameter Van der


Waals volume, free volume and the changing of the density and free volume after
swelling in supercritical CO2.

Poly- Tg Afr Vw ρ1 Vf0 ρ2 Vf1 Δρ ΔV


mer (oC) (Å) (Å3) (g/cm3) (cm3/g) (g/cm3) (cm3/g) (g/cm3) (cm3/g)
1 248 30.37 1031.648 1.317 0.2342 1.319 0.2330 - 0.002 -0,0012
2 185 32.84 1087.52 1.311 0.2384 1.295 0.2479 0.016 0,0095
3 262 30.02 1096.54 1.329 0.2035 1.322 0.2075 0.007 0,004
4 280 39.05 651.013 1.391 0.3672 1.411 0.3570 - 0.020 -0,0102
5 270 22.45 624.822 1.369 0.3817 1.366 0.3833 0.003 0,0016
269.82
6 270 24.14 654.141 1.364 0.3798 1.376 0.3734 - 0.012 -0,0064
270.56
7 218 72.53 521.673 1.259 0.2416 1.302 0.2154 - 0.043 -0,0262
8 190 72.34 553.099 1.256 0.2385 1.246 0.2441 0.010 0,0056
9 221 71.39 614.855 1.308 0.2358 1.313 0.2328 - 0.005 -0,003
10 205 28.08 614.851 1.296 0.2428 1.315 0.2317 - 0.019 -0,0111
11 210 41.86 642.950 1.299 0.2538 1.344 0.2280 - 0.045 -0,0258
12 190 40.61 721.688 1.233 0.2673 1.259 0.2506 - 0.026 -0,0167
13 280 44.77 495.140 1.383 0.171 1.378 0.174 0.005 0.002
14 270 34.23 663.816 1.360 0.198 1.335 0.212 0.025 0.014
273.8
15 270 20.48 1096.54 1.336 0.216 0.311 0.230 0.025 0.014
268.4
16 216 12.32 546.402 1.316 0.246 1.339 0.233 -0.023 -0.013
17 223 14.84 518.139 1.330 0.235 1.309 0.247 0.021 0.012
18 218 13.11 527.414 1.325 0.236 1.343 0.226 -0.018 -0.010
19 232 18.57 501.620 1.364 0.208 1.350 0.216 0.014 0.008
20 228 11.95 608.868 1.392 0.237 1.411 0.227 -0.019 -0.010
21 235 13.91 582.420 1.407 0.227 1.382 0.240 0.025 0.013
22 253 12.83 451.458 1.379 0.222 1.358 0.228 -0.021 -0.008
23 263 17.51 424.690
Tg = glass transition temperature; Afr = Kuhn segment; Vw = Van der Waals volume;
ρ1 = density before swelling; ρ2 = density after swelling; Vf0 = free volume before swelling; Vf1 = free volume
after swelling; Δρ = ρ1–ρ2 = modification of density; ΔV=Vf1–Vf0 = modification of free volume.
56 Inga A. Ronova, Lev N. Nikitin, Gennadii F. Tereschenko et al.

a b

c d

Figure 3. Dependence of glass transition temperature ( Tg ) on free volume ( Vf ) for the studied
polymers AMIC (3a), OXAD (3b), PEAM (3c), ACET (3d) and CYAN (3e).

The equilibrium coefficients of swelling with CO2 of these polyheteroarylenes are not
high (table 2). In order to find an explanation of this behavior we examine now the
dependence of glass transition temperature on the free volume which was calculated with the
equation (5) taking into consideration the Van der Waals volume and the density of the
Supercritical Carbon Dioxide Swelling of Polyheteroarylenes Synthesized… 57

studied polymers. It is known that when the free volume of the polymers increases their glass
transition temperature decreases because the conformational transitions take place easier by
heating. The macromolecular chains start moving easier towards each other and the
amorphous polymers soften.
After treatment with sc CO2 the density of the polymers modified: for some of them the
density decreased, therefore the swelling did take place, while for other polymers the density
increased. To explain such a behavior, we examine now the dependence of glass transition
temperature on free volume, and the dependence of free volume on conformational rigidity.
The degree of swelling is not high in comparison with the degree of swelling of
polyimides [4] and, therefore, the experimental measurements of the glass transition
temperature after swelling with sc CO2, by using the DSC method is not reasonable.
Figure 3 presents the dependence of glass transition temperature on the free volume for
five series of polymers before and after swelling in sc-CO2. As can be seen in figure 3a and
3d, only in the series AMIC and ACET the line showing the free volume of the polymers
after swelling in sc-CO2 is on the right side, which means that the swelling of polymers did
take place, although not in a significant degree. Polymer AMIC 1 is out of both cases. This
shows that polymers AMIC 2 and AMIC 3 contained residual solvent N-methylpyrolidone
(NMP) which was partially eliminated from these polymers by swelling in sc CO2. If this
would not have been observed, after swelling in sc-CO2 all points had been situated on the
same line.
For the polymers in series OXAD (figure 3b), the density of polymers 4 and 6 after
swelling in sc CO2 increased, and only for polymer 5 it decreased below the initial value. The
dependence of glass transition temperature on the free volume is linear, with a high
correlation coefficient. The increase of density after modification in sc CO2 could be
explained by absorption of CO2 in polymer matrix, due to the formation of hydrogen bonds
between CO2 and hydrogen atoms of amide groups. It is known that CO2 molecule is
quadruple, in the two ends having oxygen atoms with a pair of non-participating electrons.
These electrons may participate in the formation of hydrogen bonds with hydrogen atoms
from polymer matrix. In this case it can be the hydrogen of amide groups. If the distance
between hydrogen atoms of repeating unit and oxygen atom of CO2 is smaller than the sum of
Van der Waals radii, and the energy of formation of complex decreases, then the hydrogen
bond is formed. To verify this hypothesis, calculations were performed, by using the quanto-
chemical method AM1 [14]. If we consider that the formation energy of the repeating unit is
E1 and the formation energy of CO2 is E2, then the total energy of the two independent units
(repeating unit and CO2 molecule) is: E = E1 + E2 = −181.74 Kcal/mol. The calculated
formation energy of the complex of these two units was −183.39 Kcal/mol which is 1.65
Kcal/mol lower. Therefore, the formation of the complex is more probable than the existence
of separated units. On the other hand, the sum of Van der Waals radii of oxygen and
hydrogen atoms is 2.53 Å [16]. The calculations showed that the distance between the oxygen
atom of carbonyl group and the hydrogen atoms of amide group is 2.27 Å, which is 0.26 Å
shorter than the sum of Van der Waals radii of these atoms. Thus, it was proved the
possibility of formation of complexes between amide groups in polymer matrix and CO2
molecules. In such case it is understandable why a part of CO2 during decompression remains
in polymers 4 and 6: because in series OXAD only these two polymers contain amide groups.
58 Inga A. Ronova, Lev N. Nikitin, Gennadii F. Tereschenko et al.

Similar specific interaction of CO2 with polymers was also observed earlier [25]. This is
confirmed by diffusion coefficients of CO2 in these polymers (Table 2).
In the polymer series PEAM (figure 3c) the dependence of glass transition temperature
on the free volume is linear for three polymers: 7, 9 and 11. In the case of sample 10 the free
volume is significantly smaller than it should be according to the general dependence in this
series. It shows that in case of this polyheteroarylene, when the film was prepared, part of the
solvent remained inside it and did not evaporate by drying until constant weight. This part of
the free volume can be calculated. The solvent used to cast the films was N-methyl-
pyrrolidinone (NMP). We calculated its Van der Waals volume. We start from the equation
showing the dependence of glass transition temperature on the free volume and we find that
the free volume of polymer 10 before treatment with sc-CO2 is 0.262 cm3/g. The difference
between the experimental value of free volume and the calculated one is 0.0192 cm3/g, or 7.9
% of the free volume of this polymer. That is the part of free volume which is occupied by the
solvent and this is why the density of the polymer was higher. The value of the free volume
occupied by the solvent corresponds to 2.4 %. After swelling in sc CO2 the free volume of
these polymers, with the exception of 8, decreases significantly (Table 3): for polymer 9 it
decreases with 1.4%, while for polymer 11 it decreases with 10.2% It shows that the
absorption of CO2 in the polymer matrix is similar to the polymers in series OXAD, but it
occurs in different degree for each polymer. The diffusion coefficients which are given in
table 2 confirm the formation of hydrogen bonds in these polymers because they are lower in
comparison with diffusion coefficient of polymer 9. In case of this later polyheteroarylene the
diffusion coefficient is high enough and a big part of CO2 leaves the polymer matrix during
decompression.
Figure 3d shows the dependence of glass transition temperature on free volume of the
ACET polymers before and after swelling with sc CO2. It can be seen here that the line
referring to the free volume after swelling is situated in the right side which means that the
swelling of the polymers did take place, although not in the same degree. The degree of
swelling is not high in comparison with the degree of swelling of polyimides [4] and,
therefore, the experimental measurements of the glass transition temperature after swelling
with sc CO2, by using the DSC method is not reasonable. Both dependences shown in figure
3d have high correlation coefficients. The degree of swelling of the polymers in this group
differ significantly from each other. In the case of polymer 13, the free volume increases with
1.7%, while in the case of polymer 15 it increases with 6.5%. This behavior is connected with
the conformational rigidity of the polymers which decreases from 44.77 Å in the case of
polymer 13 to 20.48 Å in the case of polymer 15 (table 3). While the rigidity of the polymer
increases, their free volume decreases (figure 4). When the number of flexible bridges (Ph–
O–Ph) increases, in the case of polymers 14 and 15, and therefore the probability of
conformational transitions in polymer chains increases during treatment with sc CO2 leading
to the formation of nanocavities, the free volume of the polymer increases. The voluminous
side groups R (structures shown in table 1) have a significant role here since they do not
allow the polymer chains in the initial films to pack tightly, which increases the probability of
conformational transitions around the flexible bridges.
A completely different behavior is observed in the case of CYAN polymers when we
examine the dependence of glass transition temperatures on free volume (figure 3e). Before
swelling with CO2 the dependence of glass transition temperature on free volume (figure 3e)
and the dependence of glass transition temperature on Kuhn segment (figure 2c) divides in
Supercritical Carbon Dioxide Swelling of Polyheteroarylenes Synthesized… 59

three sub-groups: polymers 16, 17, 18 and 19 which contain diphenylketone or biphenylene
unit in the dianhydride segment; polymers 20 and 21 containing hexafluoroisopropylidene
diphthalic units in the dianhydride segment; polymers 22 and 23 containing pyromellitic unit
in the dianhydride segment. Since the polymer 23 is partially crystalline, it will not be taken
into consideration in the next discussion. The dependence of free volume on Kuhn segment
divides in the same three sub-groups (figure 4d).

a b

c d

Figure 4. Dependence of free volume on the conformational rigidity (Kuhn segment) of the studied
polymers.

The data given in figure 4 showing the dependences of the free volume before and after
swelling in CO2 upon its conformational rigidity confirm the conclusions made on the basis
of figure 3. With increasing the rigidity, the free volume decreases. Both of the dependencies
(before and after swelling) have high coefficients of correlation, but after swelling in CO2
only for polymers AMIC the correlation coefficient increases, while for the polymers OXAD,
PEAM and ACET it has a slight decrease. This is directly connected with the presence of
solvent in polymers before and after swelling, and with the presence of residual CO2
participating in the formation of hydrogen bonds with amide groups.
The swelling of the CYAN polymers takes place in the polymers 17, 19 and 21,
containing imide rings, and it increases with the increase of the flexibility (table 3). To
explain such a difference, we can refer to a previously published paper [26] presenting a study
60 Inga A. Ronova, Lev N. Nikitin, Gennadii F. Tereschenko et al.

of the IR spectra of the polyamidic acids prepared in various solvents. There, it was shown
that in the synthesis or dissolution in N-methylpyrrolidinone (NMP) followed by heating up
to 200ºC to remove the solvent, two competitive processes may take place:

− the first is the formation of crosslinks between the chains of polyamidic acids:

− the second is the formation of polymer/NMP complex:

The energy of the formation of the complex =


The energy of the formation of the complex =
–31 kJ/mol
–74 kJ/mol

Thus, the formation of crosslinks in the polyamidic acids prevents their swelling with sc
CO2. The formation of complexes between NMP and amide groups hinders the free rotation
around N–Ph bond and leads also to the formation of inter-chain bonds. Also, the low degree
of swelling of 3.8% to 7% found in polyimides 17, 19 and 21 is connected with the fact that
during the imidization of the polymers in NMP, it is possible the formation of anhydride
bridges leading to crosslinks between chains. But in the case of the polyimides such
crosslinks are less frequent than in the case of the corresponding polyamidic acids.
Now we look into the table 2 which presents the diffusion coefficients of CO2 obtained
during desorption of CO2 and equilibrium degree of swelling Zo. Both these parameters are
determined with moderate degree of precision. The diffusion coefficients and equilibrium
degree of swelling Zo of these polyheteroarylenes show the strong connection of these values
with the chemical structure and the properties of polymers. Thus, in case of series AMIC, the
polymer 1 shows the highest value of diffusion coefficient D, and a medium value of
equilibrium degree of swelling Zo , which could be explained by a lower coefficient of
molecular packing, and a higher rigidity of the polymer. The polymers in series OXAD show
Supercritical Carbon Dioxide Swelling of Polyheteroarylenes Synthesized… 61

high values of D for 4 and 6, in comparison with 5, which can be connected with the
formation of specific interaction between CO2 and amide groups. Finally, the series PEAM
shows low values of D and Zo , and among them the polymer 12 exhibits the highest
coefficient of molecular packing and one of the lowest values of D. For the ACET polymers
14 and 15, they are close to each other, regardless the significantly different rigidity. At the
same time, also very close are the values of the free volume increase which were calculated
on the basis of the change of density and Van der Waals volume of the repeating unit. The
diffusion coefficients of the CYAN polymers 17, 19 and 21 are very close to each other, with
the exception of polymer 21 which contains hexafluoroisopropylidene bridges, while the
coefficients of swelling of these three polyimides are almost identical. In the case of CYAN
polymers 16, 18 and 20 the coefficients of swelling are different and they increase with the
decrease of the rigidity. The increase of rigidity enables the increase of the number of
crosslinks due to the formation of anhydride bridges. In case of polymer 21 both diffusion
coefficient and equilibrium coefficient of swelling are high probably because of a stronger
sorption of carbon dioxide during swelling and its possible retention in the polymer matrix
due to week interactions between hydrogen in amide groups and oxygen in carbon dioxide.

4. CONCLUSIONS
This study shows that the swelling process of polyheteroarylenes with supercritical
carbon dioxide is connected with the history of the polymer film preparation: in which solvent
was the synthesis of the polymer performed and from which solvent was cast the film.
However, the conformational analysis allows to determine certain factors which influence
this process. Thus, with the increase of conformational rigidity, the degree of swelling of the
polymers is lower. The presence of voluminous side groups facilitates the swelling process.
Due to the presence of residual solvent the advanced drying of the polymer film is necessary
before treatment with supercritical carbon dioxide. After the treatment with supercritical
carbon dioxide it is also necessary to maintain the film at high temperature for some time in
order to evolve the CO2 gas.
The free volume in polymers depends on their conformational rigidity. When the films
are prepared from polymer solutions, solvents having low boiling point should be used in
order to prevent the influence of the residual solvent on the swelling process of the polymers
in supercritical CO2. The polymers containing amide groups easily form hydrogen bonds with
CO2 which hinders significantly their swelling. The study of the dependence of glass
transition temperature on free volume and on Kuhn segment, before and after swelling with
supercritical carbon dioxide, of two groups of polyheteroarylenes, ACET and CYAN,
showed that the swelling degree is low, below 7%, and it depends significantly on the
structure of the repeating units, for example on the presence of voluminous side substituents
and flexible brigdes such as Ph–O–Ph. The use of high boiling solvent N-
methylpyrrolidinone (NMP) in the synthesis of polyheteroarylenes may lead to the formation
of complexes between NMP and polymer chains which give rise to crosslinks and increase
the rigidity and thus reduce the degree of swelling with supercritical carbon dioxide.
62 Inga A. Ronova, Lev N. Nikitin, Gennadii F. Tereschenko et al.

ACKNOWLEDGMENTS
The authors are grateful for the financial support provided through by the Romanian
Research Program PNCD2 (Project no. 11008/2007) and grant of Presidium Russian
Academy of Sciences P-18.

REFERENCES
[1] Pavlova, S. S. A.; Ronova, I. A.; Timofeeva, G. I.; Dubrovina, L.V. On flexibility of
cyclochain polymers, J. Polym. Sci. Polym. Phys. Ed. 1993, 31, 1725-1757.
[2] Ronova, I. A.; Pavlova, S. S. A. The Effect of the Conformational Rigidity on Several
Physical Properties of Polymers, High Perform. Polym. 1998, 10, 309-329.
[3] Gallyamov, M. O.; Vinokur, R. A.; Nikitin, L. N.; Said-Galiyev, E. E.; Khokhlov, A. R;
Schaumburg, K. Poly(methyl methacrylate) and poly(butyl methacrylate) swelling in
supercritical carbon dioxide and the formation of a porous structure. Polym. Sci. Ser. A.
2002, 44, 581-592.
[4] Ronova, I.А.; Nikitin, L.N.; Sinitsyna, O.V.; Yaminsky, I.V. Treatment of polymers
with supercritical carbon dioxide. Efficient method to increase the free volume. Phys.
Khim. Process. Mater. 2008, 4, 54-59.
[5] Bruma, M. ; Hamciuc, E. ; Sava, I., Hamciuc, C. ; Iosip, M. D.; Robison, J. Compared
properties of polyimides based on benzophenonetetracarboxylic dianhydride, Rev.
Roum. Chim., 2003, 48: 629-638.
[6] Sava, I. and Bruma, M. Polyamide-esters containing benzonitrile or isopropylidene
units in the main chain, Rev. Roum. Chim., 2004, 49: 69−76.
[7] Bruma, M. ;Sava, I. ;Hamciuc, E., Hamciuc, C.; Damaceanu, M. D. Synthesis and
characterization of heterocyclic polyimides as high performance materials with
potential applications in RF MEMS devices processing, Romanian Journal of
Information Science and Technology, 2006, 9: 277-284.
[8] Sava, I.; Iosip, M. D.; Bruma, M.; Hamciuc, C.; Robison, J.; Okrasa, L.; Pakula, T.
Aromatic polyamides with pendant acetoxy benzamide groups and thin films made
therefrom. Eur. Polym. J. 2003, 39, 725-738.
[9] Sava, I.; Bruma, M. and Ronova I. A. Conformational parameters and some physical
properties of polyamides containing pendant acetoxy benzamide groups. Mol. Cryst.
Liq. Cryst. 2004, 416, 201-207.
[10] Sava, I.; Bruma, M. Compared properties of thin films from poly(1,3,4-oxadiazole)s.
Rev. Roum. Chim. 2005, 50, 783-790.
[11] Hamciuc, E.; Bacosca, I.; Bruma, M.; Ignat, M. Aromatic polyimides containing cyano
substituents for high performance applications. Proceedings 30th International
Semiconductor Conference, Sinaia, Romania, 2007, 2, 357-360.
[12] Bacosca, I.; Hamciuc, E.; Bruma, M.; Ronova, I. A. Study of aromatic polyimides
containing cyano groups. High. Perform. Polym., in press.
[13] Ronova, I. A. ; Nikitin, L. N. ; Sokolova, E. A. ; Sava, I. ; Bruma, M. Study of the
behavior of polyheteroarylenes treated with supercritical carbon dioxide, High.
Perform. Polym., in press.
Supercritical Carbon Dioxide Swelling of Polyheteroarylenes Synthesized… 63

[14] Dewar, M. J. S.; Zoebisch, E. F.; Healy, E. F.; Stewart, J. J. Development and use of
quantum mechanical molecular models. 76. AM1: a new general purpose quantum
mechanical molecular model, J. Am. Chem. Soc. 1985, 107, 3902-3909.
[15] Rozhkov, E. M.; Schukin, B. V.; Ronova I. A. Methods for the calculation of occupied
volumes in glassy polymers: the lattice integration and the Monte Carlo method, Eur. J.
Chem. (Central European Science Journals) 2003, 1, 402-426.
[16] Askadskii, A. A.; Kondrashchenko, V. I. Computer material science of polymers;
Nauchnyi Mir: Moscow, 1999.
[17] Nikitin, L. N.; Said-Galiyev, E. E.; Vinokur, R. A.; Khokhlov, A. R.; Gallyamov, M.
O.; Schaumburg, K. Poly(methyl methacrylate) and Poly(butyl methacrylate) Swelling
in Supercritical Carbon Dioxide, Macromolecules, 2002, 35, 934-940.
[18] Nikitin, L. N.; Marat, O.; Gallyamov, M. O.; Rostislav, A.; Vinokur, R. A.; Nikolaev,
A. Yu.; Said-Galiyev, E. E.; Khokhlov, A. R.; Jespersen, H. T.; Schaumburg, K.
Swelling and impregnation of polystyrene using supercritical carbon dioxide, J.
Supercritical Fluids 2003, 26, 263-273.
[19] Nikitin, L. N.; Nikolaev, A. Yu.; Said-Galiev, E. E.; Gamsasade, A. I.; Khokhlov, A. R.
The formation of the porosity in the polymers with help of supercritical carbon dioxide.
Supercritical fluids. The theory and practice, 2006, 1, 77-88.
[20] Berens, A. R.; Huvard, G. S.; Korsmeyer, R. W.; Kunig, F. W. Application of
compressed carbon dioxide in the incorporation of additives into polymers, J. Appl.
Polym. Sci. 1992, 46, 231-242.
[21] Webb, K. F.; Teja, A. S. Solubility and diffusion of carbon dioxide in polymers, Fluid
Phase Equilibria 1999, 158-160, 1029-1034.
[22] Von Schnitzler, J.; Eggers, R. Mass transfer in polymers in a supercritical CO2 –
atmosphere, J. Supercritical Fluids 1999, 16, 81–92.
[23] Crank, J. The mathematics of diffusion. Clarendon Press: Oxford, 1975.
[24] Ronova, I. A.; Nikitin, L. N. ; Sokolova, E. A ; Bacosca, I.; Sava, I.; Bruma, M.
Swelling of polyheteroarylenes in supercritical carbon dioxide, J. Macromol. Sci., Part
A, 46 (10), 929-936 (2009).
[25] Kazarian, S. G. ; Vincent, M.F.; Bright, F.V.; Liotta, C. L.; Eckert, C. A. Specific
Intermolecular Interaction of Carbon Dioxide with Polymers, J. Am. Chem. Soc., 1996,
118: 1729-1736.
[26] Kostina, Yu. V.; Moskvicheva, M. V.; Bondarenko, G. N.; Yablokova, M. Yu;
Alentiev, A. Yu. The influence of solvent nature on the imidization reaction of
polyamidic acid based on benzophenonetetracarboxylic dianhydride and m-phenylene
diamine. Proceedings 15th Russian Conference on “The structure and Dynamic of
molecular system” Yoshkar Ola, 2008, vol. 1, p.133-138.
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.65-73 ©2010 Nova Science Publishers, Inc.

Chapter 6

INHIBITION OF 2-HEXENAL OXIDATION BY


ESSENTIAL OILS OF GINGER, MARJORAM, JUNIPER
BERRY, BLACK AND WHITE PEPPER

T. A. Misharina*, M. B. Terenina,
N. I. Krikunova, and I. B. Medvedeva
Emanuel Institute of Biochemical Physics,
Russian Academy of Sciences ul. Moscow, Russia

ABSTRACT
The essential oils from black and white pepper (Piper nigrum L.), juniper berry
(Juniperus communis L.), marjoram (Origanum majorana L.), and ginger (Zingiber
officinale L) were studied. By the method of capillary gas-liquid chromatography the
efficiency of inhibition of autooxidation of 2-hexenal by essential oils in hexane solutions
was studied. The stability of essential oils components during the storage of hexane
solutions was determined and compared with that of pure oils.

Keywords: Essential oils; autooxidation of 2-hexenal; inhibition of oxidation; changes in


composition during storage.

To protect themselves from infections and parasites and in reply to stress, plants
synthesize low molecule volatile terpenoids, the mixture of which is called essential oils.
Essential oils of many plants possess an intensive and pleasant aroma and also are
biologically active [1-3]. Due to these properties, essential oils are actively used in medical
and pharmaceutical industries, in aromatherapy and cosmetology, and in the production of
natural effective food flavourings [3, 4]. Organoleptic properties and biological activity of
essential oils depend on their composition [5-8]. The study of individual components of

* Emanuel Institute of Biochemical Physics, Russian Academy of Sciences ul. Kosygina 4, Moscow, 119334 Russia
e-mail: Tmish@rambler.ru
66 T. A. Misharina, M. B. Terenina, N. I. Krikunovako et al.

different essential oils showed that many terpenes possess antiradical and antioxidant activity
(AOA). The activity of cyclic monoterpene hydrocarbons with two double bonds is
comparable with AOA of phenol and α -tocopherol [9-11]. Thus, α- and γ-terpinenes
inhibited the methyl linoleate oxidation [10-13]. Sabinene, terpinenes, citronellal, neral, and
geranial possess antiradical activity [10, 14 -18]. As a rule, antioxidant activity of essential
oils is higher than of their individual components. This fact indicates the existence of
synergetic effects due to the complex multicomponent composition of oils [15, 19, 20].
For the estimation of antioxidant properties of substances or their mixtures, many
different methods are used. It was showed that the value of AOA significantly depends on the
method of its estimation and on the qualitative and quantitative composition of systems under
test [6, 10, 13,20,21]. One of the simple and informative methods of the quantitative
assessment of AOA is based on the inhibition of lower aldehyde autooxidation in the presence
of antioxidant substances [7, 8, 20- 25].
The aim of the work is to study and compare antioxidant properties of 5 essential oils in
the model system of autooxidation of 2-hexenal, the assessment of the influence of essential
oils composition and concentration on their AOA, and also the study of changes in the
composition of essential oils in the process of autooxidation in solutions and in pure oils.

MATERIALS AND METHODS


The fresh samples of essential oils of black and white pepper Piper nigrum L., juniper
berry Juniperus communis L., ginger Zingiber officinale L., and marjoram Origanum
majorana L. were obtained from the company “Plant Lipids Ltd.”, India. Hexane, undecane
and trans-2-hexenal were purchased from Sigma.

Model Systems

600 μl of trans-2-hexenal (3 μl/ml) and 400 μl of n- undecane (2 μl/ml) (internal


standard) were dissolved in 200 ml of n-hexane. The solution was separated into 3-ml
aliquots, which were placed in 5-ml glass vials and then 10 μl (3.33 μl/ml), 50 μl (16.5 μl/ml)
or 210 μl (70 μl/ml) of essential oils were added. Oil was not added in the control sample.
Each sample was prepared twice, the control sample was prepared three times. The samples in
vials with stoppers were stored in light under room temperature for 60 days. Every week vials
were opened and blown with 10 ml of air with the help of a pipette. The quantitative content
of 2-hexenal and components of essential oils in vials were determined by method of capillary
gas chromatography after every 10 days.

Gas-Chromatography Analysis

The Kristall 2000M chromatograph (Russia) with a flame ionization detector and an
SPB-1 silica fused capillary column (50 m x 0.32 mm, phase layer 0.25 μm) were used for
analyses of samples. The column temperature was increased from 60 to 250oC with the rate of
Inhibition of 2-Hexenal Oxidation By Essential Oils of Ginger, Marjoram… 67

8oC/min, the temperature of detector and injector was at 250oC. The rate of carrier gas helium
through the column was 1.5 ml/min. The identification of components in oil samples was
carried out on the basis of retention indices by their comparison with literary [26] or
experimental data obtained by us. The quantitative content of 2-hexenal and essential oils
components was calculated by the ratio of peak areas, which corresponded to the substances
and internal standard. The oxidation extent of 2-hexenal and essential oils components (%)
was determined in reference to their content in initial samples.

RESULTS AND DISCUSSION


The oils that we chose differed in their quantitative and qualitative composition but
contained many common components. Due to the method of capillary gas chromatography
we could estimate changes in the content of 2-hexenal and each oil component in model
solutions with different oil concentrations and in pure oil and also to distinguish the oxidation
products of main components. The comparison of oils composition and the oxidation speeds
of their components allowed estimate some regularity, which enables us to predict and
regulate oil composition to obtain stable mixtures. This is very important because essential
oils are currently widely used in industry and medicine. Usually the recommended storage
time for oils is 1 year; however, nobody has studied yet what really happens with oils during
this period. We earlier studied the changes in the composition of coriander, laurel, marjoram,
and fennel oils in the process of storage of pure oil samples in dark and in the light but in
bottles from dark glass [27-29]. It was established that the main process was the oil
components oxidation. Thus, we found, and then it was proved in works [10, 11], that cyclic
monoterpenes hydrocarbons α- and γ- terpinenes are completely oxidized into aromatic
hydrocarbon p-cymene. We also found oxidation products of other components of essential
oils - oxides, alcohols, and aldehydes [26-29].
For the estimation of antioxidant properties of essential oils we used a model system of
autooxidation of 2-hexenal to corresponding acid. [7, 8]. As a criteria for the comparison of
antioxidant activity of essential oils we used the quantity of 2-hexenal, which remained in
model systems after 40 days in reference to the initial quantity (%). Fig. 1 presents the
obtained results of relative AOA values of 5 studied essential oils. The model systems
included marjoram and white pepper in two concentrations: 3.3 μl/ml and 16.5 μl/ml. For the
essential oils of ginger, juniper berry and black pepper we studied an additional third system
in which the content of oil was 70 μl/ml of hexane solution. As is clearly seen from Fig. 1, the
ginger oil in the systems with minimal content of (3.3 μl/ml ) had AOA more than 50%. AOA
of marjoram oil at the same concentration showed activity 48%. The essential oils of juniper
berry, black and white pepper in diluted solutions (3.3 μl/ml) had very low AOA: 20-28%. As
can see from Fig.1, there was a dependence between concentration and AOA for all oils. It is
noteworthy that the increase in activity was usually not in proportion to the growth of
essential oil concentration. So, the increase in concentration of black pepper and juniper berry
oils by five times (from 3.3 to 16.5 μl/ml) led to the increase of AOA by 2.5-3 times. The
further increase in concentration of these essential oils up to 70 μl/ml did not change AOA.
68 T. A. Misharina, M. B. Terenina, N. I. Krikunovako et al.

100

90

80

70
2-Hexenal content, %

60

50

40

30

20

10

0
1 2 3 4 5 6

Control C1 C2

Figure 1. Inhibition of 2-hexenal oxidation by essential oils in hexane solutions during 40 days storage .
1 - Control (2-hexenal). Essential oils: 2 – ginger, 3 - marjoram, 4 - juniper berry, 5 - black pepper, 6 -
white pepper. Concentration of essential oils (μl/ml): C1 is 3.3, C2 is 16.5, and C3 is 70.

AOA of ginger essential oil was studied for three model systems with oil concentrations
of 3.3, 16.5, and 70 μl/ml. In the first system, oil inhibited the oxidation of 2-hexenal by 54%,
and in the second it inhibited by 78%, and in the third it inhibited by 94% (Fig. 1). This is
very high activity taking into consideration the fact that oil does not contain phenol
derivatives. It consisted from sesquiterpene hydrocarbons, which differed in stability. The
dynamics of changes of main components of ginger essential oil for the system with oil
concentration 16.5 μl/ml is presented in Fig. 2. The main oil component – zingiberene - is
oxidized faster than all others. After 60 days of storage, its content in the first solution
decreased by 20 times, in the second it decreased by seven times, and in the third it decreased
by 2.5 times. Also, the content of bisabolenes and sesquiphellandrene decreased (Fig. 2). It is
interesting that, parallel to the decrease in zingiberene concentration, we revealed the increase
in α-curcumene content. The structure of zingiberene is similar to the structure of α -
terpinene. Both compounds have a similar hexamerous cycle with two double bonds. The
oxidation product of α - terpinene was p-cymene [27-29]. Probably, zingiberene was oxidized
in α - curcumene, which is an aromatic compound similar to p-cymene. The content of
zingiberene in ginger oil is approximately 35% and thus this oil has high AOA. In the process
of storage in the dark of pure ginger essential oil for 12 months, the content of zingiberene
decreased by two times and the content of α - curcumene increased by 1.5 times. Such change
Inhibition of 2-Hexenal Oxidation By Essential Oils of Ginger, Marjoram… 69

in the oil composition significantly worsens its organoleptic characteristics and


physicochemical and biological properties of ginger essential oil. This should be taken into
consideration while using stored ginger essential oil. The sweet marjoram essential oil
contained from 15 to 25% of linalool, terpinen-4-ol, and γ-terpinene and did not contain
phenol derivatives. With the increase in oil concentration its AOA increased by 1.5 times and
was 72% (Fig. 1). Fig.3 presents the quantity of compounds which was left after 40 days of
oxidation (in percentage from initial). During the storage of the diluted solution of oil, mono-
and sesquiterpene hydrocarbons and even terpinen-4-ol were significantly oxidized; in more
concentrated solution, only the content of α - and γ-terpinenes decreased significantly. The
content of p-cymene which was oxidation product of terpinenes increased. In both solutions
the content of geraniol changed slightly. The pure marjoram oil preserved content stability in
the dark for a year; in the light the noticeable oxidation began after 4 months of storage [28].

120
Content,% from initial

100

80

60

40
0 20 40 60
days

1 2 3 4 5

Figure 2. The content of main components (% from initial) of ginger essential oil in the oxidation
process in hexane solution: 1 - α-curcumene, 2 - zingiberene, 3 - α -bisabolene, 4 – β-bisabolene, and 5
- sesquiphellandrene.
70 T. A. Misharina, M. B. Terenina, N. I. Krikunovako et al.

100
90
80
70
Content,% from initial

60
50
40
30
20
10
0
1 2 3 4 5 6 7 8

I II
Figure 3. The influence of marjoram oil concentration (I is 3.3 μl/ml and II is 16.5 μl/ml) on the content
of its components (% from initial) after 40 days of oxidation: 1 - sabinene, 2 - myrcene, 3 - α -
terpinene, 4 - γ-terpinene, 5 - α -terpinolene, 6 - β -caryophyllene, 7 - geraniol, and 8 – terpinen-4-ol.

The significant influence of the concentration on antioxidant properties was revealed


from black pepper, white pepper, and juniper berry oils. They had practically the same
quantitative composition of components - mono- and sesquiterpene hydrocarbons. In hexane
solution they did not show antioxidant properties because the oxidation of 2-hexenal
happened to the same extend as in the control sample, which did not contain essential oils.
Under the concentration 16.5 μl/ml the black pepper essential oil inhibited the oxidation of 2-
hexenal by 58%, white pepper essential oil inhibited it by 49%, and juniper berry oil inhibited
by 65%. In the solution with a concentration of 70 μl/ml, the black pepper essential oil had
AOA 62% and that of juniper berry had 66% (Fig. 1). In the diluted solutions, during 40 days
practically all components of essential oil were oxidized. For the solution with a
concentration of 16.5 μl/ml, the changes in the content of main oil components are presented
in Fig. 4. As is seen, after 40 days the content of α - and γ-terpinenes in oils decreased by 5-6
times and that of β-caryophyllene decreased by 2.5-3 times, the content of other components
changed insignificantly. We revealed that the oxidation of caryophyllene began when the
majority of α - and γ -terpinenes was oxidized. Under the storage of pure essential oils in the
dark a similar process was detected after 8 months. By that time, the content of terpinenes
was 2-10% of the initial content and that of caryophyllene was 80-85%, whereas the content
of caryophyllene oxide increased by 2-2.5 times and that of p-cymene increased by 4 times.
Thus, the content of γ-terpinene and caryophyllene oxide may be an indicator of the freshness
Inhibition of 2-Hexenal Oxidation By Essential Oils of Ginger, Marjoram… 71

and quality of black and white pepper essential oils. The additional experiments showed that
the stability of these oils increase significantly at addition to these oils of other essential oils
such as, for example, juniper berry, garlic, or nutmeg oils.

100

90

80
Content, % from initial

70

60

50

40

30

20

10

0
1 2 3 4 5 6 7

I II III
Figure 4. The content of main components (% from initial) in black pepper (I), white pepper (II) and
juniper berry (III) in the oxidation process in hexane solution: 1 - α – and β- pinenes, 2 - sabinene, 3 -
myrcene, 4 - 3-carene, 5 - limonene, 6 – α- and γ-terpinenes, and 7 - β-caryophyllene.

Thus, conducted research show that antioxidant properties of essential oils are determined
by their composition. Antioxidant properties of essential oils, which consist of terpene
hydrocarbons and alcohols, are determined by content of α- and γ-terpinenes and their
sesquiterpene analogs. We revealed the significant influence of the concentration of such oils
on their antioxidant properties. It was established that in the oxidation of the main
components of essential oils the substances are formed, which are present in natural essential
oils. The stability of essential oil composition increased with the increase of their
concentration in model solutions. The oxidation of substances in pure essential oils happened
much more slower than in solutions.
72 T. A. Misharina, M. B. Terenina, N. I. Krikunovako et al.

REFERENCES
[1] L.H. Madsen, B.R.Nielsen, G.Bertelsen, L.H. Skibsted: Screening of antioxidative
activity of spices. A comparison between assay based on ESR spin trapping and
electrochemical measurement of oxygen consumption. Food Chem., 57, 331 (1996).
[2] S.A. Voitkevich: “Efirnye masla dlya parfyumerii i aromaterapii” (Essential Oils for
Perfumery and Arometherapy). Pishch. Prom., Moscow, 1999. 3. Flavours and
Fragrances. Chemistry, Bioprocessing and Sustainability (Ed. R.G. Berger). Springer,
New York, 2007, 43-116.
[3] K. Bauer, D. Garbe, H. Surburg: Common Fragrance and Flavor Materials, VCH
Verlag, Weinheim, 1990.
[4] G. Cervato, M. Carabelli, S. Gervasio, A. Cittera, R. Cazzola, B. Cestaro: Antioxidant
properties of oregano (Origanum vulgare L.) leaf extracts. J. Food Biochem., 24, 453
(2000).
[5] H.J.D. Dorman, A. Peltoketo, R.Hiltunen, M.J.Tikkaken: Characterisation of the
antioxidant properties of deodourised aqueous extracts from selected Lamiaceae herbs.
Food Chemistry, 83, 255 (2003).
[6] K.G. Lee, T. Shibamoto: Antioxidant property of aroma extract isolated from clove
buds. Food Chem., 74, 443, (2001).
[7] K.W. Lee, Y.J. Kim, D.-O. Kim, H.J. Lee, C.Y. Lee: Major phenolics apple and their
contribution to the total antioxidant capacity. J. Agric. Food Chem., 51, 6516, (2003).
[8] G.Litwinienko, K.U. Ingold: Solvent effects on the rates and mechanisms of reaction of
phenols with free radicals. Acc. Chem. Res., 40, 222 (2007).
[9] G. Ruberto, M. Baratta: Antioxidant activity of selected essential oil components in two
lipid model systems. Food Chem., 69, 167 (2000).
[10] M.C. Foti, K.U. Ingold: Mechanism of inhibition of lipid peroxidation by γ–terpinene,
an unusual and potentially usefull hydrocarbon antioxidant. J. Agric. Food Chem. 51,
2758 (2003).
[11] M.T. Baratta, H.J.D. Dorman, S.G. Deans, D.M.Biondi, G.Ruberto: Chemical
composition, antimicrobial and antioxidative activity of laurel, sage, rosemary, oregano
and coriander essential oils. J. Essent. Oil Res., 10, 618 (1998).
[12] S.S.Pekkarinen, H.Stocmann, K.Schwarz, M.Heinone, A.I.Hopia: Antioxidant activity
and partitioning of phenolic acids in bulk and emulsified methyl linoleate. J. Agric.
Food Chem., 47, 3036 (1999).
[13] G.Sacchetti, S.Maietti, M.Muzzoli, M.Scaglianti, S.Manferdini, M.Radice, R.Bruni:
Comparative evaluation of 11 essential oils of different origin as functional
antioxidants, antiradicals and antimicrobials in foods. Food Chem. 91, 621 (2005).
[14] G. Singh, S. Maurya, C. Catalan, M.P. De Lampasona: Studies on essential oil, Part 42.:
chemical, antifungal, antioxidant and spout suppressant studies of ginger essential oil
and its oleoresin. Flavour Fragrance. J., 20, 1 (2005).
[15] A.Wei, T. Shibamoto: Antioxidant activities and volatile constituents of various
essential oils. J. Agric. Food Chem., 55, 1737 (2007).
[16] C. Menut, J.M. Bessiere, D.Samate, A.K. Djibo, G.Buchbauer, B. Schopper: Aromatic
plants of tropical west Africa. XI. Chemical composition, antioxidant and antiradical
Inhibition of 2-Hexenal Oxidation By Essential Oils of Ginger, Marjoram… 73

properties of the essential oils of three Cymbopogon species from Burkina Faso. J.
Essent. Oil Res., 12, 207 (2000).
[17] N.Mimica-Dukic, B. Bozin, M. Sokovic, N. Simin: Antimicrobial and antioxidant
activities of Melissa officinalis L. (Lamiaceae) essential oils. J. Agric. Food Chem., 52,
2485 (2004).
[18] D.Huang, B.Ou, L.R.Prior: The chemistry behind antioxidant capacity assays. J. Agric.
Food Chem., 53, 1841 (2005).
[19] K.G. Lee, T.Shibamoto: Determination of antioxidant potential of volatile extracts
isolated from various herbs and spices. J. Agric. Food Chem., 50, 4947 (2002).
[20] K.G. Lee, T.Shibamoto: Antioxidant activities of volatile components isolated from
Eucalyptus species. J.Sci. Food Agric., 81, 1573 (2001).
[21] K.Yanagimoto, H.Ochi, K.G. Lee, T. Shibamoto: Antioxidative activities of volatile
extracts from green tea, oolong tea, and black tea. J. Agric. Food Chem., 51, 7396
(2003).
[22] C.J. Lee, T. Shibamoto, K.G. Lee: Identification of volatile components in basil
(Ocinum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant
properties. Food Chem., 91, 131 (2005).
[23] B.Bozin, N.Mimica-Dukic, G.Anachov: Characterization of the volatile composition of
essential oils of some Lamiaceae spices and the antimicrobial and antioxidant activities
of the entire oils. J. Agric. Food Chem., 54,1822 (2006).
[24] T.A. Misharina, A.L. Samusenko: Antioxidant properties of essential oils from lemon,
grapefruit, coriander, clove, and their mixtures. Applied Biochem. and Microbiol., 44,
473 (2008).
[25] W. Jennings, T.Shibamoto: Qualitative Analysis of the Flavor and Fragrance Volatiles
by Glass Capillary Gas Chromatography, Academic, New York: 1980, 130-154.
[26] T.A Misharina: Influence of the duration and conditions of storage on the composition
of the essential oil from coriander seeds. Applied Biochem. and Microbiol., 37, 622
(2001).
[27] T.A Misharina, A.N. Polshkov, E.L. Ruchkina, I.B. Medvedeva: Changes in the
composition of the essential oil of marjoram during storage. Applied Biochem. and
Microbiol., 39, 311 (2003).
[28] T.A Misharina, A.N. Polshkov: Antioxidant properties of essential oils: autoxidation of
essential oils from laurel and fennel and of their mixtures with essential oil from
coriander. Applied Biochem. and Microbiol., 41, 610 (2005
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.75-82 ©2010 Nova Science Publishers, Inc.

Chapter 7

THE ORGANOPHOSPHORUS PLANT GROWTH


REGULATOR MELAPHEN AS
ADAPTOGEN TO LOW MOISHER

I. V. Zhigacheva*, E. B. Burlakova, T. A. Misharina,


M. B. Terenina, N. I. Krikunova, I. P. Generozova
and A. G. Shugaev
N.M. Emanual Institute of Biochemical Physics,
Russian Academy of Science, Moscow, Russia

ABSTRACT
We studied the effect of insufficient watering and organophorphorus plant growth
regulator (melaphene) on peroxide oxidation of lipids (POL) in pea sprouts membranes.
Water deficiency and melaphene increase the generation of reactive oxygen species.
However, a response to a presowing melaphene treatment of pea sprouts grown under
standard condition is higher by 18.5% as compared with pea sprouts grown under
conditions of water deficiency. A decrease in the generation of reactive oxygen species in
response to the melaphene treatment in comparison with control group is associated with
changing the fatty acid content of plasmatic and organellas membranes. In sprouts
membranes, the content of unsaturated fatty acids having 18, 20 and 22 carbon atoms
decreases drastically. The melaphene treatment prevents from changes in the fatty acid
content induced by a water deficiency. We supposed that the protective effect of
melaphene in conditions of water deficiency is associated with preventing thereby
changes in the fatty acid content of membranes.

Keywords: plant growth regulators, lipid peroxidation, plazmolemma, fatty acid.

* N.M. Emanual Institute of Biochemical Physics, Russian Academy of Science, 4 Kosygin str., Moscow, 119334
Russia, Zhigacheva@mail.ru
76 I. V. Zhigacheva, E. B. Burlakova, T. A. Misharina et al.

INTRODUCTION
Survival of plants in constantly changing conditions of an environment probably only at
the plant can adapt to them. At a cellular level it is carried out by means of protective
systems. As protective molecules in low concentration molecules superoxide and peroxides of
hydrogen which concentration in a cell raises some kinds stress may operate [1]. Reactive
oxygen species (ROS) stimulates the accumulation of cyclic adenosine monophosphate and
cyclic guanosine monophosphate, causes activation of protein kinases [2]. Its, on the one
hand, act as participants of signaling cascades as a result of which occurs gene expression
supervising synthesis of the protective systems components, on other hand, its activate the
processes of lipid peroxidation in biological membranes [3,4]. As the test for course of free
radical oxidation in biological membranes we studied influence of stress on processes of lipid
peroxidation. As model of stressful influence on biological membrane we used model of low
moisture. Cellular membranes are one of the basic places where there is a damage of a cell at
water deficiency. Water deficiency modifies cellular membranes and membranes of the
organelles influencing on their functions and a metabolism of a cell [5,6]. In this connection
as object of research served the fragments of the endoplasmatic reticulum, nuclear
membranes and plasma membranes. As plant growth regulators raise stability of plants to
stressful influences it was interesting to study influence of a plant growth regulator Melaphen,
the melamine salt of bis(oxymetyl)phosphonic acid, upon studied rates. Melaphen was
synthesized in Arbuzov Institute of Organic and Physical Chemistry, Kazan’ Research
Center, Russian Academy of Sciences.

NH2
O
N
HOP(CH2OH)2
H2N N NH2

Moreover, Karimova F.G. detected some phosphothirosine protein-target for Melaphen


actions. It was proteins of photosynthetic assimilation of carbon [7].
We also studied influence of low moisture and Melaphen upon fatty acids composition
of cellular membranes as at a drought changes fatty acids composition of biological
membranes [8, 9]

MATERIALS AND METHODS


In the work, we used biological membranes isolated from Pisum sativum pea sprouts
grown under standard conditions and under low watering conditions.
Pea seeds were germinated as follows: control seeds were rinsed with soapy water and
0.01% KMnO4 solution and then kept watered for 60 min; experimental seeds were kept in a
10-7% melaphene solution for 30 min and then in water for another 30 min. In a day, half of
the control seeds and half of the melaphene-treated seeds were carried over onto a dry
filtering paper in open cuvettes. After two days of “drought”, the seeds were carried over into
The Organophosphorus Plant Growth Regulator Melaphen … 77

closed cuvettes on a periodically wetted filtering paper, where the seeds remained for 4 days.
On the fifth day, we calculated the number of germinated seeds and isolated mitochondria.
Isolation of membranes from 5-day sprouts epicotyls was performed by a method of
[10] in our modification. The epicotyls having a length of 3 to 6 cm (20-25 g) were placed
into a homogenizer cup, poured with an isolation medium in a ratio of 1:2, and then were
rapidly disintegrated with scissors and homogenized with the aid of a press. The isolation
medium comprised: 0.4 M saccharose, 5 mM EDTA, 20 mM КН2РО4 (рН 8.0), 10 mM КСl,
2 mM dithioerythritol, and 0.1% BSA (free of fatty acids).The homogenate was centrifugated
at 25000g for 5 min. The precipitate was resuspended in 8 ml of a rinsing medium and
centrifugated at 3000g for 3 min. The suspension medium comprised: 0.4 M saccharose, 20
mM КН2РО4, 0.1%. BSA (free of fatty acids) (рН 7.4).
Methylation of fatty acids. A one-step methylation of fatty acids was performed by a
method previously described in [11] and modified.
Gas chromatography analysis (GCA) of samples of hexane solutions of fatty acid
methyl esters was performed on a Kristall 2000 M chromatograph (Russia) equipped with a
flame-ionization detector and an SPB-1 quartz capillary column (50 m x 0.32 mm, phase
layer 0.25 µm). An analysis of hexane solutions of methyl esters was performed at a
programmed temperature at a rate of 4°С/min from 120° to 270 °С (50 min). The injector and
detector temperature was 250°С. The gas carrier speed was 1.5ml/min. An analysis was
performed for 2 µm samples of hexane solutions. The components in methyl esters samples
were identified from the retention indices thereof as compared with references [12] or our
experimental data obtained.
In the experiment, we used the following reagents: potassium carbonate, methanol
(Merck, Germany), hexane (Panreac, Spain), acetyl chloride (Acros, Belgium), saccharose,
BSA (free of fatty acids)(Sigma, USA).

RESULTS AND DISCUSSION


In the first series of experiments, we studied the effect of drought and melaphene on free-
radical oxidative processes in pea sprouts membranes. As a model process, a lipid
peroxydation (LP) was studied. Low moisture results in a 1.5-fold increase in the POL
products fluorescence. The melaphene treatment of the control seeds increased the POL
products fluorescence by a factor of 6.5; whereas the fluorescence of POL products in
membranes of pea sprouts subject to insufficient watering increased by a factor of 5.3 (Fig.
1). An increase in the content Н2О2 and POL products was observed also in membranes of
other plants, grown under drought conditions. [13-15]. Some authors are of opinion that an
increase in the level of reactive oxygen species, namely Н2О2, caused by stresses such as
drought is associated with the participation of Н2О2 in redox-regulation of signal cascade
during acclimatization [3,16-18]. Note that an increase in the concentration of reactive
oxygen species, mainly Н2О2 is observed also as a response to a stimulation of membrane
receptors by various agents, in particular peptide factors of growth [19, 20] and cytokines
[21.22]. In our experiments, an increase in the POL products level may be evidence for the
activation of a family of NADPH-oxidases associated with a plasmatic membrane, which
catalyze the generation of reactive oxygen species [23]. The supposition is supported by data
78 I. V. Zhigacheva, E. B. Burlakova, T. A. Misharina et al.

on increasing the microviscosity of annular lipids upon a melaphene treatment of animal and
plant cells [24]. A lowered response to the effect of the preparation on membranes of pea
sprouts subject to insufficient watering may be a result of modification of physicochemical
properties of the membranes. Therefore, in another series of experiments we studied the effect
of the water stress and melaphene treatment on a fatty acid content of the membranes.

Influenc e of Melaphen on the level of 
L P  in the peas  membrane. 
7000000

6000000
Intens ity of fluores c enc e

5000000
C ontrol
4000000
C ontrol +  Melaphen

3000000 Drought
Drought+ Melaphen
2000000

1000000

0
380 400 420 440 460 480 500 520 540
Wa ve s  le ng th in nm  
Legend : abscissa axis – Waves length in nm; ordinate axis – intensity of fluorescence.

Picture 1. INFLUENCE OF MELAPHEN ON THE LEVEL OF LIPID PEROXYDATION UN THE


PEA SPROUT MEMBRANES.

Water deficiency results in a decrease in the ratio of unsaturated and unsaturated fatty
acids in pea sprouts membranes (Table 2). In particular, there occurs a 3-fold increase in the
content of lauric acid, a 1.7-fold increase in the content of nonadecanoic acid, and a 2-fold
increase in the content of heptadecanoic and arachidonic acid (Table 1). At the same time, the
proportion of unsaturated fatty acids having 18 carbon atoms decreases. The content of
linoleic acid decreases by 10.2%; the contents of linolenic and octadecenic acids decrease by
17.6% and 24%, respectively. The content of stearic acid increases by 66% that result in a
decrease in the ratio of unsaturated fatty acids having 18 carbon atoms and stearinic acid from
20.78±0.60 to 11.30±0.40 (Table 2). Similar data on the effect of water deficiency on the fatty
acid content were obtained for potato cell membranes and membranes of Arabidopsis thaliana
and apricot leaves [8, 25-27].
Note a considerable decrease in the proportion of С20 - fatty acids. The ∑С20 ratio of
unsaturated acids/ С20:0 decreases by a factor of 3.3. Hence, unsaturated fatty acids having 22
carbon atoms completely vanish from the membrane composition. The melaphene treatment
of pea seeds prevents from drought-induced changes in the fatty acid composition of
membranes. It is safe to suppose that a decrease in the generation of reactive oxygen species,
as a response to melaphene treatment of pea seeds germinated under conditions of insufficient
watering, is a result of changes in the fatty acid content of membranes, since these changes
The Organophosphorus Plant Growth Regulator Melaphen … 79

according to some authors [28] may affect the function of receptors and regulate the number
thereof.

Table 1. Influence of Melaphen and low moisture for fatty acid composition of the peas
membrane. (Relative per-cent. Results of 6 experiments).

Fatty acid Control Control+Melaphen Drought Drought+Melaphen


12:0 0.33±0.13 0.21 ±0.10 0.99±0.15 0.36±0.10
14:0 0.67±0.03 0.45±0.02 0.64±0.20 0.65±0.20
15:0 0.37±0.15 0.65±0.12 0.51±0.020 0.35±0.05
16:1ω7 0.69±0.10 0.69±0.10 0.95±0.13 0.58±0.15
16:0 18.67±0.15 18.09±0.20 20.48±0.15 18.5 6±0.15
17:0 0.34±0.20 0.61±0.12 0.69±0.10 0.38±0.16
18:2 ω6 55.40±0.30 59.00±0.40 49.76±0.35 55.45±0.14
18:3 ω3 11.1±0.2 0 9.50±0.11 9.15±0.30 11.22±0.23
18:1 ω9 5.14±0.40 3.78±0.37 5.89±0.20 4.71±0.40
18:1 ω7 1.10±0.10 1.16±0.24 0.83±0.03 0.98±0.05
18:0 3.50±0.15 3.75±0.29 5.80±0.24 3.60±0.10
19::0 0.13±0.04 0.11±0.01 0.22±0.05 0.16±0.03
20:2 ω6 0.33±0.10 0.32±0.08 0.12±0.30 0.35±0.10
20:1 ω 9 0.35±0.03 0.24±0.01 0.26±0.03 0.35±0.03
20:1 ω15 0.20±0.01 0.10±0.01 0.14±0.10 0.25±0.01
20: 1 ω 7 0.22±0. 10 0.10±0.20 0.17±0.12 0.24±0.16
20:0 0.52±0.31 0.54±0.26 1.06±0.22 0.51±0.12
22:1 ω13 0.03±0.01 0.10±0.01 - 0.09±0.06
22:1 ω9 0.13±0.01 0.02±0.01 - 0.10±0.01
22: 1 ω 7 0.12±0.02 0.10±0.01 - 0.14±0.02
22:0 0.41±0.11 0.30±0.03 0.90±0.18 0.47±0.05
23:0 0.27±0.02 0.25±0.01 0.65±0.10 0.30±0.10

Table 2. Influence of Melaphen and low moisture for index of fatty acid composition of
pea sprout membranes. (Relative per-cent. Results of 6 experiments).

Fatty acids Control Control+Melaphen Drought Drought+Melaphen


∑ saturated fatty acids 25.205±0.108 24.965±0.120 32.735±0.140 25.340±0.106
∑ unsaturated fatty acids 74.795±0.140 75.110±0.128 67.265±0.173 74.660±0.137
18:0 3.500±0.150 3.750±0.290 5.800±0.240 3.600±0.100
∑ unsaturated fatty acids С18 72.735±0.250 73.44±0.280 65.625±0.220 72.49±0.213
∑ unsaturated С18/С18:0 3.500±0.166 3.750±0.970 5.800±0.920 3.600±1.000
Index unsaturation С18 42.950±1.619 40.384±0.857 23.000±1.089 41.800±2.029
20:0 0.520±0.310 0.540±0.260 1.060±0.220 0.510±0.120
∑ unsaturated С20 1.100±0.060 0.760±0.075 0.690±0.113 1.170±0.075
∑ unsaturated С20/С20:0 2.120±0.194 1.400±0.288 0.650±0.514 2.290±0.625
22:0 0.410±0.110 0.300±0.030 0.900±0.180 0.470±0.050
∑unsaturated C22 0.275±0.012 0.022±0.012 - 0.340±0.003
∑ unsaturated С22/С22:0 0.670±0.109 0.730±0.400 0 0.860±0.060
∑unsaturated/ ∑saturated 3.370±0.129 3.000±0.106 2.050±0.124 2.950±0.129
fatty acids
80 I. V. Zhigacheva, E. B. Burlakova, T. A. Misharina et al.

CONCLUSION
The data obtained show that insufficient watering modifies not only the ratio С18
unsaturated fatty acids to С18:0, but also affects more strongly the ratio С20 unsaturated fatty
acids to С20:0, as a result, the membranes are completely devoid of unsaturated fatty acids
having 22 carbon atoms. Evidently, melaphene prevents from changing the fatty acid content
of membranes and thus enhances the resistance of plants to insufficient watering. Moreover,
in pea sprouts membranes treated with melaphene and grown under conditions of insufficient
watering, the proportion of unsaturated fatty acids having 22 carbon atoms increases by a
factor of 1.5 as compared with pea sprouts grown under standard conditions; the effect may
obviously results in enhancement of productivity of plants grown even under drought
conditions [29].

REFERENCES
[1] Bhattacharjee S./ Reactive oxygen species and oxidative burst. Roles in stress,
senescence and signal transduction in plants.// Curent science 2005, 89, 1113-1121.
[2] 2.Rivlin J., Mendel I., Rubinstein S., Etkovitz N., Breibrant H./ Role hydrogen peroxide
in sperm capacition and acrosome reaction// Biol. Reproduction 2004, 70, 518-522.
[3] Beck E H, Fettig S, Knake C, Hartig K and Bhattarai T./ Specific and unspecific
responses of plants to cold and drought stress; //J. Biosci. 2007 32 501–510
[4] M.F. Bystrova, E.N. Budanova, Hydrogen peroxide and peroxiredoxines in redox-
regulation of intracellular signaling.// Biol. Membranes 2007, 24, 2, 115-125.
[5] Junior R. R.M., Oliveira M.S.C., Baccache M.A., F.M. de Paula/ Effect of Water deficit
and rehydratation on the polar lipid and membrane resistance leaves of Phaseolus
vulgaris L. cv. Perola.// Brasilian archives of biology and technology, 2008, V.51, N 2,
[6] Sahsah Y, Campos P., Gareil M. et all/ Enzymatic degradation of polar lipid in Vigna
uniquiculata leaves and influence of drought stress// Plant. Physiol. 1988, V. 104, P.
577-586.
[7] Karimova F.G., Vanjushina S.A., Fedina E.O., Mudarisov F.F.// Tyrosine
phosphorilation of plant proteins induced by melaphene.// Status of studies and outlook
for the future use of a plant growth regulator of the new generation – Melaphene in
farming and biotechnology 2006, Kazan’, Shkola, 50-69.
[8] A. Leone A. Costa, S. Grillo, M. Tucci and all/ Acclimation to low water potential
changes in membrane fatty acid composition and fluidity in potato cells// Plant, Cell
and Environment 1996 19, 1103-1109
[9] A. Ayoz, A. Kadioglu, A. Dogru//Leaf rolling effects on lipid and fatty acid
composition in Ctenanthe setosa (Marantaceae) subjected to water-defficit stress// Acta
Physiol.Plantarum 2001, 23, 1861-1864.
[10] V. I. Popov, E.K.Ruge, A.A. Starkov// Influence of inhibitors of electronic transfer on
formation of reactive oxygen species at oxidation of succinate be rea sprouts
mitochondria// Biochemistry (Biohimiya) 2003, 68, 7, 910-916.
[11] J. Wang, H. Sunwoo, G. Cherian, I.S. Sim// Fatty acid determination in chicken egg
yolk. A comparison of different methods// Poultary science 2000, 79, 1168-1171.
The Organophosphorus Plant Growth Regulator Melaphen … 81

[12] R.V. Golovina, T.E. Kuzmenko// Thermodynamic evaluation interaction of fatty acid
metyl esters with polar and non-polar stationary phases, based on their retention
indices chromatographia// 1977, 10 , 9, 545-546.
[13] D. Pastore, D. Trono, M. N. Laus, N. Di Fonzo and Z. Flagella// Possible plant
mitochondria involvement in cell adaptation to drought stress. A case study: durum
wheat mitochondria//J. of Experemental Botany 2005, 58, 2, 195-210.
[14] Xiaoli Liu , Xuejun Hua Ж Juan Guo , Dongmei Qi ,Lijuan Wang , Zhipeng Liu ,
Zhiping Jin, Shuangyan Chen , Gongshe Liu// Enhanced tolerance to drought stress in
transgenic tobacco plants overexpressing VTE1 for increased tocopherol production
from Arabidopsis thaliana// Biotechnol Lett. 2008, 30:1275–1280
[15] P. Valentovič, M. Luxová, L. Kolarovič, O. Gašparíková// Effect of osmotic stress on
compatible solutes content, membrane stability and water relations in two maize
cultivars// Plant Soil Environ 2006, 52, 4, 195-210
[16] Dutilleul C, Garmier M, Noctor G, Mathieu C, Chetrit P, Foye CH, et al.// Leaf
mitochondria modulate whole cell redox homeostasis, set antioxidant capacity, and
determine stress resistance through altered signaling and diurnal regulation// The Plant
Cell 2003 ,15, 1212–1226
[17] Leister D. // Genomics-based dissection of the cross-talk of chloroplasts with the
nucleus and mitochondria in Arabidopsis //. Gene 2005, 354, 110–116.
[18] Rhoads DM, Subbaiah CC.// Mitochondrial retrograde regulation in plants//
Mitochondrion 2 007, 7, 177–194.
[19] Ohba M., Shibanuba M., Kuroki T., Nose K.// Production of hydrogen peroxide by
transforming grows factor-beta I and its involvement in induction of egr-I in mouse
osteoblastic cells// J. Cell. Biol. 1994, 126, 1079-1088.
[20] Sandaresan M., Yu.Z.X. Ferrans V.J., Irani K., Finkel T.// Requirement for generation
H2O2 for platelet- derived grows factor signal transduction// Science 1995, 270, 269-
299.
[21] Sattler M., Winkler T., Verma S., Byrne S.H. Shrikhande G., Salgia R., Griffin J.D.//
Hematopoietic grows factors signal through formation of reactive oxygen species//
Blood 1999, 93, 2928-2935.
[22] Tala S., Woodhead V., Foreman J.C., Chain B.M.// The role reactive oxygen species in
triggering proliferation and IL-2 secretion in T cells// Free Rad. Biol. Med. 1999, 26,
14-23. Lanbeth J.D., Nox/Duox family of nicotinamide adenine dinucleotide
(phosphate) oxydases// Curr. Opin.Hematol. 2002, 9, 11-17.
[23] I.V. Zhigacheva, L.F. Fatkullina, E.B. Burlakova, A.G. Shugaev, I.P. Generozova, S.G.
Fattakhov, A.I. Konovalov// Influence of the organophosphorus plant grows regulator
on structural characteristics of membrane of plant and animal origin// Biological
membranes ( Biologicheskie membrany RUS) 2008,.25, № 2, . 150-156.
[24] S.P. Makarenko, Yu.M. Konstantinov, S.V. Kotimchenko, T.A. Konenkina, A.S.
Arziev, Fatty acid content of lipids of Zea mays and Elymus sibiricus.mitochondria
membranes. /Fiziologiya. rastenii 2003, .50, 4, 487-492.
[25] A. Gigon, A.R. Matos, D. Laffray, Y.Z.-Fodil, Anh-Thu Pham-Thi. Effect оf drought
on lipid metabolism in the leaves of Arabidosis Thaliana (Ecotype Columbia)// Annals
of Botany 2003, 93, 3 , 841-850.
82 I. V. Zhigacheva, E. B. Burlakova, T. A. Misharina et al.

[26] Guo Yun-ping, Li Jia-rui. Changts of fatty acids composition of membrane lipids,
tethylene release and lipoxygenase activity in leaves of apricot under drought// Journ.of
Zhejiang University (Agrical & Life Sci) 2002, .28, 5, 513-517.
[27] N.E. Kucherenko, A.V. Vasiliev// Lipidy 1985 Vyschashkola, Kiev, 247 pp.
[28] V.I. Kostin, O.V. Kostina, V.A. Isaichev// Results of stidies on use of melaphene in
crop growing. //State of studies and outlook for future use of a plant growth regulator
of the new generation – Melaphene in farming and biotechnology 2006, Kazan’,
Shkola, 27-34.
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.83-90 ©2010 Nova Science Publishers, Inc.

Chapter 8

ANTIOXIDANT PROPERTIES OF ESSENTIAL


OILS FROM CLOVE BUD, LAUREL, CARDAMOM,
NUTMEG AND MACE

T. A. Misharina*, M. B. Terenina, and N. I. Krikunova


Emanuel Institute of Biochemical Physics,
Russian Academy of Sciences ul, Moscow, Russia

ABSTRACT
The antioxidant properties and stability during the storage of hexane solutions of 5
individual essential oils from clove bud (Caryophyllus aromaticus L.), cardamom
(Elettaria cardamomum L.), laurel (Laurus nobilis L.), nutmeg (Myristica fragrans
Houtt.) and mace (Myristica fragrans Houtt), were studied by the method of capillary
gas-liquid chromatography. We assessed the antioxidant properties by the efficiency of
autooxidation inhibition of aliphatic aldehyde (trans-2-hexenal) into the according carbon
acid. The essential oil of clove bud had the maximal efficiency of inhibition of 2- hexenal
oxidation (83%) which was not depended on oil concentration in model solution.
Antioxidant properties of essential oils of nutmeg, mace and laurel was connected with a
content of substituted phenols, and depended poorly on oils concentration in model
systems. The stronger dependence the antioxidant activity on the oil concentration in
model solution was found for cardamom essential oil. We studied the changes in essential
oils composition during the storage of their hexane solutions for 100 days in the light and
compared it with the stability of pure essential oils stored for a year.

Keywords: Essential oils; autooxidation of 2-hexenal; inhibition of oxidation; changes in


composition during storage.

Essential oils of many plants possess an intensive and pleasant aroma and also are
biologically active [1-3]. Due to these properties, essential oils are actively used for a long

* Emanuel Institute of Biochemical Physics, Russian Academy of Sciences ul, Kosygina 4, Moscow, 119334
Russia; e-mail: Tmish@rambler.ru
84 T. A. Misharina, M. B. Terenina, and N. I. Krikunova

time in different industries, mainly in perfumes (fragrances and aftershaves), in food (as
flavorings and preservatives) and in pharmaceuticals [3, 4]. As a rule, essential oils are a
complex mixture of organic substances with different functional groups, mainly terpenoids
and substituted phenols. The composition of essential oils determines their organoleptic
properties and biological activity including antioxidant [5-8]. The antioxidant activity of
many essential oils is not surprising in view of the presence of phenol groups. It is well
known that almost all phenols can function as antioxidants of lipid peroxidation because they
trap the chain-carrying lipid peroxyl radicals [9-11]. Plant phenolics are multifunctional and
can act as reducing agents, hydrogen-donating antioxidants and singlet-oxygen quenchers [12
- 14]. As a rule, antioxidant activity of essential oils is higher than of their individual
components. This fact indicates the existence of synergetic effects due to the complex
composition of oils [15, 19, 20].
For the estimation of antioxidant properties of substances or their mixtures, many
different methods are used. It was showed that the value of antioxidant activity significantly
depends on the method of its estimation, which is why published data obtained by different
methods is practically not comparable. Besides, antioxidant properties of essential oils depend
on the qualitative and quantitative composition of systems under test [6, 10, 13, 20, 21]. One
of the simple and informative methods of the quantitative assessment of antioxidant activity is
based on the inhibition of speed of lower aldehyde autooxidation, for example hexanal, in the
presence of antioxidant substances [7, 8, 20, 22]. This method was used successfully for the
estimation and comparison of antioxidant properties of a number of essential oils [20, 22-25].
The aim of the work is to study and compare antioxidant properties of 5 essential oils
containing substituted phenols in the model system of autooxidation of 2-hexenal, the
assessment of the influence of essential oils composition on their antioxidant activity, and
also the study of changes in the composition of essential oils during their autooxidation in
solutions and in pure oils.

MATERIALS AND METHODS


The fresh samples of essential oils from clove bud (Caryophyllus aromaticus L.), laurel
(Laurus nobilis L.), cardamom (Elettaria cardamomum L.), nutmeg (Myristica Fragrans
Houtt.) and mace (Myristica fragrans Houtt) (company “Plant Lipids Ltd.”, India) we
studied.
For the estimation of antioxidant properties of essential oils and their mixture, 600 μl
trans-2-hexenal (3 μl /ml) and 400 μl undecane (2 μl /ml) ( an internal standard) were
dissolved in 200 ml of n-hexane. The solution was separated into 3-ml aliquots, which were
placed in 5-ml glass vials and then 10 μl (3.33 μl /ml), 50 μl (16.5 μl /ml) or 210 μl (70 μl
/ml) of essential oils were added. Oil was not added in the control sample. Each sample was
prepared twice, the control sample was prepared three times. The samples in vials with
stoppers were stored in light under room temperature for 100 days. Every week vials were
opened and blown with 10 ml of air with the help of a pipette. The quantitative content of 2-
hexenal and components of essential oils in vials were determined by method of capillary gas-
chromatography after every 10 days from the beginning of storage.
Antioxidant Properties of Essential Oils … 85

Gas-chromatography analysis of essential oils samples and control sample was carried
out on a Kristall 2000M chromatograph (Russia) with a flame ionization detector and an SPB-
1 fused silica capillary column (50 m x 0.32 mm, phase layer 0.25 μm). The samples were
analyzed at the column temperature programming from 60 to 250oC with the speed of
8oC/min under the temperature of detector and injector at 250oC. The rate of carrier gas
helium through the column was 1.5 ml/min. We analyzed 2 μl of hexane solutions at once.
The identification of components in oil samples was carried out on the basis of the retention
indexes by their comparison with literary [26] or experimental data obtained by us. The
quantitative content of 2-hexenal and essential oils components was calculated by the ratio of
peak areas, which corresponded to the substances and internal standard. The oxidation extent
of 2-hexenal and essential oils components (%) was determined in reference to their content
in initial samples.

RESULTS AND DISCUSSION


The main component of clove bud oil was eugenol (80,3%), which is responsible for its
odor and antiseptic and antioxidant properties. Other major components were eugenyl acetate
(10,1%) and caryophyllene (6,3%). Nutmeg oil was obtained by steam distillation of the
kernels of nutmeg, which are the dried fruits of Myristica Fragrans Houtt. Mace oil was
obtained from the coverings of nutmeg. The nutmeg oil contained ca. 90% monoterpene
hydrocarbons, mainly sabinene (30,7%), α -pinene (14,0%), β -pinene (10,8%), and γ -
terpinene (5,8%). Major oxygen-containing constituents were terpinen-4-ol (6,7%) and
phenol ether derivatives: safrole (0,9%), myristicin (2,4%) and elemicin (1,2%). Mace oil
contained the same substances, but their content was differed: sabinene (20,5%), α -pinene
(21,2%), β -pinene (16,0%), γ -terpinene (5,8%), terpinen-4-ol (4,6%), safrole (1,6%),
myristicin (2,8%) and elemicin (0,3%). Cardamom oil was produced from the seeds of
Elettaria cardamomum (L.) Maton (Zingiberaceae). This essential oil contained 41,2% 1.8-
cineole, 39,2 % α -terpinyl acetate. The content of monoterpene hydrocarbons, linalool,
geraniol, linalyl and geranyl acetates was ca. 1-2 %. Trace constituents like unsaturated
aliphatic aldehydes may be important for the typical cardamom aroma. Laurel leaf essential
oil was obtained from leaves of evergreen tree Laurus nobilis L. The main components of this
oil were 1.8-cineole (49,8%), α -terpinyl acetate (9,6%), eugenol (7,1%), linalool (4,4%),
sabinene (9,5%), α -pinene (5,8%), β -pinene (4,6%), α -terpineol (2,3%), and terpinen-4-ol
(1,7%). Using the method of capillary gas chromatography allowed us to estimate changes in
the content of each oil component in the model systems with different oil concentration and in
pure oil during autooxidation, and also to distinguish the oxidation products of main
components. Furthermore, due to the comparison of oil composition and the oxidation rates of
components, we managed to reveal some regularity, which enables us to predict and to
regulate oil composition to obtain stable mixtures. This is very important because essential
oils are currently widely used in industry and medicine. Usually the recommended storage
time for oils is 1 year; however, nobody has studied yet what really happens with oils during
this period. We earlier studied the changes in the composition of coriander, laurel, marjoram,
and fennel oils in the process of storage of pure oil samples in the dark and in the light but in
bottles from dark glass [27-29]. It was established that the main process is the oil components'
86 T. A. Misharina, M. B. Terenina, and N. I. Krikunova

oxidation. Thus, we found, and then it was proved in works [10, 11], that cyclic monoterpenes
hydrocarbons α- and γ- terpinenes are completely oxidized into aromatic hydrocarbon p-
cymene. We also found oxidation products of other components of essential oils - oxides,
alcohols, and aldehydes [26-29].
For the estimation of antioxidant properties of essential oils we used a model system of 2-
hexenal day-light induced autooxidation, which is used in the similar studies [7, 8, 20-25]. As
a criteria for the estimation of antioxidant properties of essential oils we used the quantity of
2-hexenal, which remained unoxidized after 40 days in reference to the initial quantity (%).
Fig. 1 presents the obtained results of relative antioxidant activity values of 5 studied essential
oils. The all model systems included essential oils in two concentrations: 3.3 μl /ml and 16.5
μl /ml. For the essential oils of cardamom and mace we studied an additional third systems in
which the content of oils was 70 μl /ml of hexane solution. As is clearly seen from Fig. 1, the
concentration of oils influenced on their antioxidant activity for all oils with the exception of
clove bud oil. It is noteworthy that the increase in activity was usually not in proportion to the
growth of essential oil concentration. The systems with minimal content of laurel, mace, and
nutmeg oils (3.3 μl/ml ) had antioxidant activity more than 50%. The increase of content of
these oils by five times was accompanied by the increase of antioxidant activity by 20-30%,
and the further increase in content of mace oil by four times (from 16.5 to 70 μl /ml) led to
the growth of oil activity by only 6 %. The cardamom oil in diluted solution (3.3 μl /ml) had
very low antioxidant activity - only 20%. The increase in concentration of this oil by five
times (from 3.3 to 16.5 μl /ml) led to the increase of activity by 3.5 times. The further
increase in concentration of cardamom essential oil up to 70 μl /ml led to the increase of
antioxidant activity only by 3 %.
The inhibition of 2-hexenal oxidation by clove bud oil was practically non-dependent on
the concentration and was 75-78%. Antioxidant activity and composition stability of clove
bud oil were high. In both hexane solutions, oil did not change the composition for 100 days.
Pure individual oil was also stable while being stored in the dark for 2 years and in the light
for 8 months. We did not notice oxidation even of traces amounts of monoterpene
hydrocarbons in stored clove bud oil.
Antioxidant activity of nutmeg and mace essential oils increased from 53 to 69% and
from 63 to 78% accordingly, with the increase of their concentration in model solutions (Fig.
1). Thus, activity of mace oil was higher to 10% than that for nutmeg oil. These oils were
close in the quantitative and qualitative content of components. The main compounds in oils
were monoterpene hydrocarbons and alcohol terpinen-4-ol. The aroma of this species is due
to phenol derivatives - safrole and myristicin, the content of which in both oils was from 0.9
to 2.6%. These compounds together with terpene hydrocarbons were correspondent for the
antioxidant activity of oils. During the autooxidation of oils in hexane solutions for 40 days,
only the content of γ-terpinene decreased by 3-4 times and the content of its oxidation
product, p-cymene, increased. During storage of these pure oils for 4 months, the content of
γ-terpinene did not change but the storage for 1 year led to practically the complete oxidation
of γ-terpinene and the oxidation of 50% of caryophyllene with the formation of caryophyllene
oxide.
Antioxidant Properties of Essential Oils … 87
 
90

80

70

2-Hexenal content, %
60

50

40

30

20

10

0
1 2 3 4 5 6

Control C1 C2 C3

Figure 1. Relative antioxidant activity of essential oils. 1 - Control (2-hexenal) Essential oils: 2 - clove
bud, 3 - nutmeg, 4 - mace, 5 - laurel, 6 - cardamom. Concentration of essential oils (μl/ml): C1 - 3.3, C2
- 16.5, and C3 - 70.

Laurel and cardamom essential oils had close composition of main components – 1,8-
cineole and terpinyl acetate; the difference in aroma was due to the presence in cardamom oil
of about 1% of neryl and linalyl acetates and nerolidol. Laurel essential oil in hexane solution
at concentration of3.3 μl /ml inhibited the oxidation of 2-hexenal 1.5 times more effectively
than cardamom essential oil (Fig. 1). Laurel oil, after 40 days of autooxidation in the light in
hexane solution, remained stable. We previously established that individual pure laurel oil did
not change its content in storage in the dark for 2 years [29]. The stability of cardamom
essential oil as well as its antioxidant activity depended on its concentration in hexane
solution. So, under the concentration 3.3 μl /ml the quantity of sabinene decreased by five
times and terpinyl acetate decreased by two times, α- and γ -terpinenes were oxidized
completely. In more concentrated solutions, only α- - and γ -terpinenes were oxidized
completely; also, the content of other components in solutions with oil concentration of 16.5
and 70 μl /ml changed in the same way. It is noteworthy that antioxidant activity of
cardamom oil was increasing with the increase of its concentration in the model system (Fig.
1). Probably, they were mainly α- and γ -terpinenes, which were in answer for the antioxidant
activity properties of this oil. During the storage of pure cardamom essential oil, after 100
days we noticed the significant oxidation of α- and γ -terpinenes; the quantity of sabinene
decreased by 65% and that of terpinyl acetate decreased by 35% (Fig.2). The reason for
changes in laurel and cardamom essential oil behavior was probably that laurel oil contained
approximately 7% of eugenol and 2% of methyl eugenol, which possesses strong
antioxidation properties. Due to their presence, the laurel oil has higher antioxidant activity
88 T. A. Misharina, M. B. Terenina, and N. I. Krikunova

and was relevant to oxidation. Adding to the cardamom essential oil at least 0.5% of clove
bud oil, which contained approximately 80% of eugenol, increased significantly the relevance
of all components to oxidation in comparison with individual cardamom oil (Fig. 2). In many
cases, the antioxidant activity of the essential oils could not be attributed to the major
compounds, and minor compounds might play a significant role in the antioxidant activity,
due to synergistic effects [15,19,20 ]. For instance, in Melaleuca species ( cajuput oil or tea-
tree oil), essential oil containing 1,8-cineole (34%) and terpinen-4-ol (19%) exhibited
stronger antioxidant activity than those with high methyleugenol (97%) or 1,8-cineole (64%)
contents [3].

100

4
90
Component content, %

80 3

70

2
60

50

40 1

30
0 20 40 60 100
days

Figure 2. The content of sabinene and α-terpinyl acetate (% from initial) in cardamom oil and its
mixture with 0.5% clove bud oil during the storage: 1 and 2 are sabinene and α-terpinyl acetate in
cardamom oil; 3 and 4 are sabinene and α-terpinyl acetate in the mixture of cardamom and 0.5% clove
bud oils.

Thus, conducted research and literary data show that essential oils are effective natural
antioxidants, which are able to compete with synthetic ones. Antioxidant properties of
essential oils are determined by their composition. Oils with high content of substituted
phenols are able to significantly inhibit oxidation processes of labile unsaturated aldehydes
even in low concentrations. Antioxidant properties of essential oils, which consist of terpene
Antioxidant Properties of Essential Oils … 89

hydrocarbons and alcohols, are determined by α- and γ- terpinenes and their sesquiterpene
analogs. We revealed the significant influence of the concentration of such oils ( cardamom,
for example) on their antioxidant properties. The stability of essential oil composition
increased with the increase of their concentration in model solutions. The oxidation of
substances in pure essential oils happened slower than in solutions.

REFERENCES
[1] L.H. Madsen, B.R.Nielsen, G.Bertelsen, L.H. Skibsted: Screening of antioxidative
activity of spices. A comparison between assay based on ESR spin trapping and
electrochemical measurement of oxygen consumption. Food Chem., 57, 331 (1996).
[2] S.A. Voitkevich: “Efirnye masla dlya parfyumerii i aromaterapii” (Essential Oils for
Perfumery and Arometherapy). Pishch. Prom., Moscow, 1999.
[3] Flavours and Fragrances. Chemistry, Bioprocessing and Sustainability (Ed. R.G.
Berger). Springer, New York, 2007, 43-116.
[4] K. Bauer, D. Garbe, H. Surburg: Common Fragrance and Flavor Materials, VCH
Verlag, Weinheim, 1990.
[5] G. Cervato, M. Carabelli, S. Gervasio, A. Cittera, R. Cazzola, B. Cestaro: Antioxidant
properties of oregano (Origanum vulgare L.) leaf extracts. J. Food Biochem., 24, 453
(2000).
[6] H.J.D. Dorman, A. Peltoketo, R.Hiltunen, M.J.Tikkaken: Characterisation of the
antioxidant properties of deodourised aqueous extracts from selected Lamiaceae herbs.
Food Chemistry, 83, 255 (2003).
[7] K.G. Lee, T. Shibamoto: Antioxidant property of aroma extract isolated from clove
buds. Food Chem., 74, 443, (2001).
[8] K.W. Lee, Y.J. Kim, D.-O. Kim, H.J. Lee, C.Y. Lee: Major phenolics apple and their
contribution to the total antioxidant capacity. J. Agric. Food Chem., 51, 6516, (2003).
[9] G.Litwinienko, K.U. Ingold: Solvent effects on the rates and mechanisms of reaction of
phenols with free radicals. Acc. Chem. Res., 40, 222 (2007).
[10] G.. Ruberto, M. Baratta: Antioxidant activity of selected essential oil components in
two lipid model systems. Food Chem., 69, 167 (2000).
[11] M.C. Foti, K.U. Ingold: Mechanism of inhibition of lipid peroxidation by γ–terpinene,
an unusual and potentially usefull hydrocarbon antioxidant. J. Agric. Food Chem. 51,
2758 (2003).
[12] M.T. Baratta, H.J.D. Dorman, S.G. Deans, D.M.Biondi, G.Ruberto: Chemical
composition, antimicrobial and antioxidative activity of laurel, sage, rosemary, oregano
and coriander essential oils. J. Essent. Oil Res., 10, 618 (1998).
[13] S.S.Pekkarinen, H.Stocmann, K.Schwarz, M.Heinone, A.I.Hopia: Antioxidant activity
and partitioning of phenolic acids in bulk and emulsified methyl linoleate. J. Agric.
Food Chem. , 47, 3036 (1999).
[14] G.Sacchetti, S.Maietti, M.Muzzoli, M.Scaglianti, S.Manferdini, M.Radice, R.Bruni:
Comparative evaluation of 11 essential oils of different origin as functional
antioxidants, antiradicals and antimicrobials in foods. Food Chem. 91, 621 (2005).
90 T. A. Misharina, M. B. Terenina, and N. I. Krikunova

[15] G. Singh, S. Maurya, C. Catalan, M.P. de Lampasona: Studies on essential oil, Part 42.:
chemical, antifungal, antioxidant and spout suppressant studies of ginger essential oil
and its oleoresin. Flavour Fragrance. J., 20, 1 (2005).
[16] A.Wei, T. Shibamoto: Antioxidant activities and volatile constituents of various
essential oils. J. Agric. Food Chem., 55, 1737 (2007).
[17] C. Menut, J.M. Bessiere, D.Samate, A.K. Djibo, G.Buchbauer, B. Schopper: Aromatic
plants of tropical west Africa. XI. Chemical composition, antioxidant and antiradical
properties of the essential oils of three Cymbopogon species from Burkina Faso. J.
Essent. Oil Res., 12, 207 (2000).
[18] N.Mimica-Dukic, B. Bozin, M. Sokovic, N. Simin: Antimicrobial and antioxidant
activities of Melissa officinalis L. (Lamiaceae) essential oils. J. Agric. Food Chem., 52,
2485 (2004).
[19] D.Huang, B.Ou, L.R.Prior: The chemistry behind antioxidant capacity assays. J. Agric.
Food Chem., 53, 1841 (2005).
[20] K.G. Lee, T.Shibamoto: Determination of antioxidant potential of volatile extracts
isolated from various herbs and spices. J. Agric. Food Chem., 50, 4947 (2002).
[21] K.G. Lee, T.Shibamoto: Antioxidant activities of volatile components isolated from
Eucalyptus species. J.Sci. Food Agric., 81, 1573 (2001).
[22] K.Yanagimoto, H.Ochi, K.G. Lee, T. Shibamoto: Antioxidative activities of volatile
extracts from green tea, oolong tea, and black tea. J. Agric. Food Chem., 51, 7396
(2003).
[23] C.J. Lee, T. Shibamoto, K.G. Lee: . Identification of volatile components in basil
(Ocinum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant
properties. Food Chem., 91, 131 (2005).
[24] B.Bozin, N.Mimica-Dukic, G.Anachov: Characterization of the volatile composition of
essential oils of some Lamiaceae spices and the antimicrobial and antioxidant activities
of the entire oils. J. Agric. Food Chem., 54,1822 (2006).
[25] T.A Misharina, A.L. Samusenko: Antioxidant properties of essential oils from lemon,
grapefruit, coriander, clove, and their mixtures. Applied Biochem. and Microbiol., 44,
473 (2008).
[26] W. Jennings, T.Shibamoto: Qualitative Analysis of the Flavor and Fragrance Volatiles
by Glass Capillary Gas Chromatography, Academic, New York: 1980, 130-154.
[27] T.A Misharina: Influence of the duration and conditions of storage on the composition
of the essential oil from coriander seeds. Applied Biochem. and Microbiol., 37, 622
(2001).
[28] T.A Misharina, A.N. Polshkov, E.L. Ruchkina, I.B. Medvedeva: Changes in the
composition of the essential oil of marjoram during storage. Applied Biochem. and
Microbiol., 39, 311 (2003).
[29] T.A Misharina, A.N. Polshkov: Antioxidant properties of essential oils: autoxidation of
essential oils from laurel and fennel and of their mixtures with essential oil from
coriander. Applied Biochem. and Microbiol., 41, 610 (2005).
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.91-121 ©2010 Nova Science Publishers, Inc.

Chapter 9

SPECIFIC PROPERTIES OF SOME BIOLOGICAL


COMPOSITE MATERIALS

N. Barbakadze*, E. Gorb and S. Gorb


Max Plank University, Shtutgart,
Germany Georgian Technical University, Tbilisi, Georgia

ABSTRACT

Miniaturisation of technical systems creates the need for today’s science and
engineering to assess the mechanical properties of small volumes of material. A specific
feature of the structure and the combination of the desirable properties across several
different length scales are fascinating using plenty of examples in biology. Determining
the extraordinary properties of natural materials at the nanometer scale is regarded as
very attractive targets for materials science. Mechanical behavior of various biological
materials such as insect and plant cuticles was studied by applying experimental
approaches of material science in order to explain their structural design and working
principles. Experiments were performed on the head articulation cuticle of the beetle
designed for friction minimisation and on the wax covered plant surfaces adapted for
attachment prevention. Both insect and plant cuticles are multifunctional composite
materials and have a multilayered structure. Gula cuticle of the beetle Pachnoda
marginata is a part of the head articulation, which is a micromechanical system similar to
a ball bearing. The surfaces in this system operate in contact and must be optimised
against wear and friction and provide high mobility within the joint. The measurements
on the gula cuticle were performed in order to understand structure and mechanical
behavior of material working for friction minimizing. The wax layer on the plant surfaces
is a barrier for the attachment system of insects. Antiattachment function could be caused
by contamination of attachment pads of insect with the wax crystalloids. Increase in
roughness due to location of the wax crystals on the plant surface causes decrease in the
real contact area between the plant surface and attachment pads of insect. These are two
of the hypotheses why insects cannot walk on the plant surfaces structured with wax.
Nanoindentations on different plant surfaces were performed in order to understand the

* Max Plank University, Shtutgart,Germany Georgian Technical University, 77, Kostava Str., 0175 Tbilisi,
Georgia, natali5@web.de
92 N. Barbakadze, E. Gorb and S. Gorb

deformation behavior of the wax layer. This study is believed to be one of the first for
mechanically testing insect cuticle and the very first for wax coated plant surfaces in
native condition.

MOTIVATION AND LITERATURE REVIEW

”Why should engineers be interested in biological materials?


One only has to look to the history of technology, starting from the first use of tools, to
see that revolution and progress are driven by the new possibilities which new materials
create.”
Julian Vincent

Natural materials have been used in engineering over twelve millennia. The first houses
and bridges were built from grass, wood and stone. The first clothes and shoes were made
from animal skins. There are still no alternatives to wood and horsehair for many musical
instruments (Wegst and Ashby, 2004). We use natural materials everyday. They are always
around us and the interest in biological systems is still growing. The natural systems inspire
the engineering sciences in the design of new materials. One example is the development of
different replacement tissues from metal or plastic (Vincent, 1990).
Most natural materials are structured fiber-matrix composites (Wainwright et al., 1976;
Vincent, 1990). The components in them are quite similar. They consist mainly of natural
polymer fibers embedded in a protein matrix. For example, cellulose, pectin and protein build
a plant cell wall (Wegst and Ashby, 2004); insect cuticle is the composite of chitin and
protein (Neville, 1975; Wainwright et al., 1976; Vincent, 1990; Vincent and Wegst, 2004).
The biological materials exhibit a hierarchical structure. Natural systems such as skin, bone,
cartilage and cuticle are layered composites with different single layer structures.
Despite the limited constituents, the natural systems display a great variety of mechanical
properties. Collagen and elastin are the main components in skin, bone and cartilage.
However, depending on the amount of the constituents and the fiber orientation, each system
has different mechanical properties (Wegst and Ashby, 2004).
The structural and mechanical design of natural systems can be mimicked to provide new
so called “intelligent” materials with “smart” structures. Skin, for instance, shows
compressive properties useful in the development of tactile sensors (Vincent, 1990). The
man-made hydrophobic surfaces have been inspirited from the “lotus effect” of the plant leaf.
The technique of the turn around of the beetle arouses the interest of robotics researchers to
design a robot being able to cope with any difficulties in movement.
In figures 1 and 2 (Vincent and Wegst, 2004) the wide spectrum of mechanical properties
of natural materials is shown. Figure 1 presents Vickers hardness for a range of natural
materials. For example, the arthropod (invertebrates) cuticle displays a great variation of
hardness values depending on its function and condition (wet or dry). In the figure 2 Young’s
modulus is plotted versus density for different natural materials. Again arthropod cuticle
shows different elastic modulus comparable with human skin, cork, wood and even
aluminum.
Determining the extraordinary properties of natural materials at the nanometer scale is
regarded as a very attractive target of materials research. However, tribological systems in
Specific Properties of Some Biological Composite Materials 93

biology have not been studied much from a materials science point of view. The majority of
work in tribology research has focused on the material properties and working mechanisms of
human and animal joints (Fung 1993; Persson 1998). It was found that articular cartilage has
a unique quality of lubrication and shock absorption that is mainly due to the multilayer
structure (Fung, 1993). Being related to bone, cartilage has a very small coefficient of friction
for relative motion between two articular pieces (Fung, 1993).

Figure 1. A compilation of hardness data for natural materials. (Figure adapted from Vincent and
Wegst, 2004, created using the Natural Materials Selector).

There are considerable data in the literature about ultrastructural architecture and
mechanical properties of the arthropod cuticle (Fraenkel and Rudal, 1940; Neville, 1975;
Hepburn and Joffe, 1976; Andersen, 1979; Andersen et al., 1996; Vincent, 1980; 2002;
Binnington and Retnakaran, 1991; Gorb, 1997; Arzt et al., 2002; Enders et al., 2004; Vincent
and Wegst, 2004). It is the most widely distributed high-performance composite material after
plant cell and wood (Vincent, 2002). Much work has been published on the correlation of
microtribological properties of biological systems with its material structure (Scherge and
Gorb 2000; Gorb and Scherege 2000; Gorb et al. 2001; Dai et al. 2002; Gorb and Perez
Goodwyn 2003; Perez Goodwyn and Gorb 2004).
Wax coated plant surfaces were studied in relation to insect attachment systems over the
last century (Kerner von Marilaun, 1898). Great work has been done on the structure and the
chemical composition of the wax crystals (Hallam and Chambers, 1976; Bianchi et al., 1978;
Haas and Schonherr, 1979; Baker, 1982; Avato et al., 1984; Jeffree, 1986; Baker and Gaskin,
1987; Walton, 1990; Barthlott, 1990; Bianchi, 1995; Barthlott et al., 1998; Gorb, 2001;
Eigenbrode and Jetter, 2002; Gorb and Gorb, 2002). However little is known about the
mechanical properties of the wax layer.
This study aims to explore mechanical performance of insect and plant cuticle using the
material sciences approach. Head articulation cuticle of the beetle (Pachnoda marginata)
designed for friction minimization and wax covered pea (Pisum sativum) plant surfaces
adapted for insect attachment prevention have been studied. Mechanical properties were
determined by nanoindentation.
94 N. Barbakadze, E. Gorb and S. Gorb

Figure 2. Young’s modulus versus density chart for natural materials. (Figure adapted from Vincent and
Wegst, 2004, created using the Natural Materials Selector).

HEAD ARTICULATION OF THE BEETLE


The body of insects is completely covered by cuticle, which is a multifunctional interface
between the animal and the environment (Hepburn and Joffe, 1976; Gorb, 2001). It serves
primarily as an exoskeleton that gives the body its shape and stability. The cuticle is also a
main barrier against evaporation of water from the body and protects the insects against
desiccation (Locke, 1964; Binnington and Retnakaran, 1992).
Like most biological materials, cuticle is a fiber composite (Hepburn and Chandler,
1980). The fibers consist of chitin and the matrix is formed by proteins. Chitin is a natural
polymer composed of 300 nm long and 3 nm thick nanofibrils (Vincent, 1980; 2002). Each
Specific Properties of Some Biological Composite Materials 95

nanofibril contains about 19 molecular chains (Fraenkel and Rudall, 1940; Vincent, 1980;
2002) running antiparallel to one another (i.e. alpha chitin) (Neville, 1975; Vincent, 1980).
Chitin filaments are distributed in the protein matrix (Andersen et al., 1996) which stabilises
the chitin. Water contained in the proteins (about 90 % of the protein matrix) presumably
functions to separate the two main components of the cuticle from each other (Vincent, 1980).
Arthropod (invertebrates – animals without backbones) cuticle has a multilayer structure
(Neville, 1975; Andersen, 1979) (figure 3). It typically consists of three main layers:
epicuticle, exocuticle and endocuticle. The latter two layers form the procuticle. In some
insects there is a mesocuticle between the endocuticle and the exocuticle (Neville, 1975;
Andersen, 1979; Binnington and Retnakaran, 1992). The nonchitinous, tanned lipoproteinous
epicuticle is the outer layer of the cuticle, which is very thin and has a relatively high tensile
strength. The surface of the epicuticle is coated with wax and lipids. The hard and stiff solid
part has a dense chitin-protein structure and forms the exocuticle. The endocuticle is the
thickest region of the cuticle and has low chitin content (Locke 1964; Neville 1975,
Binnington and Retnakaran 1992).
From a mechanical point of view, there are two main types of insect cuticle: soft and
hard. In larvae the cuticle is soft and colorless. A simultaneous hardening and darkening,
called sclerotisation (Fraenkel and Rudall, 1940), forms a hard and colored integument in
most adults. There is a great variation in kinds of the cuticle depending on the proportions of
the main components and, accordingly, mechanical properties (Hepburn and Chandler, 1976).
In various animal groups and on different body parts there are different types of cuticle from
very hard and brittle to soft, ductile and also rubber-like (Jensen and Weis-Fogh, 1962).

- epicuticle
- exocuticle

procuticle

cuticle
- endocuticle

- epidermal cell layer

Figure 3. Schematic of the multilayered structure in the insect cuticle. The shaded areas represent the
single cuticle layers: outer surface layer, epicuticle which does not contain chitin fibers; procuticle with
two layers: exocuticle and endocuticle. Last two cuticle layers contain chitin fibers oriented nearly
always parallel to the surface. The endocuticle is connected with epidermis cell structure.

The wide spectrum of mechanical properties of the insect cuticle suggests that each body
part is optimised to its function. Any movement involving contact between two surfaces or
between a surface and a medium has to deal with the resistance of the surfaces or medium.
This resistance is a friction phenomenon, believed to have a great influence on the design of
biological structures (Gorb, 2001). Living microsystems have developed different ways to
save muscular energy by the use of friction-optimised systems. Surface pairs in biological
objects are designed to maximise or to minimise contact forces within joints. In biology they
96 N. Barbakadze, E. Gorb and S. Gorb

are defined as friction and anti-friction systems respectively. Working principles of friction
and anti-friction systems are based on mechanical interlocking and a maximisation or a
minimisation of the contact area (Gorb, 2001). Frictional systems require high friction to
fixate body parts to one another (Gorb, 2001). The surface pair can be predefined as in the
wing-locking mechanisms of beetles (Hammond, 1989; Gorb, 1997; Gorb, 1999b) and the
head-arresting system of dragonflies (Gorb, 1999a; Gorb, 2001). Among various cases of
contact pairs in biology, anti-friction systems always have a predefined pair of surfaces like
the head articulation system in beetles (Arzt et al., 2002; Enders et al., 2004) or the
hemelytra-hindwing locking mechanism in bugs (Perez Goodwyn and Gorb, 2004).
An interesting example of anti-friction system is a head articulation of the beetle
Pachnoda marginata. The surfaces in this system operate in contact and must be optimised
against wear and friction to provide high mobility within the joint. A predefined, functionally
corresponding pair of the contacting surfaces forms a tribological system with specific
ultrastructural architecture and properties. However, each contacting piece has a different
structure. The rather smooth surface of the hemispherical head-part or gula is believed to
contribute to the friction minimisation in contact with its counterpart in the ventral part of the
prothorax, covered by asymmetric outgrowths (Enders et al., 2004). In living conditions the
organic substances on the cuticle surface, such as lipids and waxes are expected to serve as
lubrication materials to reduce the friction in this biological microsystem. For this study a
head part (gula) cuticle of the head articulation of the beetle Pachnoda marginata was
selected. Both the structure and the mechanical properties of this material are largely
unknown.
The goal of these investigations was to obtain detailed information about the cuticle
structure in the gula and to determine its mechanical properties. Assembly of the head
articulation and structure of both contacting surfaces were studied by means of computer
tomography and scanning electron microscopy (SEM). SEM images were also used for the
evaluation of nanoindentation tests. Hardness and elastic modulus were measured using the
nanoindentation technique. The samples were mechanically tested in the fresh, the dry and the
chemically treated conditions in order to understand the influence of desiccation (fresh versus
dry condition) and removal of an outer wax layer (chemically treated condition). The
questions asked were: What are the structure and the local mechanical properties of the head
articulation cuticle? How does the liquid content influence the mechanics of the joint
material? How do surface waxes influence hardness and elastic modulus of the gula? Since
the structure and the mechanical properties of the material are very important for tribological
performance, this study aims to make a contribution to exploring the working principle of the
gula cuticle.

MATERIALS AND SAMPLE PREPARATION


Samples were prepared from the head articulation system of the beetle Pachnoda
marginata. The beetles were anesthetized with CO2. Heads were dissected from the body and
freed of soft tissue. To prevent the drying of the specimens, fresh gula cuticles were tested
immediately (in 3-5 min after dissecting from the body). Dry samples were prepared from
fresh cuticles by drying. To obtain the same degree of desiccation, fresh samples were dried
Specific Properties of Some Biological Composite Materials 97

in an oven for 24 h at 40°C. Chemically treated samples were prepared from the dry gula
cuticles. In order to remove waxes from the surface, dry samples were treated in a solution of
chloroform and methanol (2:1) for 50-60 min and air-dried.

Figure 4. X-Ray tomography (µ-CT system) images in transversal (A) and sagittal (B) planes of the
head articulation of the beetle Pachnoda marginata.

SHAPE AND STRUCTURE OF THE CUTICLE


To observe the working position and the shape of the contacting surfaces, the anatomy of
the head articulation system of the beetle Pachnoda marginata was explored with high
98 N. Barbakadze, E. Gorb and S. Gorb

resolution computer tomography (X-Ray µ-CT system, model 1072 tomograph by Sky Scan
at the Institute für Kunststoffprüfung (IKP) at the University of Stuttgart, Germany). This
method allows non-invasive imaging of the internal structure. The measurements were
performed on dry samples. The images of the head articulation of the beetle were taken in two
directions: transversal and sagittal (magnification × 30).
Images showing cross sections of the contact parts (gula and prothorax) of the head
articulation were taken in dry insects in two directions: transversal (figure 4 A) and sagittal
(figure 4 B). The hemispherical convex shape of the gula cuticle can be seen in the cross
section image in both transversal and sagittal direction. As can be distinguished in the figures,
the head articulation system is an open joint. In the transversal image, the head articulation of
the beetle is reminiscent of a technical ball bearing system.
The surfaces of the contacting pair of the head articulation system were studied in the
scanning electron microscopy (SEM). The samples were fixed in 2.5% glutaraldehyde in a
phosphate buffer (pH = 7.3). The specimens were dehydrated in an ascending row of ethanol
and then critical-point dried. Pieces of the dried material were fractured using a razor blade.
The prepared samples were mounted on holders, sputter-coated with gold-palladium (10 nm
thickness) and examined in a Hitachi S-800 scanning electron microscope (SEM) at 20 kV.

A
prothorax
gula

50 µm 50 µm
B

C 5 µm D 5 µm

Figure 5. SEM images of the gula (A, C) and prothorax (B, D) surfaces. On the gula surface pores (A,
C) and dried organic substances (C) can be distinguished. The figures B and D show cuticlar
outgrowths on the prothorax surfaces. The gula and the prothorax surfaces operate in contact (shown
with arrows on the figures A and B) in the head articulation system of the beetle Pachnoda marginata.
Specific Properties of Some Biological Composite Materials 99

Figure 5 shows the surface structures of the gula plate (figure 5A and 5C) and its
counterpart (prothorax) (figures 5B and 5D). The gula is rather smooth while the prothorax is
covered by cuticular outgrowths. The arrows in the figures 5 A and 5 B show that these
surfaces contact each other in the head articulation of the beetle.

EPI 
gula  EXO 

ENDO 

A 200 µm B 20 µm

2 µm
C 20 µm
D
Figure 6. SEM images of the gula surfaces. A: shows entire surface of the gule. B: cross section of the
gula cuticle showing the single cuticle layer: EPI – epicuticle, EXO – exocuticle and ENDO –
endocuticle. Perpendicular orientation of the chitin fibers can be distinguished in the exocuticle layer.
The well-defined pores, dried organic substances and cracks can be seen on the cuticle surface (C and
D).

SEM images in the figure 6 provide information on the shape of the gula cuticle and the
ultrastructural architecture within a single cuticle layer. The gula has a hemispherical smooth
surface (figure 6 A). Island-like places, occurring on the surface, are, presumably, dried
secretory substances, delivered to the surface by well-defined pores, which run deep into the
material and apparently are connected with epidermal cells (figure 6C and 6D). Cracks found
on the surface are probably caused by material desiccation (figure 6D). The cross-section of
the cuticle shows the layered structure of the fiber composite (figure 6B). The thickness of the
gula cuticle is about 80 µm. A very thin (7-8 µm) and dense epicuticle can be distinguished.
100 N. Barbakadze, E. Gorb and S. Gorb

Procuticle is present with two layers: dense exocuticle, which is about 22 µm thick and the
endocuticle, which is very thick (about 50 µm) and less dense compared to the exocuticle
layer. As can be distinguished in figure 6 B, chitin fibres are oriented nearly perpendicular to
the surface in the exocuticle layer and parallel to the surface in the endocuticle.
The exocuticle structure of the gula differs from the layered pattern of regular cuticle,
where the orientation of the chitin fibers is parallel to the surface (Neville, 1975; Wainwright
et al., 1976; Vincent, 1990). The fibers in the exocuticle are oriented perpendicular or at some
angles to the surface. A similar fiber orientation has previously been found in attachment pads
of grasshopper (Gorb and Scherge, 2000). However, the advantage of such structures is not
understood at present. Further investigations are required to clarify the reason for this
perpendicular fiber orientation.

SURFACE ROUGHNESS
The sample surface was studied using a white-light interferometer (Zygo New View 500,
Zygo Corporation, USA). This technique can be used to obtain the average surface roughness
(Ra), the average absolute value of ten-point height (Rz) or root mean square (rms)
representing the height profile’s roughness.
The results of surface roughness measurements are summarized in table 1 and shown in
the figure 7. In the fresh condition, the surface of the hemispherical convex gula cuticle is
extraordinarily smooth for a biological material (Ra = 0.033 ± 0.005 µm, rms = 0.038 ± 0.007
µm) (figure 7 A). After desiccation, the outer layer of the cuticle builds up high roughness
(figure 7 B). The water loss is thought to cause the constriction of the structure in inner layers
and folding of the outer layer. These structural changes could be the reason why dry samples
exhibit a rougher surfaces (Ra = 0.161 ± 0.011 µm, rms = 0.197 ± 0.015 µm). After the
chemical treatment, the surface roughness becomes lower (Ra = 0.103 ± 0.008 µm, rms =
0.117 ± 0.011 µm) (figure 7 C). It could be caused by dissolving the dry organic substances
or structure on cuticle surface. However, the roughness of the fresh samples remains lower
than that of dry and chemically treated ones.

Table 1. Summary of surface roughness parameters, Ra and rms, obtained by white-


light interference microscopy for fresh, dry and chemically treated gula cuticles. The
surface roughness measurements were carried out on three samples for each condition
(SD = standard deviation).

Surface roughness parameter


Ra rms
Sample SD SD
(nm) (nm)
condition
fresh 33 ±5 38 ±7
dry 161 ± 11 197 ± 15
chemically treated 103 ±8 117 ± 11
Specific Properties of Some Biological Composite Materials 101

Figure 7. Surface profiles of fresh (A), dry (B) and chemically treated (C) cuticles. The images were
obtained by white light interference microscopy.
102 N. Barbakadze, E. Gorb and S. Gorb

MECHANICAL PROPERTIES
Nanoindentation provides a fast and reliable technique for evaluating local mechanical
properties, such as hardness and elastic modulus, of very small volumes of the material
(Oliver and Pharr, 1992; Bhushan and Li, 2003). During the past decade, this method has
become an important tool in materials characterization (details about this method see in
Barbakadze 2005; Barbakadze et al., 2006).
In figures 8 A and 8 B hardness and elastic modulus results are plotted vs. displacement
(indentation depth into the sample surface). Each data point is the average value of
approximately 150 indents (10 beetle heads with 15 indents on each, 150 indents in all).

Figure 8. The results of hardness and elastic modulus from indentation tests on the gula-cuticle:
hardness (A) and elastic modulus (B) are plotted versus displacement for fresh, dry and chemically
treated samples. Hardness and elastic modulus were calculated by using the CSM technique from the
projected contact area during indentation. Each data point corresponds to the mean value of
approximately 150 measurements.
Specific Properties of Some Biological Composite Materials 103

The measurements revealed a strong dependence of the mechanical behavior on the


preparation conditions of biological samples. The significance of the differences due to the
conditions was checked by means of statistical calculations. The experimental data were
compared with a one-way ANOVA and a Tukey post tests (Origin 7 SR2). Hardness and
elastic modulus values of the fresh, the dry and the chemically treated samples were
compared with each other by different displacements of 250, 500, 1000 and 1500 nm. The
results of the statistical calculations are summarised in table 2. Hardness and elastic modulus
differ significantly for the fresh, dry and chemically treated conditions. Exceptions are the
elastic modulus values of the dry and chemically treated samples at indentation depths of
1000 and 1500 nm. The effect of the chemical treatment on the sample stiffness at these
depths can hardly be observed. Statistical calculations show no significant difference between
elastic moduli of the dry and chemically treated samples at indentation depths higher than
1000 nm.
The average hardness value (H=0.10±0.07 GPa) of the fresh samples is significantly
lower than that of the dry (H=0.49±0.14 GPa) and the chemically treated (H=0.52±0.15 GPa)
ones (figure 8B) (ANOVA p<0.0005, Tukey post test p<0.0005). The difference between the
hardness results of the dry (H=0.49±0.14 GPa) and chemically treated samples (H=0.52±0.15
GPa) is lower but also significant (statistic: ANOVA p<0.0005), especially in the
displacement range of 200-1000 nm.
The same tendency was observed for the elastic modulus (figure 8C). The dry
(E=7.50±1.80 GPa) and chemically treated (E=7.70±1.90 GPa) samples are significantly
stiffer than fresh ones (E=1.50±0.80 GPa) (statistic: ANOVA p<0.0005, Tukey post test
p<0.0005). In this case the chemical treatment causes a slight increase of the elastic modulus
below 1000 nm indentation depth into the sample surface. At a depth higher than 1000 nm,
the elastic modulus values of the dry and chemically treated samples are not significantly
different.
The maximum displacement for the fresh samples was 3000 nm, but for the dry and
chemically treated samples only 2000 nm, because the maximum load of the instrument had
been reached. High values of the hardness and elastic modulus in the first 50 nm of
indentation are erroneous data caused by contact building between the indenter tip and sample
due to the roughness and contamination of the sample’s surface. The hardness and elastic
modulus for all samples slowly decreases with indentation depth. However, after 1700 nm,
both parameters drop rapidly for the dry and chemically treated specimens.
In general, soft and compliant cuticle contains more water than hard and stiff (Vincent
and Wegst, 2004). Since the gula cuticle belongs to the hard and brown type of cuticle, it has
a low content of water. But even so, the results show that desiccation has a great influence on
the mechanical behavior of the cuticle tested. After drying, the gula cuticle becomes about 5
times harder and stiffer (H = 0.49 ± 0.14 GPa, E = 7.50 ± 1.80 GPa) than in the fresh state
(H = 0.10 ± 0.07 GPa, E = 1.50 ± 0.80 GPa). Water content seems to be the crucial factor,
which influences the mechanical properties such as hardness and elastic modulus. According
literature data, humidity loss changes the mechanical behavior of biological materials
significantly (Andersen et al., 1996; Arzt et al., 2002; Enders et al., 2004; Vincent and Wegst,
2004). In the fresh cuticle, chitin filaments are spaced within a protein-matrix containing
about 90 % water (Andersen et al., 1996; Vincent, 1980). Therefore absence of water, one of
the main components of the cuticle, is expected to lead to some changes of the structure.
There are literature data on the structural changes depending on water reduction in the cuticle
104 N. Barbakadze, E. Gorb and S. Gorb

during sclerotisation (Andersen et al., 1996), but nothing is known about how drying alter the
cuticle structure. However it is obvious that the water content makes cuticle soft and
compliant. The increase in hardness and elastic modulus, especially of the cuticle layer near
the surface up to 1700 nm, is thought to be caused by structural changes due to water loss.
Indentation tests on various biological surfaces (insects and plants) display a considerable
difference in the mechanical behavior between fresh/hydrated and dehydrated materials
(Hillerton et al., 1982; Arzt et al., 2002; Enders et al., 2004; Vincent and Wegst, 2004).

Table 2. Results of statistical calculations of the significance (One-Way-ANOVA and


Tukey post tests) of differences between the hardness and elastic modulus for various
indentation depths (250 nm, 500 nm, 1000 nm and 1500 nm) in fresh, dry and
chemically treated conditions. + means that there is a significant difference between the
values compared; - means that there is no significant difference between the values
compared. There is no significant difference in the elastic modulus values between the
dry and chemically treated samples an indentation depth of 1000 nm and 1500 nm.

Hardness
Elastic modulus
+ +
Fresh
+ +
+ +

250
Dry
+ +
+ +
Chem. treated
+ +
+ +
Fresh
+ +
+ +
Dry
+ + 500

Depth, nm
+ +
Chem. treated
+ +
+ +
Fresh
+ +
+ +
1000

dry —
+ —
+ +
Chem. treated ——
+
+ +
Fresh
+ +
+ +
1500

Dry —
+ —
+ +
Chem. treated ——
+ —
Condition Fresh Dry Chem. treated
Specific Properties of Some Biological Composite Materials 105

Removal of surface waxes and lipids causes only small changes in the indentation results
(slight increase in hardness and stiffness), especially at a depth below 1000 nm. Without
water, the mechanical behavior of the gula cuticle is influenced only a little by the outer
wax/lipid layer and is presumably determined mainly by proteins and chitin, which could not
be removed by the chemical treatment used in this study. In order to understand how the two
main components (proteins and chitin) contribute to the mechanical properties of the cuticle,
further studies are necessary.
SEM images were helpful to evaluate indentation data. The maximum indentation depth
in our experiments was 3 µm. Therefore, measurements of the mechanical properties were
done in the epicuticle (figure 6 B), which is about 7 µm thick. According to several models
(Jönsson and Hogmark, 1984; Burnett and Rickerby, 1987; McGurck et al., 1994; Rother and
Jehn, 1996; Korsunsky et al., 1998) concerning the hardness determination for thin metal
films, the results obtained could also be influenced by layers of the exocuticle. After Bueckle
(1965) and Bhushan (1996), hardness measured up to an indentation depth of 0.7 µm
(displacement < 1/10 of the layer thickness) can be assumed to characterize the epicuticle.
With larger displacements the hardness will be characteristic of the whole composite of the
epicuticle/exocuticle layers. This suggestion is true only for hardness measurement. The
elastic modulus measurements will be influenced by the whole composite since it is a long-
range effect.
The mechanical behavior of the gula cuticle can be regarded as that of a soft substrate
coated by a hard film. A decrease in hardness and elastic modulus with increasing indentation
depth is caused by the underlying layers. As already mentioned, deeper layers in cuticle are
softer and more compliant than the outer ones. The influence of the underlying layers on the
mechanical properties is stronger on dry and chemically treated samples than on fresh ones.
Without water, surface layers appear to become harder and stiffer than the deeper layers
showing a lower density and higher protein content (Locke 1964; Neville 1975, Binnington
and Retnakaran 1992).
Several studies of elastic modulus and hardness of insect cuticle using different methods
can be found in the literature. Almost all experiments were performed on dry or rehydrated
samples. There are only a few measurements of the mechanical properties of cuticle made by
indentation. The average hardness (0.10 - 0.52 GPa) and elastic modulus (1.50 - 7.70 GPa)
values obtained in this study are similar to those of various sclerotised cuticles (Vickers
hardness 0.2-0.5 GPa and Young’s modulus 1 - 10 GPa (Vincent and Wegst, 2004)).
However the hardness (0.10 GPa) of the fresh samples is below this range (0.2 - 0.5 GPa). It
is believed that these are possibly the first measurements of hardness and elastic modulus on
the insect cuticle in native condition.
Comparable hardness values were obtained on other cuticle elements. For example, the
dry wing membrane cuticle of the dragonfly Aeshna cyanea tested with a nanohardness-test-
machine (Hysitron TriboScope) exhibited a hardness of 0.2 GPa (Kreuz et al., 1999). The
different parts of the dehydrated locust cuticle measured by means of a Leitz Miniload
hardness tester (using a Vickers diamond) (Hillerton et al., 1982) revealed H = 0.24-0.33
GPa. However the dry samples of the beetle head tested in this study (H = 0.49 GPa) were
still harder than the cuticles mentioned above.
The elastic modulus values (E = 7.50 ± 1.80 GPa) obtained for the dry beetle cuticle is
very high when compared to the elastic modulus from tensile test of the dry wing membrane
of the dragonfly Aeshna cyanea, where E = 1.5 ± 0.5 GPa (Kreuz et al., 1999). The reduced
106 N. Barbakadze, E. Gorb and S. Gorb

modulus for different body parts of the dehydrated dragonfly (Odonata, Anisoptera) were
calculated from quasistatic nanoindentation experiments (Hysitron Inc., Minneapolis, MN)
(Kempf, 2000). The mean values were lower (1.5 – 4.7 GPa) as well than elastic modulus
obtained for dry samples in this study. Recent nanoscale study (quasistatic nanoindentation
testing instrument, Hysitron Inc., Minneapolis, MN) of Drosophila melanogaster integument
during development (Kohane et al., 2003) shows that the thickness and the development stage
of the cuticle was very important for stiffness measurements. The average reduced elastic
modulus (Er) of 0.41 MPa, 15.43 MPa and 4.37 MPa were determined by in vivo experiments
for the cuticles of the larval, puparial and adult insects respectively. Thickness of the cuticles
of Drosophila melanogaster was very low (2.3-17.7 µm) when compared to that of gula
cuticle (about 80 µm). However, the mechanical properties of the cuticle are strongly
influenced also by proportions of the main components (chitin and proteins) (Fraenkel and
Rudal, 1940; Locke, 1964; Neville, 1975; Hepburn and Chandler, 1976; Binnington and
Retnakaran, 1992).

Figure 9. AFM images of the cuticle surface after indentation test showing the residual deformation.
The image of the indent shows elastic recovery, which is characteristic for the elastic-plastic contact in
most engineering materials. However such shape of the indent here is thought to be caused by visco-
elastic relaxation expected for biological materials.

The nanoindentation experiments in this study were carried out in the epicuticle, which is
a nonchitinous layer. It contains proteins and is covered with lipids and surface waxes
(Neville, 1975; Binnington and Retnakaran, 1992). Surprisingly the elastic modulus of the
Specific Properties of Some Biological Composite Materials 107

fresh gula cuticle (E = 1.50 ± 0.80 GPa) is comparable to that of the chitin filament (1.97 ±
0.07 GPa) (Joffe and Hepburn, 1973). There are contradictory data in the literature about
which component (chitin or protein) is responsible for the elastic modulus of the cuticle.
According to Fraenkel and Rudall (1940), the mechanical properties of the cuticle are
determined by chitin, whereas Vincent (1980; 2002) suggests that the protein matrix is
decisive for its mechanical behavior. Elasticity which is expected to be a long-range effect is
influenced by the underlying exocuticle layer consisting of a high amount of chitin in a
protein matrix (Neville, 1975; Binnington and Retnakaran, 1992). Nevertheless, according to
our experimental results, it can be concluded that the elastic modulus in fresh cuticle is not
only influenced by only chitin or only the protein-matrix, but by both components.
After an indentation test the surface profiles of the samples were investigated by atomic
force microscopy (AFM) (DME, DualScope™ C-21 with scanner DS 45-40 BIO). Figure 9
shows the surface profile of the gula cuticle. Residual deformation of the cuticle surface after
indentation seems similar to elastic-plastic contact, which is typical of most engineering
materials (Barbakadze 2005). An elastic recovery on the indent-image can be distinguished.
This is due to a visco-elastic relaxation expected for soft biological materials (Wainright et
al., 1976; Vincent, 1990; Kohane et al., 2003).

WAX COATED PLANT SURFACES


The cuticle is an essential element of a plant. Being the outer surface layer, it is of
fundamental functional and ecological importance for the interaction between plants and their
environment (Barthlott, 1990; Bianchi, 1995; Gorb, 2001). The plant cuticles display different
surfaces. They may be pigmented or colorless, smooth, textured or hairy, dry or covered with
epicuticular secretions (Jeffree, 1986; Gorb, 2001). The extraordinary diversity of the plant
surface structure is a reflection of their different environment (Jeffree, 1986). Preventing
desiccation and allowing controlled exchange of gases are the important functions of
epicuticular components (Baker, 1982; Jeffree, 1986; Barthlott, 1990).
The plant cuticle is a multilayered composite consisting of a polymeric cutin matrix and
the cuticular waxes (Eigenbrode, 2002). Figure 10 shows the generalized structure of the
plant cuticle (Gorb, 2001). An outer wall of epidermal cells is covered by a pectinaceous
cuticle membrane, which is protected by a layer made up of cellulose microfibrils. On the
cuticle proper consisting of a lamellate layer, cutin and the epicuticular waxes are deposited
(Bianchi, 1995; Gorb, 2001). Cutin is high molecular weight lipid polyester (Bianchi, 1995).
The epicuticle waxes are surface lipids (Bianchi, 1995). They display considerable
ultrastructural and chemical diversity. The plant waxes are a complex mixture of very long-
chain aliphatics including different chemical compounds, such as hydro-carbons, wax esters,
primary and secondary alcohols, fatty acids, aldehydes, ketones, ß-diketones, triterpenoids
and flavonoids (Walton, 1990; Eigenbrode, 2002). A current classification of plant
epicuticular waxes, based on the studies of 13 000 plant species using high resolution
scanning electron microscopy (SEM), distinguishes 23 types of wax crystals (Barthlott, 1990;
Barthlott et al., 1998). The most common types of crystal shapes are tubes, solid rodlets,
filaments, plates, ribbons and granules (Baker, 1982; Jeffree, 1986; Bianchi, 1995; Barthlott
et al., 1998). The wax crystalloid structure correlates with its chemical composition
108 N. Barbakadze, E. Gorb and S. Gorb

(Barthlott, 1990; Bianchi, 1995; Barthlott et al., 1998). For example, aldehydes are very
important for the formation of wax filaments, which is characteristic of the pea Pisum sativum
leave surface (Gorb, 2001). The eucalyptus epicuticular wax, containing ß-diketones and
primary alcohols, exhibits mixed tubes and plates structure (Hallam and Chambers, 1976;
Jeffree, 1986).

Figure 10. The generalised structure of a plant cuticle: W - epicuticular wax; CP - cuticle proper; CL -
cuticular layer of reticulate region traversed by cellulose micro fibrils; P - pectinaceous cuticle
membrane and middle lamella; CW - cell wall; PL - plasma lemma (taken from Gorb, 2001).

All plant surfaces carry a partial or a continuous layer of the amorphous wax as the
obligatory final layer of the cuticle (Baker, 1982, Barthlott et al., 1998). However some plants
consist of even two amorphous wax layers. Upon the amorphous layer, wax crystals of
different shapes emerge and grow. The wax crystal layer varies widely in thickness from 1 to
20 µm. The thickness of the wax crystalloids lies in the nanometer range (Gorb, 2001; Enders
et al., 2004). Wax morphology varies with the environmental conditions. The thickness of the
layer, the size, the orientation and the density of wax crystals can be significantly modified by
temperature and humidity (Baker, 1982). The epicuticular waxes occur in plants primarily on
the surface of leaves, stems and fruits (Bianchi et al., 1978; Avato et al., 1984; Baker and
Gaskin, 1987; Barthlott, 1990; Bianchi, 1995; Barthlott et al., 1998; Gorb, 2001). On the
surfaces of many plant species, such as Eucalyptus leaves, the wax is continuously
regenerated. In this way the surface is protected and kept in a fully functional state. However,
the epicuticular wax cannot be replaced after having been removed by abrasion from some
plant surfaces (Bianchi, 1995).
Although no study on mechanical properties of the wax layer has been carried out, it is
believed in biology that the plant wax and the underlying layers display different mechanical
properties. A cuticularisation of the thick outer epidermal walls contributes to mechanical
stability of the surface. The wax covering is mechanically very unstable and in biology
Specific Properties of Some Biological Composite Materials 109

known as “soft film” (Barthlott, 1990, Barthlott et al., 1998). Being fragile, the waxes are
easily removed by rubbing (Barber, 1955; Jeffree, 1986). The wax crystalloids increase the
roughness. Together with the wax chemistry, the roughness makes the plant surface
hydrophobic and decreases its wettability. The hydrophobic constituents of the wax and
surface microroughness reduce the adhesion of the dust and other particles on the outer cell
wall. This “anticontamination function” called “lotus effect” is thought to be the most
important ecological function of the water-repellent plants (Baker, 1982; Barthlott, 1990;
Barthlott and Neinhuis, 1997).
An interaction between the plant surfaces and their environment also involves the
complex and very important relation between plants and insects (Juniper and Southwood,
1986). Insects possess the attachment devices allowing the hold and the movement anywhere
and on nearly any kind of surfaces (Gorb, 2001). The structures of attachment pads adapted
for the natural, mainly plant surfaces enable the insects to move freely on any technical
surfaces as well. However, some surfaces even in nature raise difficulties for their visitors.
The wax layer on the plant is a barrier for the attachment systems of insects. Walking on the
plant surface structured with the wax crystals was found to be impossible for insects
(Eigenbrode, 1996; Gaume et al., 2002; Gorb and Gorb, 2002). Even though the interaction
between the wax crystalloid layer and the insect has been of great interest to researchers for
about a century (Kerner von Marilaun, 1898; Haberlandt, 1909; Knoll, 1914), the function of
such epicuticular layer preventing adhesion remains largely unknown. There are only a few
experimental studies reporting some hypotheses, why the insects cannot attach on the wax
coated plant surfaces (Eigenbrode, 1996; Gorb and Gorb, 2002; Gaume et al., 2002): (1) the
location of wax crystals on the plant cuticle may be responsible for an increase in the surface
microroughness; this can be the reason for reduced real contact area between the plant surface
and the attachment system of insects (Stork, 1980); (2) the wax crystals are mechanically
unstable and fragile; attachment can be prevented by the contamination of the attachment
system of insects by the adhesion of the wax crystals to its pads (Juniper and Burras, 1962);
(3) the possible reaction between the wax layer and the secretion liquid of the attachment
system of insects (Gaume et al., 2002).
The interaction between the insect attachment systems and the plant anti-attachment
surfaces motivated this work. The goal was to study the mechanical behavior of plant surfaces
coated with wax crystals to understand the anti-attachment mechanism of the wax and to
clarify the first two hypotheses. The leaves of two pea (Pisum sativum) mutants were selected
for this study. The wild type pea leaves are strongly covered with wax crystals (filaments). In
“glossy” mutation, the amount of waxy bloom is greatly reduced (Eigenbrode and Espelie,
1995; Eigenbrode and Jetter, 2002). The samples in fresh and dry conditions were
mechanically tested using the Nano indenter® SA2. The structure of plant surfaces was
explored by means of scanning electron microscopy (SEM). This study is believed to be the
first attempt to characterize the mechanical properties of the wax crystals of plant surfaces.

MATERIALS AND SAMPLE PREPARATION


The experiments were performed with the pea Pisum sativum. The wild and glossy
mutants of the pea plant were planted and grown in the laboratory. The samples were
110 N. Barbakadze, E. Gorb and S. Gorb

prepared from the pea leaves of both mutants: the wild type with normal wax layer and the
glossy type with reduced waxy bloom. The top and bottom sides of the leaves were tested in
the untreated condition. The nanoindentations were performed on the specimens in the fresh
and dry conditions. To prepare dry samples, the fresh cuts of leaves were air dried at room
temperature for 48 h.

A B

20 20 µm

C
D

2 µm 2 µm

E F

2 1

Figure 11. SEM images of the wax crystalloid layer on the wild pea leaf surface. A and C show the
surface of the top side; B and D show the surface of the bottom side. Images A and B show cell
structure of the plant leaf. On the C and D images wax crystal covering the leaf can be distinguished.
The bottom side (D) of the pea leaf appears to have more dense waxy bloom than the top side (C). E
and F show the top side of the wild pea leaf.
Specific Properties of Some Biological Composite Materials 111

STRUCTURE OF WAX LAYER


The surfaces of the pea leaves were observed using scanning electron microscopy (SEM).
The samples, 7×7 mm in size, were cut from the leaves and mounted on an aluminum holder.
The surface structure of the wild and the glossy plants were tested at 5 kV. The observations
of top and bottom surfaces of both pea mutants were performed in the untreated, fresh
condition.

A B

20 µm 20 µm

C
D

2 µm 2 µm

E F

2 µm 1 µm

Figure 12. SEM images of the wax crystalloid layer on the glossy pea leaf surface. A and C show the
surface of the top side; B and D show the surface of the bottom side. Images A and B show cell
structure of the plant leaf. On the C and D images single wax crystals on the leaf can be distinguished.
The top side (C) of the glossy pea leaf appears to have more wax crystals than the bottom side (D). E
and F show the top side of the glossy pea leaf.
112 N. Barbakadze, E. Gorb and S. Gorb

Figures 11 and 12 show the surface structure of the wild and glossy pea leaves
respectively. The images are taken on the top and the bottom side of the leaf for each plant
type. The cell structure of the plant surfaces can be distinguished in figures 11 A, B and 12 A,
B. The openings on the figures 11 A, B and 12 A, B are stomata for gaseous exchange. There
is a great difference in the leaf surface between the wild and glossy pea. The wild pea samples
are completely covered with wax crystals (figures 11 C, D, E, F), while the glossy pea surface
only shows single wax crystals (figures 12 C, D, E, F). The comparison of SEM images of the
two plant mutants (figures 11 C, D and 12 C, D) leads to the estimate that the surface of the
glossy pea leaf bears only about one third of wax crystals. There is a small difference in the
wax covering of the top and bottom side of the same plant species as well. The bottom side of
the wild pea sample shows a denser wax crystal structure than the top side (figure 11 C, D).
The single wax crystals appear to be larger on the top surface of the glossy pea leaf than on
the bottom side (figure 12 C, D). The single wax crystals can be estimated to be 1 – 2 µm
long with a diameter of 100 - 200 nm (figures 11 E, F and 12 E, F). As can be seen in the
figures 11 E, F and 12 E, F, the wax crystals are oriented at different angles to the surface.

MECHANICAL PROPERTIES
Table 3 and 4 summarise the results of mechanical properties, hardness and elastic
modulus. The mechanical behaviors of the sample surfaces differ presumably depending on
the amount of wax crystalloids in the dry, but not in the fresh condition. It is surprising that
there is no difference in the mechanical properties (hardness and elastic modulus) between the
fresh samples with normal and with reduced wax layer. The maximum load corresponding to
the maximum displacement (3 µm) is about 3.5 mN for all fresh samples.
The wax layer appears to have an influence on the mechanical behavior of the dry plants.
The top and bottom surface of the same plant differ due to the different density and amount of
the wax crystals as well. In general, the surface with more wax shows lower hardness and
elastic modulus values (figure 13 and 14). The wax layer is believed to make the plant cuticle
surface soft and compliant.

Table 3. Summary of mechanical properties obtained at the displacement of 100 and


500 nm for fresh plant samples.

Hardness (GPa) Elastic modulus (GPa)


Plant species by indentation depth of by indentation depth of
100 nm 500 nm 100 nm 500 nm
wild top 0.7 ± 0.5 0.7 ± 0.2 3.2 ± 0.8 1.1 ± 0.2
wild bottom 0.7 ± 0.4 0.6 ± 0.4 2.7 ± 0.3 1.0 ± 0.3
glossy top 0.5 ± 0.5 0.5 ± 0.3 2.2 ± 0.5 1.0 ± 0.2
glossy bottom 0.4 ± 0.5 0.6 ± 0.2 2.6 ± 0.7 1.1 ± 0.1
Specific Properties of Some Biological Composite Materials 113

Table 4. Summary of mechanical properties obtained at the displacement of 100 and


500 nm for dry plant samples.

Hardness (MPa) Elastic modulus (MPa)


Plant species by indentation depth of by indentation depth of
100 nm 500 nm 100 nm 500 nm
wild top 5.2 ± 2.8 2.3 ± 0.9 285.2 ± 153.1 238.3 ± 194.2
wild bottom 1.2 ± 0.8 0.3 ± 0.1 168.2 ± 90.1 96.4 ± 59.3
glossy top 50.7 ± 41.1 23.7 ± 19.3 799.3 ± 569.1 570.4 ± 381.2
glossy bottom 7.5 ± 6.1 5.7 ± 3.9 526.8 ± 382.7 403.1 ± 291.1

In the figure 13 and 14, hardness and elastic modulus (mean values of approximately 100
measurements are shown) are plotted versus displacement for all fresh (figure 13 A and 14 A)
and all dry (figure 13 B and 14 B) samples. By looking at the curves showing hardness values
at the beginning of the indentation, a little influence of the wax layer could be observed only
at the first 200 nm of indentation depth. In this range the fresh samples display a slightly
different behavior depending on the presence of the wax layer. Fresh plant samples with
normal wax layer tend to be harder only at the first 200 nm of indentation depth than those
with reduced wax. In the first 200 nm the indenter tip obviously contacts single wax crystals.
This is thought to be caused by high mechanical stability of single wax crystalloids that build
a very unstable and soft wax layer (Barthlott, 1990). However, a difference in the elastic
modulus values between the fresh samples is not noticeable.

(B)

Figure 13. Hardness versus displacement curves of all fresh (A) and all dry (B) pea leaves. The mean
results of approximately 100 measurements are shown up to 1 µm.
114 N. Barbakadze, E. Gorb and S. Gorb

(A)

Figure 14. Elastic modulus versus displacement curves of all fresh (A) and all dry (B) pea leaves. The
mean results of approximately 100 measurements are shown up to 1 µm.

All fresh samples exhibit the same mechanical behavior at larger displacements
apparently due to the influence of the underlying layers. The plant cuticle has a multilayered
structure (Eigenbrode and Jetter, 2002), which consists of the mechanically stable
cuticularised epidermis covered by unstable and soft wax layer (Barthlott, 1990; Barthlott et
al., 1998). The hardness and the elastic modulus values corresponding to the fresh samples
are expected to be strongly influenced by cell walls and pressure of the cell fluid. However,
the cell wall itself appears to be soft and compliant (dry samples are softer and more
compliant than fresh ones). The cell structure, filled by fluid, appears to be influenced by
internal pressure of the cell fluid and therefore, is hard and stiff. This is why the effect of the
wax layer cannot be detected. A schematic of the cross section of the plant sample during the
indentation is shown in figure 15.
In the dry condition, the influence of the cell fluid is minimized or removed and there is a
great difference in the mechanical behavior between the plant surfaces with normal and with
reduced wax layer. The dry samples with normal wax layer are softer and more compliant
than the dry ones with reduced wax layer. In addition, the same mechanical behavior was
observed in the case of pitcher plant Nepenthes alata, eucalyptus Eucalyptus guinnii and red
cabbage Brasica (Barbakadze, unpublished results).
The desiccation greatly influences the mechanical behavior of biological materials (Gorb,
2001; Vincent and Wegst, 2004). However the effect of water loss is different for insect and
plant cuticles. Fresh insect cuticles are softer and more compliant than dry ones. By contrast,
the hardness and the elastic modulus values of fresh plant cuticle surfaces are higher than that
of dry ones.
Specific Properties of Some Biological Composite Materials 115

indente
cuticle 

cell wall 

cell fluid 

cell structure
Figure 15. Schematic of the cross section of the plant during indentation experiment. The arrows show
pressure of the cell fluid under the indenter.

WAX COVERINGS AND INSECT ATTACHMENT PADS


The attachment abilities of insects were recently tested on the plant surfaces investigated
in this study. The leaves of the wild pea (Pisum sativum) (Eigenbrode and Jetter, 2002) were
difficult to cope for insects. While walking on these surfaces, the insects often stopped and
cleaned their feet whereas they had less difficulty on the surface of the glossy mutant with
reduced waxy bloom.
To elucidate the hypotheses of reduced contact area and contamination effect of the wax
crystals, let us consider the results obtained in connection with the blowfly Calliphora vicina
(Niederegger et al., 2002), which is the model for most studies of the structure and the
attachment behavior of pads. What would the hardness and elastic modulus values obtained in
this study mean for this insect? The blowfly C. vicina weighs about 4 mN and possesses
attachment pads structured with a few thousand (4000 - 6000) flexible (1 N/m spring
constant) cuticular outgrowths called seta. A single spatula (tip of the seta) has an area of
1 µm2 (Niederegger et al., 2002). Thickness of the wax crystals lie in a few hundred
nanometer range. By walking, three feet of the insect with one third of the whole amount of
setae come in contact. However, the area of a spatula increases in contact by about 35 %
(Niederegger et al., 2002). An estimation of the load exerted by one single seta in contact
with the plant surface gives values ranging between 134 and 400 nN.
In the nanoindentation experiments, the hardness and elastic modulus values
corresponding to this load range were obtained at displacements of 20-40 nm: for the wild
pea, H = 50-340 MPa and E = 2.50 - 8.00 GPa (figure 16). With these considerations, one can
assume that wax crystals remain stable under insect pads and contribute to a decrease of the
116 N. Barbakadze, E. Gorb and S. Gorb

contact area between the insect attachment pad and the plant surface (hypothesis 1). However,
the wax crystals are not oriented perpendicularly to the surface but at different angles. The
insects do not only push down the plant surface but also use lateral forces while walking.
These considerations make the estimation more difficult. Regarding all these factors,
contamination of the insect pads (hypothesis 2) due to the breaking and adhesion of wax
crystals to the pad surface cannot be excluded. Further measurements on the isolated cuticle
(without cell structure) surface are necessary.

Figure 16. Hardness and elastic modulus of the wild pea samples (top side of the leaf) in the
displacement range of 0 – 80 nm. Grey area shows the displacement area of 20 to 40 nm, corresponding
to the load range of 134 – 400 nN. This load range is estimated to act during the walking of the blowfly
C. vicina on the plant surface.

SUMMARY
The mechanical behavior of various biological materials such as insect and plant cuticles
was studied by applying experimental approaches of material science. A head articulation
cuticle of the beetle designed for friction minimisation and the wax covered plant surfaces
adapted for attachment prevention have been chosen for this study. Both insect and plant
cuticles are multifunctional composite materials and have a multilayered structure. The gula
cuticle of the beetle Pachnoda marginata is a part of the head articulation, which is a
micromechanical device similar to a technical ball bearing. The surfaces in this system
operate in contact; they must be optimised against wear and friction and must provide high
mobility within the joint. The measurements on the gula cuticle were performed in order to
understand structure and tribo-mechanical behavior of material working for friction
minimizing. The plants carry amorphous wax layer which by some species is covered by wax
Specific Properties of Some Biological Composite Materials 117

crystals. The insects cannot attach on such plant surfaces. The location of the wax crystals on
the plant cuticle increases the surface microroughness. This could decrease the real contact
area between the plant surface and attachment pads of insect. Attachment can be prevented by
contamination of attachment pads of insect with the wax crystalloids. In order to understand
the deformation behavior of the wax layer two pea plant (Pisum sativum) mutants (wild type
with normal wax layer and glossy with reduced waxy bloom) were studied. To observe the
effect of desiccation all samples (insect and plant cuticles) were tested in fresh and dry
conditions. To understand the influence of an outer wax/lipid layer, tribo-mechanical
experiments on the gula cuticle were performed as well in a chemically treated condition.
This study is believed to be one of the first for mechanically testing of insect cuticle and the
very first for wax coated plant surfaces in native condition.
Mechanical properties were determined by using the Nano Indenter® SA2 (MTS Systems
Corporation, Oak Ridge, USA). A high damping coefficient and a high resonant frequency of
the indenter in this system allows determining mechanical properties of soft and structured
materials with low contact stiffness. The biological samples can be successfully tested in
native condition. Employing continuous stiffness measurement (CSM) technique allows
measuring contact stiffness continuously during indentation and obtaining the depth-
dependence of the hardness and elastic modulus. This is very important for biological
materials because their mechanical properties vary with depth.
The mechanical behavior of all samples was found to be greatly influenced by drying.
However changes in hardness and elastic modulus were different for insect and plant cuticles.
Desiccation made gula cuticle of the beetle harder and stiffer but the plant surface softer and
more compliant. After drying, gula cuticles became harder and stiffer by a factor of about 5
compared to the samples in the fresh state. Chemical treatment caused further hardening of
the material. Increase in stiffness could be observed only in first 1 µm of the indentation
depth. There was no significant difference between elastic modulus of the dry and chemically
treated samples at indentation depth higher than 1 µm.
Hardness and elastic modulus values of the fresh plant samples were significantly higher
than that of the dry ones. There was almost no difference between samples with normal and
with reduced wax layer in fresh condition. All fresh samples exhibited the same mechanical
behavior apparently due to the influence of cell walls and especially pressure of the cell-fluid.
In dry condition the effect of the cell-fluid was removed and the samples showed a great
difference in mechanical properties between the plant surfaces with normal and with reduced
wax layer. Dry samples with normal wax layer were softer and more compliant than with
reduced waxy bloom.

REFERENCES
Andersen, S.O. (1979) Insect cuticle. Annual Revue of Entomology, 24: 29-61.
Andersen, S.O., Peter, M.G. and Roepstorff, P. (1996) Cuticular Sclerotization in Insects.
Comparative Biochemistry and Physiology, 113B (4): 689-705.
Arzt, E., Enders, S. and Gorb, S. (2002) Towards a micromechanical understanding of
biological surface devices. Zeitschrift für Metallkunde, 93 (5): 345-351.
118 N. Barbakadze, E. Gorb and S. Gorb

Avato, P., Bianchi, G. and Mariani, G. (1984) Epicuticular waxes of Sorghum and some
compositional changes with plant age. Phytochemistry, 23: 2843-2846.
Baker, E.A. (1982) Chemistry and morphology of plant epicuticular waxes. In: The plant
cuticle. Cutler, D.F., Alvin, K.L., Price, C.E. (eds.) Academic Press, London, pp. 139-
165.
Baker, E.A. and Gaskin, R.E. (1987) Composition of leaf epicuticular waxes of Pteridium
subspecies. Phytochemistry, 26: 2847-2848.
Barbakadze, N. (2005) Micro/nanomechanical Measurements on Insect and Plant Cuticles.
Dissertation. University Stuttgart.
Barbakadze, N., S. Enders, S. Gorb and E. Arzt (2006) Local mechanical properties of the
head articulation cuticle in the beetle Pachnoda marginata (Coleoptera, Scarabaeidae).
Journal of Experimental Biology, 209: 722-730.
Barber, N.H. (1955) Adaptive gene substitutions in Tasmanian Eucalyptus. I. Genes
controlling glaucousness. Evolution, 9: 1-14.
Barthlott, W. (1990) Scanning electron microscopy of the epidermal surface in plants. In:
Scanning electron microscopy in taxonomy and functional morphology. Claugher, D.
(ed.) Clarendonpress, Oxford, pp. 69 - 83.
Barthlott, W. and Neinhuis, C. (1997) Purity of the sacred lotus or escape from contamination
in biological surfaces. Planta, 202: 1-8.
Barthlott, W., Neinhuis, C., Cutler, D., Ditsch, F., Meusel, I., Theisen, I. and Wilhelmi, H.
(1998) Classification and terminology of plant epicuticular waxes. Botanical Journal of
the Linnean Society, 126 (3): 237-260.
Bianchi, G., Avato, P., Bertorelli, P. and Mariani, G. (1978) Epicuticular waxes of two
sorghum varieties. Phitochemistry, 17: 999-1001.
Bianchi, G. (1995) Plant waxes. In: Waxes: chemistry, molecular biology and functions.
Hamilton, R. J. (ed.). The Oily Press: Dundee, pp. 177-222.
Bhushan, B. (1996) Tribology and mechanics of magnetic storage devices. Springer-Verlag,
New York.
Bhushan, B. and Li, X. (2003) Nanomechanical characterization of solid surfaces and thin
films. International Materials Reviews, 48 (3): 125-164.
Binnington, K. and Retnakaran, A. (1992) Physiology of the insect epidermis. CSIRO,
Australia, Melbourne.
Bueckle, H. (1965) Mikrohaertepruefung und ihre Anwendung. Stuttgart, Berliner Union.
Burnett, P. J. and Rickerby, D. S. (1987) The mechanical properties of wear-resistant
coatings: I: Modelling of hardness behaviour. Thin Solid Films, 148: 41-50.
Burnett, P. J. and Rickerby, D. S. (1987) The mechanical properties of wear-resistant
coatings: II: Experimental studies and interpretation of hardness. Thin Solid Films,
148: 51-65.
Dai, Z., Gorb, S. N. and Schwarz, U. (2002) Roughness-dependent friction force of the tarsal
claw system in the beetle Pachnoda marginata (Coleoptera, Scarabaeidae). Journal of
Experimental Biology, 205: 2479-2488.
Eigenbrode, S. D. and Espilie, K. E. (1995) Effects of plant epicuticular lipids on insect
herbivores. Annual Review of Entomology, 40: 171-194.
Eigenbrode, S. D. (1996) Plant surface waxes and insect behaviour. In: Plant cuticles - an
integral functional approach. BIOS Scientific Publishers Ltd, Oxford, pp. 201-222.
Specific Properties of Some Biological Composite Materials 119

Eigenbrode, S.D. and Jetter, R. (2002) Attachment to Plant Surface Waxes by an Insect
Predator. Integrative and Comparative Biology, 42: 1091-1099.
Enders, S., Barbakadze, N., Gorb, S. N. and Arzt, E. (2004) Exploring biological surfaces by
nanoindentation. Journal of Materials Research, 19 (3): 880-887.
Fraenkel, G. and Rudall, K. M. (1940) A study of the physical and chemical properties of the
insect cuticle. Proceedings of the Royal Society, B 129: 1-34.
Fung, Y., C. (1993) Biomechanics, Mechanical Properties of Living Tissues. Springer-Verlag,
pp. 500-544.
Gorb, S. N. (1997) Ultrastructural architecture of the microtrichia of the insect cuticle.
Journal of Morphology, 234: 1-10.
Gorb, S. N. (1999a) Evolution of the dragonfly head-arresting system. Proceedings of the
Royal Society of London, B 266: 525-535.
Gorb, S.N. (1999b) Ultrastructure of the thoracic dorso-medial field (TDM) in the elytra-to-
body arresting mechanism in tenebrionid beetles (Coleoptera: Tenebrionidae). Journal of
Morphology, 240: 101-113.
Gorb, S.N. and Scherge, M. (2000) Biological microtribology: anisotropy in frictional forces
of orthopteran attachment pads reflects the ultrastructure of a highly deformable
material. Proceedings of the Royal Society of London, B 267: 1239-1244.
Gorb, S.N. (2001) Attachment devices of the insect cuticle. Kluwer Academic Publishers, The
Netherlands.
Gorb, S. N., Gorb, E. and Kastner, V. (2001) Scale effects on the attachment pads and friction
forces in syrphid flies (Diptera, Syrphidae). Journal of Experimental Biology, 204:
1421-1431.
Gorb, E.V. and Gorb, S. N. (2002) Attachment ability of the beetle Chrysolina fastuosa on
various plant surfaces. Entomologia Experimentalis et Applicata, 105: 13-28.
Gorb, E. and Gorb, S. (2002) Contact separation force of the fruit burrs in four plant species
adapted to dispersal by mechanical interlocking. Plant Physiology and Biochemistry,
40: 373-381.
Gorb, S.N. and Perez Goodwyn, P.J. (2003) Wing locking mechanisms in aquatic
Heteroptera. Journal of Morphology, 257 (2): 127-146.
Haberlandt, G. (1909) Physiologische Pflanzenanatomie. Engelmann, Leipzig.
Hallam, N. D. and Chambers, T. C. (1976) The leaf waxes of the genus Eucalyptus
L’Heritier. Australian Journal of Botany, 18: 335-386.
Hammond, P.M. (1989) Wing-folding mechanism of beetles, with special reference to
investigations of adephagen phylogeny (Coleoptera). In: Ervin, T., Ball, G. E. and
Whitehead, D.R. (eds.) Carabid beetles: their evolution, natural history and
classification. Boston, London: Junk Publishers: The Hague, pp. 113-180.
Hepburn, H. R. and Joffe, I. (1976) On the material properties of insect exoskeletons. In: The
insect integument. Hepburn, H.R. (ed.), Elsevier, Amsterdam, pp. 207-235.
Hepburn, H.R. and Chandler, H.D. (1976) Material properties of arthropod cuticles: the
arthropodial membranes. Journal of Comparative Physiology, 109: 177-198.
Hepburn, H. R. and Chandler, D. (1980) Materials Testing of Arthropod Cuticle Preparations.
In: Cuticle technologies in arthropods. Miller T.A. (ed.). New York, Springer Verlag,
pp. 1-44.
Hillerton, J. E., Reynolds, S. E. and Vincent, J. V. (1982) On the indentation hardness of the
insect cuticle. The Journal of Experimental Biology, 96: 45-52.
120 N. Barbakadze, E. Gorb and S. Gorb

Jeffree, C. E. (1986) The cuticle, epicuticular waxes and trichomes of plant, with references
to their structure, functions and evolution. In: Insects and the plant surface. Juniper, B.
E., Southwood, T. R. E. (eds.) Edward Arnold: London, pp. 23-64.
Jensen, M. and Weis-Fogh, T. (1962) Strength and elasticity of locust cuticle. In: Biology and
Physics of locust flight. Philosophical Transactions of the Royal Society, B 245 pp. 137-
169.
Joffe, I. and Hepburn, H. R. (1973) Observations on regenerated chitin films. Journal of
Materials Science, 8: 1751–1754.
Jönsson, B. and Hogmark, S. (1984) Hardness measurements of thin films. Thin Solid Films,
114: 257-269.
Juniper, B.E. and Burras, J.K. (1962) How pitcher plants trap insects. New Scientist.
13: 75-77.
Juniper, B. E. and Southwood, R. (1986) Insects and the Plant Surfaces. London, UK:
Edward Arnold.
Kempf, M. (2000) Biological materials, determination of Young’s moduli of the insect cuticle
(dragonflies; Anisoptera). Application note, Hysitron Inc, www.hysitron.com.
Kerner von Marilaun, A. (1898) Pflanzenleben 2. Bibliographisches Institut, Leipzig/Wien.
Knoll, F. (1914) Über die Ursache des Ausgleitens der Insektenbeine an wachsbedekten
Pflanzendteilen. Jahrbuch für Wissenschaftliche Botanik, 54: 448-497.
Kohane, M., Daugela, A., Kutomi, H., Charlson, L., Wyrobek, A. and Wyrobek, J. (2003)
Nanoscale in vivo evaluation of the stiffness of Drosophila melanogaster integument
during development. Wiley Periodicals, Inc.: 633-642.
Korsunsky, A. M., McGurk, M. r., Bull, S. J. and Page, T. F. (1998) On the hardness of
coated systems. Surface Coatings Technology, 99: 171-183.
Kreuz, P., Kesel, A., Kempf, M., Göken, M., Vehoff, H. and Nachtigall, W. (1999)
Mechanische Eigenschaften biologischer Materialien am Beispiel Insektenflügel. BIONA
report, 14: 201-202.
Locke, M. (1964) The structure and formation of the integument in insects. In: The
physiology of Insecta. Rockstein M. (ed.), Academic Press, New York, pp. 123-213.
McGurk, M. R., Chandler, H. W., Twigg, P. C. and Page, T. F. (1994) Modelling the
hardness response of coated systems: the plate bending approach. Surface Coatings
Technology, 68/69: 576-581.
Neville, A. C. (1975) Biology of the arthropod cuticle. Springer Verlag, Berlin, Germany.
Niederegger, S., Gorb, S. and Jiao, Y. (2002) Contact behaviour of tenent setae in attachment
pads of the blowfly Calliphora vicina (Diptera, Calliphoridae). Journal of Comparative
Physiology, A 187: 961-970.
Oliver, W. C. and Pharr, G. M. (1992) An improved technique for determining hardness and
elastic modulus using load and displacement sensing indentation experiments. Journal of
Materials Research, 7 (6): 1564-1583.
Perez Goodwyn, P. J. and Gorb, S. N. (2004) Anti-frictional properties of contacting surfaces
in the hemelytra-hindwing locking mechanism in the bug Coreus Marginatus
(Heteroptera, Coreidae). Journal of Morphology (in press).
Persson, B. N. J. (1998) Sliding friction. Springer: Berlin, Heidelberg, New York.
Rother, B. and Jehn, H. A. (1996) Coating and interface characterization by depth-sensing
indentation experiments. Surface and Coatings Technology, 85: 183-188.
Specific Properties of Some Biological Composite Materials 121

Scherge, M. and Gorb, S. N. (2000) Microtribology of biological materials. Tribology Letters,


8: 1-7.
Stork, N. E. (1980) Role of wax blooms in preventing attachment to brassicas by the mustard
beetle, Phaedon cochleariae. Entomologia Experimentalis et Applicata, 28: 100-107.
Vincent, J. F. V. (1980) Insect cuticle - a paradigm for natural composites. In: The
mechanical properties of biological materials. Symposium of the Society Experimental
Biology, 34: 181-210.
Vincent, J. F. V. (1990) Structural Biomaterials. The University Press, Princeton.
Vincent, J. F. V. (2002) Arthropod cuticle –a natural composite shell system. Composite Part
A - Applied Science and Manufacturing, 33 (10): 1311-1315.
Vincent, J. F. V. and Wegst, U.G. K. (2004) Design and mechanical properties of insect
cuticle. Arthropod Structure & Development, 33: 187-199.
Wainwright, S. A., Biggs, W. D., Currey, J. D. and Gosline, J. M. (1976) Mechanical Design
in Organisms. Princeton University Press.
Walton, T. J. (1990) Waxes, cutin and suberin. In: Lipids, membranes and aspects of
photobiology, Dey, P. M., Harborne, J. B. (eds.), pp. 105-158.
Wegst, U. G. K. and Ashby, M. F. (2004) The mechanical efficiency of natural materials.
Philosophical Magazine, 84, 21: 2167-2181.
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.123-153 ©2010 Nova Science Publishers, Inc.

Chapter 10

PROPERTIES AND APPLICATIONS OF AMINOXYL


RADICALS IN POLYMER CHEMISTRY

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii*,


T. V. Pokholok, and G. E. Zaikov
N.M. Emanuel Institute of Biochemical physics,
Russian Academy of Sciences, Moscow, Russia

ABSTRACT
The review includies the information about the structure and physical properties of
stable radicals, ESR spectroscopy of stable aminoxyl radicals (ARs), chemical properties
and applications of ARs, generation of ARs in reactions of spin trapping and synthesis of
polymers containing stable ARs.

Keywords: aminoxyl radicals, electron spin resonance, polymers with stable radicals.

INTRODUCTION
Free radical reactions initiated by NO2 and other nitrogen oxides in polymers are
accompanied by generating stable nitrogen-containing radicals. Analysis of their structures
and kinetics of formation provides information on free radical stages of complex processes of
the polymer nitration under various conditions [1]. These studies can be used in synthetic
chemistry in particular for the development of methods of the polymer modification, for
example, for the preparation of spin-labelled macromolecules. The generation of spin labels
thus occurs in consecutive reactions including formation and conversions of specific
intermediate molecular products and active free radicals.
Most of the traditional developing methods of preparing spin labelled macromolecules
are based on use of organic compounds capable of interacting with polymers to form stable

* pgb@sky.chph.ras.ru , chembio@sky.chph.ras.ru
124 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

aminoxyl radicals (AR). Grafting these radicals can be carried out by means of nitroso
compounds and nitrons which capture active radicals. ARs represent a vast class of stable
radicals widely used now in chemistry. Numerous articles and monographs are devoted to
synthesis and properties of these radicals [2, 3]. The insertion of AR to macromolecules
allows essentially changing physical, chemical and operational characteristics of polymers.
Generally this effect is conditioned by the presence of >N−O• fragments in polymer chains
and the interaction of these radical centers with components of polymeric materials. In the
present chapter, structures and properties of ARs and ways of the stable radical generation
using reactions of organic synthesis are considered.

1. STRUCTURE AND PHYSICAL PROPERTIES OF STABLE RADICALS


Within the framework of the resonance theory, the AR structure can usually be
represented by the following resonant structures [4]:

R1 R1
N O N O (II.1)
R2 R2

The schemes of the electronic shell formation of the paramagnetic centre from AR and
levels of the molecular orbitals of AR are respectively shown in Fig. 1 and Fig. 2.

z z
y

N O x

Figure 1.
Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 125

π∗

2pz
2pz n
2py

N N O O

Figure 2.

The length of N−O bond in all types of AR makes up 1.27-1.30 Å. This value
corresponds to the length of a three-electronic bond, i. e. to such electronic structure in which
binding σ and π orbitals are occupied by electron pairs, whereas the unpaired electron
occupies nonbinding π* orbital formed by pz orbitals of nitrogen and oxygen atoms and is
located approximately in equal parts between them. It is accepted that the delocalization of
unpaired electron between N and O atoms with lowering its energetic level (134 kJ⋅mol-1) is
the main reason of high stability of AR [3].
The geometrical structure of the radical centre appreciably depends on character of R1
and R2 groups. The angle between CNC plane and N−O bond in different radicals varies from
0o up to 30o [3]. The deviation of nitrogen atom from coplanarity does not result in any
changes of energy, but the hybridization of nitrogen atom determines the characteristics of
ESR spectra of AR. Most of AR do not form dimers > N−O−O−N < in a wide range of
temperatures and various solvents [5]. The AR dimerization is the thermodynamically
inefficient process connected with loss of the stabilization energy of two radical fragments
ARs have two absorption bands in their electronic spectrum. Di-t-alkylaminoxyls have
bands at 240 nm (ε = 3000 l mol −1 cm −1) and at 410-460 nm (ε = 5 l mol −1 cm −1). The first
is caused by π → π* transition, the second belongs to n → π* transition (Fig. II.2). Because of
conjugation of an aromatic ring with aminoxyl groups, the bathochrome shift occurs for
absorption bands. The shifts up to 335-340 and 490-510 nm are observed for diarylaminoxyls
[3]. The vibration frequencies of AR should be appeared in IR spectra at 1340-1370 cm −1 [3].
Unfortunately there are bands in this region frequently connected with vibrations of alkyl
groups. According to the studies of solid and solution IR and Raman spectra of 1,1,3,3-
tetramethylisoindolin-2-yloxyl radicals [6], the N−O• stretching frequency is 1431cm−1. This
apparently anomalous peak position was confirmed by isotopic substitution studies and ab
initio density functional theory calculations. Therefore, IR spectroscopy can be carefully
applied for identification of AR. The dissociation energy of C−N bond determined by the
electronic impact method makes up 121.5 kJ mol-1 [7]. The rather low energy makes feasible
the decay of AR due to breaking C−N bond in certain conditions. The ionization potential of
AR is high enough (7 eV) [8]. This makes the increased stability of AR in reactions in which
126 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

cations and radical cations are primary active centers, namely, in reactions induced by
irradiation and oxidation.

2. ESR SPECTROSCOPY OF AMINOXYL RADICALS


The ESR spectra of AR are quantitatively described by spin Hamiltonian [2]:
H = −β H 0 gS + STI
(2)

where β is Bohr magneton, H 0 is the external magnetic field intensity, g is g-tensor, S is


the electron spin operator, T is the tensor of hyperfine interaction (HFI), I is the nuclear
spin operator. Spin Hamiltonian consists of isotropic and anisotropic parts:

H = Hi + Ha (3)

H i = − βg H 0 S + a IS (4)

H a = −β H 0 g ' S + ST ' I (5)

where g and a are isotropic values of g-factor and HFI constant. They are determined by
the following equations:

g = 1 / 3( g x + g y + g z ) = 1 / 3r g ' (6)

a = 1 / 3(Tx + T y + Tz ) = 1 / 3rT ' (7)

where g ' and T ' are diagonal tensors. The constituents of these tensors are connected with
common g ' - and T ' - tensors by the equations:

g i = g i '+ g (8)

Ti = Ti' + a (9)

(i = x+ y+ z)
Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 127

In solutions with low viscosity, ARs are rapidly rotated with total averaging g -and T -
tensors. Therefore ESR spectra of AR in solutions are characterized by triplet signal with the
component intensity ratio of 1:1:1 owing to interaction of unpaired electron with the 14N
nucleus having I = 1. This triplet splits because of interactions with magnetic nucleuses in α−
and β− positions. The isotropic HFI constants aN dependent on the AR structure are given
below [9]:

aN, mT
Dialkylaminoxyl 1.4-1.7
Alkylarylaminoxyl 1.1-1.4
Diarylaminoxyl 09-1.1
Acylaminoxyl 0.67-1.1
Alkoxyalkylaminoxyl 2.4-2.8
Alkoxyarylaminoxyl 1.3-1.5

The analysis of HFI using quantum-chemical calculations has allowed determining spin
density ρ on N and O atoms. For example, the ρ-value in di-t-alkylaminoxyls is distributed
practically fifty-fifty between N and O atoms, at that an unpaired electron is almost
completely located on a radical fragment [10]. In alkylaryl- and diarylaminoxyl radicals there
is the capability of an unpaired electron to be delocalized onto aryl fragment with
considerable decreasing ρ on N atom. Therefore, the constant of splitting aN in these radicals
is less than in dialkylaminoxyl radicals. In viscous liquids, the rotation of radicals becomes
slower, and both g- and HFI tensors are not completely averaged. As a result, HFS
components are broadened out, and the high-field component shows this broadening to a
greater extent. For various components ( M = 0,±1 ) of AR spectrum one can obtain the
following equations for the correlation time τ of radical rotation [2]:

⎛ ΔH 1 ΔH −1 ⎞ 15π 3ΔH 0
τ' = ⎜⎜ − ⎟⎟ (10)
⎝ Δ H 0 ΔH 0 ⎠ 8 HbΔγ
⎛ ΔH 1 ΔH −1 ⎞ 4π 3h
τ' ' = ⎜⎜ − − 2 ⎟⎟ (11)
⎝ Δ H 0 ΔH 0
2
⎠ 8βb

where ΔH i is the width of components Mi, b = 2 / 3[T2 − 1 / 2(T x + T y )] and


β
Δγ = [ g z − 1 / 2( g x + g y )]
=
−11
The equations (10) and (11) are valid for τ = 5 ⋅ 10 − 5 ⋅ 10 −9 s. The correlation
time is connected with radius of rotating molecules and viscosity of the solvent η by the
Stokes equation:
128 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

4 πηr 3
τ= (12)
3kT

For determining τ in the range of slow movements ( τ > 5⋅10−9 s), the dependence of τ on
shift of high-field line Δ relative to its position in ESR spectrum of can be used [11]:

Δ = const ⋅ τ 3 / 2 (13)

−9 −7
By the equation (13) one can calculate τ in the range of 5 ⋅ 10 − 5 ⋅ 10 s and use
these values in studies of the rotational diffusion of macromolecules.
The correlation time can be expressed as function of the parameter S = Az ' / Az where
Az ' is half of distance between extremes of outside lines of triplet ESR spectrum and Az is
the same value in conditions of extremely slow movements of AR [12]:

τ = a (1 − S ) b (14)

where a and b depend on diffusion model. The equation (14) is convenient for using in the
−8 −6
range of 10 < τ < 10 s.

3. CHEMICAL PROPERTIES OF AMINOXYL RADICALS


The stability of AR is conditioned by tautomeric conversions and depends on chemical
structure of substituents at N atom, temperatures and solvents [5, 9]. The general mechanism
of the AR decay is a disproportionation with formation of nitrons and hydroxylamines. ARs
having primary or secondary alkyl groups are short-living species because they easily
undergo disproportionation by the scheme:

OH O
O
2 RCH2NR1 RCH2NR1 + RCH=NR1 (II.15)

In a number of cases, the arising products, for example, hydroxylamines can be rather
effective acceptors of short-living radicals. They are easily oxidized by nitroso compounds,
aminoxyl radicals, oxygen or other oxidizers, which are accumulated as a result of side
reactions. In this case one can observe the post - accumulation of radical adducts [13, 14].
Nitrons formed by the reaction (15) represent itself spin traps and can accept AR. In some
cases such adducts can be more stable than radical adducts of the initial generation, so only
adducts of the second and third generations will be observed in ESR experiments. ARs of
high stability are formed when the nitrogen is connected with tertiary carbon atom and the
disproportionation is excluded [3]. Such radicals are, for example, di-t-butylaminoxyl (I),
Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 129

derivatives of 4,4-dimethyloxazolidineoxyl (II), 2,2,5,5,-tetramethylpyrrolinoxyl (III) and


derivatives of piperidine-1-oxyl (IV).

O
R1
(CH3)3C-N-C(CH3)3
O N O
N N
R1 R2
O O
(I) (II) (III) (IV)

Changes in structure of nitrogen substituents make AR susceptible to dimerization, if thus


the electron delocalization degree is increased and, hence, there is an opportunity for
reactions by other centers. So tert-butylphenylaminoxyl (V) is much less stable than radical I
due to the unpaired electron delocalization to an aromatic ring. From the point of view of
chemical properties, the delocalization makes possible an attack of p-position of phenyl ring
by second AR:

O O
H
(CH3)3C-N-C6H5 + N C(CH3)3

(V)
O
(CH3)3C-NH-C(CH3)3 + O N C(CH3)3 (II.16)

Although the mechanisms of decay of AR of this type in something differ, they always
include attack of aminoxyl groups to o- or p- positions of aromatic rings. The o-substituents
stabilize AR owing to violation of the coplanarity and the decrease of spin density in an
aromatic ring [15]. The bulky substituents in p- and m- positions also stabilize AR as in this
case steric hindrances arise for radical attack [16]. The triphenyl-t-butylaminoxyl (VI)
unusually decays. The spatial strain of radical centre results in N−C dissociation in AR [17]:

(C6H5)3C-N-C(CH3)3 (C6H5)3C + O=NC(CH3)3 (II.17)


(VI)

The solvents essentially influence on the decay rate, and rate constants in polar solvents
are less because of AR blocking as a result of the formation of hydrogen bonds with solvents.
In conditions, when the hydrogen atom abstraction from molecules of surroundings is
difficult, aminoxyls are stable up to 200-220o [18]. AR can accept one radical with the
formation of diamagnetic compounds:
130 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

N−O• + R• → >N−O−R (18)

The rate constants of reactions of AR with solvated electrons, H atoms, OH•, CH3•
amount to 109 − 1010 l⋅mol−1⋅s−1 and 108⋅l mol−1⋅s−1 with radicals C•HOH [19]. This property
of AR serves as the basis for their use as counters of radicals. The unique property of AR is
their capability of reacting without participation of unpaired electrons with retention of
paramagnetism. Such reactions are widely used for synthesis of new AR with various
substituents [3], for synthesis of metalorganic radicals containing Tl, Hg, Fe etc. [20]. By this
way, polyradicals were obtained in which paramagnetic fragments are interconnected in the
uniform molecular system [21]. These reactions represent a method of spin labels used in
chemistry, biochemistry and molecular biology [22].
The properties of AR as oxidizers can be shown by the example of their interaction with
hydrocarbons [23]:

>N−O• + RH → >N−OH + R•

>N−O• + R• → >N−OR (19)

By the voltammeter method, ionization potentials of oxidation of a number of ARs were


measured in acetonitrile [24]. The conclusion was made that for oxidation of AR the rather
strong oxidizers are required. Bromine and chlorine easily and quantitatively oxidize AR into
reactive oxoammonium salts [25, 26]. Stable diarylaminoxyls under the action of halogens
also form oxoammonium salts, and the ease of oxidation of the radicals is determined by
nature of substituents:

R R R R
O O
3/2X2
R'' N R'' R'' N R''X3 (II.20)

R R' R R'
(VII)

If R = R′ = H, R′′ = OCH3, then radicals VII are oxidized by bromine. In the case of R =
R′ = R′′ = OCH3, radicals VII are easily oxidized even by iodine. The basic products of
vigorous reaction of radicals I with ozone are nitro-t-butane and oxygen [27]. The oxidation
of alcohols into carbonyl compounds can be carried out via the interaction of piperidinoxyl
with Cu (II) [28]:

>N O + Cu2+ >N=O + Cu


+

>N=O + CH3OH >N-OH + CH2OH (II.21)

CH2OH HCHO + H+
Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 131

Under the action of strong acids, the protonation of AR takes place [3]:

H+ +
>N O >N OH (II.22)

The protonation of ARs is the first stage of their interaction with mineral acids, which
results in products of disproportionation [29]:

HX + + H
2 >N O >N O X + >N X (II.23)
OH

In such a manner, ARs of piperidine, hydrogenated pyrrole and nitronylaminoxyls


disproportionate. Aminoxyls also disproportionate under the action of allyl and benzyl
bromide by the following scheme [30]:

2 >N O + RBr >N=OBr + >N OR (II.24)

Along with reactions without the participation of the radical centre, ARs react as typical
radicals. At the elevated temperatures they abstract hydrogen atoms, chlorine, bromine and
other elements. There are examples of sufficiently reactive AR in H-atom abstraction at
ordinary temperatures. The benzotriazole-N-oxyl (BTNO) generated by the oxidation of 1-
hydroxybenzotriazole (HBT) with a CeIV salt in acetonitrile spontaneously decays with a first-
order rate constant of 6.3⋅10-3 s-1 at room temperature [31]. The decay of this aminoxyls is
strongly accelerated in the presence of H-donor substrates such as alkylarenes, benzyl and
allyl alcohols:

ArCH2OH HBT
N N
Ce(IV)
N N ArCH2OH (II. 25)
- H+
N N
HBT OH BTNO O

The kinetic isotope effect confirms the H-abstraction step as rate-determining.


ARs recombine with many radicals participating in chain chemical reactions and add to
multiple bonds. Dialkylaminoxyl radicals actively react with alkyl radicals [5, 32], sulfur-
containing radicals [33], solvated electrons and by radicals generated by γ−radiolysis of
organic compounds [34]. ARs recombine with hydroxyl radicals, but do not react with HO2•
radicals [35].
As distinct from dialkylaminoxyl radicals, aromatic ARs react with peroxide radicals
[36]. If alkyl radicals or hydrogen atoms participate in reactions, the basic products of such
reactions are the corresponding ethers and hydroxylamines [34]. Hydroxyalkyl radicals are
captured by AR [34] with the formation of unstable ethers, which are decomposed to yield
aldehyde and hydroxylamine:
132 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

>N O + CH2OH >N O CH2OH >N OH + CH2O (II.25)

ARs are useful "counters" of active alkyl radicals [37] and inhibitors of radical
polymerization [38]. It should be noted that aliphatic and aromatic ARs have approximately
identical reactivity in inhibition. These radicals are similar to quinones and considerably
exceed nitroso compounds as radical inhibitors. The comparison of reactivity of spin traps
and AR shows that ARs are 2-5 orders of magnitude more effective radical acceptors than
nitrons and nitroso compounds. Therefore, new effective acceptors of radicals are generated
already at early stages of the short-living radical trapping.
Calculations in the framework of density functional theory (DFT) [39] for model AR
H2NO• indicate that addition to the carbonyl carbon is exothermic by 18.7 kcal·mol−1 [40].
This prediction was tested experimentally in reactions of AR IV with ketenes [41]. In this
case facile reaction occurred, and on the basis of the theoretical as well as kinetic and product
studies the reactions were interpreted as proceeding through attack of one IV at the carbonyl
carbon forming a α−acyl radical intermediate. Then the intermediate radical reacts with
another IV at Cβ :

O
IV
C O C O N

O
IV
C O N (II.26)
O

The adducts of AR with organic free radicals have attracted considerable attention
because of their potential utility as free radical initiators, and because of the important role of
reversible dissociation of adducts of IV in living radical polymerization.
The wide development is observed in study of participation of ARs in various
photochemical reactions, phototransfer of electrons and electronic energy. For some radicals
the basic process is the dissociation with the nitric oxide detachment, while other types of AR
mainly abstract hydrogen atoms from solvents. The quantum yield of such process is very
high (~0.5) [42]. Di-t-alkylaminoxyls are poorly stable under the exposure to UV light. So the
photolysis of radicals IV (R1 = OH) by light with λ = 350 nm in toluene completely converts
them into equal quantities of hydroxylamine and benzyl ether of hydroxylamine [43]. Thus,
the capability of some excited ARs to abstract a hydrogen atom with the subsequent
recombination of formed radicals and AR provides a method of functionalization
macromolecules.
The radical III (R=CONH2) decays during photolysis with breaking N−C bonds [44]:
Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 133

CONH2 CONH2
hν (II.27)
+ NO
N
O

The photochemical transformations of AR depend not only on a type of a radical, but also
on chemical properties of the solvent [42, 45]. The radical I in pentane dissociates with the
detachment of t-butyl groups during photolysis by light with λ < 300 nm in the band of π→π*
transitions:

O OC(CH3)3

(CH3)3C-N-C(CH3)3 (CH3)3CN=O + (CH3)3C-N-C(CH3)3 (II.28)
C5H12
70% 25%

In the solution of radicals I in carbon tetrachloride there is an absorption in the range of


300-400 nm corresponding to the charge-transfer band. The irradiation of the solution by light
with 313 < λ < 360 nm results in the radical I decomposition with the quantum yield of 1.7:

O

(CH3)3C-N-C(CH3)3 (CH3)3CN=O + (CH3)3CCl +
CCl4

OCCl3 OCl

(CH3)3C-N-C(CH3)3 + (CH ) C-N-C(CH ) + (CH3)2C=CH2 (II.29)


3 3 3 3

By this is meant that the decay of radicals I takes place both under the action of light and
in secondary reactions with products of the solvent photolysis, in particular with CCl3•
radicals.

4. APPLICATIONS OF AMINOXYL RADICALS


Stable radicals named also as spin labels find wide applications in various areas of
scientific researches and manufacture. The area of ARs applications includes organic
chemistry and photochemistry, chemical kinetics and catalysis, analytical chemistry,
chemistry of polymeric materials, molecular biology and medicine. In experimental chemistry
ARs are applied to recognize the mechanism of chemical reactions, structures of active
radicals in a wide temperature range [46]. The important feature of AR is the regular change
of their ESR spectra depending on mobility, nature of surrounding molecules and mutual
distances. They are widely applied in researches of physics and chemistry of polymers [18].
For these purposes, a small quantity of spin labels is introduced into the studied polymer so
134 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

that 200-600 monomer units account for one stable radical. In these conditions, the widening
of ESR spectra caused by a spin exchange is excluded.
With the help of spin labels one can determine parameters of molecular movements and
their change under the various external effects, study the dynamics of conformations of
macromolecules in solutions, investigate the molecular dynamics in solid polymers, carry out
the analysis of compatibility of components of complex polymer blends, investigate cross-
linked and filled polymers. The important area of applications of spin labels is the study of
the mechanism and kinetics of reactions in heterogeneous systems, interfaces, defects of
packing and so on [18]. The capability of recombining with other active particles provides a
way for the AR application as inhibitors and regulators of polymerization, effective stabilizers
of polymer oxidation, thermal, mechanical and photo degradation of polymers. Kinetic
features of the inhibited oxidation of polypropylene and polyethylene by 2, 2, 6, 6-
tetramethyl-4-benzoyloxypiperidine-1-oxyl have been studied [47, 48].
AR in the grafted form can be also used as inhibitors. Rubbers containing one aminoxyl
group per 1000-3000 monomer units show increasing induction periods of the oxidation at
140o a several times [49]. High-molecular-weight inhibitors are favourable for high-
temperature stabilization and for polymers with the high molecular mobility. These conditions
provide more homogeneous distribution of such stabilizers and show the basic advantage of
them connected with their nonvolatility [50].
The increase of the nitrocellulose working life in the presence of 2,2,6,6-tetramethyl-4-
ethyl-4-oxypiperidine-1-oxyl was observed during mechanical actions. Additives of this
stabilizer in the concentration of 0.3 weight % increase durability of the polymer by a factor
of hundred. At that, the breaking strength is increased in several times, and the creep rate of
the material decreases in 100 times [51]. These results were confirmed by investigations of
mechanical degradation of polypropylene in the presence of radicals IV (R=OOCNHC6H5)
[52]. AR grafted on polypropylene considerably increases its stability during treatment in the
stirrer [53, 54].
ARs can be used as effective quenchers of the exited states and controlling agents
photochemical and radiating processes. ARs have been used as photostabilizers of films and
fibers [54, 55]. These radicals have been used in synthesis of polymers with strong magnetic
properties namely polyradicals. On the basis of polyacetylene containing AR, the polymer
ferromagnetic having residual magnetization of 1 G has been prepared [56].
ARs are used in molecular biology for obtaining spin-labelled macromolecules. These
labels register slightest changes in the macromolecule states [22]. With the help of spin labels,
the conformational transformations of biopolymer macromolecules as well as changes in the
structure of biomembranes and nucleic acids have been studied.

5. GENERATION OF AMINOXYL RADICALS IN REACTIONS OF


SPIN TRAPPING
The possibility in principle to stabilize short-living radicals has been shown for the first
using their reactions with nitroso compounds and nitrons [57, 58]:
Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 135

O
R1 N=O + R R1 N R (II.30)
O O
R
R1 C N R3 + R R1 C N R3 (II.31)
R2 R2

All aliphatic nitroso compounds are dimers in a solid phase, but they dissociate in
solutions and a gas phase. The monomer form of nitroso compounds accepts radicals. The
aliphatic nitroso compounds form enough stable adducts with short-living radicals of the very
different structure. The character of ESR spectra of radical adducts with tertiary nitroso
compounds is practically identical for all spin traps of the given type and is determined by a
number of β−hydrogen atoms or other atoms for >N−O• fragments. The most frequently used
spin trap is t-nitroso butane (TNB). The irradiation of a reacting mixture during
photochemical generation of radicals is always accompanied by the formation of some
symmetric AR by the following way [59]:


(CH3)3СN=O ⎯⎯→ (CH3)3CNO* → (CH3)3C• + NO (32)

O
(CH3)3C + (CH3)3CN=O (CH3)3C N C(CH3)3
(33)

Usually concentrations of symmetric AR are less than those for basic radicals, but the
superposition of spectra of two radicals frequently complicates the interpretation.
Alkyl hydroperoxides react with nitroso compounds giving AR [60]:

OOR'

R'OOH + (CH3)3CNO (CH3)3C-N-OH

O O
TNB
R' + (CH3)3C-N-OH R'-O-N-C(CH3)3 (II.34)
O

Nitroso compounds also easily react with certain anions [61] with the formation of
oxyanions, which are oxidized into AR either by nitroso compound itself or traces of O2:

O O
TNB
R + (CH3)3CNO (CH3)3C-N-R (CH3)3C-N-R (II.35)

Therefore the interpretation of data of the radical accepting by TNB in the presence of
oxidizers and in electron donor media is complicated. The 2-methyl-2-nitosobutanone-3 is
136 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

close to TNB in chemical properties and ESR spectra of radical adducts [59], and sometimes
it is more effective acceptor of radicals.
The defect of TNB connected with its sensitivity to light, oxidizers, strong acids and
some anions limits to the application of this trap. Some advantages in comparison with
aliphatic have aromatic nitroso compounds. The majority aromatic nitroso compounds
excepting 2,4,6-tri-t-butylnitroso benzene (BNB) are dimers which dissociate in solutions The
monomer form of aromatic nitroso compounds accepts radicals. The character of ESR spectra
of aromatic nitroso compounds is determined by a number of the substituents in aromatic
rings and β−hydrogen atoms in a radical fragment. They form enough stable adducts with
many short-living radicals, but do not form stable adducts with RO•, RO2•, •OH radicals and
halogen atoms. The enough detailed consideration of the capability of aromatic nitroso
compounds for detection and identification of metalorganic radicals containing Co, Mo, Fe,
V, Mn, Re, Cr, Os is given in the works [62, 63].
Among aromatic nitoso compounds, nitroso benzene (NB) has found the greatest
application. It is more accessible and not sensitive to visible and near UV-light. Only the light
with λ < 310 nm gives rise to the formation of diphenylaminoxyl. Spin adducts with NB are
usually stable at room temperature. The basic imperfection of NB is the complexity of the
analysis of ESR spectra because of additional lines from protons of phenyl groups. Other
essential restriction for the NB applications is the impossibility of its use in solutions
containing alkali, alcoholates and other electron donors. In similar conditions, the stable
radical anions of NB C6H5NO• − are formed [64].
As a spin trap, nitroso durene (ND) is also used. The main advantage of ND is the
simplicity of ESR spectra of radical adducts, the insensitivity to UV-light and high stability of
the adducts at room temperature. The drawback of ND is the bad solubility in many solvents,
the broadening of lines because of interaction with protons of methyl groups and instability of
adducts with RO• radicals [65]. The certain advantages for studying reactions of spin trapping
are inherent to BNB [66]. It is well dissolved in many solvents and exists in the active
monomer form in solid state and solutions. BNB is the bifunctional trap, and radicals join
both to nitrogen and oxygen atoms. The properties of substituted NB such as 2,4,6,
trimethylcarbonylnitrosobenzene, 2,4,6-trimethoxynitrosobenzene and
pentafluoronitrosobenzene have been investigated [65].
Nitrons are also widely used as spin traps. Nitrons have the much greater thermal and
photochemical stability than nitroso compounds. They are monomers and have activity to free
radicals even in a solid state. As a result of accepting of radicals by reaction (31), ARs are
formed. As a rule, the fragment R3 of these AR represents tertiary alkyl group, and the
fragment R1 is the substituted aromatic group. Inconvenience of using of nitrons as spin traps
is the absence in most cases HFS from atoms of the attached radical in ESR spectra of formed
AR.The information on a structure of the captured radical, as well as in a case of nitroso
β
compounds, can be obtained from aN and a H constants. Generally three most accessible
nitrons are applied as spin traps:

C6H5-CH=N-C6H5
diphenylnitron (DPN) [67, 68]
Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 137

CH2=N-C(CH3)3
methylene-t-butylnitron (MN) [69-71]

C6H5-CH=N-C(CH3)3
С-phenyl-N-t-butylnitron (PBN) [72, 73]

Obtained from DPN, ARs are rather reactive and transform into diamagnetic molecules
by recombining with radicals. This disadvantage has caused a small applicability of DPN as a
spin trap. MN [69-71] is more active trap than other nitrons. The carbon atom, which is
attacked by short-living radicals is less shielded. MN forms considerably more stable adducts
with •ОН, НО2• and (СН3)3СОО• radicals [74]. Essential advantage of MN in comparison
with TNB is that this nitron catches aminoradicals, whereas nitroso compounds do not add
them [69]. Alongside with TNB, PBN is the basic trap widely used in studies of short-living
free radicals [75]:

O R O

C6H5-CH=N-C(CH3)3 + R C6H5-CH-N-C(CH3)3 (II.36)

PBN is stable to the action of light, O2 water and well dissolved in many solvents.
Radical adducts with PBN are stable at room temperature and in a number of cases can be
isolated in the pure state.. PBN catches more extensive variety of radicals than TNB, for
example, F•, Cl•.
The insertion of two functional groups OH and >C=N→O into structure of one molecule
has been carried out in the work [76]. As a spin trap, С-(3,5-di-t-butyl-4-hydroxyphenyl)N-t-
butylnitron was used. This bifunctionsl trap forms phenoxyl radicals in reaction with radicals
having pronounced oxidizing properties (RO•, R•C=O, PhCOO•) and ketones in triplet-exited
states. The radicals with unpaired electron on carbon atoms add to β− carbon of the nitron
forming AR:

R O
O R
HO CH-N-C(CH3)3 (II.37)
HO CH=N-C(CH3)3

O
R
O CH=N-C(CH3)3 (II.38)
138 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

The study of oxidation by the spin trap method is associated with identification of
adducts of nitrons with RO2• radicals [77, 78]. Adducts of RO2• radicals with PBN are
unstable and even at 263 K rapidly decay. During decomposition of these adducts, the
products of interaction with RO• radicals are formed:

O O
O
C6H5-CH-N-C(CH3)3 C6H5-CH-N-C(CH3)3 + (R2)R1 CH (II.39)
O R1
OR2(R1)
O CH
R2

Thus, though the RO2• radicals cannot be fixed in concrete conditions of oxidation of
hydrocarbons, the formation of PBN adducts with RO• radicals is the qualitative indication on
occurrence of peroxide radicals in the reacting system.
The composition of products of interaction of aliphatic nitrons with OH• radicals can be very
various. For nitrons containing aromatic groups, for example PBN, three paths of the
reactions are possible inclusive of the hydrogen atom abstraction from alkyl groups, addition
of OH• to aromatic rings and nitron groups with formation of AR [79, 80]:

C6H5-CH=N-C(CH3)2CH2 (II.40)
O O

C6H5-CH=N-C(CH3)3 + OH CH=N-C(CH3)3 (II.41)


HO
O

C-N-C(CH3)3 (II.42)
OH

Nitrons can accept various atoms and radicals. The convincing evidence of hydrogen
atom addition to nitrons has been obtained by the example of PBN [19]. The attachment of H
atoms to aromatic rings gives cyclohexadienyl radicals [81]. Adducts of PBN with H atoms
are rather unstable and are not observed in non-polar solvents. The spin trapping of
fluorinated radicals and Cl atoms by PBN takes place [82, 83].
A series of 3-aryl-2H-benzo[1,4]oxazin-4-oxides

N X
O
Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 139

were prepared, and their ability to trap free radicals was investigated by ESR spectroscopy
[84]. In organic solvents, these compounds were able to efficiently scavenge all carbon- and
oxygen-centered radicals tested, giving very persistent aminoxyls, except with superoxide
anion whose spin adducts were unstable. The main feature of these nitrones as spin traps lies
in the possibility to recognize the initial radical trapped. In fact, besides a g-factor and
aminoxyl nitrogen coupling constant dependent on the species trapped, the ESR spectra also
show different patterns due to hyperfine splitting characteristic of the radical scavenged. This
last important feature was investigated by means of density functional theory calculations.
The enough overall summary of the ESR characteristics of various AR produced in nitrons is
given in the works [19, 72]. In these studies the various aspects and features of application of
the spin trap method in studying of the mechanism of chemical reactions are considered.

6. SYNTHESIS OF POLYMERS CONTAINING STABLE


AMINOXYL RADICALS
The chemical properties of polymeric AR are determined by the >N O• fragment and
basically similar to those of low-molecular AR. They are characterized by the high chemical
stability in a wide range of pH in water solutions, thermal stability up to 180o and oxidative
stabilities. The life time of such radicals makes up many years and practically is not limited in
inert media. The interaction of AR having functional groups with those of macromolecules is
the most widespread procedure of obtaining aminoxyl-containing polymers [18]. The AR
preparation methods are given in the monographs [2, 3]. As the example of such reactions one
can consider the graft of AR III (R=COOH or COOCH3) to polyethylene glycol having end
hydroxyl groups [85]. The first stage of the reaction includes the treatment of radicals III by
sulfochloride to obtain AR containing chloranhydride groups. Then the attachment of
synthesized aminoxyl to the polymer is carried out by the following scheme:

O
Cl C
pyridine O
CH2OCH2CH2OH + CH2OCH2CH2O C (II.43)
N
O
N
O

Polyvinylacetate with AR was obtained by the reaction with radicals III (R=COOH)
[86]. Macromolecules of modified polyvinylacetate contains from 1 to 10 AR.
Polyacrylates, polymethylacrylates and polymethylmetacrylate with AR are synthesized
by reaction of copolymer containing chloranhydride groups with a radicals IV (R=OH) [87]:

CH3 CH3 CH3 CH3


IV(R=OH)
CH2 C CH2 C CH2 C CH2 C
pyridine
COCl COOCH3 C O N O (II.44)
COOCH3
O
140 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

Synthesis of spin-labelled polystyrene has been carried out by the reaction of the polymer
containing chlormethyl groups with radicals IV (R=OH) [88, 89]:

IV (R=OH) H
H2C CH CH2 H2C C CH2
pyridine (II.45)

CH2Cl CH2O N O

AR (VIII) was introduced to macromolecules of polyvinylacetate by the partial


saponification [90]:

Cl N
NH N O
CH2-CH-CH2 CH +
N N
OCOCH3 OCOCH3
Cl (VIII)

CH2-CH-CH2 CH (II.46)
N
OCOCH3 O NH N O
N N

Cl

Polyethylene films containing carbonylhydrazide groups were treated by radicals IV (R =


−N=C=S) [91]. The spin-labelled polyethylene was obtained by the following reaction:

ethanol
H2C CH + S=C=N N O
35o
C O
NHNH2

H2C CH
C O
NHNH C NH N O (II.47)
S
Synthesis of spin-labelled copolymer of styrene with maleic anhydride has been carried
out on heating a solution of the copolymer and radical IV (R=NH2) in anhydrous THF [92]:
Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 141

+ THF
CH-C-CH2 CH NH2 N O

O O
O

CH-C-CH2 CH CH CH CH2 CH II.48)


COOH C O
O O
O NH

N
O

However, the most of synthetic polymers have no suitable reactive groups. AR can be
inserted into such polymers only via copolymerization or chemical modification of
macromolecules. Using reactions of AR without participation of unpaired electrons, polymers
were synthesized from monomers containing one or two free-radical fragments [93]. The
paramagnetism of such polymers amounts to 1.5-2.1 1021 spin⋅g−1. AR (IX) on the basis of
radical-containing diacetylenes

HO C C C C OH

N N
O O
(IX)

undergoes polymerization converting into polymeric polycrystals under the action of light or
on heating (80-100o ) [56, 94]. A polyacethylene chain with stable radical substituents (R•) is
R R R R

formally similar to the hypothetical polyene polyraducal which


serves a theoretical model for a “ferromagnetic” macroradicals. The solid-state
polymerization of such diacetylenes was thought to be a perspective or at least feasible route
to conjugated polyradicals. Indeed, several features of the polymerization approach seem to
be advantageous. Owing to the monomer lattice control, exact stereo specificity of the
polyradical may be guaranteed. Crystalline nature of the polymerization products should
facilitate characterization of the latter. Since no catalysts are used for the polymerization,
probability of the reaction products contamination is reduced. Based on GPH analysis, the
polymerization products are oligomers with polymerization degree n = 4 – 10 [94]. A broad
structureless absorption band in their optical spectra, peaking at λmax = 380 nm, is indicative
for rather short conjugation length. After the thermal treatment, X – ray analysis has revealed
almost total amorphisation of the polymerization products of some monomers similar to IX.
142 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

ESR showed also that magnetic behavior of the products was typical for a spin labelled
oligomer chain (triplet signal and a typical τ − correlation times vs molecular mass
dependence in solution) without any signs of ferromagnetic ordering of spins. Thus,
ferromagnetic properties of polyconjugated polyradicals produced from aminoxyl-substituted
diacetylenes seem to be illusive.
AR (X)

C2H5OOC COOC2H5

N
O (X)

has been incorporated into the main chain of "living" polystyrene obtained by anionic
polymerization in the presence of butyl lithium [95]:

Bu-(CH2-CH)n-CH2-CH Li
X
BuLi
C6H6

O O
H2C CH C C CH CH2 (II.49)

N
O

By the same method of “living“ radical polymerization, a series block copolymers of


poly(ethylene oxide-styrene) with narrow polydispersity were synthesized by the following
two step approach [96]. At first, “living” anionic polymerization of ethylene oxide with
sodium-4-oxy-2,2,6,6-tetramethyl-1-piperidinoxyl as initiator yields polyethylene oxide with
AR at chain end:

60o
O N ONa + n O N O ( CH2CH2O )n H (II.50)
O

Then a stable free radical polymerization of styrene gives a block copolymer:


Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 143

AIBN, 120o
O N O ( CH2CH2O )n H

C ( CH2CH )m O N O ( CH2CH2O )n H (II.51)


CN

By the polycondensation of 1,10-decandiol with chloranhydride of terephthalic acid in


the presence of AR (XI),

HO (H2C)4 (CH2)4 OH
N
O (XI)

the polymer of the following structure has been obtained [97]:

(H2C)10 O C C O (H2C)4 (CH2)4


N
O O
O

However, not always polymerization or copolymerization of monomers with AR occurs


without participation of unpaired electrons. For example, attempts to synthesize polymers
from monomers containing radicals IV [R = −O (C=O) C (CH3) =CH2] have not been
successful, because radicals of growing chains actively react with aminoxyl groups [98, 99].
Therefore, for preparation of spin-labelled polymers one can use monomers containing
appropriate amines with the subsequent oxidizing the polymer. In such a manner the spin-
labelled polymer has been synthesized [100] from the monomers:

CH3
H 2C C C X NH where X = NH, O
O

The oxidation of polyacrylamides having diphenylamine groups by lead dioxide gives


polymer with AR [101]:
144 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

CR-CH2
O C
HN N
O

where R = H, CH3.
Aminoxyl-containing polyvinylppyrrolidone and polyvinylcaprolactame have been
obtained by the copolymerization of corresponding monomers and amines

CH=CH2 CH=CH2
HO
H2C OH

,
N
H N
H

with the further oxidation of amine groups in the copolymers [102]:

OH
N N O N H2C OH N O
O O
,
N
O N
O

O OH O O
N N O N H2C OH N
,
N
O N
O

The graft of AR to polyethylene has been performed in the reaction of copolymers


containing carbonyl groups (0.5 %) with 2-amine-2-methyl propanol [103]:
Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 145

CH3
[O]
CH2-C-CH2 + H2N CH3 CH2-C-CH2
OH NH -H2O
CH2
O CH3
HO HO H2C C
CH3

CH2-C-CH2 (II.50)
O N O

The polymer inclusive of nitron and aminoxyl groups has been obtained by the reaction
of polystyrene containing formaldehyde groups in p-position of phenyl rings with 2,3-
bis(hydroxylamino)-2,3-dimethylbutan [104]:

CH3
CH3OH PbO2
CH-CH2 + H3C C NHOH CH-CH2
H3C C NHOH
CH3

CHO HON NOH

(II.51)
CH-CH2

O N N O

The polymers with grafted AR can be also produced in reactions of macroradicals formed
by mechanodegradation, photolysis or radiolysis in the presence of aminoxyl biradicals. By
this method, for example, AR (XII)

COOR
,R= N O
COOR
(XII)

has been introduced into macromolecules of polyolefines in the course of their


mechanodestruction [105]. Biradicals are grafted upon macromolecules by one of two radical
fragments. By ESR method [106], the nature of polymeric AR formed during photooxidation
146 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

of films of isotactic polypropylene containing bis-(2,2,6,6-tetramethyl-4-piperidyne)-


sebacinate has been studied:

O O
OC(CH2)8CO

N N
H H

Only one of amine groups of this stabilizer is oxidized to AR during photolysis. The part
of monoradicals formed recombines with alkyl macroradicals of polypropylene, and then the
second amine group is converted into AR.
To obtain spin-labelled polymers, various nitroso compounds are widely used. The wide
set of reactions represents a variety of methods of the polymer modification by these
compounds. In the most cases, AR can be generated by reactions of nitroso compounds with
metalorganic reactants:

R-NO + R1-M R N O M
+ hydrolysis R N (II.52)
O
oxidation
R1 R1

When ARs are introduced into phenyl rings of polystyrene, the first stage is mercuration
of the polymer. Under the action of nitrosyl chloride, the mercurated polystyrene is converted
into that containing nitroso groups. The synthesis of modified polystyrene includes also
stages of the polymer treatment by phenylmagnesium bromide and silver oxide [107]:

NOCl C6H5MgBr
H2C CH H2C CH
THF

HgOAc N=O
AgO
H2C CH H2C CH (II.53)

NOH N O
Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 147

The spin-labelled polystyrene contains one AR per 1000 monomer units. If t-


butylmagnesium chloride is used instead of phenylmagnesium bromide, the polymer with t-
butylaminoxyl groups can be obtained [108]:

HC CH2

(H3C)3C N O

It should be noted that the synthesis via the scheme (53) has a number of drawbacks. The
mercurated polystyrene is in cline to cross-linking, and the polymer containing nitroso groups
is the unstable compound. The more suitable method of synthesis of the spin-labelled
polystyrene is given below [109]:

J2,HJO3 C4H9Li
H2C CH o
H2C CH
90 ,C6H5NO2 C6H6

TNB Ag2O
H2C CH H2C CH H2C CH (II.54)
CH3OH

+
Li (H3C)3C N O Li (H3C)3C N O

This synthesis is not accompanied by the destruction or cross-linking of macromolecules.


TNB can be used use as a trap for carbanions which are converted into AR by the
following reaction:

hydrolysis
R + (CH3)3CN=O (H3C)3C N R (H3C)3C N R (II.55)
oxidation
O O

The choice of suitable initiating system allows of preparing polystyrene containing AR


on one (n-butyl lithium) or two (naphthalate sodium) ends of macromolecules. By this
method, aminoxyl-containing polymethylstyrene, poly-2-vinylpiridine and copolymer of
styrene with methylstyrene have been obtained [61].
Polymethylmethacrylate with the end t-butylaminoxyl groups was synthesized by the
similar method [110]. However, the anionic polymerization of methylmethacrylate is
accompanied by some side reactions influencing on efficiency of the process especially on a
place of the label attachment. The optimal conditions of the synthesis include THF as the
148 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

solvent, 70o and initiators: n-butyl lithium, sodium naphthalate or 9-lithium fluorine. In these
conditions one can obtained polymethylmethacrylate with the end AR:

CH3 CH3 But


TNB CH3OH
H2C C H2C C N
O
COOCH3 COOCH3

CH3 But CH3 But


oxidation (II.56)
H2C C N H2C C N
OH O
COOCH3 COOCH3

The important reaction of nitroso compounds resulting in spin-labelled polymers is the


radical capture. Polystyrene containing t-butyl aminoxyl was obtained by radical
polymerization in the presence of TNB and t-butylperoxyoxalate [111]:

PhCH=CH2
(CH3)3C O + PhCH=CH2 PhCHCH2CH2OC(CH3)3

(CH3)CNO
PhCHCH2(PhCHCH2)nCH2OC(CH3)3

PhCHCH2(PhCHCH2)nCH2OC(CH3)3 (II.57)
(H3C)3CN O

AR can be introduced into macromolecules also by the reaction of nitroso compounds


with macroradicals generated by mechanodestruction, thermal degradation or photolysis of
polymers. The experimental data and schemes of TNB conversions in reactions with alkyl
macroradicals of polypropylene under shearing forces or photolysis are represented in the
work [53]. During polyethylene grinding in a mixture with NB at 77 K, ARs are appeared on
a surface of particles of the polymer as a result of interactions of the spin trap with
macroradicals [112]. The multiple treatment of cross-linked rubbers containing TNB by
repeated swelling-cooling leads to occurrence of AR as a result of bond scissions with the
formation of macroradicals [113]. The synthesis of polyethylene containing ARs has also
been performed by thermal destruction of the polymer films with BNB additives [114]. The
films with BNB were prepared by combined dissolutions of the polymer and BNB in benzene
with subsequent removal of the solvent.
Spin-labelled rubbers (polybutadiene, polyisoprene, copolymer of isobutylene with
isoprene) were prepared by reactions with 2,6-dichloronitrosobenzene in toluene solutions
[115]. The ESR signal of AR occurs immediately after mixing solutions of rubbers and the
nitroso compound. AR formed are stable for months. The method proposed allows of
obtaining various spin-labelled polymers having > C=C < bonds.
Spin labels were grafted on polyethylene under the γ−radiolysis in the presence of PBN
[116]:
Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 149

+ Ph-CH=N-C(CH3) CH2-CH-CH2 (II.58)


CH2-CH-CH2
CH-Ph
(H3C)C N O
O

The γ−radiolysis at −196o generates alkyl macroadicals ~ CH2-С•Н-CH2~ detected by


the ESR spectrum. The formation of AR was observed at – 70o via the reaction 58.
The peculiar example of spin-labelling is 1,3-dipolar addition of paramagnetic nitrons
(XIV) to double bonds of rubbers [117]:

+ N O
CH=CH CH-CH (II.59)
O
N
(XIV) HC N
O

N
O

Like that spin-labelled methylvinylpyridine, chloroprene and divinyl rubbers have been
obtained.
The method of incorporation of AR into the polymer matrix has been demonstrated by
the example of polymerization of pyrrole [118]. In the presence of protons, radicals IV cause
polymerization of pyrrole to polypyrrole with incorporated reduced (hydroxylamine) form
and oxidized (nitrosonium ion) form of AR in polymer matrix. In electrochemical oxidation
of pyrrole in the presence of AR, a coupled electrochemical-chemical synthesis produces
polypyrrole films with incorporated nitrosonium ions. They can by reduced to AR by partial
film reduction.
The successful use of spin labels in analysis of functionalization of macromolecules has
been demonstrated by the example of the copolymer of styrene – divinylbenzene with
different content of carboxy groups. The radical IV (R1 = OH) and 6-(3-aminophenyl)-2,4-
diphenylverdazil

H2N

N
N N
N

were selected as spin labels, considering their distinct ESR spectra and the presence of
reactive hydroxyl and amino groups. Carboxy-containing copolymer was converted into the
corresponding acid chloride. The resin was then treated with DMA solutions of IV and
verdazyl spin labels in various concentrations. It turned out that concentrations of radicals in
150 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

the spin labelled polymer measured by ESR method and initial taken concentrations of
radicals have a discrepancy of about 1 – 1.5 %. Hence, some polymer, containing functional
groups (–COOH, −NCO, epoxy, etc) capable of binding the functional groups of the spin
labels, can be analyzed rapidly and accurately using the considered procedure.
Thus, the different ways are suitable for the AR formation in polymers including purely
chemical synthetic methods as well as radiation-initiated, photo- and mechano-chemically
initiated processes. The choice of one or other way depends on chemical structure of the
polymer, availability or lack of reactive functional groups in macromolecules.

REFERENCES
[1] Pariiskii G. B., Gaponova I. S., Davydov E. Ya. Russ. Chem. Rev. 2000, 69, 985-999
[2] Tormala P., Lindberg J. J. In Structural Studies of Macromolecules by Spectroscopic
methods; Ivin K. J.; Ed.; Khimiya: Moscow, 1980, 236-253.
[3] Rozantsev E. G., Sholle V. D. Organic Chemistry of Free Radicals. Khimiya: Moscow,
1979.
[4] Nonhebel D. C., Tedder J. M., Walton J. C. Radicals. Cambridge University Press:
Cambridge, 1979.
[5] Rozantsev E. G. Free Nitroxyl Radicals. Plenum: New York, 1970.
[6] Rintoul L., Micallef A. S., Reid D. A., Bottle S. E. Spectrochim. Acta. A. 2006. 63. 398-
402
[7] Carmichael P. J., Govenlock B. G., Johnson C. A. F. J. Chem. Soc. Perkin Trans. 2.
1973, 1853-1856.
[8] Lipey M. M., Potapov V. K., Kostyanovsky R. G. Khim. Vysok. Energiy. 1974. 8, 496-
499.
[9] Buchachenko A. L., Wasserman A. M.. Stable Radicals. Khimiya: Moscow, 1973.
[10] Aurich H. G., Hahn K., Stork K., Weiss W. Tetrahedron. 1977, 33, 969-975.
[11] McCelley R.C., Shimshick E. J., McConnell H. M. Chem. Phys. Lett. 1972, 13, 115-
119.
[12] Goldman S. A., Bruno G. V., Freed J. H. J. Phys. Chem. 1972, 76, 1858-1960.
[13] Bluhm A., Weinstein J. J. Amer. Chem. Soc. 1970. 92, 1444-1446.
[14] Doba T., Noda S., Yoshuda H. Bull. Chem. Soc. Japan. 1979. 52, 21-24.
[15] Forrester A. R., Hepburn S. P. J. Chem. Soc. C. 1970. 1277-1280.
[16] Calder A., Forrester A. R. J. Chem. Soc. C. 1969. 1459-1463.
[17] Mender O. W., Janzen E. J. J. Org. Chem. 1969 34, 4072-4082.
[18] Wasserman A. M., Kowarsky A. L. Spin Labels and Probes in Physical Chemistry of
Polymers. Nauka: Moscow, 1986.
[19] Zubarev V. E., Belewsky V. N., Bugaenko L. T. Russ. Chem. Rev. 1979, 48, 1361-
1392.
[20] Shapiro A. B., Rozantsev E. G.. Izv. AN SSSR ser. chimich. 1966, 1650-1652.
[21] Neiman M. B., Rozantsev E. G., Golubev V. A. Izv. AN SSSR. ser. chimich. 1965, 548-
550.
[22] Berliner L. J. Spin Labeling Theory and Applications. Academic Press: New York,
1974.
Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 151

[23] Buchachenko A. L., Tkacheva O. P. Kinetika i Kataliz. 1966. 7, 777-787.


[24] Summermann W., Deffner U. Tetrahedron. 1975. 31, 593-596.
[25] Golubev V. A., Zjdanov R. I., Rozantsev E. G. Izv. AN SSSR. ser. chimich. 1970, 184-
185.
[26] Zjdanov R. I., Golubev V. A., Rozantsev E. G. Izv. AN SSS. ser. chimich. 1970,186-
187.
[27] Bailey P. S., Keller J. E. J. Org. Chem. 1970, 35, 2782-2785.
[28] Keana J. F. W. Chem. Rev. 1978, 78, 37-63.
[29] Golubev V. A., Zjdanov R. I., Gida V. M., Rozantsev E. G. Izv. AN SSSR. ser. chimich.
1971, 853-855.
[30] Low H., Paterson I., Tedder J. M. J. Chem. Soc. Chem. Commun. 1977, 171-172.
[31] Brandi P., Galli C., Gentili P. J. Org. Chem. 2005, 70, 9521-9528.
[32] Beckwith A. L. J., Polle J. S. J. Am. Chem. Soc. 2002, 124, 9489 -9497.
[33] Murayama K., Yoshioka T. Bull. Chem. Soc. Japan. 1969, 42, 1942-1947.
[34] Nigan S., Asmus K. D., Willson R. L. J. Chem. Soc., Faraday Trans.11976, 2324-2328.
[35] Skuratova S. I., Kozlov Yu. N., Zakatova N. V., Sharpaty V. A. Zjurn. Fizich. Khimii.
1971, 45, 1821-1823.
[36] Karpukhin O. N., Pokholok T. V., Shlyapintokh V. Ya. Vysokomolek. Soed. A. 1971,
13, 22-27.
[37] Khloplyankina M. S., Buchachenko A. L., Neiman M. B., Vasil’eva A. G. Kinetika i
Kataliz. 1965, 6, 394-398.
[38] Ruban L. V., Buchachenko A. L., Neiman M. B., Kohanov Yu. V. Vysokomolek. Soed.
A. 1971, 8, 1642-1646.
[39] Becke A. D. Phys. Rev. 1988, 38, 3098-3100.
[40] Huang H., Henry-Riyad H., Tidwell T. T. J. Am. Chem. Soc. 1999, 121, 3939-3943.
[41] Allen A. D., Cheng B., Fenwick M. H., Givehchi B., Henry-Riyad H., Nikolaev V. A.,
Shikhova E. A., Tahmassebi D., Tidwell T. T., Wang S. J. Org. Chem. 2001, 66, 2611-
2617.
[42] Melnikov M. Ya., Smirnov V. A. Photochemistry of Organic Radicals. Moscow State
University: Moscow, 1994.
[43] Keana J. F. W., Dinerstein R. J., Baitis F. J. Org. Chem. 1971, 36, 209-211.
[44] Keana J. F. W., Baitis F. Tetrahedron Lett. 1968, 365-369.
[45] Bogatyreva A. I., Buchachenko A. L. Russ. Chem. Rev. 1975, 44, 2171-2204.
[46] Maslov S. A., Zaikov G. E. Russ. Chem. Rev. 1977, 46, 1253-1272.
[47] Shilov Yu. B., Batalova R. M., Denisov E. T. Doklady AN SSSR. 1972, 207, 388-389.
[48] Shilov Yu. B., Denisov R. E. T. Vysokomolek. Soed. A. 1974, 16, 2313-2316.
[49] Ilieva T. G., Potapov E. E., Polenov E. A. Kautchuk i Rezina. 1989, 39-40.
[50] Emanuel N. M., Buchachenko A. L. Chemical Physics of Polymer Degradation and
Stabilization. Science Press: Utrecht, NET, 1987.
[51] Malchevsky V. A., Zakrevsky V. A.. Mekhanka Polymerov. 1978, 2, 342-344.
[52] Rogov Yu. N., Deyun F. V., Anisin V. A., Smirnov L. P., Manelis G. B. Doklady AN
SSSR. 1983, 230, 637-642.
[53] Chakraborty K. B., Scott G. Y. H. J. Polym. Sci. Polym. Lett. Ed. 1984, 22, 553- 558.
[54] Chakraborty K. B., Scott G. Y. H. J. Appl. Polym. Sci. 1985, 30, 3267-3284.
[55] ElMalaika S., Scott G. In Degradation and Stabilization of Polyolefins; Allen N. S. L.;
Ed.; Appl. Sci. Publ.: New York, 1983, p. 219.
152 E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

[56] Korshak Yu. V., Ovchinnikov A. A., Shapiro A. B., Medvedeva T. V., Spector V. N.
Exp. Theor. Phys. Lett. 1986, 43, 309-311.
[57] Iwamura M., Inamoto N. Bull. Chem. Soc. Japan. 1967, 40, 703.
[58] Mackor A., Wajer T. A., De Boer T. J., Van Voorat J. D. Tetrahedron Lett. 1967, 2757-
2761.
[59] Forshult S., Laggercrant Z. C., Torselle K. Acta Chem. Scand. 1969, 23, 522-530.
[60] Terabe S., Kanoka R. J. Chem. Soc. Perkin. Trans. 1972, 2, 2163-2172.
[61] Forrester A. R., Hepburn S. P. J. Chem. Soc. C. 1971, 701-703.
[62] Rehorec D., Hennig H. Can. J. Chem. 1982, 60, 1565-1573.
[63] Hadson A., Leppert M. F., Lednor P. W. J. Chem. Soc. Dalton Trans. 1981, 2159-2163.
[64] Russell G., Geels J., Smentowsti F. J. J. Am. Chem. Soc. 1967, 89, 3821-3828.
[65] Terabe S., Kuruma K., Konaka R. J. Chem. Soc. Perkin Trans. 2. 1973, 1252-1258.
[66] Terabe S., Konaka R. J. Am. Chem. Soc. 1971, 93, 4306-4307.
[67] Iwamura M., Inamoto N. Bull. Chem. Soc. Japan. 1970, 43, 756-760.
[68] Bluhm A., Weinstein J. J. Org. Chem. 1972, 37, 1748-1753.
[69] Anderson N., Norman R. J. Chem. Soc.B. 1971, 993-1003.
[70] Janzen E. G., Lopp I. J. Magnet. Resonance. 1972, 7, 107-110.
[71] Baldwin J., Qureshi A. R., Sklarz B. J. Chem. Soc. C. 1969, 1073-1079.
[72] Janzen E. G. Acc. Chem. Res. 1971. 4, 31-40.
[73] Janzen E. G., Blackburn B. J. J. Am. Chem. Soc. 1969. 91. 4481-4490.
[74] Konaka R., Terabe S., Mizuta T., Sakata S. Can. J. Chem. 1982, 60, 1532-1541.
[75] Church D. F. J. Am. Chem. Soc. 1984. 106, 5073-5079.
[76] Paifici J. G., Browning H. L. J. Am. Chem. Soc. 1970, 92, 5231-5233.
[77] Howard J. A., Tait J. C. Can. J. Chem. 1978, 56, 176-178.
[78] Zubarev V. E., Belevsky V. N., Yarkov S. P. Doklady AN SSSR. 1979, 244, 1392-1396.
[79] Greenstock C. L., Wiebe R. H. Can. J. Chem. 1982, 60, 1560-1564.
[80] Neta P., Steenken S., Janzen E. G. J. Phys. Chem. 1980, 84, 532-535.
[81] Pryor W. A., Lin T. H., Stanley J. P. J. Am. Chem. Soc. 1973, 95, 6993-6998.
[82] Janzen E. G., Knauer B., Gerlock J. J. Phys. Chem. 1970, 74, 2037-2038.
[83] Janzen E. G., Knauer B., William S. L. J. Phys. Chem. 1970, 74, 3025-3028.
[84] . Astolfi P., Marini M., Stipa P. J. Org. Chem. 2007, 72, 8677 -8682.
[85] Tormala P., Lattila H., Lindberg J. J. Polymer. 1973, 14, 481-487.
[86] Bluhm F. D., Dikson J. E., Miller W. G. J. Polym. Sci. Polym. Phys. Ed. 1984, 22, 211-
221.
[87] Bullock A. T., Cameron G. G., Krajewski V. J. Phys. Chem. 1976, 80, 1792-1797.
[88] Regan S. L. J. Am. Chem. Soc. 1974, 96, 5275-5276.
[89] Regan S. L. J. Am. Chem. Soc. 1975, 97, 3108-3112.
[90] Aleksandrova T. A., Wasserman A. M., Tager A. A. Vysokomolek. Soed. A. 1977, 19,
137- 141.
[91] Rasmussen J. R., Bergbreiter D. E., Whitesides. G. M. J. Am. Chem. Soc. 1977, 99,
4746- 4756.
[92] Kulikov N. V., Likhtenshtein G. I., Rozantsev E. G., Suskina V. I., Shapiro A. B.
Biofizika. 1972, 17, 42-48.
[93] Rozantsev E. G., Novelko G. Ph. Vysokomolek. Soed. B. 1967, 9, 866.
[94] Lazareva O. L., Motyakin M. V., Shapiro A. B., Shchegolikhin A. N. Synthetic Metals.
1997, 85, 1685-1686.
Properties and Applications of Aminoxyl Radicals in Polymer Chemistry 153

[95] Freidrich C., Noel C., Ramassuel R., Rassat A. Polymer. 1980, 21, 232-235.
[96] Hua F. J., Yang Y. L. Polymer. 2001, 42, 1361-1369.
[97] Sundholm F., M. Wasserman A., Barashkova I. I., Timofeev V. P. Eur. Polym. J. 1984,
20, 733-738.
[98] Griffith O. H., Keana W. J. P., Rottshalffer S., Warlick T. A. J. Am. Chem. Soc. 1967,
89, 5072-5074.
[99] Forrester A. R., Hepburn P.. J. Chem. Soc. C. 1971, 20, 3322-3328.
[100] Kurusaki A. K., Lee K. W., Okawara M. J. Polym. Sci. Polym. Chem. Ed. 1972, 10,
3295- 3297.
[101] [101]. Braun D., Hauge S. Makromol. Chem. B. 1971, 150, 57-62.
[102] [102]. Wasserman A. M., Timofeev V. P., Aleksandrova T. A., Shapiro A. B. Eur.
Polym. J. 1983, 19, 333-339.
[103] Bullok A. T., Cameron G. G., Smith P. N. Eur. Polym. J. 1975, 11, 617-624.
[104] Miura Y., Nakai K., Kinoshita M. Makromol. Chem. B. 1973, 172, 685-687.
[105] Radzig V. A., Shapiro A. B., Rozantsev E. G. Vysokomolek. Soed. B. 1972, 14, 685-
687.
[106] Hodgeman D. K. C. J. Polym. Sci. Polym. Chem. Ed. 1980, 18, 533-539.
[107] Drefahl G., Horhold H. H., Hofmann K. D. J. Prakt. Chem. B. 1968, 37, 137-142.
[108] Bullok A. T., Cameron G. G., Smith P. M. J. Phys. Chem. 1973, 77, 1635-1639.
[109] Bullok A. T., Cameron G. G., P. M. Smith. Polymer. 1972, 13, 89-90.
[110] Bullok A. T., Cameron G. G., Elson J. M. Polymer. 1974, 15, 74-76.
[111] Chalfont G. R., Perkins M. J. J. Am. Chem. Soc. 1968, 90, 7141-7142.
[112] Vivatpanachart S., Nomura H., Miyahara Y. Polymer. 1981, 22, 896-898.
[113] Zander P., Karl-Friedrich A. Plaste und Kautch. 1986, 33, 288-290.
[114] Kitahara T., Shimada S., Kashiwabara H. Polymer. 1980, 21, 1299-1303.
[115] Miclos G., Rockenbauer A., Tüdos P. Macromolecules. 1987, 20, 3083-3086.
[116] Kusumoto N., Sakai T. Polymer. 1979, 20, 1175-1177.
[117] Barashkova I. I., Martin V. V., Anfimov B. N., Volodarsky L. B. Vysokomolek. Soed.
B. 1987, 29, 354-357.
[118] Rapta P., Bartl A., Dunsch L. Synthetic Metals. 1997, 84, 187-188.
[119] Katritzky A. R., Belyakov S.A., Strab S., Cage B., Dalal N. S. Tetrahedron Lett. 1999,
407-410.
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.155-164 ©2010 Nova Science Publishers, Inc.

Chapter 11

SYNTHESIS OF FLEXIBLE MANUFACTURINGS FOR


PHOSPHORIC INDUSTRY WASTE UTILIZATION
BASED ON THE CALS-CONCEPT

A. M. Bessarabov*1, A. V. Kvasyuk1 and G. E. Zaikov 2


1
State Scientific-Research Institute of Chemical Reagents and
High Purity Chemical Substances (IREA), Moscow, Russia
2
Institute of Biochemical Physics Russian Academy of sciences,
Moscow, Russia

ABSTRACT
CALS-projects of sodium phosphite and hypophosphite technologies – products of
phosphoric sludge utilization (the most aggressive waste of phosphoric industry) were
developed. CALS-technology of the flexible two-grocery production was developed
based on the theory of the flexible scheme synthesis

Keywords: flexible manufacturing, CALS-technologies, phosphoric industry, phosphorus


sludge, sodium phosphite, sodium hypophosphite

BACKGROUND
Utilization of phosphorus sludge formed in large amounts in the phosphoric industry was
considered. The corresponding task was formulated as development of a closed-circuit
technological process at which phosphorus sludge will re-enter processing for the maximal
extraction of useful products from its composition [1].

*State Scientific-Research Institute of Chemical Reagents and High Purity Chemical Substances (IREA),
Bogorodsky Val, 3, 107076, Moscow, Russia, E-mail: bessarabov@irea.org.ru
156 A. M. Bessarabov, A. V. Kvasyuk and G. E. Zaikov

AIMS
Aim of the work is synthesis of flexible manufacturing of sodium phosphite and sodium
hypophosphite for phosphoric industry waste utilization based on the CALS-concept.

EXPERIMENTAL PART
At the first stage the nomenclature of the phosphorus-containing compounds entering the
complex phosphorus sludge processing flow sheet (circuit) is examined. In the circuit four
target products are considered: sodium hypophosphite, sodium phosphite and obtained
through its further processing dibasic lead phosphite and phosphorous acid (fig. 1).

Phosphorus slag (sludge)

SODIUM PHOSPHITE SODIUM HYPOPHOSPHITE


Na2HPO3 or Na2HPO3×5H2O Na H2PO2 or (NaH2PO2*H2O)

PHOSPHOROUS ACID DIBASIC LEAD PHOSPHITE


2Pb*PbHPO3*0.5H2O
H3PO3

Figure 1. Scheme of multiassortment (4-product) process for phosphorus sludge utilization

Development of processes for phosphorus sludge recovery was conducted within the
limits of the most modern and perspective system of computer support - CALS-technologies
(Continuous Acquisition and Life cycle Support - continuous information support of life
cycle of a product) [2]. The CALS concept is based on the complex of uniform information
models, standardization of ways of access to the information and its correct interpretation in
accordance with international standards. Thus uniform ways of process control and
interaction of all participants of development are provided. A key idea of CALS concept is
increasing of product life cycle due to increase of efficiency of control of the information on a
product. CALS task is transformation of a product life cycle into highly automated process
through re-structuring of its component business-processes [3].
Within the limits of CALS concept two basic circuits of phosphorus sludge processing
have been considered: with sodium phosphite [4] and sodium hypophosphite [5] as final
products. The yielded circuits were implemented in the CALS-project. In the CALS-project
used is the typical computer structure of initial data for designing which includes the
following subcategories [6]: the general data on technology (01); characteristics of the
Synthesis of Flexible Manufacturings … 157

executed research and experimental work (02); Feasibility study of a recommended


production method (03); a patent card (04); characteristics of feedstock, auxiliary materials
(05); physical and chemical constants and properties of initial, intermediate and a final
products (06); chemical, physicochemical bases and basic production flow sheet (07);
operating technological parameters of production process (08); the mass balance of
production process (09); characteristics of by-products and solid waste (10); the mathematical
description of technological processes and devices (11); data for calculation, designing, a
choice of the basic production equipment (12); recommendations for process automation (13);
the analytical control of production process (14); methods and technological parameters of
purification from chemical and industrial pollutants (15); safety data sheet including fire and
explosion hazard data, fire fighting procedures, health hazard data, regulatory information
(16); the list of reports and recommended literature on considered technology (17).
On the basis of the offered typical structure of initial data for designing, the CALS-
project for sodium phosphite production technology has been developed. Shown in Fig. 2
block diagram includes a preparatory stage and 4 basic production stages: phosphorus sludge
decomposition in the reactor, filtering of a mineral part, correction of solution density,
neutralization of excess of alkali in solution:

Figure 2. An element of the CALS-project «Flow diagram of sodium phosphite production».

1. The Preparatory stage. The phosphorus sludge is classified in a grinder to particle


size optimal for interaction with sodium alkali (NaOH) and then a solution is
prepared. In parallel alkali solution is prepared through diluting alkali liquor to
concentration NaOH = 31 % using water as solvent. All stages of the corresponding
158 A. M. Bessarabov, A. V. Kvasyuk and G. E. Zaikov

technological block with characteristics of the equipment as well as additional


information are put in the CALS-project and are used by both developers of
technology, and engineering personnel (analysts, etc.).
2. Decomposition of phosphorus sludge in a reactor. In the reactor (1) NaOH solution is
fed and simultaneously phosphorus sludge is charged. The reaction is conducted at a
temperature of 100oC. Upon interaction of phosphorus sludge with sodium alkali
from the reactor the phosphine-hydrogen mix leaves. The obtained product solution
is directed to the next stage (2). The corresponding technological block is included in
the CALS-project with all necessary characteristics of the used equipment.
3. Filtering of a mineral part. The solution directed from reactor (1) to vessel (2) is
filtered. A deposit remaining on the filter is a mineral part of phosphorus sludge and
is used as a mineral fertilizer. The solution passed through filters (2) enters the next
stage (3). Drawings of the filter, input and output parameters and other major
characteristics are included in the CALS-project.
4. Correction of sodium phosphite solution density. The obtained sodium phosphite
solution is diluted with water to a necessary concentration. The ratio of components,
temperature, characteristics of the equipment are included in the CALS-project.
5. Neutralization of excess of alkali. After a stage (3) sodium phosphite enters a stage
(4) where neutralization of excess of sodium alkali (NaOH) with phosphorous acid
(H3PO3) solution is carried out.

Finally sodium phosphite goes to the packing stage. Types of packing and its
characteristics are included in the CALS-project. The database of the CALS-project also
contains the basic documents on sodium phosphite production: certificates, process
regulations, characteristics of final products, performance characteristics of the used
equipment, etc.
Similarly to production of sodium phosphate, the typical structure of the CALS-project
has been developed for production of sodium hypophosphite. The corresponding developed
block diagram shown in Fig. 3 includes a preparatory stage and 9 basic production stages.
The scheme includes the following blocks:

1. The Preparatory stage. Phosphorus sludge with content of phosphorus 30-50%, in a


liquid state, heated to the temperature 700С, is pumped to storage tanks (receivers).
For prevention of phosphorus sludge stratification in storage tanks the latter are fitted
with agitators. The dosage of phosphorus sludge from the tanks is afforded by
forcing with water.
2. Preparation of calcium hydroxide suspension (5) and sodium hydroxide (4) is
conducted in two parallel tanks - mixers of suspension (40 м3 volume each) heated
with external pipe coil to the temperature 500С. Sodium hydroxide with impeller
pump is pumped from intermediate storehouse to receiver tank. When the required
amount of sodium hydroxide is fed, the agitator is started and charging of calcium
oxide hydrate (slaked lime) begins. The dosage of 12 tones (one batch) takes one
hour. Then at constant stirring 12 м3 of water is added and this mix within 8 hours is
agitated in the receiver tank (6). Preparation of suspension proceeds with intense
reaction and foaming. After eight-hour stirring the mix is considered ready for
application.
Synthesis of Flexible Manufacturings … 159

3. Decomposition of phosphorus sludge in a reactor (1). The phosphorus sludge is


loaded into reactor from position (3) along with the obtained solution from the mixer
(6). After some time to reactor (1) the solution of isopropanol (7) is fed that is
necessary for fuller extraction of phosphorus from phosphorus sludge. The reaction is
conducted at a temperature of 85-90oC. Upon interaction of phosphorus sludge with
sodium alkali from the reactor the phosphine-hydrogen mix leaves. The obtained
product solution is directed to the next stage (2).
4. Further phosphorus sludge decomposition in an additional reactor (2): from the rector
(1) the obtained mix is directed to rector (2) thus mixing with a mother solution after
sodium hypophosphite centrifuging. After the end of reaction, the solution from an
additional reactor (2) enters vacuum filters (11).
5. Filtering in drum-type vacuum filters. The obtained solution from a reactor (2) is
filtered off in drum-type vacuum filters (11). A deposit formed at filtering is
collected and used as fertilizer in the agrarian industry. The solution passed through
vacuum filters (11) enters the neutralizer (12).
6. Neutralization of excess of sodium alkali. After passage the drum-type vacuum filters
a solution consisting from NaH2PO2 (8 %), Na2HPO3 (9 %) and СaHPO3 (25 %)
enters the neutralizer (12). Neutralization of excess of sodium alkali is afforded by
dilution with hypophosphorous acid which is stored in vessel (15).
7. Preparation of a hypophosphorous acid (H3PO2). The obtained at stage 5 product
solution (calcium hypophosphite) with concentration of 12% is mixed with oxalic
acid. The obtained solution is filtered (14) and the formed hypophosphorous acid is
stored in vessel (15) and is used as required in the neutralization stage (12).
8. Concentrating of sodium hypophosphite. Sodium hypophosphite is concentrated (16)
by evaporation for the further fine filtration which is carried out in vessel (17). The
formed sodium alkali and sodium hypophosphite are directed to recycling.
9. Crystallization of sodium hypophosphite (17) and centrifuging of suspension (18).
After crystallization (17) suspension goes on filtration to a centrifuge. The formed
mother solution goes to recycling through an additional reactor (2). After
centrifuging (18) sodium hypophosphite is dried (19) for removal of excessive
moisture from the final product.

After the production cycle is finished, the product goes for packing (20). The operation
mode and constructional characteristics for each stage are included in the corresponding
sections of the CALS-project. The CALS-project also contains marketing analysis results. It
is shown, that the considered products are in great demand. Sodium phosphite is one of the
scarcest salts of phosphorus. It is widely used: in electroplating, as a reagent for synthesis of
dibasic lead phosphite - the best stabilizer of PVC-compositions and also as a reducer in
inorganic syntheses [4]. Sodium hypophosphite is used as a reducer at depositing nickel,
cobalt and tin coatings on metals and plastic; as antioxidant preventing discoloration of alkyd
resins upon their preparation etc. [5].
160 A. M. Bessarabov, A. V. Kvasyuk and G. E. Zaikov

Figure 3. An element of the CALS-project «Flow diagram of sodium hypophosphite production»).

RESULTS AND DISCUSSION


As the considered processes have many related attributes, we were faced with a task of
their association in uniform production unit for sodium phosphite and sodium hypophosphite.
For optimum development of two-product production, the developed by our group theory of
synthesis of flexible multiassortment chemical engineering systems (Fig. 4) is used [7, 8].

Workshop Organizational IV
Joint flexibility of 2-nd step level
Department Organizational IIII
Multi-assortment flexibility of 1-st step level
CTS II
Structural flexibility
level
Nomenclature
Technological I
p. h.p.А1
flexibility level
p.f.a. h.pА2
c.p. h.p.А3

Figure 4. Hierarchical structure of synthesis of flexible chemical engineering systems.


Synthesis of Flexible Manufacturings … 161

For similar multiassortment schemes four levels of system analysis are considered:
nomenclature, productional-technological, organizational-technological and organizational-
productional.
Attribute of the top (4-th) technological level is a separate shop as the complex cybernetic
system. Associated problems: stabilization of material and information streams between
aggregated sections; distribution of raw material, power and manpower resources. Attribute
of the 3-rd - organizational level is the aggregated section. Associated problems: optimization
of equipment arrangement and minimization of a production cycle.
The bottom (1-st) level is the nomenclature level. Its characteristic attributes: a single
product or one technological stage. The primary goals: expansion of a set of available grades
for this one product or variation of available capacity of a technological stage. Functioning of
the bottom (1-st) level is provided by technological flexibility which is determined by
possibility of carrying out several technological tasks using the existing equipment due to
flexible scheme adaptable for production of a given product (under the nomenclature) either
with insignificant expenses for readjustment of the equipment (washing, fitting of pipelines
etc).
The greatest interest for us represents the second technological level. Its characteristic
attribute – multiassortment technology. Associated problems: optimum use of intermediate
products and the common initial reagents; using of elements of flexibility with the purpose of
assortment expansion; variation of capacity of all technological process.
This approach we applied to development of the block diagram of flexible two-product
scheme of sodium hypophosphite and sodium phosphite synthesis. Developed production is
the basic unit for a full complex of phosphorus sludge processing (Fig. 1). Incorporated into
this complex individual production processes for dibasic lead phosphite and phosphorous acid
employ sodium phosphite obtained using flexible scheme as raw material.
To substantiate feasibility of uniting two processes in one flexible scheme it is necessary
to carry out the analysis of existing individual production processes with the purpose of
specifying groups of technologies which are suitable for organizing by a flexible principle.
The first stage of this analysis is decomposition of the considered product assortment using
hierarchical approach based on two basic attributes: technological and chemical similarity.
Each of the pointed attributes has the gradation levels. So, technological similarity is
subdivided into similarity of raw material preparation methods (dissolution, filtration,
crushing etc.), production methods (type of transformation of raw material to a main product,
uniformity of technological operations and the used equipment), and packing methods.
Chemical similarity is determined, first of all, by belonging of substances to the same class
(acid, base, salt, ether etc.) inside which class sublevels are isolated on the basis of physical
and chemical properties of substances. For example, salts are classified according to the
character of the anion (acid residue) - nitrates, sulphates, phosphates etc.
The analyzed production processes for sodium hypophosphite and sodium phosphite meet
both attributes of the theory of flexible chemical engineering systems as possessing
technological and chemical similarity. This allowed us to carry out synthesis of the flexible
two-product scheme (Fig. 5).
162 A. M. Bessarabov, A. V. Kvasyuk and G. E. Zaikov

Figure 5. The flexible scheme of sodium hypophosphite and sodium phosphite production.

The developed optimal scheme includes 23 blocks: 9 combined blocks containing


operations used in both production processes for sodium phosphite and sodium
Synthesis of Flexible Manufacturings … 163

hypophosphite (solid line); 3 blocks applied only to production of sodium phosphite (dot
line); 11 blocks concerning only production of sodium hypophosphite (dashed line).

Figure 6. An element of the CALS-project «Initial data for designing» (The flexible scheme of sodium
hypophosphite and sodium phosphite of production).

For transferring from one product to another, in the scheme 2 flexible switching units are
included: Flexible Unit of Switching-1 (FUS-1) and Flexible Unit of Switching-2 (FUS-2).
The unit FUS-1 is responsible for switching of streams: either directing sodium alkali from
the position (4) directly to reactor (1) in production of sodium phosphite, or to position (6) for
mixing with lime hydrate in the production of sodium hypophosphite. After the reactor (1) in
case of sodium phosphite synthesis in the unit FUS-2 switching to position (10) is suggested
for filtering of the obtained reaction mixture, or switching to position (2) for phosphorus
sludge further decomposition and mixing with mother liquor in case of sodium hypophosphite
synthesis. Flexible switching units allow producing sodium hypophosphite and sodium
phosphite at minimal controlling acts.
The developed flexible scheme (Fig. 5) is included in the CALS-project (Fig. 6) with all
operation characteristics, drawings of the used equipment etc. Each element of equipment
included in CALS information system has one of three identification attributes: the unit used
only for production of sodium phosphite; the unit used for production of sodium
hypophosphite; unit which will be probably used for production of sodium hypophosphite and
sodium phosphite. In the pilot CALS-project drawings of all apparatuses included into the
block diagram (Fig.6) are given. If necessary it is possible to consider separately the drawing
or an element of interest in a subsection №12 «Data for calculation, designing and industrial
164 A. M. Bessarabov, A. V. Kvasyuk and G. E. Zaikov

application». For example, in the subsection №12.01 (Reactor) there are drawings of a
reactor, the maintenance instruction, certificates of conformity etc.
Fig. 6.
Development of the design documentation was carried out using the specialized software
for the computerized designing «AutoCAD». For convenience of data storage and searching
time reduction of, some big drawings and block diagrams have been transformed in PDF-files
(Fig. 6). The same was used for storage of large text documents prepared in Word.

CONCLUSIONS
The modern level of innovative production development is intimately connected with
CALS-technologies, that is with use of uniform information space at all stages of the product
life cycle - from designing and utilization to disposal (recovery). Introduction of information
CALS-technologies for designing flexible production of sodium phosphite and sodium
hypophosphite allows to obtain both salts not only with high characteristics, but also to
provide full post-sale support including documentation in the electronic form.

REFERENCES
[1] A.Bessarabov, L.Puigjaner, E.Koltsova E., T.Ogorodnikova: The system analysis of
multiassortmental manufacturing of phosphorus-containing products based on CALS-
technologies. 6th European Congress of Chemical Engineering, ECCE-6, Copenhagen,
Bella Center, 1, 445-446, (2007).
[2] A.N.Davydov, V.V.Barabanov, E.V.Sudov: CALS Technology: Future Developments
and Directions, Stand. Kach., 7, 12-18, (2002).
[3] A.M.Bessarabov, A.N.Ponomarenko, M.Ya.Ivanov: CALS Information Technologies
(ISO-10 303 STEP) in Development of Plasmochemical Processes for Synthesis of
Ultrapure Ultradispersed Oxides. Russian Journal of Applied Chemistry, 80(1), 13-18,
(2007).
[4] A.J.Strugatskaya, E.M.Koltsova: Synthesis of sodium phosphite from phosphorus
sludge at presence of oxygen. Jurnal Prikladnoi Khimii, 68(7), 1602-1604, (1995)
[5] E.M.Morgunova, T.D.Averbuch: Studying of process of synthesis of sodium
hypophosphite. Jurnal Prikladnoi Khimii, 40(2), 274-284, (1967).
[6] A.M.Bessarabov, A.N.Afanas’ev: CALS Technology in Devising Promising Chemical
Processes, Khim. Tekhnol., 3, 26-30, (2002).
[7] A.M.Bessarabov: Cybernetic Approaches in the Technology of Chemical Reagents and
High-Purity Substances, Khim. Prom-st., 10, 666-671, (1996).
[8] A.M.Bessarabov, A.V.Avseev, E.M.Koltsova, L.Puigjaner: Modernization of multi-
assortment manufacturing of ultra pure materials, 4th European Congress of Chemical
Engineering, ECCE-4, Granada, Spain, 10(9), 9-11, (2003).
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.165-189 ©2010 Nova Science Publishers, Inc.

Chapter 12

PRACTICAL HINTS ON THE APPLICATION OF


NANOSILVERS IN ANTIBACTERIAL
COATING OF TEXTILES

S. Dadvara, A. Oroume a and A. K. Haghi*,b


a
Department of Textile Engineering, Isfahan University of Technology, Isfahan, Iran
b
Department of Textile Engineering, Faculty of Engineering,
University of Guilan, Rasht, Iran

ABSTRACT1
Nanotechnology is defined as the contour and manufacture of materials, devices and
systems with control at nanometer dimensions and is considered as a concept in which
everything in the world is considered from the viewpoint of atomic or molecular building
blocks, is already influencing a very broad range of human technological activity. When
a bulk material is reduced to small size particles with one or more dimension within the
nanometer range, the material will show properties that are drastically different from
those of the bulk material. Recently, nanosized organic and inorganic particles have
drawn increased attention in medical textile applications due to their amenability to
biological functionalization. The nanoscience and nanotechnology make excellent
developments in textile science as well as others sciences. The development of new
clothing products based on the immobilization of nanoparticles on textile fibers has
recently received a growing interest from both academic and industrial sectors. These
products impart unrivaled properties, especially antibacterial activity, to the treated
fabrics. In the last few decades there has been growing interest in reducing the
availability of commercial textile containing chemical antibacterial agents due to the
environmental pollution, poisonous nature, and irritant property; so the new types of safe
and commodious biocidal materials need to be replaced with these chemical agents. A
wide range of nano-structures such as nanosilvers can be immobilized on fibers, which
brings antibacterial properties to the final clothing product. This review introduces the
application of nanosilvers in antibacterial coating of textiles in details up to 2008.

* Corresponding author. Tel.: +98 911 1318290. E-mail address: Haghi@Canada.com (A. K. Haghi).
166 S. Dadvar, A. Oroume and A. K. Haghi

Keywords: Nanosilvers, Synthesizing methods, Microorganism, Antibacterial coating,


Textiles.

1. INTRODUCTION
Norio Taniguchi was the first person who used the term nanotechnology in 1974. This
term is defined as the contour and manufacture of materials, devices and systems at the
molecular and atomic levels to attain unique properties which can be suitably manipulated for
the desired applications. In fact, nanotechnology is considered as a concept in which
everything in the world is already influencing a very broad range of human technological
activity [1-3]. One nanometer being equivalent to the width of three or four atoms, and is
equal to 10-9m or one millionth of a millimeter; in this range, groups of atoms bound
covalently together and electrons display special behavior in which nanotechnology is verily
aimed at harnessing this behavior. In the nanoscale, the nano-objects must be contained tens
or hundreds of atoms to achieve the requisite size in so far as each nanoparticle should
contain only about 3×107 atoms/molecules [3]. Thereupon, the internationally acclaimed
range for research and study for the nanotechnology is between 0.1 nm and 100 nm [4].
Practically, when a bulk material is reduced to small size particles with one or more
dimension within the nanometer range, the material will show properties that are drastically
different from those of the bulk material [3, 5]. Nanomaterials can be obtained by two
methods, namely ‘bottom-up’ and ‘top-down’ approaches. The bottom-up approach is
referred to produce nanomaterials through assembling molecule by molecule and atom by
atom but the top-down approach, basically belong to manufacturing nanomaterials through
miniaturizing of bulk materials [3, 6]. Nanotechnology provides the ability to engineer the
properties of materials by controlling their size, and this has driven research towards a
multitude of potential uses for nanomaterials [2, 7, 8]. Presently, the nanosized organic and
inorganic particles are also finding increasing attention particularly in textile applications due
to their considerable properties in the nanoscale. A huge variety of different types of
nanoparticles are already available, ranging from simple UV absorbers used in sunscreens to
highly sophisticated and polyfunctional particles used to control drug delivery, and in solar
panels to harvest sunlight and convert it into electric current [1, 3]. Nanosized heavy metals
and metal oxides such as Ag, Cu, Zn, Hg, TiO2, Al2O3, ZnO and MgO possess unrivaled
properties such as antibacterial activity, photo-catalytic ability, self-cleaning properties,
electrical conductivity, UV-absorption/blockage properties, and photo-oxidizing capacity [7,
9-11]. Nowadays, one of the most interesting properties of these nanoparticles is antibacterial
activity. Among the above nanoparticles, silver is the best natural inorganic metal which is
capable to kill bacteria and fungi and generally can be appeared in the form of powder and
colloidal solution. Silver nano-powders and colloidal nanosilvers are effective antibacterial
agents due to their effective action adversely affecting the cellular metabolism and inhibiting
cell growth. The chemistry has revealed that silver nanoparticles isn’t toxic to human cells in
vivo and is reported to be biocompatible [4, 7, 8, 10, 12, 13].
With the advent of nanotechnology a wide range of nanoparticles can be coated on
textiles, which imparts unrivaled properties to the treated textiles [9, 14-18]. The most
developed nanotechnology application for textiles is currently in the area of coating
Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 167

particularly antibacterial coating. Antibacterial coatings are applied to textiles for four major
reasons: to control the spread of disease and the danger of infection following injury, to
control the infestation by microbes, to control the development of odor from perspiration,
stains, and other soil on textile materials, and to control the deterioration of textiles, specially
fabrics made from natural fibers, caused by mildew [7].
Recently, the new techniques for the modification of textile fibers using antibacterial
nanosized silver particles were introduced by Dubas et al. [9, 18], Ki et al. [19], Yuranova et
al. [12], Chen et al. [20], and Lee et al. [7, 11] which the fibers or fabrics coated using
nanosilvers possessing antibacterial activity. In this review, the application of silver
nanoparticles in antibacterial coating of textiles in recent years up to 2008 is aimed to be
reported in a detailed way. The most popular synthesis procedures of silver nanoparticles,
antibacterial properties of silver nanoparticles, silver nanoparticles-microorganisms
interaction, and assessment of antibacterial activity are the most important issues which are
discussed in this review.

Figure 1. Cubical silver nanoparticles which synthesized by reduction of silver nitrate in the presence of
ethylene glycol [38]

2. KINDS OF SYNTHESIS PROCEDURES OF SILVER NANOPARTICLES


Several synthesizing procedures were reported for the preparation of the silver
nanoparticles in the literature and most of them were based on chemical reduction, Tollens
process, UV light reduction, UV light and chemical reduction concurrently and biological
process. Among these methods, chemical reduction methods were widely studied due to fact
that it is a valid, economic and convenient method [1, 5, 10, 21-37]. For chemical reduction
methods, the choice of the reducing agent is of course the major factor; γ-radiation,
hydrazine, sodium boron hydride, sodium citrate, potassium bitartarate, dimethyl formamide,
ascorbic acid, and alcohols are some of the reducing agents that have been successfully used
168 S. Dadvar, A. Oroume and A. K. Haghi

[5, 37]. The reducing ability will determine the formation kinetics and hence reaction
temperature. The reaction can be carried out in either aqueous solution or in organic solvent
such as the polyol process. All these methods involve the reduction of relevant metal salts,
usually silver nitrate or silver acetate, in the presence of a suitable protecting agent, which is
necessary in controlling the growth of metal colloids through agglomeration [37]; long-chain
n-alkanethiols are the most common protective agents employed to stabilize silver colloids,
even though aromatic amines such as aniline, carboxylic acids, and polymers have been also
employed. However, these methods can lead to significantly different results in terms of size
and morphology depending not only on the choice of the reducing agent and stabilizer but
also on the reaction conditions [5].
Preparation of silver nanocubes (Figure 1) with reduction of silver nitrate using ethylene
glycol was reported to be noteworthy by Sun and Xia [38]. In this preparation, ethylene
glycol serves both as reducing agent and solvent, whereas poly(vinylpyrrolidone) is used as a
capping agent. They show that by controlling experimental conditions, such as temperature,
metal salt concentration, metal/stabilizer ratio, and growth time, the size and morphology of
the silver nanocrystals can be easily tuned, and large quantities of highly symmetric silver
nanocubes of various dimensions can be obtained. In another study, a new procedure for the
preparation of highly monodisperse myristate-capped silver nanoparticles has been reported.
This method involves the suspension of silver myristate in triethylamine followed by gentle
heating at 80◦C for 2h, which gradually produces a solution of uniformly spherical silver
nanoparticles that can be precipitated by addition of acetone. Thus, the nanoparticles can be
isolated as solid materials and re-dispersed in nonpolar solvents where they are stable for up
to 1 week. The particle size and size distribution are affected by the alkyl chain length of the
carboxylate ligand and the tertiary amine. For example, the particles with silver stearate are
highly monodispersed with sizes between 1.9-3.5nm, whereas the particle distribution with
silver octanoate is broad and the sizes are larger (between 5.5 and 31.5nm). With octylamine
in the place of triethylamine, the particle size decreases, but the size distribution is also
broader. This procedure is of great promise for scale-up because of the ease of preparation,
mild conditions, and use of relatively nontoxic reagents [5]. Shrivastava et al. [1] introduced a
novel procedure for the preparation of silver nano particles. In this method, a solution of 0.01
M Ag+ was prepared by dissolving 0.017 g AgNO3 in 100 ml of de-ionized water. During the
process additives like ammonia (30%) were added drop-wise, so that silver ions formed a
stable soluble complex. The solution obtained was used as the precursor for the silver
nanoparticles. A blend of reducing agents like D-glucose and hydrazine was used during the
synthesis of the nanoparticles. Blending was essential to control the rate of reduction resulting
in an achieved optimum rate. A higher reducing rate was shown to form clusters of silver
nanoparticles with reduced stability. About 110 ml of such a blend of reducing agents (at a
concentration of 0.01 M) was incorporated into 100 ml of silver nitrate stock solution (0.01
M) with continuous stirring. This ensured complete reduction of the silver ions to form silver
nanoparticles at a concentration of 0.005 M in aqueous media. The pH of the nanoparticles
formed by that way was maintained at 7.4 with citric acid (1 M). The brown solutions of Ag
nanoparticles were stored in closed glass vials under ambient conditions for future
experiments. Sarkar et al. [26] demonstrated a facile and faster method of preparation of
silver nanocrystals. In this method, silver nitrate solution (0.02 M) was prepared by
dissolving the required amount of AgNO3 in (1:1) ammonia. Similarly, the aldehyde solution
and the surfactant solution having equal concentration to that of the silver nitrate solution
Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 169

were prepared in dehydrated ethanol and water, respectively. Then 1 ml silver nitrate solution
was taken in a well-cleaned dry beaker; 1 ml SDS solution was added to it and it was mixed
well for a few minutes (5 min for each set) by continuous stirring. 1 ml aldehyde solution was
finally added to this mixture. A light yellow color appeared at room temperature (30◦C). The
solution was heated on a water-bath and the temperature was recorded. When the temperature
of the solution reached 80◦C, the light yellow color of the solution started turning into deep
yellow. Then the color gradually changed through yellowish brown (82◦C) to reddish brown
(84◦C) and finally it turned into brownish black (86◦C) (Figure 2). On further increasing the
temperature of the solution, no perceptible change in color was observed. It is interesting to
note that the range of reduction temperature is nearly identical to that reported by Chu et al.
for the case of Au. The gradual change of color in this temperature range indicated the
formation of Ag nano particles of different dimensions. Thus, the study was taken one step
further by preparing five different set solutions at the above-mentioned temperatures (i.e. at
30, 80, 82, 84 and 86◦C).

Figure 2. The temperature-dependent color change during the progression of the reaction [26]

In other study, Yu [27] stated that the AgNO3 was used as precursor in the preparation of
the silver nanoparticles. Na+-poly(γ-glutamic acid) (PGA) served as a linear homo-
polypeptide made of only glutamic acid residuals (both D-form and L-form) in γ-peptide
bond linkages with the degree of polymerization ranging from 1000 to 12000. The reaction
scheme was similar to the silver mirror reaction. Briefly, 0.001 M AgNO3, 0.01 M NaOH,
and 0.02 M NH4OH mixed thoroughly together for 1h. Then, the 1ml of silver alkali solution
was added into 10 ml of 0.5-2wt% PGA solution and subsequently added 1 ml of 10 wt% of
dextrose. The reaction temperature was maintained at 60◦C by a water bath under dark
conditions. The resulted samples were defined as PGA/Ag0, depending on the used
concentration of PGA. For example, 2PGA/Ag0 means that silver nanoparticle stabilized by 2
wt% PGA under chemical reduction by reducing agent-dextrose in comparison with that of
2PGA/Ag+ in the absence of dextrose.
In an innovative procedure, green synthesis of silver nanoparticles was reported by Chen
[31]. Microwave is employed to synthesize silver nanoparticles and a green reagent,
carboxymethyl cellulose sodium (CMS) is also involved. CMS is a kind of biomaterial,
widely applied in many fields, such as food, medicament, daily chemistry, petroleum, paper
making, textile and architecture, due to its innocuity and great performance on thickening,
dispersing, emulsifying and steadying. In this method, CMS can work as both a reducing and
170 S. Dadvar, A. Oroume and A. K. Haghi

a stabilizing reagent in the synthesis of silver nanoparticles. Furthermore, no other agent is


needed in the reaction except AgNO3. In a typical reaction, 10 ml of 0.01M AgNO3 aqueous
solution was mixed with certain volume of 0.1% CMS aqueous solution under stirring. The
mixed solution was adjusted to 200 ml by adding different volumes of water. Then it was
conveyed into a round flask, and the flask was fixed into the microwave reactor. After turning
on the electromagnetic stirrer and microwave reactor, silver nanoparticles were then obtained
gradually.
Another method that is of great potential for the synthesis of silver nanocrystals was
described by Sondi et al. [39] which talks about obtaining smaller particles with higher
specific surface area and narrower size distribution. In a typical experiment, the silver
hydrosols were prepared by adding, under agitation, 10 cm3 of an aqueous 1 mol dm−3
ascorbic acid solution at a flow rate of 3 cm3 min−1 into 90 cm3 of an aqueous solution
containing 5 wt% of Daxad 19 and 0.33 mol dm−3 AgNO3. The reacting solutions were
agitated with a stirrer at 900 rpm at room temperature. In order to remove the surfactant and
excess silver ions, the resulting silver precipitate was washed five times with de-ionized
water. Finally, the nanosize silver was obtained as a dried powder by freeze drying and kept
for future experiments. The obtained powder was fully re-dispersed in de-ionized water by
sonication and therefore aqueous dispersions of silver nanoparticles at the desired
concentrations were easily made.
Zhang et al. [34] prepared colloidal silver nanoparticles in water-in-oil micro-emulsion.
Silver nitrate was used as the starting material for the silver nanoparticles and hydrazine
hydrate (50.0%) was used as the reducing agent. The micro-emulsion system used in the
study consisted of dodecane as the continuous oil phase, sodium bis(2-ethylhexyl)
sulphosuccinate (AOT) as the surfactant, an aqueous solution as the dispersed phase, without
addition of any co-surfactant. In a typical procedure, the micro-emulsions were prepared by
mixing the same volume of aqueous solution of silver nitrate (0.2 M) and hydrazine hydrate
(0.6 M) to the 0.2 M AOT/dodecane solution. Similar method has been reported to synthesize
the colloidal silver nanoparticles in AOT micro-emulsion. However, the surfactant which had
been used as the emulsifier was a mixture of Ag (AOT) and AOT. The used solvents are also
short chain hydrocarbons, e.g., isooctane and cyclohexane. The overall molar concentrations
of silver nitrate and hydrazine hydrate were 4×10-4 M and 1.2×10-3 M, respectively. The
molar ratio of hydrazine hydrate and silver nitrate was held constant for all experiments at a
value of 3. The water-to-AOT molar ratio, W, was kept the same in all cases and was equal to
7.5. The micro-emulsion containing hydrazine hydrate was added into another micro-
emulsion containing silver nitrate drop by drop. After all the hydrazine hydrate micro-
emulsion was added, the vigorous magnetic stirring was maintained for 2 h, the resulting
micro-emulsion mixtures changed to a stable light yellow color after the reaction, indicating
the formation of Ag nanoparticles.

3. CLASSIFICATION OF MICROORGANISMS
Microbes are the tiniest creatures not seen by the naked eye. They include a variety of
microorganisms like Bacteria, Fungi, Algae and viruses. Bacteria are unicellular organisms
which grow very rapidly under warmth and moisture. Furthermore, bacteria family can be
Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 171

classified into two categories, namely, gram-positive (Staphylococcus aurous) and gram-
negative (Escherichia coli). Some specific types of bacteria are pathogenic and cause cross-
infection. Fungi, molds or mildew are complex organisms with slow growth rate. They stain
the fabric and decrease the performance properties of the fabrics. Fungi are active at a pH
level of 6.5. Algae are typical microorganisms which are either fungal or bacterial. Algae
require continuous sources of water and sun light to grow and develop darker stains on the
fabrics. Algae are active in the PH range of 7-8. Dust mites are eight legged creatures and
they occupy the household textiles such as blankets bed linen, pillows, mattresses and carpets.
The dust mites feed on human skin cells and liberated waste products can cause allergic
reactions and respiratory disorders. Some harmful species of the bacteria and fungi are listed
in Table 1 [40].

Table 1. Some harmful species of microorganisms [7, 40]

Bacteria Fungi
gram-positive bacteria gram-negative bacteria Cloth damaging fungi Crop damaging fungi
Staphylococcus aureus or Escherichia coli Aspergillus niger Fusarium species
pyogens
Staphylococcus epidermidis Klebsiella pneumoniae Trichoderma viride Rhizoctonia solani
Streptococcus group A Proteus vulgaris Curvularia lunota Sclerotium rolfsii
Salmonella typhi
Salmonella-Shigella
Pseudomonas aeruginosa

3.1. Inhibition of Microorganisms Using Chemical Antibacterial Agents and


Silver Nanoparticles

Negative effect on the vitality of the microorganisms is generally referred as


antibacterial. The activity which affects the bacteria is known as antibacterial and that of
fungi is antimycotic [40]. Oxidizing agents such as aldehydes, halogens and proxy
compounds attack the cell membrane, get into the cytoplasm and affect the enzymes of the
microorganisms. Radical formers like halogens, isothiazones and peroxy compounds are
highly reactive due to the presence of free electrons. These compounds virtually react with all
organic structures in particular oxidizing thiols in amino acids. Even at the lowest level of
concentrations, these substances pose particular risk to nucleic acids by triggering mutations
and dimerization. Quaternary ammonium compounds exert their influence external to the
microorganisms by disruption of the delicate cell membranes and therefore don’t need to be
absorbed in solution to produce their bacterial killing. One of the most durable type of
antibacterial products is based on a diphenyl ether (bis-phenyl) derivative known as either 2,
4, 4'-trichloro-2' hydroxy diphenyl ether or 5-chloro-2-(2, 4-dichloro phenoxyl) phenol.
Triclosan products have been used for more than 25 years in hospitals and personal care
products such as antibacterial soap, toothpaste and deodorants. Triclosan inhibits growth of
microorganisms by using an electro chemical mode of action to penetrate and disrupt their
cell walls. When the cell walls are penetrated, leakage of metabolites occurs and other cell
functions are disabled, thereby preventing the organism from functioning or reproducing. The
Triclosan when incorporated within a polymer migrates to the surface, where it is bound.
172 S. Dadvar, A. Oroume and A. K. Haghi

Because, it isn’t water-soluble, it does not leach out, and it continuously inhibits the growth
of bacteria in contact with the surface using barrier or blocking action. Quaternary ammonium
compounds, biguanides, amines and glucoprotamine show poly cationic, porous and
absorbent properties. Materials treated with these substances bind microorganisms to their
cell membrane and disrupt the lipopolysaccharide (LPS) structure resulting in the breakdown
of the cell. Complexing metallic compounds based on metals like cadmium, silver, copper
and mercury cause inhibition of the active enzyme centers (inhibition of metabolism) [40-42].
In other words, silver ions function in adversely affecting cellular metabolism to inhibit
bacterial cell growth. Since the silver ions are absorbed into bacterial cells, silver ions stop
respiration, basal metabolism of the electron transfer system and transport of substrate in the
microbial cell membrane; also, silver ions inhibit the bacterial growth by producing active
oxygen on the surface of silver powder. The mechanism of antibacterial action of silver ions
is closely related to their interaction with proteins, particularly at thiol groups (–SH). It was
considered that silver ions attack to the protein molecules and bind protein molecules together
by forming bridges along them. Through this way the cellular metabolism of enzymes is
inhibited and the microorganism dies; i.e. when the metallic silver is in contact with an
oxygen metabolic enzyme of a microorganism, it becomes ionized. As a result, DNA
molecules become condensed and lose their ability to replicate upon the infiltration of Ag
ions. The silver ions also interact with the thiol groups of proteins, which induces the
inactivation of bacterial proteins. As shown in the below reaction (Figure 3), the silver ion
interacts with the thiol groups (–SH) of the enzyme in the microorganism and forms an -SAg
linkage with the enzyme, which effectively blocks the enzyme activity [4, 19, 39, 43, 44].

SH SAg

Enzyme + 2Ag+ Enzyme + 2H+

SH SAg
Figure 3. Interaction of silver ions with the enzyme in the microorganism [4]

As already mentioned, the total surface area of the nanosized silver particles is larger as
compared with the bulk silver particles in the same volume, so the antibacterial ability of the
first state is more effective than the latter [19]. It was speculated that the behavior of silver
nanoparticles is widely similar to that of silver ion. The mechanism of antibacterial actions of
silver nanoparticles is still not well understood. In a previous report on the bactericidal
activity of silver nanoparticles, it was shown that the interaction between silver nanoparticles
and constituents of the bacterial membrane caused structural alterations in and damage to
membranes, finally leading to cell death. It has been suggested that disruption of membrane
morphology may cause a significant increase in permeability, leading to uncontrolled
transport through the plasma membrane and, finally, cell death. The differences between
gram-positive and gram-negative bacteria essentially rest in the structure of their respective
cell walls. The gram-negative bacteria have a layer of LPS composed of covalently linked
lipids and polysaccharides with the poor strength and rigidity at the exterior, followed
Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 173

underneath by a thin layer of peptidoglycan with the thickness of about 7-8 nm. Negative
charges on the LPSs are attracted towards weak positive charges available on silver
nanoparticles. In contrast, the cell wall in gram-positive bacteria is principally composed of a
thick layer of peptidoglycan with the thickness of about 20-80 nm, consisting of linear
polysaccharide chains cross-linked by short peptides to form a 3D rigid structure. The rigidity
and extended cross-linking not only endow the cell walls with fewer anchoring sites for the
silver nanoparticles but also make them difficult to penetrate. The extent of inhibition of
bacterial growth was dependent on the concentration of nanoparticles in the medium.
Interaction between nanoparticles and the cell wall of bacteria would be facilitated by the
relative abundance of negative charges on the gram-negative bacteria, which was congenial to
the fact that growth of gram-negative bacteria was more vigorously affected by the silver
nanoparticles than that of the gram-positive microorganisms. Silver as a soft acid has a
greater tendency to react with sulphur- or phosphorus- containing soft bases, such as R-S-R,
R-SH, RS-, or PR3. Thus, sulfur-containing proteins in the membrane or inside the cells and
phosphorus-containing elements like DNA are likely to be the preferential sites for silver
nanoparticle binding. As demonstrated by electron microscopy, interaction with nanoparticles
resulted in pits in the cell wall and in the protein de-naturation, contributing to the
antibacterial effects of the nanoparticles.

Figure 4. Escherichia coli treated with silver nanoplates in which nanosized silver particles appear as
dark irregular pits on the cell surface [43]

Figure 4 shows the image of a part of a vigorously damaged cell membrane treated with
silver nanoparticles. Results of the re-culture experiments were consistent with the entry of
nanoparticles inside bacterial cells and strong agglomeration with bacterial cellular
components. Once inside the cell, nanoparticles would interfere with the bacterial growth
signaling pathway by moderating tyrosine phosphorylation of putative peptide substrates
critical for cell viability and division [1, 39, 43].
174 S. Dadvar, A. Oroume and A. K. Haghi

Figure 5. Transmission electron micrograph of Escherichia coli cell treated with 50μg cm−3 of silver
nanoparticles in liquid Luria–Bertani (LB) medium for 1h (a) and close up view of the membrane of
this cell (b) [39]

The TEM analysis (Figure 5) and the existence of elementary silver in the membranes of
treated bacteria, detected by energy dispersive analysis X-ray (EDAX) (Figure 6), confirm the
incorporation of silver nanoparticles into the membrane structure [39].

Figure 6. EDAX spectra of native Escherichia coli (a) and Escherichia coli treated with 50 μg cm−3 of
silver nanoparticles in liquid Luria–Bertani (LB) medium for 4 h (b) [39]

A similar effect was described by some workers [39] when Escherichia Coli bacteria
were treated with highly reactive metal oxide nanoparticles. A bacterial membrane with this
morphology discloses a significant increase in permeability, leaving the bacterial cells
incapable of properly regulating transport through the plasma membrane and, finally, causing
cell death. It is well known that the outer membrane of Escherichia Coli cells is
predominantly constructed from tightly packed LPS molecules, which provide an effective
Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 175

permeability barrier. Recently, in other study workers have shown that metal depletion may
cause the formation of irregular-shaped pits in the outer membrane and changed membrane
permeability, which is caused by progressive release of LPS molecules and membrane
proteins (Figure 7). It can be speculated that a similar mechanism causes the degradation of
the membrane structure of Escherichia Coli during treatment with silver nanoparticles.
Extensive investigations directed to better understanding of interaction between silver
nanoparticles and bacterial components should shed light on the mode of action of this
nanomaterial as a biocidal material [39].

Figure 7. Scanning electron micrographs of native Escherichia coli cells (a) and cells treated with 50 μg
cm−3 of silver nanoparticles in liquid Luria–Bertani (LB) medium for 4 h (b) [39]

4. ANTIBACTERIAL COATING
Textiles, especially those made of natural fibers, are an excellent medium for the growth
of microorganisms when the basic requirements such as nutrients, moisture, oxygen and
appropriate temperature are present. The large surface area and ability to retain moisture of
textiles also assist the growth of microorganisms on the fabric. Therefore, there is a great
demand for antimicrobial coatings of textiles to control the growth of microorganisms, such
as bacteria, fungi or mildew, and prevent the textile from deterioration of strength and quality,
staining, odors, and health concerns caused by microorganisms [45]. An awareness of general
sanitation, contact disease transmission, and personal protection has led to the development of
antibacterial fibers to protect wearers against the spread of bacteria and diseases rather than to
protect the quality and durability of the textile material [46]. From the standpoints of a
propensity toward tidiness as a social manner and a demand for advanced medical
technology, antibacterial goods have recently received a growing interest. Figure 8
statistically shows the development of several antibacterial textiles, both treated antibacterial
fabrics and fabrics with antibacterial fibers, in the west Europe until 2000 [47, 48]. Textiles
have a wide use in healthcare and medical fields, and most of them currently used in clinics
and hospitals, such as laboratory coats, medical cloths, wound dressings, sanitary products,
medical disposables, and women hygiene products, are conducive to cross-infection or
transmission of diseases caused by microorganisms. The inherent properties of the textile
fibers provide room for the growth of microorganisms; besides, the structure of the substrates
and the chemical processes may induce the growth of microbes [4, 18, 40, 44, 49].
176 S. Dadvar, A. Oroume and A. K. Haghi

Figure 8. Statistically development of antibacterial textiles, both treated antibacterial fabrics (a) and
fabrics with antibacterial fibers (b), in the west Europe until 2000 [50]

The need for the chemical antibacterial coatings as mentioned previously can be
contributed to four items: to control the spread of disease and the danger of infection
following injury, to control the infestation by microbes, to control the development of odor
from perspiration, stains, and other soil on textile materials, and to control the deterioration of
textiles, specially fabrics made from natural fibers, caused by mildew [7, 11]. In order to
carry out this particular coating on textiles and garments, this point should be attended that all
types of textiles such as shirts, hosiery, blouses, diapers, and underwear are more susceptible
to wear and tear. It is important to take into account the impact of stress strain, thermal and
mechanical effects on the coated materials; so, some requirements such as durability to
washing, dry cleaning and hot pressing, selective activity to undesirable microorganisms, no
harmful effects to the manufacturer, user and the environment, compatibility with the
chemical processes, easy method of application, no deterioration of fabric quality, resistant to
body fluids and resistant to disinfections, compatibility with the statutory requirements of
regulating agencies, need to be satisfied to obtain maximum benefits out of the coating [40,
44]. Chemical antibacterial coating of textiles first appeared in 1941 [46]. The primary
objective of the chemical antibacterial coating was to protect textiles from being affected by
microorganisms. Basically, the application of the chemical antibacterial coating is extended to
textiles used for outdoor, healthcare sector and sports. The greater use of synthetic fibers and
blends in such items as shirts, hosiery, blouses, and underwear has accelerated the need for
bacteriostatic coatings on clothing. The moisture-transport characteristics of such blends tend
to cause a greater degree of ‘perspiration wetness’ than occurs with fibers of wholly natural
Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 177

fibers. The chemical antibacterial substances function in different ways. In the conventional
leaching type of coating, the species diffuse and poison the microbes to kill. This type of
coating shows poor durability and may cause health problems. The non-leaching type of
coating shows good durability and may not provoke any health problems. A large number of
textiles with chemical antibacterial coating function by diffusion type. The rate of diffusion
has a direct effect on the effectiveness of the coating. For example, in the ion exchange
process, the release of the active substances is at a slower rate compared to direct diffusion
and hence, has a weaker effect. Similarly, in the case of antibacterial modifications, the active
substances aren’t released from the fiber surface; so this type of antibacterial coating is less
effective. These substances are active only when they come in contact with microorganisms.
These so called new technologies have been developed by considering the medical,
toxicological and ecological principles. The antibacterial textiles can be classified into two
categories, namely, passive and active based on their activity against microorganisms. Passive
materials don’t contain any active substances but their surface structure produces negative
effect on the living conditions of microorganisms (Lotus effect or Anti-adhesive effect).
Materials containing active antibacterial substances act upon either in or on the cell. Many
chemical antibacterial agents used in the textile industry are incorporated with textile
substrates comparatively at lower concentrations. It must be ensured that these substances
aren’t only permanently effective but also that they are compatible with skin and the
environment. A wide palette of antibacterial compounds is now in use but differs in their
mode of action. Materials with active coatings contain specific active antibacterial substances,
which act upon microorganisms either on the cell, during the metabolism or within the core
substance (genome). However, due to the very specific nature of their effect, it is important to
make a clear distinction between chemical antibacterial agents and other active substances
which have a broad range of uses [40]. Hence, the antibacterial properties of such textile
materials can be grouped into two categories, temporarily or durably functional fabrics.
Temporary biocidal properties of fabrics are easy to achieve in coating, but easy to lose in
laundering. Durability has generally been accomplished by a common technology, a slow-
releasing method. According to this method, sufficient antibacterial agents are incorporated
into fibers or fabrics by means of a wet coating process. The treated fabrics deactivate
bacteria by slowly releasing the biocide from the materials. However, the antibacterial agents
will vanish completely if they are impregnated in materials without covalent bond linkages
[49].
Traditionally, textile materials have been imparted antibacterial properties by chemically
or physically incorporating chemical agents onto fabrics through the coating processes [41,
42, 45, 49]. After World War II fungicides used on cotton fabrics were compounds such as 8-
hydroxygiunoline salts, copper naphthenate, copper ammonium fluoride and chlorinated
phenals [40]. Generally, the antibacterial agents can be applied to the textile substrates by
exhaust, pad-dry-cure, coating, spray and foam techniques. The substances can also be
applied by directly adding into the fiber spinning dope. A number of methods for
incorporating antibacterial functions into textile materials have been developed elsewhere;
these methods such as insolubilisation of the active substances in/on the fiber, treating the
fiber with resin, condensates or cross linking agents ( e.g. the attachment of chitosan to cotton
fabric via cross linking agents [46, 51, 52]), micro encapsulation of the antibacterial agents
with the fiber matrix, use of graft polymers, homo polymers and/or copolymerization on to
the fiber (e.g. graft polymerization of N-halamide monomers onto cellulosic substrates [46]),
178 S. Dadvar, A. Oroume and A. K. Haghi

and chemical modification of the fiber by covalent bond formation (e.g. placement of
quaternary ammonium salts onto cotton fabrics using a covalently bound adduct or covalent
attachment of a chloromelamine derivatives [46, 53]) have been developed for improving the
durability of the chemical antibacterial coating process [40, 42]. Antibacterial textile
materials based on helamine chemistry showed biocidal properties against a wide range of
pathogens, and are also non-toxic and environmentally friendly. For example, in a typical
approach, antibacterial cellulosic fabrics were developed by means of the use of 1,2,3,4-
butanetetracarboxylic acid and citric acid, together with subsequent oxygen bleaching.
Carboxylic acids have been converted to peroxy acids by being reacted with hydrogen
peroxide under acidic conditions, while carboxylic acid groups can be incorporated into
cellulose fabrics. In another procedure, a dimethylol hydantoin derivative, dimethylol-5,5-
dimethylhydanation, was used in chemical treatment of cellulose, and subsequent chlorine
bleaching can convert unreacted amide or imide bonds in the hydantoin [42, 49].
Generally, chemical antibacterial agents used to control the growth of microorganisms on
textile fabrics and several bacteriostatic textiles coatings exist for personal wears, but poor
activity against microorganisms, lack of wash durability, environmental pollution, and
inadequate safety data to meet current requirements or a combination of these factors has
limited their use [7] and makes them not suitable for applications in health foods, filters, and
textiles [44]. A common problem in disinfection or bacteriostasis is the selection of an agent
that kills all organisms in the shortest time without damaging the contaminated materials.
Antibacterial agents should always be diluted exactly as specified by the manufacturer.
Solutions that are too weak may be ineffective, and those that are too strong can be dangerous
for human body [7]. Consequently, a safe, wash-resistant textile coating capable of inhibiting
the growth of both bacteria and fungi is required.
The wave of nanotechnology has shown a huge potential in the textile and clothing
industry and has provided a new area for futuristic research in science and technology. Using
nanotechnology to improve existing material performances and developing unrivaled
functions on textile materials are flourishing. As discussed, commercial textiles containing
chemical antibacterial agents are sorely poison and irritant for human body; hence the new
types of safe and commodious biocidal materials need to be replaced with these chemical
agents [39]. By this reason, the new antibacterial coating by taking advantages of
nanotechnology have been developed. The development of new clothing products based on
the immobilization of nanoparticles on textile fibers has recently received a growing interest
from both the academic and industrial sectors. Nowadays, a wide range of nanoparticles and
nano-structures can be immobilized on fibers, which brings new properties to the final
clothing product. Numerous patents were developed for the surface modification of fibers
with nanoparticles, including blending of the nanoparticles in the polymer matrix before
spinning or chemical grafting of the desired functional groups onto the fibers; for example,
Japanese researchers revealed an antibacterial cloth used for washing breasts of milk cow.
Chinese researchers showed a new method for making antibacterial fabric with long lasting
broad-spectrum antibacterial effect against more than 40 bacteria. The fabric is manufactured
by dissolving silver nitrate in water, adding ammonia water into the solution to form silver-
ammonia complex ion, adding glucose to form a nanosized silver particles as a treating agent,
adding fabric into the treating agent, and ironing the fabric by electric iron or heat-rolling
machine [4, 11].
Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 179

Kin et al. [19] imparted antibacterial properties to the wool fabrics using sulfur nano-
silver ethanol based colloid supplied from NP-Tech Co., Ltd., Korea with the particle size
of average 4.2 nm. The antibacterial wool textiles were prepared by a general padding
method with the diluted sulphur nano-silver colloidal solution. Before the coating process,
untreated wool fibers were cleaned with dichloromethane (40◦C, 30 min), rinsed with ethanol
and water twice (25◦C, 10 min), and equilibrated in a conditioned room (20◦C, 60% R.H.).
The fibers were treated with nano-silver colloid by a conventional pad-dry-cure method.
Practically, the wool fabrics were immersed in a fresh colloidal bath for 10 min and squeezed
using a laboratory padder at the constant pressure. The samples were dried at room
temperature for prevention of thermo-migration of metal particles for 30 min, and then the
curing process of samples was performed at 120◦C, for 5 min. Figure 9 discloses the
scanning electron microscopy of wool fiber treated using 100 ppm of sulphur nano-silver
colloidal solution. They concluded that the antibacterial efficacy on wool fibers was easily
achieved by the conventional pad-dry-cure method using the nanosized silver colloid
including sulfur compound insofar as the manufactured wool textiles using the treated fibers
with the silver particle were showed as excellent antibacterial products.

Figure 9. Scanning electron microscopy photograph of wool fiber treated using sulphur nano-silver
colloid; (a) 5000 magnifications (b) 30000 magnifications [19]

Dubas et al. [9] disclosed the new procedure in which antimicrobial silver nanoparticles
were immobilized on nylon or silk fibers by following the layer-by-layer deposition
method. In this method the sequential dipping of nylon or silk fibers in dilute solutions of
poly(diallyldimethylammonium chloride) and silver nanoparticles capped with
poly(methacrylic acid) led to the formation of a colored thin film possessing
antimicrobial properties (Figure 10). Silver nanoparticles were prepared through the photo-
reduction of silver nitrate under UV light in a dilute solution of poly(methacrylic acid). As a
result, upon exposure of a silver nitrate/poly(methacrylic acid) mixture to UV light, the
solution quickly turned pink and finally red after several hours. Practically, nylon or silk
fibers were wrapped around a rectangular aluminum holder, 2.5×3.5 cm2 and spun in various
solutions using a small dc motor. A home-built robotic platform, accommodating eight 100
ml beakers, was programmed to successively expose the fibers to either polyelectrolyte or
silver nanoparticle solutions followed by three 1 min water rinses and repeated as many times
as needed. At the end of the deposition process, the samples were allowed to dry overnight
180 S. Dadvar, A. Oroume and A. K. Haghi

and then wrapped on a plastic holder before measurements with the spectrophotometer. In a
typical experiment setup, the polyelectrolyte multilayers (PEM) were composed of 20 layers
by alternatively dipping the fibers in 1 mM poly(diallyldimethylammonium chloride) and
silver nanoparticles solution for 2 min. The sequential dipping of the fibers in
poly(diallyldimethylammonium chloride) and poly(methacrylic acid) capped silver
nanoparticles solutions led to the appearance of a red color onto the fiber. The color is due to
the immobilization of the silver nanoparticles onto the fibers. Also, this point should be
attended that the pH of all solution was set to a value of 7. Finally, they concluded that the
fibers coated with the nanoparticles exhibit antimicrobial activity, which could render them
useful in applications such as water sanitation or antimicrobial fabrics.

Figure 10. Scanning electron microscopy of nylon (left picture) and silk (middle and right pictures)
fibers coated with poly(diallyldimethylammonium chloride) and silver nanoparticles capped with
poly(methacrylic acid) [9]

Potiyaraj et al. [18] synthesized the silver chloride nanocrystals on silk fiber. The
growth of the nanocrystal was achieved by sequential dipping of the silk fibers in
alternating solution of either silver nitrate or sodium chloride followed by a rinse step.
The resulting fiber coated with nano-AgCl crystals could be used as a photo-catalyst in
water splitting applications or as an antibacterial agent. Figure 11 obviously shows all
steps describing the preparation of the silver chloride nanocrystal by sequential dipping
in silver nitrate and sodium chloride. Practically, the silk fibers similarly mentioned in [9]
were wrapped around a rectangular aluminum holder, 2.5×3.5 cm2 and spun in various
solutions using a small DC motor. A home-built robotic platform, accommodating eight 100
ml beakers, was programmed to successively expose the silk fibers to either silver nitrate or
sodium chloride solutions followed by three water rinses of 1 min each. The number of
dipping steps was fixed to 20 meaning that the fiber was dipped 10 times in silver nitrate and
10 times in sodium chloride solutions. At the end of the deposition process the samples were
allowed to dry overnight. In order to confirm the formation of nanocrystal at the surface of
the silk fiber, scanning electron microscopy image of the surface of the silk fiber coated
using AgCl nanoparticles has been taken (Figure 12). Finally, they concluded that the
resulting AgCl crystals generally could be used as an antibacterial agent or if assembled on
conducting fibers, could be used in water splitting application.
Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 181

Figure 11. Steps describing the preparation of the silver chloride nanocrystal by sequential dipping in
silver nitrate and sodium chloride [18]

Figure 12. Scanning electron microscopy image of the AgCl nanoparticles formed at the surface of
the silk fiber. The AgCl nanoparticles were synthesized by 20 alternate dipping in AgNO3 and
NaCl solutions followed by a rinse in water [18]

Lee et al. [7] manifested the method in which the polyester nonwovens were
incorporated with colloidal silver nanoparticles supplied by NP-Tech Co., Ltd., Korea. In
this method typically the polyester nonwovens were immersed in a colloidal silver
nanoparticles bath for 1 minute and squeezed to 100% wet pick-up with a laboratory pad
at a constant pressure. Subsequently the treated polyester nonwovens were dried at 120◦C
for 5 minutes. Figure 13 discloses the antibacterial coating process of the textile fabrics. They
varied the mean size of silver nanoparticles to improve the antibacterial effect of nonwovens
and demonstrated that the growth of bacterial colonies was absolutely inhibited using low
concentration of colloidal silver nanoparticles with the mean size smaller than 5 nm. In other
words, the smaller particle sizes had better antibacterial effects on silver-padded nonwoven
fabrics. They also stated that higher concentration of silver colloids have better bacteriostasis
because bacterial reductions decreased when the silver concentrations in the pad bath
decreased.
182 S. Dadvar, A. Oroume and A. K. Haghi

Figure 13. Schematic of antibacterial coating process for polyester nonwovens [7]

In other study, Lee et al. [11] produced antibacterial woven cotton and polyester fabrics
using the same colloidal silver nanoparticles supplied by NP-Tech Co., Ltd., Korea.
Figure 14 shows clearly shows the well dispersed of silver nanoparticles on fiber surfaces in
each fabric. In this procedure, woven cotton and polyester fabrics were padded through a
certain concentration of silver colloids and squeezed to 83% wet pick-up with a laboratory
pad at a constant pressure. They demonstrated that antibacterial efficacy and good laundering
durability on textile fabrics can be easily achieved with using nanosized silver colloidal
solution through padding process.

(a) (b)

Figure 14. Scanning electron microscopy (SEM) images of silver nanoparticles on (a) cotton and (b)
polyester fibers [11]

Jeong et al. [44] prepared PE/PP nonwovens using various kinds of nanosized silver
colloids. They employed three different types of nano-sized silver colloids supplied by NP-
Tech Co., Ltd., Korea: nano-sized silver particles dispersed in water (NSW), nano-sized
silver particles dispersed in ethanol (NSE), and nano-sized silver/sulfur composite particles
dispersed in ethanol (SNSE) to prevent aggregation of the particles; Figure 15 presents
TEM images of the nano-sized spherical silver colloid. The mean diameter of silver
nanoparticles in the water-based colloid was 8.11 nm, and that of the ethanol-based
Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 183

colloids was 3 nm. In this method, PE/PP nonwovens were padded using colloid
solutions at the pressure of 3 kgf/cm2 by an auto fade mangle. Subsequently, the samples
were immediately dried at 120◦C for 3 min. As a result, they observed that the
nonwovens treated using NSW (the water-based silver nanoparticles) had the deepest
color at the same concentration but the color of the colloid interestingly was almost
invisible in the nonwovens treated using SNSE (the ethanol-based nano-sized silver/sulfur
composite particles); according to this observation nonwovens treated using SNSE may be
a good antibacterial coating agent that will not influence the intrinsic color of the fabric.
Furthermore, the SNSE coated fabrics also exhibited the highest antibacterial efficacy.

(a) (b)

Figure 15. TEM images of silver nanoparticle (×200 K): (a) nano-sized silver particles dispersed in
water (NSW), (b) nano-sized silver particles dispersed in ethanol (NSE) [44]

5. ASSESMENT OF ANTIBACTERIAL ACTIVITY


‘Quantitative tests’ for antibacterial activity of textiles usually involved sterilization of
fabric, followed by its inoculation with a test organism and incubation prior to determination
of bacteria remaining on the fabric. ‘Qualitative tests’ for such activity consist of visual
observation of microbial growth on the fabric after exposure to the test organism [54].
The disadvantages of quantitative methods can be listed as being time-consuming,
expensive, and they have not been assessed (with the exception of the AATCC-100 method)
for inter-laboratory correlation of test results. Three quantitative methods are currently being
used for determining antibacterial activity:

• AATCC-100 test method: In this method fabrics are sterilized in an autoclave or with
ethylene oxide, inoculated with either gram-positive (Staphylococcus aureus) or
gram-negative (Klebsiella pneumoniae) bacteria, and incubated in an agar medium;
bacteriostatic activity of fabric swatches (treated and untreated controls) is calculated
as percent reduction of bacteria on the fabric [55].
184 S. Dadvar, A. Oroume and A. K. Haghi

• Quinn test method: This test method permits direct enumeration of bacterial colonies
on the fabric surface, but the test isn’t easy to run, colonies aren’t easily visible, and
the possibilities for diffusion of the antimicrobial agent have not been minimized
[40]. In this method fabrics that are free from microorganisms are prepared by
laundering or sterilizing; the fabrics are then inoculated with one or several test
organisms (Micrococcus, Pseudomonas, Lactobacillus, Escherichia coli,
Staphylococcus aureus, or Klebsiella pneumoniae) and dried under conditions of
known relative humidity. They are then placed on sterile agar plates, covered with a
thin layer of agar, and incubated. After incubation, the bacteriostatic activity of the
fabric is determined by counting the remaining bacteria colonies with a low-power
microscope [54].
• Lashen test method: In this method sample is suspended tautly and horizontally in a
Petri dish by means of a wire hanger with hooks (Figure 12). After autoclaving, 0.4
ml of molten AATCC agar containing 20µg of 2, 3, 5-triphenyl-2H-tetrazolium
chloride (TTC) per ml and 2 million bacterial cells is applied slowly and uniformly to
the fabric surface. To prevent drying of the agar, approximately 5 ml of sterile
distilled water is added to the Petri dish bottom. After 48 hr of incubation at 37 C, a
colony count is made on both sides of the white fabric surface. The TTC is reduced
by bacterial dehydrogenase enzymes to form a red insoluble dye
(triphenylformazan), which stains bacterial colonies red. A low-power stereoscopic
microscope may be used to be certain all colonies are counted. Untreated control
samples with 2 million cells are included in the test, but the colonies which develop
are too numerous to count. These samples appear pinkish due to numerous
microscopic red colonies. However, a countable sample of 200 colonies is obtained
by applying a 1:10000 agar dilution of the seeded agar to untreated fabric [56].

Table 2. Some of the antibacterial activity tests [50]

Code Number Test Title Test Procedure


SN 195920-1992 Textile fabrics: Determination of the antibacterial activity: Agar
diffusion plate test
SN 195921-1992 Textile fabrics: Determination of the antimycotic activity: Agar
diffusion plate test
Agar diffusion
AATCC 30-1993 Antifungal activity, assessment of textile materials: Mildew and rot
tests
resistance of textile materials
semi-quantitative
AATCC 147-1993 Antibacterial assessment of textile materials: Parallel streak methods
AATCC 90-1982 Antibacterial activity of fabrics, detection of: Agar plate method
AATCC 174-1993 Antimicrobial activity assessment of carpets
JIS L 1902-1998 Testing method for antibacterial textiles
AATCC 100-1993 Antibacterial finishes on textile materials
SN 195924-1983 Textile fabrics: Determination of the antibacterial activity: Germ count
Challenge tests
method
quantitative
ISO 846-1997 Plastics-Evaluation of the action of microorganisms
ISO 11721-1-2001 Textiles-Determination of resistance of cellulose containing
New Methods ISO TC38 WG23: Testing for antibacterial activity
CEN TC248 WG13: Textiles-Determination of the antibacterial activity – Agar plate
diffusion test
Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 185

In qualitative tests, antibacterial activity of fibers is affected by the ability of the chemical
agent to diffuse off the fiber into the culture medium. Although these tests are inexpensive
and may be performed rapidly, they lack applicability to durable antibacterial fiber coatings,
for the high agent-diffusibility that results in a positive test also results in coatings with low
durability to laundering. In AATCC 147-1977 test method, bacteriostatic activity is measured
by observing growth-free areas around fabric specimens placed perpendicular to streaks of
agar inoculum. The parallel-streak test has been adopted as the qualitative method of choice
over the agar-plate method (AATCC 90-1977), because its results can be correlated in inter-
laboratory tests and it is less dependent on diffusion. However, control samples are
recommended for this test, since some investigators have noted false-positive results on
untreated fabrics. Both the parallel streak and agar-plate methods use Staphylococcus aurous
and Klebsiella pneumoniae as test microorganisms [40, 54]. Table 2 shows that some of the
classified antibacterial activity test methods are used in textile researching [50].
Generally, Bacteriostatic activity of colloidal silver nanoparticles was evaluated after
certain contact time and calculated percent reduction of bacteria using the following equation:

A− B
R (%) = ×100
A

Where R = the reduction rate, A = the number of bacterial colonies from untreated
fabrics, and B = the numbers of bacterial colonies from treated fabrics [10, 11, 19, 45].

6. CONCLUSION
Textiles, especially those made of natural fibers, are an excellent medium for the growth
of microorganisms; therefore, there is a great demand for antimicrobial coatings of textiles to
control the growth of microorganisms. In this review, an overview on application of silver
nanoparticles in antibacterial coating of textiles has been presented. Silver nanoparticle as a
novel antibacterial agent has advantages over conventional chemical antibacterial agents;
because utilizing it in the antibacterial coating process is very convenient. Silver
nanoparticles are skin friendly and don’t cause skin irritation. Some antimicrobial agents are
extremely irritant and toxic to human body but the nano-sized silver particles have been well
known as non-toxic in spite of being claimed to kill many different disease organisms. Some
advantages and disadvantages of colloidal silver nanoparticles which can be used as an
alternative antibacterial agent in textile antibacterial coating may be counted as follows:

Advantages:

• Outstanding antibacterial performance


• Skin friendly, non-allergenic and non-toxic to the skin
• Not irritating or sensitizing to the skin
• Ineffective against mammalian cell membranes
• Strongly effective against microorganisms
• Selective activity to undesirable microorganisms
186 S. Dadvar, A. Oroume and A. K. Haghi

• Long lasting broad-spectrum antibacterial effect


• Compatible with the chemical processes
• Durable to washing, dry cleaning and hot pressing
• Cheap and easy method of application
• Complying with the statutory requirements of regulating agencies Ecofriendly
• Ecofriendly

Disadvantages:

• Color change of fabrics treated using colloidal silver nanoparticles


• Commonly time-consuming and costly synthesis procedures of silver nanoparticles
• Destroying of beneficial bacteria in such cases

REFERENCES
[1] S. Shrivastava, T. Bera, A. Roy, G. Singh, P. Ramachandrarao, and D. Dash,
“Characterization of enhanced antibacterial effects of novel silver nanoparticles”,
Nanotechnol, 18, 1 (2007).
[2] J. L. Elechiguerra, J. L. Burt, J. R. Morones, A. Camacho-Bragado, X. Gao, H. H. Lara,
and M. J. Yacaman, “Interaction of silver nanoparticles with HIV-1”, J
Nanobiotechnol, 3(No.6), 1 (2005).
[3] J. J. Ramsden, “What is nanotechnology?”, Nanotechnol Perceptions, 1, 3 (2005).
[4] Yan, J. and J. Cheng, “Antimicrobial yarn having nanosilver particles and methods for
manufacturing the same”, US Patent No. 6,979,491 B2 (2005).
[5] O. Masala and R. Seshadri, “Synthesis routes for large volumes of nanoparticles”, Annu
Rev Mater Res, 34, 41 (2004).
[6] D. K. Tiwari, J. Behari, and P. Sen, “Application of nanoparticles in waste water
treatment”, World Appl Sci J, 3(No.3), 417 (2008).
[7] H. J. Lee and S. H. Jeong, “Bacteriostasis of nanosized colloidal silver on polyester
nonwovens”, Text Res J, 74(No.5), 442 (2004).
[8] N. Perkas, G. Amirian, S. Dubinsky, S. Gazit, and A. Gedanken, “Ultrasound-assisted
coating of nylon 6,6 with silver nanoparticles and its antibacterial activity”, J Appl
Polym Sci, 104, 1423 (2007).
[9] S. T. Dubas, P. Kumlangdudsana, and P. Potiyaraj, “Layer-by-layer deposition of
antimicrobial silver nanoparticles on textile fibers”, Colloid Surface A: Physicochem
Eng Aspects, 289, 105 (2006).
[10] N. Durán, P. D. Marcato, G. I. H. De Souza, O. L. Alves, and E. Esposito,
“Antibacterial effect of silver nanoparticles produced by fungal process on textile
fabrics and their effluent treatment”, J Biomed Nanotechnol, 3, 203 (2007).
[11] H. J. Lee, S. Y. Yeo, and S. H. Jeong, “Antibacterial effect of nanosized silver colloidal
solution on textile fabrics”, J Mater Sci, 38, 2199 (2003).
[12] T. Yuranova, A. G. Rincon, C. Pulgarin, D. Laub, N. Xantopoulos, H. J. Mathieu, and
J. Kiwi, “Performance and characterization of Ag–cotton and Ag/TiO2 loaded textiles
Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 187

during the abatement of E. coli”, J Photochem Photobiol A: Chemistry, 181, 363


(2006).
[13] C. Damm, H. Münstedt, and A. Rösch, “Long-term antimicrobial polyamide 6/silver-
nanocomposites”, J Mater Sci, 42(No.15), 6067 (2007).
[14] S. Wang, W. Hou, L. Wei, H. Jia, X. Liu, and B. Xu, “Antibacterial activity of nano-
SiO2 antibacterial agent grafted on wool surface”, Surf Coat Tech, 202, 460 (2007).
[15] N. Vigneshwaran, K. S., A. A. Kathe, P. V. Varadarajan, and V. Prasad, “Functional
finishing of cotton fabrics using zinc oxide–soluble starch nanocomposites”,
Nanotechnol, 17, 5087 (2006).
[16] J. Scholz, G. Nocke, F. Hollstein, and A. Weissbach, “Investigations on fabrics coated
with precious metals using the magnetron sputter technique with regard to their anti-
microbial properties”, Surf Coat Tech, 192, 252 (2005).
[17] D. Hua, K. Cheuk, Z. Wei-ning, W. Chen, and X. Chang-fa, “Low temperature
preparation of nano TiO2 and its application as antibacterial agents”, Trans Nonferrous
Met Soc China, 17, 700 (2007).
[18] P. Potiyaraj, P. Kumlangdudsana, and S. T. Dubas, “Synthesis of silver chloride
nanocrystal on silk fibers”, Mater Lett, 61, 2464 (2007).
[19] H. Y. Ki, J. H. Kim, S. C. Kwon, and S. H. Jeong, “A study on multifunctional wool
textiles treated with nano-sized silver”, J Mater Sci, 42, 8020 (2006).
[20] C. Y. Chen and C. L. Chiang, “Preparation of cotton fibers with antibacterial silver
nanoparticles”, Mater Lett, 62, 3607 (2008).
[21] A. Šileikaitė, I. Prosyčevas, J. Puišo, A. Juraitis, and A. Guobienė, “Analysis of silver
nanoparticles produced by chemical reduction of silver salt solution”, Mater Sci,
12(No.4), 287 (2006).
[22] N. Niño-Martínez, G. A. Martínez-Castañón, A. Aragón-Piña, F. Martínez-Gutierrez, J.
R. Martínez-Mendoza, and F. Ruiz, “Characterization of silver nanoparticles
synthesized on titanium dioxide fine particles”, Nanotechnol, 19, 1 (2008).
[23] M. O. Elhag Ahmed and W. Kee Leong, “Colloidal silver nanoparticles stabilized by a
water-soluble triosmium cluster”, J Organomet Chem, 691, 1055 (2006).
[24] A. Frattini, N. Pellegri, D. Nicastro, and O. de Sanctis, “Effect of amine groups in the
synthesis of Ag nanoparticles using aminosilanes”, Mater Chem Phys, 94, 148 (2005).
[25] R. P. Bagwe and K. C. Khilar, “Effects of intermicellar exchange rate on the formation
of silver nanoparticles in reverse microemulsions of AOT”, Langmuir, 16, 905 (2000).
[26] S. Sarkar, A. D. Jana, S. K. Samanta, and G. Mostafa, “Facile synthesis of silver nano
particles with highly efficient anti-microbial property”, Polyhedron, 26, 4419 (2007).
[27] D. G. Yu, “Formation of colloidal silver nanoparticles stabilized by Na+–poly(γ-
glutamic acid)–silver nitrate complex via chemical reduction process”, Colloid Surface
B: Biointerfaces, 59, 171 (2007).
[28] Z. J. Jiang, C. Y. Liu, and Y. Liu, “Formation of silver nanoparticles in an acid-
catalyzed silica colloidal solution”, Appl Surf Sci, 233, 135 (2004).
[29] P. Mukherjee, M. Roy, B. P. Mandal, G. K. Dey, P. K. Mukherjee, J. Ghatak, A. K.
Tyagi, and S. P. Kale, “Green synthesis of highly stabilized nanocrystalline silver
particles by a non-pathogenic and agriculturally important fungus T. asperellum”,
Nanotechnol, 19, 1 (2008).
188 S. Dadvar, A. Oroume and A. K. Haghi

[30] Y. H. Chen and C. S. Yeh, “Laser ablation method: use of surfactants to form the
dispersed Ag nanoparticles”, Colloid Surface A: Physicochem Eng Aspects, 197, 133
(2002).
[31] J. Chen, J. Wang, X. Zhang, and Y. Jin, “Microwave-assisted green synthesis of silver
nanoparticles by carboxymethyl cellulose sodium and silver nitrate”, Mater Chem Phys,
108, 421 (2008).
[32] Y. Tan, Y. Li, and D. Zhu, “Preparation of silver nanocrystals in the presence of
aniline”, J Colloid Interf Sci, 258, 244 (2003).
[33] W. S. Liao, T. Yang, E. T. Castellana, S. Kataoka, and P. S. Cremer, “A rapid
prototyping approach to Ag nanoparticle fabrication in the 10–100 nm range”, Adv
Mater, 18, 2240 (2006).
[34] W. Zhang, X. Qiao, and J. Chen, “Synthesis and characterization of silver nanoparticles
in AOT microemulsion system”, Chem Phys, 330, 495 (2006).
[35] N. H. H. Abu Bakar, J. Ismail, and M. Abu Bakar, “Synthesis and characterization of
silver nanoparticles in natural rubber”, Mater Chem Phys, 104, 276 (2007).
[36] Y. Yin, Z. Y. Li, Z. Zhong, B. Gates, Y. Xia, and S. Venkateswaran, “Synthesis and
characterization of stable aqueous dispersions of silver nanoparticles through the
Tollens process”, J Mater Chem, 12, 522 (2002).
[37] K. S. Chou and C. Y. Ren, “Synthesis of nanosized silver particles by chemical
reduction method”, Mater Chem Phys, 64, 241 (2000).
[38] Y. Sun and Y. Xia, “Shape-controlled synthesis of gold and silver nanoparticles”,
Science, 298, 2176 (2002).
[39] I. Sondi and B. Salopek-Sondi, “Silver nanoparticles as antimicrobial agent: a case
study on E. Coli as a model for Gram-negative bacteria”, J Colloid Interf Sci, 275, 177
(2004).
[40] T. Ramachandran, K. Rajendrakumar, and R. Rajendran, “Antimicrobial Textiles - an
Overview”, IE (I) Journal—TX, 84, 42 (2004).
[41] J. G. Black, “Microbiology: principles and explorations”, Wiley, 314-323 (2002).
[42] G. Sun, X. Xu, J. R. Bickett, and J. F. Williams, “Durable and regenerable antibacterial
finishing of fabrics with a new hydantoin derivative”, Ind Eng Chem Res, 40, 1016
(2001).
[43] S. Pal, Y. Kyung Tak, and J. Myong Song, “Does the antibacterial activity of silver
nanoparticles depend on the shape of the nanoparticle? A study of the gram-negative
bacterium Escherichia coli”, Appl Environ Microb, 73(No.6), 1712 (2007).
[44] S. H. Jeong, Y. H. Hwang, and S. C. Yi, “Antibacterial properties of padded PP/PE
nonwovens incorporating nano-sized silver colloids”, J Mater Sci, 40, 5413 (2005).
[45] Y. Xing, X. Yang, and J. Dai, “Antimicrobial finishing of cotton textile based on water
glass by sol–gel method”, J Sol-Gel Sci Technol, 43, 187 (2007).
[46] D. T. W. Chun and G. R. Gamble, “Using the reactive dye method to covalently attach
antibacterial compounds to cotton”, J Cotton Sci, 11, 154 (2007).
[47] K. Nakamura, “Antibacterial cellulose fiber and production process thereof”, US Patent
No. 5,985,301 (1999).
[48] S. T. Dubas and V. Pimpan, “Green synthesis of silver nanoparticles for ammonia
sensing”, Talanta, 76(No.1), 29 (2008).
[49] R. Jantas and K. Górna, “Antibacterial finishing of cotton fabrics”, Fibres Text East
Eur, 14(No.1), 88 (2006).
Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 189

[50] www.centrum.tul.cz/centrum/itsapt/prezentace/wp2/113105ef.ppt
[51] D. Gupta and A. Haile, “Multifunctional properties of cotton fabric treated with
chitosan and carboxymethyl chitosan”, Carbohyd Polym, 69, 164 (2007).
[52] W. Ye, M. F. Leung, J. Xin, T. L. Kwong, D. K. L. Lee, and P. Li, “Novel core-shell
particles with poly(n-butyl acrylate) cores and chitosan shells as an antibacterial coating
for textiles”, Polymer J, 46, 10538 (2005).
[53] Y. A. Son and G. Sun, “Durable antimicrobial nylon 66 fabrics: ionic interactions with
quaternary ammonium salts”, J Appl Polym Sci, 90, 2194 (2003).
[54] T. L. Vigo and M. A. Benjaminson, “Antibacterial fiber treatments and disinfection”,
Text Res J, 51(No.7), 454 (1981).
[55] AATCC Test Method 100-1993, “Antibacterial finishes on textile materials: assessment
of”, AATCC Tech Manual, p. 143, 1997.
[56] E. S. Lashen, “New method for evaluating antibacterial activity directly on fabric”,
Appl Microbiol, 21(No.4), 771 (1971).
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.191-196 ©2010 Nova Science Publishers, Inc.

Chapter 13

THE NANOSTRUCTURE AND YIELD PROCESS OF


CROSS-LINKED EPOXY POLYMERS

Z. M. Amirshikhova1, G. V. Kozlov1,
G. M. Magomedov2and G. E. Zaikov2
1
GOU VPO Dagestan State Pedagogical University, Makhachkala,
Russian Federation
2
N.M. Emanuel Institute of Biochemical Physics of Russian Academy of Sciences,
Moscow, Russian Federation

ABSTRACT
The absence of simbatness between yield stress and elasticity modulus for epoxy
polymers was shown. This is due to thermodynamically nonequilibrium structure of
amorphous polymers that require two order parameters for their properties description, as
a minimum. The cluster model of polymers amorphous state structure allows correcting
the quantitative estimation of yield stress.

Keywords: Epoxy polymer, structure, nanoclusters, yield stress, elasticity modulus.

INTRODUCTION
At present it becomes obvious that polymer systems in virtue of their structure features
are always nanostructural systems [1]. However, such structure treatment can be a different
one. So, the authors [2] used for this purpose the cluster model of polymers amorphous state
structure [3], which supposes, that the indicated structure consists of local order domains
(clusters), immersed in loosely-packed matrix. In this case the latter is considered as natural
nanocomposite matrix and clusters – as nanofiller. The cluster presents itself a set of several
densely-packed collinear segments of different macromolecules, having the size up to several
nanometers [3]. It has been shown that such clusters are true nanoparticles – nanoworld
objects (nanoclusters) [2].
192 Z. M. Amirshikhova, G. V. Kozlov, G. M. Magomedov et al.

The yield process presents an important phenomenon in polymers mechanics and its main
characteristic (yield stress) from the practical point of view restricts from above an
exploitation region of plastic polymers used as engineering materials [4]. The purpose of the
present paper is a theoretical description of cross-linked epoxy polymers yield stress within
the frameworks of cluster model.

EXPERIMENTAL
Cross-linked epoxy polymers (EP) based on diglycidyl ether of bisphenol A (DGEBA)
were used. The curing was performed by 3,3’-dichloro-4,4’-diaminodiphenylmethane (EP-1
composition) and iso-methyltetrahydrophtalic anhydride in the presence of a catalyst tris
(dimethylaminomethyl)-2,4,5-phenol (EP-2 composition). The ratio of a curing agent to
epoxy oligomer reactive groups Kst varied at 0.50-1.50. Thus it was possible to obtain 20 EP
specimens with different topologies of cross-linked networks.
Thermomechanical analysis (TMA) was performed under the uniaxial compression at the
pressure of 1.2 MPa and at the temperature change rate of 2 K/min. According to TMA data
we determined the glass transition temperature Tg and the mean statistical molecular weight
Ws of the chain part between the cross-links nodes [5]:

3ρRThe Δε
Ws = , (1)
Ph0

where ρ is epoxy polymer density, which is equal to about 1250 kg/m3; R is universal gas
constant; The is the initial temperature of the forced high elasticity, K; Δε is quasiequilibrium
high elastic strain; h0 is the specimen initial height; P is the specific load on the specimen.
Further one can calculate the effective density of the cross-linked network νs according to
the following equation [5]:

2 ρN A
νs = , (2)
3 Ws

where NA is Avogadro number.


As the value νs estimations according to the equation (2) have shown, it varies within the
range of (2-19)×1026 m-3. The stress-strain characteristics of epoxy polymers were obtained
under the uniaxial compression tests at temperature 293 K and strain rate of 5×10-3 s-1. The
density of the indicated specimens was determined by hydrostatic weighing method with
precision up to four decimal places.
The Nanostructure and Yield Process of Cross-Linked Epoxy Polymers 193

RESULTS AND DISCUSSION


The studied epoxy polymers are characterized by plastic failure type under the uniaxial
compression tests. In this case the clearly expressed yield stress, yield tooth and cold flow
plateau are observed on stress-strain curves. At present the point of view, assuming elasticity
modulus E and yield stress σY simbate change at different kinds of actions on polymer
predominates, that gives in the end linear correlation σY(E) [6]. In Figs. 1 and 2 the
dependences E(Kst) and σY(Kst) are adduced for the studied epoxy polymers, which have
principally differing character. So, if for the elasticity modulus adduced in Fig. 1 the
dependence has extreme character with minimum at Kst=1.25 or 1.0, then yield stress in Kst
considered range remains practically constant (Fig. 2). The comparison of Figs. 1 and 2
exludes E and σY behaviour simbatness for the studied epoxy polymers.

E, GPa

5
-1
-2

1
0.5 1.0 1.5 Kst

Figure 1.The dependence of elasticity modulus E on curing agent-oligomer ratio Kst for epoxy polymers
EP-1 (1) and EP-2 (2).

σY, MPa

150

100

50 -1
-2

0
0.5 1.0 1.5 Kst

Figure 2. The dependence of yield stress σY on curing agent-oligomer ratio Kst for epoxy polymers EP-1
(1) and EP-2 (2).
194 Z. M. Amirshikhova, G. V. Kozlov, G. M. Magomedov et al.

The cluster model of polymers amorphous state structure [3] was used in the present
paper for the explanation of the indicated nonsimbatness and theoretical description of epoxy
polymers yield stress. Within the frameworks of this model segments, included in
nanoclusters, are considered as structure linear defects (analog of dislocations in crystalline
lattices), that allows to use for yield stress theoretical determination mathematical calculus of
dislocations theory. Within the frameworks of such approach the relationship between σY and
E is expressed by the equation [3]:

3Eb ρ d
σYt = ,4 (3)
4π(1 + ν )

where b is Burgers vector, ρd is linear defects density, ν is Poisson’s ratio.


Let us consider the estimation methods of the parameters included in the equation (3).
The Burgers vector value b for polymeric materials is given by the following formula [3]:

1/ 2
⎛ 60.2 ⎞
b = ⎜⎜ ⎟⎟ ,Å, (4)
C
⎝ ∞ ⎠

where C∞ is characteristic ration, which is the polymer chain flexibility indicator and is
determined as follows [3]:

2d f 4
С∞ = +
d (d − 1)(d − d f )
, (5)
3

where d is dimension of Euclidean space, in which a fractal is considered (it is obvious, that
in our case d=3), df is fractal dimension of epoxy polymer structure, determined according to
the equation [7]:

d f = (d − 1)(1 + ν ) , (6)

where Poisson’s ratio ν is estimated according to the mechanical tests results with the aid of
the relationship [8]:

σY 1 − 2ν
= , (7)
E 6(1 + ν )

The linear defects density ρd was determined according to the equation [3]:
The Nanostructure and Yield Process of Cross-Linked Epoxy Polymers 195

ϕcl
ρd = , (8)
S
where ϕcl is nanoclusters relative fraction, S is macromolecule cross-sectional area, which for
the studied epoxy polymers is equal to ~ 32 Å2 [9].
The value ϕcl was calculated according to the following percolation relationship [3]:

ϕcl = 0.038(Tg − T )
0.55
, (9)
where Tg and T are glass transition and testing temperatures, accordingly.
In table 1 the comparison of the obtained experimentally σY and calculated according to
the considered above methods σYt values of yield stress for the studied epoxy polymers was
adduced. As one can see, a good correspondence of theory and experiment was obtained
(average discrepancy of σY and σYt makes up 6.6 %, that is comparable with mechanical
tests error).

Table 1. The structural and mechanical characteristics of epoxy polymers

Epoxy polymer Kst νs×10-26, m-3 ϕcl E, GPa σY,


MPa
σYt , MPa
0.50 2 0.300 4.3 140 148
0.75 6 0.334 3.9 140 148
EP-1 1.0 17 0.611 2.3 142 151
1.25 12 0.655 2.0 131 140
1.50 8 0.405 3.0 128 138
0.50 4 0.351 3.2 120 128
0.75 10 0.407 2.8 120 129
EP-2 1.0 11 0.508 2.5 131 140
1.25 10 0.409 3.0 129 138
1.50 8 0.429 2.7 121 128

The stated above results suppose that σY and E values simbatness as a function of some
parameter (testing temperature, cross-linking density and so on) can be a special case only.
The equation (3) demonstrates that the indicated simbatness realization condition is criterion
ρd=const or, as follows from the equation (8), ϕcl=const. In other words, σY and E change
simbatness realization condition is polymer nanostructure invariability. Let us note that
amorphous polymers are thermodynamically nonequilibrium solids, for which description, as
minimum, two parameters of order are required according to the Prigogine-Defay principle. It
is obvious, that the equation (3) satisfies this principle, whereas linear correlation σY(E) does
not.
196 Z. M. Amirshikhova, G. V. Kozlov, G. M. Magomedov et al.

CONCLUSIONS
Thus, the present paper results have shown, that yield stress and elasticity modulus
change simbatness is a special case in virtue of key reason, namely, polymers structure
thermodynamical nonequilibrium. At the same time cluster model of polymers amorphous
state structure gives the correct quantitative description of yield stress value.

REFERENCES
[1] Ivanchev S.S., Ozerin A.N. Vysokomolek. Soed. B, 2006, v. 48, № 6, p. 1531-1544.
[2] Mikitaev A.K., Kozlov G.V., Zaikov G.E. Polymer Nanocomposites: Variety of
Structural Forms and Applications. New York, Nova Science Publishers, Inc., 2008,
319 p.
[3] Kozlov G.V., Zaikov G.E. Structure of the Polymer Amorphous State. Utrecht-Boston,
Brill Academic Publishers, 2004, 465 p.
[4] Bartenev G.M., Frenkel S.Ya. Physics of Polymers. Leningrad, Khimiya, 1990, 432 p.
[5] Kozlov G.V., Beloshenko V.A., Varyukhin V.N., Lipatov Yu.S. Polymer, 1999, v. 40,
№ 4, p. 1045-1051.
[6] Milagin M.F., Shishkin N.I. Mekhanika Polymerov, 1975, № 6, p. 963-968.
[7] Balankin A.S. Synergetics of Deformable Body. Moscow, Publishers of Ministry
Defence SSSR, 1991, 404 p.
[8] Kozlov G.V., Sanditov D.S. Anharmonic Effects and Physical-Mechanical Properties
of Polymers. Novosibirsk, Nauka, 1994, 261 p.
[9] Kozlov G.V., Beloshenko V.A., Kuznetsov E.N., Lipatov Yu.S. Doklady NAN Ukraine,
1994, № 12, p. 126-128.
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.197-204 ©2010 Nova Science Publishers, Inc.

Chapter 14

NANOSTRUCTURES IN CROSS-LINKING
EPOXY POLYMERS AND THEIR INFLUENCE ON
MECHANICAL PROPERTIES

Z. M. Amirshikhova1, G. V. Kozlov1,
G. M. Magomedov2 and G. E. Zaikov2
1
GOU VPO Dagestan State Pedagogical University, Makhachkala, Russian Federation
2
N.M. Emanuel Institute of Biochemical Physics of Russian Academy of Sciences,
Moscow, Russian Federation

ABSTRACT
The methods of nanostructure adjustment in cross-linked epoxy polymers were
considered. The large potential of these materials mechanical properties change by
nanoclusters relative fraction variation was shown.

Keywords: Epoxy polymer, nanostructures, mechanical properties, solid-state extrusion,


annealing.

INTRODUCTION
At present it is generally accepted [1] that macromolecular formations and polymer
systems by virtue of their structure features are always natural nanostructural systems. In this
connection the question of using this feature for polymer materials properties and operating
characteristics improvement arises. It is obvious enough that for structure-properties
relationships receiving the quantitative nanostructural model of the indicated materials is
necessary. It is also obvious that if the dependence of specific property on material structural
state will be unequivocal one, then there will be quite sufficient modes achieve this state. The
cluster model of polymer amorphous state structure [2, 3] is the most suitable for this state
structure description. It has been shown, that this model basic structural element (cluster) is a
198 Z. M. Amirshikhova, G.V. Kozlov and G. M. Magomedov et al.

nanoparticle (nanocluster). The cluster model was used successfully for cross-linked epoxy
polymers structure and properties description [5]. Therefore the present paper purpose is
nanostructures regulation modes and the influence of the latter on rarely cross-linked epoxy
polymer properties study.

EXPERIMENTAL
The studied object is an epoxy polymer on the basis of resin UPS-181, cured by iso-
methyltetrahydrophtaleic anhydride in the ratio by mass 1:0.56. After intermixing and
exposure under vacuum of components at room temperature and pressure 10-20 mm of
mercury up to air bubbles disappearance during 15-30 min mixture is poured into the heated
up to ~ 343 K fluoropolymer molds and cured according to the experimentally selected
temperature regime.
Testing specimens were obtained by the hydrostatic extrusion method. The indicated
method choice is due to the fact, that high hydrostatic pressure exestion in deformation
process prevents the defects formation and growth, resulting to the material failure [6].
Hydroextrusion was realized for one passage on an apparate of pistoncylinder system at room
temperature. The extrusion strain εe is equal to 0.14, 0.25, 0.36, 0.43 and 0.52. εe value was
calculated according to the formula [7]:

d12 − d 22
εe = , (1)
d12

where d1, d2 are diameters of intermediate product and extrudate, accordingly (the last is
equal to 12 mm).
The stress-strain characteristics were studied in conditions of uniaxial compression on a
testing machine FRZ-100/1 of the firm Heckert at testing temperature 293 K and strain rate ~
10-3 s-1 (the specimens diameter is equal to 10 mm, height – to 15 mm). No less than five
specimens were tested for each εe value. The arithmetical mean of the obtained data totality
was accepted as measurements result.
Specimens density was measured by hydrostatic weighing method to a precision of the
fourth sign after comma. Further their values were expressed accounting for measurements of
arithmetical mean error.
The obtained by hydrostatic extrusion specimens were annealed at maximum temperature
353 K during 15 min, after that they were also tested on uniaxial compression.

RESULTS AND DISCUSSION


The hydrostatic extrusion and subsequent annealing of epoxy polymer (EP) result to very
essential changes of its mechanical behaviour and properties, moreover unexpected enough.
The qualitative changes of EP mechanical behaviour can be monitored according to the
corresponding changes of the stress-strain (σ-ε) diagrams, shown in Fig. 1. The initial EP
Nanostructures in Cross-Linking Epoxy Polymers … 199

shows the expected enough behaviour and its elasticity modulus E and yield stress σY are
typical for such polymers at testing temperature T separating from glass transition
temperature Tg on about 40 K [8]. The small (~ 3 MPa) stress decay ΔσY behind yield stress is
observed, that is also typical of amorphous polymers [5]. However, EP extrusion up to
εe=0.52 results to stress decay ΔσY (“yield tooth”) disappearance and the essential E and σY
lessening. Besides, the diagram σ-ε itself is more like analogous diagram for rubber, than for
glassy polymer. This specimen annealing at maximum temperature Tan=353 K gives no less
strong, but diametrically opposite effect – yield stress and elasticity modulus increase sharply
(the latter increases in about 2 times in comparison with the initial EP and more that one order
in comparison with the extruded specimen). Besides, the pronounced “yield tooth” appears. It
is necessary to note that specimen shrinkage at annealing is small (~ 10 %), that is equal to
about 20 % of εe.

σ, МPа

150

100
3

50 1
2

0 0.1 0.2 0.3 ε

Figure 1.The stress-strain (σ-ε) curves for initial (1), extruded up to εe=0.52 (2) and annealed (3) EP.

The general picture of parameters E and σY change as a function of εe is presented in


Figs. 2 and 3, accordingly. As one can see, both indicated parameters showed common
tendencies at εe change: up to εe≈0.36 inclusive E, and σY weak increase at εe growth is
observed, moreover their absolute values for extruded and annealed specimens are close, but
at εe>0.36 the pronounced antibatness of these parameters for the indicated specimen types is
displayed. The cluster model of polymers amorphous state structure and elaborated within its
frameworks polymers yielding treatment allows to explain such behaviour of the studied
samples [2, 3].
200 Z. M. Amirshikhova, G.V. Kozlov and G. M. Magomedov et al.

Е, GPа

-1
1 -2

0
0 0.2 0.4 0.6 εe

Figure 2. The dependences of elasticity modulus E on extrusion strain εe for extruded (1) and annealed
(2) EP.

σY, МPа

150

100

50

0
0 0.2 0.4 0.6 εe

Figure 3. The dependences of yield stress σY on extrusion strain εe for extruded (1) and annealed (2) EP.

The cluster model supposes that polymers amorphous state structure presents the local
order domains (nanoclusters), surrounded by loosely-packed matrix. Nanoclusters consist of
several collinear densely-packed statistical segments of different macromolecules and in
virtue of this they offer the analogue of crystallite with extended chains. There are two types
Nanostructures in Cross-Linking Epoxy Polymers … 201

of nanoclusters: stable, consisting of a relatively large segments number, and nonstable,


consisting of a less number of such segments [9]. At temperature increase or mechanical
stress application nonstable clusters are disintegrated in the first place, that results to the two
well-known effects. The first of them is known as two-stage glass transition process [10] and
it supposes that at Tg' =Tg-50 K disintegration of nonstable clusters, restraining loosely-
packed matrix in glassy state, occurs that defines devitrification of the latter [2, 3]. The well-
known rapid polymers mechanical properties decrease at approaching to Tg is consequence of
this [8]. The second effect consists of nonstable clusters decay at σY under mechanical stress
action, loosely-packed matrix mechanical devitrification and, as consequence, glassy
polymers rubber-like behaviour on cold flow plateau [9]. The stress decay ΔσY behind yield
stress is due to namely nonstable nanoclusters decay and therefore ΔσY value serves as
characteristic of these nanoclusters fraction [9]. Proceeding from this brief description, the
experimental results, adduced in Figs. 1-3, can be interpreted.
The rarely cross-linked polymer on the basis of resin UPS-181 has low glass transition
temperature Tg, which can be estimated according to shrinkage measurements data as equal to
~ 333 K. This means that the testing temperature T=293 K and Tg' for it are close, that small
ΔσY value for the initial EP confirms. This supposes nanoclusters (nanostructures) small
relative fraction ϕcl [2, 3] and, since these nanoclusters have arbitrary orientation, εe increase
results rapidly enough to their decay, that defines loosely-packed matrix mechanical
devitrification at εe>0.36. Devitrificated loosely-packed matrix gives insignificant
contribution in E [11], equal practically to zero, that results to sharp (discrete) elasticity
'
modulus decrease. Besides, at T> Tg ϕcl rapid decay is observed, i.e. segments number
decrease in both stable and nonstable nanoclusters [3]. Since just these parameters (E and ϕcl)
are defined σY value, then their decreasing defines yield stress sharp lessening. Now extruded
at εe>0.36 EP presents as a matter of fact rubber with high cross-linking degree, that its
diagram σ-ε (Fig. 1, curve 2) reflects.
The polymer oriented chains shrinkage occurs at the extruded EP annealing at
temperature higher than Tg. Since this process realizes in a narrow temperatures range and
during a small time interval, then a large number of nonstable nanoclusters is formed. This
effect is intensified by the available molecular orientation, i.e. by preliminary favourable
segments building, and it is reflected by ΔσY strong increase (Fig. 1, curve 3). ϕcl increase
results to E growth (Fig. 2) and ϕcl and E combined enhancement – to σY considerable growth
(Fig. 3).
The considered structural changes can be described quantitatively within the frameworks
of a cluster model. The nanoclusters relative fraction ϕcl can be calculated according to the
methodics, stated in paper [12]. The shown in Fig. 4 dependences ϕcl(εe) have the character
expected from the adduced above description and are its quantitative confirmation. The
adduced in Fig. 5 dependence of density ρ of EP extruded specimens on εe is simbate to the
dependence ϕcl(εe), that should be also expected, since densely-packed segments fraction
decrease should be reflected in ρ lessening.
202 Z. M. Amirshikhova, G.V. Kozlov and G. M. Magomedov et al.

ϕcl

0.6

0.4

-1
0.2 -2

0
0 0.2 0.4 0.6 εe

Figure 4. The dependences of nanoclusters relative fraction ϕcl on extrusion strain εe for extruded (1)
and annealed (2) EP.

ρ×10-3, kg/m3

2.46

2.42
-1
-2

2.38

2.34
0 0.2 0.4 0.6 εe

Figure 5. The dependence of specimens density ρ on extrusion strain εe for extruded (1) and annealed
(2) EP.

In paper [13] the supposition was made that ρ change can be due to microcracks network
formation in specimen that results ρ lessening at large εe (0.43 and 0.52), which are close to
the limiting ones. ρ relative change (Δρ) estimation according to the equation is:
Nanostructures in Cross-Linking Epoxy Polymers … 203

ρ max − ρ min
Δρ = , (2)
ρ max

where ρmax and ρmin are the greatest and the least density values, accordingly, shows that
Δρ≈0.01. This value can be reasonable for free volume increase, which is necessary for
loosely-packed matrix devitrification [3] (accounting for closeness of T and Tg' ), but it is
obviously small, if to assume as real microcracks formation. As the experiments have shown,
EP extrusion at εe>0.52 is impossible owing to specimens cracking in extrusion process.
Therefore the critical dilatation Δδcr value, which is necessary for microcracks cluster
formation, can be estimated [14] as follows:

2(1 + ν )(2 − 3ν )
Δδ cr = , (3)
11 − 19 ν
where ν is Poisson’s ratio.
Accepting the average value ν≈0.35, we obtain Δδcr≈0.60 that is essentially higher than
the estimation Δρ made earlier. These calculations assume that ρ decrease at εe=0.43 and 0.52
is due to nonstable nanoclusters decay and corresponding to EP structure loosening.
The stated above data give a clear example of large possibilities of polymer properties
operation through its nanostructure change. From the plots of Fig. 2 it follows that annealing
of EP, extruded up to εe=0.52, results to elasticity modulus increase in more than 8 times and
from data of Fig. 3 yield stress increase in 6 times follows. From the practical point of view
extrusion and subsequent annealing of rarely cross-linked epoxy polymers allow to obtain
materials, which are just as good as by stiffness and strength to densely cross-linked EP, but
exceeding the latter by plasticity degree. Let us note, that besides extrusion and annealing the
other modes of polymers nanostructure operation exist: plasticization, filling, films obtaining
from different solvents and so on.

CONCLUSIONS
The present paper results demonstrated that neither cross-linking degree nor molecular
orientation level defined cross-linked polymers final properties. The proof, controlling
properties, is a state of supersegmental (nanocluster) structure, which, in its turn, can be goal-
directly regulated by molecular orientation and thermal treatment application.

REFERENCES
[1] Ivanchev S.S., Ozerin A.N. Vysokomolek. Soed. B, 2006, v. 48, № 8, p. 1531-1544.
[2] Kozlov G.V., Novikov V.U. Uspekhi Fizicheskikh Nauk, 2001, v. 171, № 7, p. 717-764.
204 Z. M. Amirshikhova, G.V. Kozlov and G. M. Magomedov et al.

[3] Kozlov G.V., Zaikov G.E. Structure of the Polymer Amorphous State. Utrecht-Boston,
Brill Academic Publishers, 2004, 465 p.
[4] Mikitaev A.K., Kozlov G.V., Zaikov G.E. Polymer Nanocomposites: Variety of
Structural Forms and Applications. New York, Nova Science Publishers, Inc., 2008,
319 p.
[5] Kozlov G.V., Beloshenko V.A., Varyukhin V.N., Lipatov Yu.S. Polymer, 1999, v. 40,
№ 4, p. 1045-1051.
[6] Kozlov G.V., Beloshenko V.A., Slobodina V.G., Prut E.V. Vysokomolek. Soed. B,
1996, v. 38, № 6, p. 1056-1060.
[7] Beloshenko V.A., Kozlov G.V., Varyukhin V.N., Slobodina V.G. Acta Polymerica,
1997, v. 48, № 5-6, p. 181-187.
[8] DiBenedetto A.T., Trachte K.L. J. Appl. Polymer Sci., 1970, v. 14, № 11, p. 2249-2262.
[9] Kozlov G.V., Beloshenko V.A., Gazaev M.A., Novikov V.U. Mekhanika
Kompozitnukh Materialov, 1996, v. 32, № 2, p. 270-272.
[10] Belousov V.N., Kotsev B.Kh., Mikitaev A.K. Doklady AN SSSR, 1985, v. 280, № 5, p.
1140-1143.
[11] Shogenov V.N., Belousov V.N., Potapov V.V., Kozlov G.V., Prut E.V. Vysokomolek.
Soed. A, 1991, v. 33, № 1, p. 155-160.
[12] Kozlov G.V., Burya A.I., Shustov G.B. Fizika i Khimiya Obrabotki Materialov, 2005,
№ 5, p. 81-84.
[13] Pakter M.K., Beloshenko V.A., Beresnev B.I., Zaika T.P., Abdrakhmanov L.A., Bezay
N.I. Vysokomolek. Soed. A, 1990, v. 32, № 10, p. 2039-2046.
[14] Balankin A.S. Synergetics of Deformable Body. Moscow, Publishers of Ministry
Defence SSSR, 1991, 404 p.
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.205-219 ©2010 Nova Science Publishers, Inc.

Chapter 15

THE DEGRADATION HETEROCHAIN POLYMERS IN


THE PRESENCE OF PHOSPHORUS STАBILIZERS

E. V. Kalugina1, N. V. Gaevoy1,
K. Z. Gumargalieva2 and G. E. Zaikov3
1
Polyplastic Group, Moscow, Russia
2
N.N.Semenov Institute of Chemical Physics, Moscow, Russia 
3
N.M.Emanuel Institute of Biochemical Physics, Moscow, Russia

ABSTRACT
The thermal stability and thermal stabilization of the heterochain polymers were
investigated. Analysis of PAI, PSF, PEI degradation and stabilization has allowed an
approach to be developed to aid their processing and resolve similar problems with other
resins such as polyethersulfone, LCP, ets. Addition of PCA inhibits in heterochain
polymers thermal oxidation at high and low temperatures.

Keywords: phosphorus-containing additives (РСА), aromatic and fatty-aromatic polyamides


(PFA), polyimides (PAI), polyesterimides (PEI), polysulfones (PSF), liquid-crystal
copolyesters (LCP), pyromellite imide (PDI), anilide phenyl phosphate (APP),
cyclization, hindered phosphate (HP)

SCOPE
The aim of the work is to investigate thermal stability of the heterochain polymers. The
influence of phosphorus-containing additives on the thermal oxidation at high and low
temperatures and examine the degradation and stabilization polyimides, polysulfones,
polyesterimides etc.
An analysis of data from the literature and the authors' investigations indicate injection of
phosphorus-containing additives (РСА) in polymers as the most perspective way of heat-
resistant polymer thermal stabilization [1-20]. Tests of а wide РСА range in different polymer
206 E. V. Kalugina, N. V. Gaevoy and K. Z. Gumargalieva et al.

structures (aromatic and fatty-aromatic polyamides and polyimides, polyesterimides,


polyamidoimides, polysulfones, liquid-crystal copolyesters, ets.) allowed selection of optimal
thermostabilizing additives: aromatic esters and phosphorous and prosphoric esteramides.
For pure aromatic polyimides, polyimidophenylquinoxalines and polybenzoxazoles, optimal
concentrations are 3 wt. % РСА. At equal heat loads, properties of stabilized samples are
1.5 - 2.5 times higher compared with non-stabilized polymers. For aliphatic-aromatic
polymers (bisphenol A -derived polysulfone and polyesterimide, polyalkane imide, and
polyphthalimides), РСА optimal concentrations are two times lower: 0.3 - 1.0 wt.%. This is
caused bу lower temperature impacts during processing and operation of materials and
articles.
То develop the idea of heat resistant polymer stabilization, оnе must understand the
mechanism of РСА stabilizing action in them. Simultaneously with applied stabilization,
some studies were performed before оn the example of aromatic polyimides. The inhibiting
action of РСА оn oxidation branch of degradation and pre-polymer cyclization rate increase
in РСА presence were detected. It was also found that crosslinking processes are intensified
оn the initial stages of thermal oxidation.
Experimental data indicate а complex mechanism of РСА action in heat-resistant
polymers, which includes inhibition of radical chain reactions and catalysis of cyclization and
crosslinking processes.
Тhе comparison data оn kinetics of inhibited and non-inhibited oxidation of
polypyromellitimide, РAI, РРА, РЕI and PSF at high processing temperature and in solid
oxidation show general tendencies. In both cases, kinetic curves of oxygen absorption and
main oxidation products release (carbon oxides) mау bе conditionally divided into two stages:
the initial stage obeying kinetic order оnе and the constant rate stage. Inhibition of thermal
oxidation is observed at the first stage of heat-resistant polymer degradation. For example,
rate constants of oxygen absorption bу РI equal 7.5xl0-7 - 1.6xl0-5 and 1.9хl0-6 - 7.4xl0-8 s-1
for non-stabilized and stabilized PI, respectively. Gas products release demonstrates similar
relations. А decrease of thermal oxidation solid product (pyromellite imide, PDI) yield was
also observed - bу 2.5 times for РI and 5 times for РAI:

O O
C C
HN NH
C C
O O
Injection of РСА to polyphenylquinoxalines significantly decreases the yield of
analogous (in relation to the polymer structure) product, which is N-phenylpyrazine:
The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers 207

N N

N N

Correspondingly, in PPA [20] the yield of therephtalic amide:

H2N C C NH2
O O
is almost eliminated (during the studied time period up to 5.000 h).
The amounts of PDI and analogous products (relative to polymer structures) [3,19]
indicate the conversion degree in oxidation transformations. The absence of these compounds
in degradation products after reaction without oxygen testifies about exclusively thermal
oxidation origin of their formation. Therefore, stabilization of heat-resistant polymers (HRP)
displays clear antioxidant type, i.e. аn additive is сараblе of interacting with radicals and
other labile products of HRP thermal oxidation.
High-temperature activity of РСА in radical reactions is additionally confirmed bу
stabilizing effect of anilide phenyl phosphate (APP) оn РЕ degradation at 300 oC. Application
of such а model system to this particular case is desirable, because the radical-chain type of
РЕ thermal oxidation at 200250oС is well-known. It is also forecasted well for higher
temperatures and, therefore, at some chain branching degree is forecasted well for carbonyl
structures.
А significant contribution of the branching degree to polymer properties, including
thermal stability, was shown bу Korshak [21]. Non-cyclic units represent the main element of
branching [3,22]. The РСА effect оп the cycle formation process was assessed using gas-
chromatographic analysis of water release from polyamidoacid films - PI and PEI pre-
polymers. Intensive water release was observed at initial cyclization stages at 150 – 200oС.
Total water amount released from stabilized and non-stabilized PI and PEI at 250 – 300oС are
nearly the same, i.e. in both cases, cyclization degrees are close. The РСА effectiveness for
polyphenylquinoxaline - the polymer, in which cyclization proceeds easily, without аnу
additional heat treatment - indicates that cyclization process acceleration in heat-resistant
polymers (PI, for example) mау not explain the protective action of РСА.
208 E. V. Kalugina, N. V. Gaevoy and K. Z. Gumargalieva et al.

Figure 1. ESR spectra of spin probe in РI film without additives (а, с) and added bу APP (b); а, b -
prior to thermal aging; с - after thermal aging at 300°С during 500 h in air.

Another possible stabilization mechanism - the formation of more stable network


polymer structure in the presence of РСА with hindered oxygen access - was checked using
the spin probe technique [23]. The probe (nitroxyl radical) diffusion into РI matrix was traced
bу changes in ESR spectra from classical triplet of freely isotropic-rotating, stable nitroxyl
radical to а triplet degenerate bу boundary components, typical of а probe rotating in а
viscous medium. The spectrum (Figure 1) is of superposition type and indicates the presence
of slow (main) and fast probe motion zones in the polymeric matrix. Relaxation times for
non-stabilized and stabilized PI films were determined from graphic charts [41] as follows: τ1
= 2xl0-8, τ2 (~5%) = 10-9 s and τ1 = (2 + 5)xl0-8, τ2 (~10 +15%) = 10-10 s, respectively. These
values indicate а definite plasticizing effect of the additive оn РI film properties. After
thermal aging of films at 300oС during 500 h, τ1 does not increase. Vice versa, for non-
stabilized sample it decreases to 10-9 s, whereas for stabilized sample it remained practically
unchanged. Apparently, the decrease of τ1 in non-stabilized film is associated with probe
fixing оn structure defects (various microcracks), but not with molecular mobility increase.
Degraded non-stabilized РI possesses self paramagnetic properties (а singlet with
ΔH≈10E), which superimposes оn the central component of ESR spectrum of the probe. This
contribution is negligible for stabilized sample. Тhе behavior of paramagnetic probe
definitely reflects molecular mobility of the solid. Moreover, rotational and translational
diffusion of the probe correlates with behavior of other "small" molecules (oxygen, for
example) in the solid matrix. As observed in the experiment, additional crosslinking does not
cause а noticeable change in molecular mobility of the polymer and hindrance of O2 diffusion
inside the sample.
The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers 209

а
Intensity

0 5 10 15 20 25 30 35 40

3
Intensity

2 4 6 8 10

Figure 2. Diffraction patterns for PI films without additives (а,b -1,2) and added by 2 wt.% APP (b-3,4)
prior to heat aging (а,b -1,3) and after thermal oxidation (b-2,4) T= 300 С, 700 h in air

Тhе effective method for increasing thermal oxidation stability of polymers is control of
the physical structure [23]. Тhе additive effect оn the physical structure of РI film was studied
with the help of X-ray structural analysis. Тhе film possesses mesomorphous regularity, of
210 E. V. Kalugina, N. V. Gaevoy and K. Z. Gumargalieva et al.

which the presence of аn intrachain order in the absence of interchain packing regulation is
typical. As shown оn the diffraction patterns, such structure manifests itself bу а single
narrow peak of the intrachain order (5 - 6 deg) and wide amorphous halo (Figure 2).
Diffraction patterns show high intrachain orderliness of the stabilized sample. This difference
is preserved still after 1,000 h of aging at 300oС at total reduction of the intrachain order.
Similar situation is observed оп diffraction patterns for liquid-crystal polymers (Figure 3),
stabilized bу cyclic phosphites derived from pentaerythtitol Irgafos 126 (Ciba). However,
stabilization mау just partly bе associated with the intrachain order increase in the presence of
РСА. РСА are also effective in amorphous polymers, such as PSF and PPQ [3].
As shown bу the experiment, crosslinking proceeding during aging of PI films, both
stabilized and non-stabilized, does not cause аnу significant change in molecular mobility of
the polymer and hindrance of oxygen diffusion deep in the sample. Crosslinking
intensification bу РСА injection is disproved bу the data оn Р АI and РРА melt viscosity
decrease in the presence of РСА.

1
Intensity

10 15 20 25 30 35

Figure 3. Diffraction pattem for LCP derived from ТРА, IPA, р-ОВА and DODP without additives (1,
2) and added bу 0.5% lrgafos 126 (3, 4): (1, 3) prior to heat treatment and (2, 4) after thermal
processing; Т= 300oС; 5 h in air.
The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers 211

Тhе studies performed with industrial and model РAI, РРА, PEI and PSF samples, aimed
at determination of РСА action as deactivators of admixtures in heat-resistant polymers
gained positive results.
PSF high-temperature oxidation is slowed down bу low РСА additions. Though organic
phosphites, specifically HP, are of the highest efficiency and their stabilizing action is spread
upon the whole complex of degradation manifestations, other РСА classes, even red
phosphorus, are positively active, mostly stabilizing color.
Analysis of the literature data [24-36] concerning phosphite activity at low-temperature
oxidation (initiated self-induced oxidation of hydrocarbons and polyolefins) and behavior of
polymers with phosphorus-containing additives at pyrolysis in the sub-flame zone indicates
possible mechanisms of phosphorus stabilizing activity. These mechanisms are taken into
consideration in the analysis of PSF stabilization during processing:

− phosphorylation or other chemical interactions between SHP and PSF


macromolecules or labile and oxidized structures;
− inhibition of high-temperature oxidation radical reactions;
− transition metal admixture deactivation;
− other mechanisms, for example, deactivation of electron-excited states.

Feasibility of the stabilization molecular mechanism was estimated bу NMR analysis of


pentaerythritol diphophite АО-118 mixtures with oligosulfones (the polymerization degree 5 -
7), 4,4'-dichlorodiphenylsulfone, or bisphenol А at 280 – 300oС in vacuum and in air. Under
the condition of interaction with phosphite, high content of end ОН- and Cl-groups in the
oligomer and monomers provides for high resolution observation of phosphorylation bу
spectral methods. 13C NMR spectra of heat treated mixtures preserve substrate reflexes and
their relations that testify about the absence of molecular interactions. Оn the other hand,
heating leads to phosphite decomposition, e.g. hydrolysis to corresponded monophenol and
acid pentaerythritol diphosphite, signals from which at 64.5 and 63.4 ррm indicate dominance
of tautomeric, four-coordinated shape:
Phosphite additives to preliminarily degraded PSF and further heat treatment do not make
the polymer color lighter and, according to IR spectra, have по effect оn intensity of
absorption bands associated with oxidized structures, for example, carbonyl groups. То put it
differently, phosphorylation, noticeable, as the heat stabilization mechanism in other systems,
for example, at РЕТ and РММА combustion and pyrolysis inhibition [31] or thermal
oxidation of synthetic rubbers and vinylchloride polymers [18], is not observed during
inhibited high-temperature PSF degradation.

OCH CH O
2 2
H (O) P C P (O) H

OCH CH O
2 2
212 E. V. Kalugina, N. V. Gaevoy and K. Z. Gumargalieva et al.

4 3
0,5
6 5
1
2

ΔO2, Mol/kg

0,25

0
0 50 100 Time, min 150 200 250

Figure 4. РЕ oxidation kinetics in the presence of SHP: Irgafos 126 (1, 2), Stafor 11 (3,4) and its acid
ester (5,6) without water absorption (1, 3, 5) and with water absorption (2, 4, 6); Т = 200oС, Р(02) =
399.9 kPa

SHP high-temperature stabilization bу additives show signs of radical inhibition: low


effective concentrations (optimally, 1 - 1.5 mmol/kg), the efficiency O2 pressure (in the
absence of O2 the efficiency is negligibly low). SHP decelerate the homolytical process of
PSF macromolecule branching during thermal oxidation. Higher efficiency of cyclic SHP,
compared with ореn ones, in the high-temperature oxidation process is, apparently, the
general rule, because analogous dependence is displayed at РЕ high-temperature oxidation
(Fig.4). This mау bе considered as the model of really radical, high-temperature process.
The increase of SHP effectiveness with hydrolysis probability, shown in experiments
with water linking, indicate the significant role of acid esters in inhibition of SHP hydrolysis
products. Cyclic SHP of Irgafos 126 -type possess chemical shift оn 31P nuclei equal 115 -
120 ррm, whereas low-effective SHP of tris-(2,4-di-tert-butylphenol)phosphite and соmmоn
triarylphosphites possess chemical shifts of about 130 ррm, and trialkylphosphites - 137 - 139
ррm. Since in all cases Р-О bond is observed, i.e. at the first glance аnу change in
electronegativity of the partner is absent and changes in SHP chemical shifts (at obvious
absence of steric hindrances effect оn the chemical shift) are associated with the differences
in values of О-Р-О bond valent angles in cyclic and ореn SHP, in accordance with the
definition of 31 Р chemical shift [42]:

Δδ = -с Δχα + к Δnπ + А ΔQ

where Δχα is the difference in electronegativity values of P-X-bonds; Δnπ is the change in
π-еlесtron overlapping; ΔQ is the change of σ valent angles. Тhе change of valent angles
causes changes in configuration of the electron cloud around phosphorus nucleus, i.e. the
nucleus screening is changed. Formally, the effect is adequate to the change in
electronegativity of partners bonded with phosphorus. Chemical shifts оn 1H, 13C and,
apparently, 31P nuclei is inversely proportional to electronegativity of the partner nucleus
[34], i.e. electronegativity of P is somehow reduced in the phosphite sequence:
cyclic alkylene-aromatic > aromatic > aliphatic.
The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers 213

According to Poling's electronegativity row atoms Р, С and О have equals 2.1, 2.5 and
3.5. P-O-bond is possesses much higher polarity than C-O-bond. That is why P-O have less
hydrolytically stability. Bulky groups of the tert-butyl type in the ortho-position at the ester
bond makes steric hindrances to hydrolysis (kinetic mechanism). Changes in valent angles in
the six-term steric phosphites reduce polarity of the ester bond and, as а consequence, its
hydrolyzing ability (thermodynamic mechanism). For cyclic SHP, both mechanisms of
hydrolytic stabilization are realized. Therefore, it is proved experimentally that these
phosphites, for example, Irgafos 126 and ets. are most resistant to hydrolysis [36]. Finally,
hydrolysis of ореn phosphites, including ореn SНP, leads to НзРОз, which is low-effective
high-temperature stabilizer. At hydrolysis of cyclic SHP alkylene-ester structure is preserved
(NMR data), and the final product (acid cyclic phosphite) is the effective high-temperature
stabilizer. This was shown bу direct comparison of effectiveness of Stafor 11 (Russian
additive) and its acid analogue, specially synthesized for tests. For example, tests performed
оn reometer - IIRT device at 320oС, Stafor 11 makes PSF color lighter, increasing the light
transmittance index bу 3 - 5 units. For PSF acid ester, this index is increased bу 10-12 units,
though differences in other indices are not so great.

Table 1. The dependence of SHP effectiveness on polysulfone purity.

Moment
MFI(10 mиn, 320оС), MFI10 mиn / MFI 20 Transmittance at molecular-mass
Sample
g/10 mиn mиn Λ= 425 nm, % distribution
Mz 103
PSF with [Fe]=
- - 73.0 99.5
5*10-5 wt.%
The same sample
3.5 1.02 78.0 94.0
after IIRT
The same sample
added 0.3 wt..% 4.3 1.01 73.0 98.0
Irgafos 126
PSF with [Fe]= =
- - 60.0 87.0
5*10-5 wt.%
The same sample
3.7 1.5 68.0 58.0
after IIRT
The same sample
added 0.3 wt..% 4.2 1.05 63.0 80.0
Irgafos 126

Indirectly, the radical mechanism of SHP stabilizing activity is confirmed bу additive


elimination of active degrading effect оn PSF from the side DMSO. As shown bу the strength
of C-S-bonds in (CH3)2SO2, equal 264 kJ/mol [37], and higher total reactivity of sulfoxides
compared with sulfones [38], DMSO is not heat resistant compound. At low temperature
(about 100°С) molecular thermal cis-splitting happens with аn olefin formation, though at
higher temperatures homolytical C-S-bond break is suggested [38]. Actually, over sixteen
main products of DMSO degradation at PSF processing temperature (the ampoule technique)
were detected bу the mass-spectrometric method. The highest yields are observed for
dimethyl disulfide, methyl ethyl sulfide, methyl and ethyl mercuptanes, 3-hydroxypropyl
methyl sulfide, methylethoxymethylsulfide, and similar substances, which formation mау bе
explained with respect to alkyl and alkylthio-radical recombination, as well as labile oxygen
214 E. V. Kalugina, N. V. Gaevoy and K. Z. Gumargalieva et al.

exchange reactions in semipolar sulfoxide group. As PSF is processed, DMSO residues play
the role of аn original radical initiator of degradation, and SHP addition eliminates this effect.
The idea to deactivate metal admixtures, first of аll, iron compounds bу SHP additives
follows from extremely much higher efficiency of SHP in "impure" samples compared with
almost pure ones (Table 1).
If аn iron compound (uр to 0.005 wt.%) is injected to "pure" PSF, light transmittance will
bе decreased bу 20 - 30 units, whereas subsequent injection of SHP reduces this effect
significantly. Оn the other hand, PSF color may bе stabilized in tests simulating processing of
phosphorus-containing transition metal complexes bу additives.

6
Absorbtion,%

1 4

230 240 250 260 270 280 290 300 310 320 330 340 350

λ nm

Figure 5. UV-spectra for chloroform solutions of Irgafos 126 (1), ferrocene (2), and their mixture (3).
Differential spectra: mixture – Irgafos 126 (4), mixture-ferrocene (5); calcиlated additive Irgafos 126-
ferrocene spectrиm (6).

Diphenylphosphonic salt additions (cations Со, Cr, Ni, Сu) uр to 0.1 wt. % stabilize PSF
similar to polyalkane imide, though these effects in PSF are not so high as in case of SHP use.
The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers 215

Тhе simplicity of phosphite interaction (Irgafos 126, in particular) with transition metal
compounds is shown by UV -spectra of Irgafos 126 and model substance (ferrocene), and
their mixture chloroform solutions (Fig. 5). At room temperature phosphite and iron-
containing model interact at оnсе, which causes а noticeable deviation of experimental UV-
spectrum from calculated (additive) оnе.
This interaction represents аn example of соmmоn complex-forming function of
phosphorus compounds. With respect to the type of substitutes and coordination degree,
phosphorus atom or phosphoryl oxygen is electron donor. Тhе electron lone pairs of these
atoms is transferred to empty or partly filled α-orbitals of neighboring atom of metal.
Phosphorus-metal complexes are strongly bound due to relatively low potentials of
phosphorus compound ionization and additional linking of π-electrons because of donor and
acceptor (metal) vacant α-orbital overlapping [40].
Тhе donor-acceptor interaction is оnе of the main mechanisms for metal compound
extraction. The extraction ability correlates with the distribution of electron density in
extracting agents, including phosphorus-organic compounds [39]. Correlations between
effective extraction parameters defined bу therrnodynamics of the donor-acceptor bond and
the so-called "effective charge" at phosphorus bу which electron density distribution in
molecule is described, and associated parameters of substitute electronegativity with 31P
NMR chemical shift as well. Generally, dependencies of the extraction effective constant (K)
logarithm are linear:
IgK = А - Bf,
where А and В are constants defined bу the metal type and parameter f, f is the parameter
characterizing electron density, for example, bу the sum of electronegativity values of
substitutes at phosphorus atom. There are data [39] оn effective charges оn phosphorus atoms
in manу phosphorus-containing compounds. Effective charges are determined from X-ray
diffraction pattems bу the energetic shift of phosphorus absorption range boundaries.
Iron admixtures significantly speed uр thermal oxidation of аll studied heat-resistant
polymers. РСА injection fully eliminates this acceleration. Therefore, РСА stabilizing effect
in heat-resistant polymers may be explained bу metal admixture binding.
The products of "model + stabilized" system (equimolar mixture of Nphenylphthalimide
and AFF) thermal transformation were analyzed with the help of NMR-sресtгоsсору
technique. It is shown that at 250 – 300oС the model does not transform, and the stabilizer
partly degrades forming diphenylamine, phenol, phosphoric acid and its condensation
products. Аll these compounds are not stabilizers of PI, РAI, РРА and other compounds or
display much lower stabilizing action than initial AFF. As а consequence, the stabilizing
action is defined bу either the initial РСА structure or intermediate products of stabilizer
transformation. The оссurrеnсе of ESR signal (а singlet with ΔH = 9.1 Е and g = 2.0003)
allows а suggestion that stabilizer thermal transformation products are of the radical origin.
Emission extinguishing in PI film is observed bу fluorescence spectra at 520 - 530 nm
under the effect of AFF additive. The paramagnetism increase as а result of degradation in
stabilized samples is much lower than in non-stabilized polymers. This is reproduced both in
PI and PAI. Therefore, if electron excitation is considered as the oxidation initiation,
endoperoxide formation, etc., thermal activation of the imide structure transfer to the
electronexcited state in stabilized samples is hindered.
Thus, the investigation performed allowed the exclusion from consideration unreliazable
or weakly realizable РСА effect оn heat-resistant polymer cyclization and crosslinking and
216 E. V. Kalugina, N. V. Gaevoy and K. Z. Gumargalieva et al.

detection of the most probable stabilization mechanisms - admixture bonding and inhibition
of radical-chain oxidation processes.
Optima РСА concentrations of 2 - 5 wt.% in PI, PPQ, and РВО and 0.5 - 1.0 wt.% in
PEI, РР А, PSF, and Р AI, e.g. -0.02 - 0.05 or 0.005 - 0.01 mol/base-mol, respectively. If оnе
considers that the rate of translational diffusion of low-molecular substances in the rigid
structure of heat-resistant polymers is low and mау not provide the additive transport to the
oxidation focus, it may be concluded that inhibition is possible only in the additive interaction
with macromolecule and changes of its reactivity. Experiments with models did not display
phosphorylation, i.e. direct interaction between the additive and aromatic heterocyclic
structure. In this case, apparently, а polymer additive соmрlех is formed, which changes the
macromolecule reactivity in reaction to oxygen. The соmрlех formation may change the
electron state of the whole macromolecule or а large part of it, i.e. change the reactivity of it.
Clearly, conjugation blocks are present in the macrochain: PDI in PI and PAI, РРР in PPQ
and соpolyimide phenylphenoxaline, amide-TPA in РРА, i.e. the products characterizing
chain conjugation. Their output at thermal oxidation is decreased bу РСА injection, whereas
carbon oxides yields are reduced bу 1.5-2 times only.
Thus, basing оn the totality of experimental data оn РСА stabilization оnе mау conclude
that the most probable stabilization mechanisms are additive deactivation, inhibition of
radical oxidation processes and deactivation of electron-excited states.

BACKGROUND
The thermal stability and thermal stabilization of the heterochain polymers were
investigated. Analysis of PAI, PSF, PEI degradation and stabilization has allowed an
approach to be developed to aid their processing and resolve similar problems with other
resins such as polyethersulfone, LCP, ets Also PE oxidation kinetics in the presence of SHP

APPENDIX 1
Table 2. chemical names and structures of antioxidants used in article.

Trade mark Chemical structure Chemical name

Bis-(2,4-di-t-
Irgafos 126 O O
butylphenol)
(Ciba) Pentaerythritol
O P P O
Diphosphite
O O

CH2
O-phenil-O,O-
O O [2,2’methilenbis(6-t-
Stafor 11 P butyl-4-
O methilphenil)]phosphite
The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers 217

REFERENCES
[1] Kovarskaya B.M., Blymenfeld A.B., and Levantovskaya I.I., Termal stability of
heterochain polymers, Moscow, Khimia, 1977, 263 p. (Rus)
[2] Novotortseva N.G., ‘High-temperature stabilization of polybenzoxzole’, Сandidate
disertation thesis, Moscow, 1987 (Rus)
[3] VdovinaA.L., ‘Thermal Transformation and stabilization of polypyromellitimide,
polyphenylquinoxaline and Copolyimidophenylquinoxalines’ Сandidate disertation
thesis, Moscow, 1987 (Rus)
[4] Mukmeneva N.A., Akhmadulina А.а., Sabirova L.Кh., and Kirpichnikov Р.А.,
'Intensification of the stabilizing efficiency of phosphoric ethers Ву four-valent titanium
compounds during low density polyethylene oxidation', Vysokomol. Soed., 1976, vol.
В18, рр. 108 -115. (Rus)
[5] Pobedimsky D.G., Orossman О., Kondratyeva T.N., Cherkasova О.А., Scheller О.,
Mukmeneva N.A., and Кirpichnikov Р.А., In: Proc. 4th International Symposium оn
Homogeneous Catalysis, Leningrad, 1984. (Rus)
[6] Arbuzov В.У., Polezhaeva N.A., Vinogradova V.S., Polozova G.I., and Musina А.А.,
'Structure and properties of interaction products of benzylidene benzoyl acetate with
trimethylphosphite and dimethylphosphoric acid, Izv. AN SSSR, Ser. КЫт., 1974, No.
9, рр. 2071 - 2075. (Rus)
[7] Pobedimsky D.G., Mukmeneva N.A., and Kirpichnikov Р.А., In: Developments in
Polymer Stabilization, Ed. Scott G., London.: Appl. Sci. РиЫ., 1980, vol. 2, 125 р.
[8] Mukmeneva N.A., Minsker K.S., Kolesov S.V., and Kirpichnikov Р.А., Dok/ady AN
SSSR, 1984, vol. 274(6), рр. 1393 - 1396. (Rus)
[9] Kirpichnikov Р.А., Mukmeneva N.A., and Pobedimsky D.G., 'Phosphorus-organic
stabilizers of polymers - efficiency and echanism', Uspekhi КЫти, 1983, vol. 52(11),
рр. 1831-1851. (Rus)
[10] Mukmeneva N.A., Gol'denberg A.L., and Lazareva N.P., 'Interaction between
phosphoric acid ethers and carboxylic groups in polyethylene', Vysokomo/. Soed., 1983,
vol. А25(6),рр. 1302 - 1306. (Rus)
[11] Pobedimsky D.G., Кirpichnikov Р.А., and Denisov Е.Т., 'About reactions of
phosphorus-organic inhibitors with hydroperoxide groups and polyethylene peroxide
radicals', Vysokomo/. Soed., 1976, vol. А18, рр. 2650 - 2658. (Rus)
[12] Hudson R., Structure and Mechanism о/ Reactions with Phosphorus-Organic
Compounds, Moscow, Mir, 1967,361 р. (Rus)
[13] Mukmeneva N.A., Sharifulin A.Sh., Eliseeva L.A., and Iskhakov О.А., 'Phosphorus-
organic inhibitors of polymeric material combustion', Proc. 61h All-Union Соп!
Combustion о/ Po/ymeric Materia/s, Suzdal', Nov. 29 - Оес. 01, 1988, Rep. Thes.,
Moscow, 1988, рр. 156 - 157. (Rus)
[14] Ruger С., Konig Т., and Schwetlick. К., 'Phosphororganische Antioxidatien. 6. Einflus
Cyclischer Phosphite auf die Radikalisch initierte Oxidation уоп Kolenwasserstoffen
und Polymeren', Acta Po/ymerica, 1986.-Bd. 37(7), S. 435 - 438.
[15] Schwetlick К., Konig Т., Ruger С., Pionteck J., and Habicher W.D., 'Chain-breaking
antioxidant activity of phosphite ester' , Ро/ут. Degrad. Stability, 1986, vol. 15, р. 97 -
108.
218 E. V. Kalugina, N. V. Gaevoy and K. Z. Gumargalieva et al.

[16] Schwetlick К., Konig Т., Pionteck J., Sasse D., and Habicher W.D., 'Organophosphorus
antioxidants. 9. Inhibition of the oxidation of hydrocarbons Ьу hindered aryl
phosphites', Ро/ут. Degrad. StabiZity, 1988, vol. 22(4), рр. 357 - 373.
[17] Lebedeva L.P. and Levin Р .1., 'Antioxidant efficiency of phosphites and their
mixtures', Vysokoтo/. Soed., 1982, vol. В24(5), рр. 379 - 383. (Rus)
[18] Mukmeneva N .А., 'Phosphorylation as the way of increasing stability of polymers',
Proc. 8th All-Uпioп Schoo/-Seтiпar оп Orgaпoeтeп tCoтpouпds, Moscow, INEOS AN
SSSR, 1984,22 р. (Rus)
[19] Kalugina Е.У., 'Thermal transformations and stabilization ot" some heat-resistant
heterochain polymers', Candidate Dissertation Thesis, Moscow, 1992.
[20] Andreeva М.В., 'Thermal transformations and stabilization of aliphatic-aromatic
polyamides and derived mixtures', Candidate Dissertation Thesis, Moscow, 2002. (Rus)
[21] Korshak V.V., Different Unit Composition оf Polymers, Moscow, Nauka, 1977,302 р.
(Rus)
[22] Kandratiev V.N. and Nikitin Е.Е., Cheтica/ Processes in Gases, Moscow, Nauka,
1981,262 р. (Rus)
[23] Emanuel N.M. and Buchachenko A.L., Cheтical Physics оf Polyтer Aging and
Stabilization, Moscow, Nauka, 1982,359 р. (Rus)
[24] Gamino О., Martinasso О., and Costa L., 'Thermal degradation of pentaeritritol
diphosphat model compound for fire retardant intumescent systems. 1. Overall thermal
degradation, Polyт. Degrad. Stab., 1990, vol. 27(2), рр. 285 - 269.
[25] Suebsaeng Т., Wilkie С.А., Burger У.Т., Carter J., and Brown С.Е., 'Solid products
from thermal decomposition of polyethylenterephtalate of investigation Ьу CPIМass,
I3C-NMR and Fourier transform IR-spectroscopy', Eur. Polyт. J., 1981, vol.17(2), рр.
1259 - 1263.
[26] Becher С.Н., Troer К., and Croleva А., 'Thermal properties P-contents PETF', Eur.
Polyт. J., 1981, vol.17(2), рр. 1259 - 1263.
[27] Troer к., Grozeva А., and Borisov О., 'Introduction of phosphorus into the PET-
molecule via 1,2-dicarbomethoxyethyl phosphate', J. Appl. Polyт. Sci., 1981, vol.
17(1), рр. 27 - 33.
[28] Wilkie С., Pettegrew J., and Brown С., 'Pyrolysis reactions of poly(methyl
methacrylate) and red phosphorus: ап investigation with cross-polarization, magic angle
NMR-spectroscopy' J. Polyт. Sci.: Polyт. Lett. Ed., 1981, vol. 19, рр. 409 - 414.
[29] Day М. and Wiles D., 'Temperature influence оп thermal degradation of fiber PETF,
опе fire retardant tris(2,3-dibromopropyl)-phosphate', J. Aпal. aпd Appl. Pyrol., 1984,
No. 7, рр. 65 - 82.
[30] Inagaku N., Sakurai S., and Katsuura К., 'Affect tris(2,3-bromo-propyl) phosphate with
flame retardant of polystyrene', J. Appl. Polyт. Sci., 1979, vol. 23, рр. 2023 - 2030.
[31] Brown С., Wilkie с., Smukalla J., and Cody В., 'Inhibition Ьу red phosphorus of
unimolecular thermal chain scission in poly(methyl methacrylate): investigation Ьу
NMR, FT-IR and laser decompositionl Fourier transform mass spectroscopy, J. Polyт.
Sci.: Polyт. Cheт. Ed., 1986, vol. 24, рр. 1297 - 1311.
[32] Day М. and Willes D., 'Combustion and pyrolysis of poly(ethylene terephthalate). 1.
ТЬе role of flame retardants in products pyrolysis', J Appl. Polyт. Sci., 1981, vol. 26,
рр. 3085 – 3091
The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers 219

[33] Gorestein О., Phosphorus-31 NMR- Principles and Applications, N.Y.: Academic
Press, 1984, 14 р.
[34] Ionin B.I., Ershov В.А., and Kol'tsov A.I., NMR-Spectroscopy in Organic Cheтis('J:
Leningrad, Khimia, 1983,269 р. (Rus)
[35] Gordon А. and Ford R, Chemist Companion, Moscow, Mir, 1976,541 р. (Rus)
[36] Spivack J., Pastor S., and Patrl А., Ро/ут. St. J., 1984, рр. 247 – 257.
[37] Energies оf Chemical Bond Break, Ionization Potentials and Affinity tо Electron,
Moscow, Nauka, 1974,351 р. (Rus)
[38] Sigeru Оае, Cheтistry ofSulfur Orgaпic Coтpouпds, Moscow, Khimia, 1975, Ch. 6.
(Rus)
[39] Mazalov L.N. and Dyumatov У.О., E/ectroпic Structure о/ Extrageпts, Novosibirsk,
Nauka, 1984, 196 р. (Rus)
[40] Gur'yanova E.N., Gol'dstein I.P., and Romm I.P., The Doпor-Acceptor Boпd, Moscow,
Khimia, 1973, 338 р. (Rus)
[41] Atlas of ESR Spectra - Spin Labels and Probes, Ed. A.L. Buchachenko, Moscow,
Nauka, 1977, 159 р. (Rus)
[42] Gorestein D., Phosphorus-31 NMR- principles and applications. NY: Academic Press,
1984, 14p.
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.221-225 ©2010 Nova Science Publishers, Inc.

Chapter 16

QUANTUM-CHEMICAL CALCULATION OF OLEFINS


OF CATIONIC POLYMERIZATION BRANCHING IN γ-,δ-
AND ε− POSITION ON RELATIONS TO DOUBLE
CONNECTION BY METHOD MNDO

V. A. Babkin1, D. S. Andreev*1,
T. V. Peresypkina and G. E. Zaikov±2
1
403343 SF VolgSABU, c. Mikhailovka, region Volgograd, s.Michurina 21
2
Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia

ABSTRACT
Quantum-chemical calculation of olefins of cationic polymerization branching in γ-
,δ- and ε− position on relations to double connection of molecules of 5-methylgepten-1,
5-methyloсtene-1, 6-methylokten-1 was done by method MNDO. Optimized by all
parameters by standard gradient method. Optimized geometrical and electronic structure
of this compound is received. The universal factor of acidity was calculated (pKa=35).
Molecules of 5-methylgepten-1, 5-methyloсtene-1, 6- methyloсtene-1 pertain to class of
very weak Н-acids (рКа>14).

Keywords: quantum-chemical calculations, method MNDO, 5-methylgepten-1

AIMS AND BACKGROUNDS


The aim of this work is a study of electronic structure of olefins cationic polymerization
branching in γ-,δ- end ε− position on relations to double connection of molecules of
molecules 5-methylgepten-1, 5-methyloсtene-1, 6- methyloсtene-1 and theoretical estimation

* 403343 SF VolgSABU, c. Mikhailovka, region Volgograd, s.Michurina 21, E-mail:sfi@reg.avtlg.ru


222 V. A. Babkin, D. S. Andreev and T. V. Peresypkina et al.

of its acid power by quantum-chemical method MNDO. The calculation was done with
optimization of all parameters by standard gradient method built-in in PC GAMESS [1]. The
calculation was executed in approach the insulated molecule in gas phase. Program
MacMolPlt was used for visual presentation of the model of the molecule. [3].

RESULTS OF COLCULATIONS
Geometric and electronic structures, general and electronic energies of olefins cationic
polymerization branching in γ-,δ- end ε− position on relations to double connection of
molecules 5-methylgepten-1, 5-methyloсtene-1, 6- methyloсtene-1 was received by method
MNDO and are shown on fig. 1-3, and in tabl.1-4 . The universal factor of acidity was
calculated by formula: pKa = 42.11-147.18*qmaxH+ [2] (where, qmaxH+ − a maximum positive
charge on atom of the hydrogen (by Milliken [1]) R=0.97, R− a coefficient of correlations,
qmaxH+=+0,05 (for 5-methylgepten-1, 5-methyloсtene-1, 6- methyloсtene-1 qmaxH+ alike
tabl.1)). pKa=35.

CONCLUSIONS
Quantum-chemical calculation of molecule 5-methylgepten-1, 5-methyloсtene-1, 6-
methyloсtene-1 by method MNDO was executed for the first time. Optimized geometric and
electronic structures of these connections were received. Acid force of molecules 5-
methylgepten-1, 5-methyloсtene-1, 6-methyloсtene-1 was theoretically evaluated (pKa=35).
These connections pertain to class of very weak Н- acids (рКа>14).

Figure 1. Geometric and electronic molecule structure of 5-methylgepten-1.(Е0= -105377 kDg/mol, Еel=
-496918 kDg/mol).

± Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygin Street, 117334 Moscow, Russia.E-
mail:chembio@sky.chph.ras.ru
Quantum-Chemical Calculation of Olefins of Cationic Polymerization Branching… 223

Table 1. Optimized bond lengths, valence corners and charges on atoms of the molecule
of 5-methylgepten-1.

Bond lengths R,A Valence corners Grad Atom Charge (by Milliken)
C(1) -0.05
C(1)-C(2) 1,34 C(2) -0.12
C(2)-C(3) 1,51 C(1)-C(2)-C(3) 127 C(3) +0.03
C(3)-C(4) 1,54 C(2)-C(3)-C(4) 113 C(4) +0.01
C(4)-C(5) 1,55 C(3)-C(4)-C(5) 118 C(5) -0.07
C(5)-C(6) 1,54 C(4)-C(5)-C(6) 114 C(6) +0.04
C(7)-C(5) 1,54 C(4)-C(5)-C(7) 114 C(7) +0.04
H(8)-C(1) 1,09 C(2)-C(1)-H(8) 122 H(8) +0.04
H(9)-C(1) 1,09 C(2)-C(1)-H(9) 124 H(9) +0.04
H(10)-C(2) 1,10 C(1)-C(2)-H(10) 119 H(10) +0.05
H(11)-C(3) 1,11 C(2)-C(3)-H(11) 108 H(11) +0.01
H(12)-C(3) 1,11 C(2)-C(3)-H(12) 110 H(12) 0.00
H(13)-C(4) 1,12 C(3)-C(4)-H(13) 109 H(13) 0.00
H(14)-C(4) 1,12 C(3)-C(4)-H(14) 108 H(14) 0.00
H(15)-C(5) 1,12 C(4)-C(5)-H(15) 104 H(15) +0.01
H(16)-C(6) 1,09 C(5)-C(6)-H(16) 111 H(16) -0.01
H(17)-C(6) 1,10 C(5)-C(6)-H(17) 111 H(17) -0.01
H(18)-C(6) 1,11 C(5)-C(6)-H(18) 113 H(18) -0.01
H(19)-C(5) 1,11 C(5)-C(7)-H(19) 111 H(19) -0.01
H(20)-C(5) 1,11 C(5)-C(7)-H(20) 113 H(20) -0.01
H(21)-C(5) 1,11 C(5)-C(7)-H(21) 111 H(21) -0.01

Figure 2. Geometric and electronic molecule structure of 5- methyloсtene-1. (Е0= -120428 kDg/mol,
Еel= -613019 kDg/mol).
224 V. A. Babkin, D. S. Andreev and T. V. Peresypkina et al.

Table 2. Optimized bond lengths, valence corners and charges on atoms of the molecule
of 5- methylo tene-1.

Bond lengths R,A Valence corners Grad Atom Charge (by Milliken)
C(1) -0.05
C(1)-C(2) 1,34 C(2) -0.12
C(2)-C(3) 1,51 C(1)-C(2)-C(3) 126 C(3) +0.03
C(3)-C(4) 1,54 C(2)-C(3)-C(4) 113 C(4) +0.01
C(4)-C(5) 1,55 C(3)-C(4)-C(5) 119 C(5) -0.05
C(5)-C(6) 1,54 C(4)-C(5)-C(6) 113 C(6) +0.04
C(7)-C(5) 1,55 C(4)-C(5)-C(7) 116 C(7) -0.01
H(8)-C(1) 1,09 C(2)-C(1)-H(8) 122 H(8) +0.04
H(9)-C(1) 1,09 C(2)-C(1)-H(9) 124 H(9) +0.04
H(10)-C(2) 1,10 C(1)-C(2)-H(10) 119 H(10) +0.05
H(11)-C(3) 1,11 C(2)-C(3)-H(11) 108 H(11) +0.01
H(12)-C(3) 1,11 C(2)-C(3)-H(12) 110 H(12) +0.01
H(13)-C(4) 1,12 C(3)-C(4)-H(13) 108 H(13) 0.00
H(14)-C(4) 1,11 C(3)-C(4)-H(14) 108 H(14) 0.00
H(15)-C(5) 1,12 C(4)-C(5)-H(15) 104 H(15) 0.00
H(16)-C(6) 1,11 C(5)-C(6)-H(16) 111 H(16) -0.01
H(17)-C(6) 1,11 C(5)-C(6)-H(17) 111 H(17) -0.01
H(18)-C(6) 1,11 C(5)-C(6)-H(18) 113 H(18) 0.00
H(19)-C(7) 1,12 C(5)-C(7)-H(19) 118 H(19) 0.00
H(20)-C(7) 1,11 C(5)-C(7)-H(20) 110 H(20) +0.01
C(21)-C(7) 1,53 C(5)-C(7)-C(21) 117 C(21) +0.03
H(22)-C(21) 1,11 C(7)-C(21)-H(22) 112 H(22) -0.01
H(23)-C(21) 1,11 C(7)-C(21)-H(23) 110 H(23) -0.01
H(24)-C(21) 1,11 C(7)-C(21)-H(24) 112 H(24) 0.00

Figure 3. Geometric and electronic molecule structure of 6-methyloсtene-1. (Е0= -120454 kDg/mol,
Еel= -605340 kDg/mol).
Quantum-Chemical Calculation of Olefins of Cationic Polymerization Branching… 225

Table 3. Optimized bond lengths, valence corners and charges on atoms of the molecule
of 6- methylo tene-1.

Bond lengths R,A Valence corners Grad Atom Charge (by Milliken)
C(1) -0.05
C(1)-C(2) 1,34 C(2) -0.12
C(2)-C(3) 1,51 C(1)-C(2)-C(3) 127 C(3) +0.03
C(3)-C(4) 1,54 C(2)-C(3)-C(4) 113 C(4) -0.01
C(4)-C(5) 1,54 C(3)-C(4)-C(5) 116 C(5) +0.01
C(5)-C(6) 1,55 C(4)-C(5)-C(6) 118 C(6) -0.07
H(7)-C(6) 1,09 C(2)-C(1)-H(7) 122 C(7) +0.04
H(8)-C(1) 1,09 C(2)-C(1)-H(8) 124 H(8) +0.04
H(9)-C(2) 1,10 C(1)-C(2)-H(9) 119 H(9) +0.05
H(10)-C(3) 1,12 C(2)-C(3)-H(10) 108 H(10) 0.00
H(11)-C(3) 1,11 C(2)-C(3)-H(11) 110 H(11) 0.00
H(12)-C(4) 1,12 C(3)-C(4)-H(12) 108 H(12) 0.00
H(13)-C(4) 1,12 C(3)-C(4)-H(13) 109 H(13) +0.01
H(14)-C(5) 1,12 C(4)-C(5)-H(14) 107 H(14) 0.00
H(15)-C(5) 1,12 C(4)-C(5)-H(15) 108 H(15) 0.00
H(16)-C(6) 1,12 C(5)-C(6)-H(16) 108 H(16) +0.01
C(17)-C(6) 1,54 C(5)-C(6)-C(17) 114 H(17) +0.04
H(18)-C(17) 1,11 C(6)-C(17)-H(18) 112 H(18) -0.01
H(19)-C(17) 1,11 C(6)-C(17)-H(19) 111 H(19) -0.01
H(20)-C(17) 1,11 C(6)-C(17)-H(20) 112 H(20) 0.00
C(21)-C(6) 1,54 C(5)-C(6)-C(21) 111 C(21) +0.04
H(22)-C(21) 1,11 C(6)-C(21)-H(22) 111 H(22) 0.00
H(23)-C(21) 1,11 C(6)-C(21)-H(23) 111 H(23) 0.00
H(24)-C(21) 1,11 C(6)-C(21)-H(24) 112 H(24) 0.00

The Table 4. Total energy ( 0), electronic energy( el), maximal charge on atom of
hydrogen(qmaxH+), universal parameter of acidity(рКа) of molecules of 5-methylgepten-
1, 5-methylo tene-1, 6-methylokten-1

№ Molecule -Е0, kDg/mol -Есв, kDg/mol qmaxH+ рКа


1 5-methylgepten-1 105377 496918 +0.05 35
2 5- methyloсtene-1 120428 613019 +0.05 35
3 6- methyloсtene-1 120454 605340 +0.05 35

REFERENCES
[1] M.W.Shmidt, K.K.Baldrosge, J.A. Elbert, M.S. Gordon, J.H. Enseh, S.Koseki,
N.Matsvnaga., K.A. Nguyen, S. J. Su, and others. J. Comput. Chem.14, 1347-1363,
(1993).
[2] Babkin V.A., Fedunov R.G., Minsker K.S. and anothers. Oxidation communication,
2002, №1, 25, 21-47.
[3] Bode, B. M. and Gordon, M. S. J. Mol. Graphics Mod., 16, 1998, 133-138.
In: Chemical Reactions in Gas, Liquid and Solid Phases…: ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.227-243 ©2010 Nova Science Publishers, Inc.

Chapter 17

THERMODYNAMICS FOR CATALASE AND HYDROGEN


PEROXIDE INTERACTION

A. A. Turovsky, A. R. Kytsya, L. I. Bazylyak and G. E. Zaikov


N.M. Emanuel Institute of Biochemical physics,
Russian Academy of Sciences, Moscow, Russia

ABSTRACT
Thermodynamic analysis of the elementary reactions of the enzymic catalysis of
hydrogen peroxide by catalase was carried out with the application of quantum-chemical
calculations. A mechanism of the enzymic catalysis of hydrogen peroxide by catalase
based on the Chorner scheme was proposed. Hypotheses about Michaelis’s complex
[catalase + H2O2] forming which further decay conditioned by the low ionization
potential of iron atom in the catalase were formed. It is shown that bioSAA (ramnolipid)
forms the thermodynamically efficient complexes with the hydrogen peroxide. On basis
of calculated thermodynamical and geometrical parameters it is shown that the catalase’s
catalytic specificity causes by the hydroperoxides dimensions.

1. INTRODUCTION
Live organisms can exist because of there ability to kinetic controlling of the chemical
reactions thus depress the thermodynamic equilibrium achievement. The key role at such
processes performs the biological catalysts (enzymes). The modern developments of the
biological catalysis shows, that the enzymatic reaction proceeds accordingly to the general
laws of the regular chemical interactions. Difference between the enzymic catalysis and the
chemical catalysis is only the complicated structure of the first.
Structure of the enzymes is the same as the proteins. From the point of view of
thermodynamics the terms “primary structure, secondary structure, tertiary structure of the
protein” designates the existence only one or bounded set of the states when free energy as a
function of spatial structure (and as a function of non-covalent interactions between amino
acid residuals of polypeptide chain correspondingly) is the lowest. Sub-isolated globules can
228 A. A. Turovsky, A. R. Kytsya, L. I. Bazylyak et al.

be combined into the quaternary structure of the protein as a result of the interaction of the
whole series of weak forces (hydrogen bond, hydrophobic or ion interactions). Prosthetic
group may be connected up to protein part (apoenzyme) by covalent or weaker (like a
hydrogen bond, hydrophobic or ion bonds) bonds. Such interactions orientate prosthetic
grouping the space.
Cofactor, which act the catalytic function, have to stay chemically invariable at the
catalytic reaction. When the cofactor’s role plays the prosthetic group then the last carries out
the whole catalytic cycle and is connected to the same enzyme molecule. At the some times
cofactor may come into the more complicated interactions and play the role of the connecting
chain between the molecules of enzyme and ensure the unity of enzyme system. Such
cofactors terms the coenzymes.
A structural peculiarity of the surface layer of protein globules allows to concentrate a lot
of chemically different functional groups at the active centre. Such functional groups have an
opportunity both to sorb the substrate and to chemical interaction with substrate.
Formation of the strong chelate complexes is possible due to the fact that polypeptide
chains did not connected strongly to the surface and characterize by some mobility. As a
result of such mobility is the capability to stratification of certain sorption fragments of the
globule upon the corresponding binding fragments of the molecule. Conformational changes
of the enzymes were detected by the different chemical and physical techniques [1].
Increased microviscosity at the surface layer, induced by the decreasing of polypeptide
chain mobility, play important role at the complexation.
It is shown [2] that thermodynamical efficiency of enzyme catalysis determined by the
values of free energies of intermolecular (in case of Michaelis’s complex formation) or
intramolecular (at the transition state of reaction) bond E–R. Here E is an enzyme and R is
radical of substrate forming the complex. Motivity of the catalysis is the energy of interaction
of E and R at the transition state not at intermediate complex. However the rate of enzyme
reaction will be increasing if the intermolecular interaction E–R at the activated state will be
more preffered.
So, the investigations of complexation thermodynamic can give the important
information about Michaelis’s complexes structure. Unfortunately information connected to
thermodynamic investigations of complexation reactions almost missing.
Catalase was taken out as an subject of investigations. Unfortunately there is no sufficient
information about its complete structure. Reliable information is connected only to the haem
of iron. But exists the assumption that the haem of iron is an active center of catalase and
exactly one is crucial for the chemical reaction of hydrogen peroxide decomposition. At the
same time protein helixes only minimize the free energy of the three-dimensional structure of
enzyme.
Taking into account above-mentioned assumptions the aim of presented work were i) the
investigations of the most verisimilar reaction of the complex “catalase – hydrogen peroxide”
formation; ii) qualitative assessment of the processes which takes place at the active centre of
catalase; iii) investigations of influence of surface-active substances (using the rhamnolipid as
example) as a promoter; iv) valuation of thermodynamic characteristics above-mentioned
processes with using of the quantum-chemical calculations.
Thermodynamics for Catalase and Hydrogen Peroxide Interaction 229

2. EXPERIMENTAL
Quantum-chemical calculations were done using the MOPAC2009 program having
applied the PM6 method. For the result’s visualisation was used the program Jmol.

3. RESULTS AND DISCUSSION


3.1. Interaction of Catalase and Hydrogen Peroxide

It is assumed that cofactor of catalase is the verily prosthetic group (haem of iron),
performs the whole catalytic cycle and is strongly connected to the enzyme molecule.
However, in fact the protein molecule can be connected to any iron’s addend molecule and
can conformationally affect to catalytic properties of the complex due to the interactions of
catalytically active groups (–О–, –С(О)–, –NH–, –S– etc.) which are included into the protein
molecule. Also we cannot accept the ionic bonds formation between the protein molecule and
atom of iron.

Figure 1. Space pattern of the haem of iron.

Since the composition of the nearest to atom of iron protein component is unknown we
have to assume that its influence onto the activity of catalase is minimum. But it is obvious
that protein parts of enzyme have a significant influence onto its stability and decrease the
free energy of the system. At the same time activity of the enzyme can increase because the
spatial interactions of the nearest fragments of polypeptide chain which contains heteroatoms
and can form coordination bond with the central atom of iron due to undivided electron pairs.
230 A. A. Turovsky, A. R. Kytsya, L. I. Bazylyak et al.

At the first approximation we neglected such interactions, though in this case proposed model
is not thorough. Since the coordination number of iron is 6 we can present the haem of iron as
follows (Fig. 1).
Since the atom of iron is charged positively and OH-group is charged negatively then
haem of iron will be indicate as [Catalase × H2O]+ OH–. Such indication did not mean the ion
pair formation because the OH-group exists not so far from iron (see Table 1) and forms a
strong bond with the one. Such assumption can be confirmed by the value of complex’s
constant of dissociation:

[Catalase × H2O]+ OH– –> [Catalase × H2O]+ + OH– (ΔF = +191,5 kcal/mol),
K = exp (–ΔF/RT) = 6,1 × 10–141.

Thus we can affirm that haem did not dissociate. It is mean that OH-group sit at the
internal coordination sphere. Such assumption can be also confirmed by fact that the solution
of catalase did not carry current and did not form precipitate Ba(OH)2 after interaction with
BaCl2.
From the Table 1 we can see that distances Fe–N at the complex [Catalase × H2O]+ OH–
are the same. Such fact testifies that the all bonds in the molecule are hybrid and are not both
coordinating and covalent bonds. Such statement also confirms by a small values of charges
of nitrogen atoms. The charge of atom of iron is equal to +1,143 that is the charge is heavily
delocalized. Let us suppose that the coordinating bonds Fe–N forms due to presence of
unshared electron pairs at the atom of nitrogen which are displaced up to atom of iron. The
charges of oxygen’s atoms both in OH-group and Н2О are sufficiently great.

Table 1. Geometrical adjectives of complexes of catalase

[Catalase × H2O]+ [Catalase × H2O2]+


Catalase (OH)2–
OH– OH–
Distance Fe–N (5), Å 1.99 2.07 2.00
Distance Fe–N (6), Å 1.99 1.95 2.00
Distance Fe–N (7), Å 1.99 1.95 2.00
Distance Fe–N (8), Å 1.99 2.03 2.00
Distance Fe–O (OH), Å 1.77 1.78 1.81
Distance Fe–O (H2O), Å 2.09 1.81 1.82
Charge at Fe 1.143 1.495 1.621
Charge at N (5) –0.033 –0.035 –0.189
Charge at N (6) –0.036 –0.269 –0.039
Charge at N (7) –0.029 –0.257 –0.183
Charge at N (8) –0.038 –0.016 –0.061
Charge at O (OH) –0.501 –0.696 –0.745
Charge at O (H2O) –0.629 –0.584 –0.758

Certain intermediate complex forms as a result of interaction between catalase and


hydrogen peroxide. Let us suppose that the first stage of catalytic reaction can be showed as
follow:

[Catalase × H2O]+ OH– + H2O2 –> [Catalase × H2O2]+ OH– + H2O (1)
Thermodynamics for Catalase and Hydrogen Peroxide Interaction 231

Calculated thermodynamic parameters of the complexes are shown at the Table 2.


Let us consider the thermodynamic of complexing. Values of ΔH, ΔS and ΔF of reaction
(1) are equal to –37,1 kcal/mol, 24,5 cal/mol K and –43,9 kcal/mol accordingly. Calculated
data shows that reaction of formation the complex between catalase and hydrogen peroxide is
an exothermic and energetically efficient. The main geometrical adjectives of complex
[Catalase × H2O2]+ OH– are presented at the Table 1 and its structure is shown at the Fig. 2.

Figure 2. Space pattern of the complex [Catalase×H2O2]+OH–.

It is necessary to notice that the distances Fe–N for fifth and eighth atoms of nitrogen a
little increased while for sixth and seventh atoms a little decreased. Also the distance Fe–O
(H2O2) is noticeably shorter in compare with such one for H2O. This fact evidences that the
interaction between atom of iron and atom of oxygen for hydrogen peroxide is more strongly
than such for water. The value of charge for iron’s atom is significantly increased too. It is
means that the delocalization of the charge is too smaller. The charge of atom of oxygen for
OH-group noticeably increased, it is means that the electrostatic bond Fe–OH is more
strongly. It is necessary to notice that the charges of nitrogen’s atoms of iron’s addend are
also changed. Such fact possibly can be explained by some changing of the geometrical
adjectives of the reactive center.
232 A. A. Turovsky, A. R. Kytsya, L. I. Bazylyak et al.

Table 2. Calculated thermodynamic and quantum-chemical parameters of parent


materials, complexes of catalase and complexes of iron

ΔH0, ΔS0, ΔF0, Ionization Electron affinity,


№ Substance (complex)
kcal/mol cal/mol К kcal/mol potential, eV eV
H2O2 –24.0 53.7 –40.0 9.890 0.115
H2O2 × 2 H2O –154.8 94.4 –182.9 10.208 –0.099
Rhamnolipid –519.5 197.8 –578.4 10.537 0.042
Rhamnolipid + Н2О2 –551.4 280.4 –635.0 10.603 –0.179
H2O –54.3 45.0 –67.7 11.906 4.068
OH– –33.0 40.7 –45.1 2.309 15.127
H3O+ 147.2 44.8 133.8 22.131 –6.124
OH● 13.3 41.2 1.0 10.485 –1.795
[Catalase × H2O]+ OH– –190.6 204.5 –251.5 6.730 –0.792
[Catalase ×H2O2]+ OH– –197.4 237.7 –268.2 7.731 –1.106
Catalase (OH)2– –261.4 248.9 –335.6 3.683 2.039
C3O2H4N –64.7 80.1 –88.6 10.343 –0.374
[Catalase ×C3O2H4N]+ OH– –210.8 209.9 –273.4 7.532 –0.943
CH3OОH –23.8 66.7 –43.7 9.854 0.304
CH3O● 0.9 55.1 –15.5 9.361 –1.124
[Catalase ×CH3OОH]+ OH– –163.2 266.2 –242.5 7.297 –0.821
(CH3)3COОH –47.4 88.0 –73.6 9.741 0.523
(CH3)3CO● –20.5 73.6 –42.4 9.386 –1.267
[Catalase ×(CH3)3COОH]+
–181.9 271.1 –262.7 7.303 –0.939
OH–
Catalase + 53.7 230.2 –14.9 10.684 –4.801
Fe(III)–E –131.5 197.8 –190.4 7.359 –1.036
O=Fe(IV)–E(+) –174.9 241.8 –247.0 7.658 –1.082
[Catalase × H2O2×2 H2O]+
–322.8 276.9 –405.3 7.802 –1.076
OH–
[Catalase × H2O2 ×
–671.2 344.2 –773.8 7.389 –0.784
Rhamnolipid]+ OH–
[Fe2+ × 6 H2O] 51.5 118.6 –16.2 16.781 –6.090
[Fe2+ × 5 H2O × H2O2] 39.5 130.2 0.7 16.861 –9.191
[Fe3+ × 5 H2O] OH– 38.2 109.8 5.5 17.354 –6.884
[Fe3+ × 6 H2O] 370.8 109.1 338.3 23.024 –12.527

We can consider that interaction between catalase and hydrogen peroxide is a redox
reaction (Chorner’s scheme) [3]. Catalase is a reducing agent and H2O2 is an oxidant. Such
reaction can be presented as follow (2):

[Catalase×H2O]+OH– + H2O2 –> [Catalase×H2O2]+OH– –> Catalase+ – OH– + OH– + OH•(2)

But probably intermediate substance Catalase (OH)2– (Fig. 3) obtains due to box effect.
Such intermediate substance Catalase (OH)2– easily react with proton H3O+ and turns into the
initial substance. These interactions we can write as follow:

[Catalase×H2O2]+OH– –> Catalase (OH)2– + OH• (3)

Catalase (OH)2– + H3O+ –> [Catalase × H2O]+OH– + H2O


Thermodynamics for Catalase and Hydrogen Peroxide Interaction 233

(ΔH = –130,7 kcal/mol, ΔS = –44,2 cal/mol K, ΔF = –117,4 kcal/mol) (4)

Let us consider the thermodynamics of the reaction (3). The values of ΔH, ΔS and ΔF of
reaction are equal to –63,0 kcal/mol, 290,1 cal/mol K and –66,4 kcal/mol correspondingly.
Presented data indicate that such process is exothermic and thermodynamically possible.

Figure 3. Space pattern of the complex Catalase (OH)2–.

It is necessary to consider some quantum-chemical characteristics of the process in detail.


Presented in the Table 2 data show that ionisation potential of the substance [Catalase ×
H2O]+OH– is equal to 6,730 eV and electron affinity of hydrogen peroxide is equal to –0,099
eV. As a measure of interaction [Catalase × H2O]+OH– + H2O2 with intermediate complex
forming let us use the value of orbital energy [4]. Taking into account the fact that upper
occupied orbital of catalase and lowest unoccupied orbital of hydrogen peroxide are
stereospecific we can evaluate the value of orbital energy of complex [Catalase × H2O2]+OH–
const
forming with using the equation . Here I is an ionisation potential and E is an electron
I−E
affinity. For this reaction the value of orbital energy is equal to 0,0015*const eV.
The value of orbital energy of forming the intermediate complex Catalase (OH)2–
(reaction (3)) is a shade higher and equal to 0,0017*const eV.
The lengths of bonds Fe–N of substance Catalase (OH)2– are rather more equal (Table 1)
than the same at complex [Catalase×H2O2]+OH–. This fact testify that the space pattern of the
intermediate complex Catalase (OH)2– rather more tend to the configuration of initial
molecule of catalase. The bond Fe–O (OН) of the intermediate complex Catalase (OH)2– is
234 A. A. Turovsky, A. R. Kytsya, L. I. Bazylyak et al.

longer then the the same at complex [Catalase×H2O2]+OH– and the value of charge of the iron
is a noticeably greater.
Formation of the intermediate complex Catalase (OH)2– may be presented as a result of
interaction of the intermediate reagent Catalase+–OH– and OH–anion. The ionisation potential
of OH–anion is sufficiently small and equal to 2,309 eV (see Table 2). This implies that anion
easily donate the electron to the oxidated form of iron and form the complex Catalase (OH)2–.
Free radicals OH• can recombine or disproportionate and form the corresponding products of
reaction. Let us consider the processes of formations of such products.
On the basis of data of Table 2 we can calculate the thermodynamic parameters of the
reactions (5) and (6).

OH• + OH• –> H2O2 ; (5)

(ΔH = +2,6 kcal/mol, ΔS = 28,7 cal/mol K, ΔF = –38,0 kcal/mol)

OH• + OH• –> H2O +O••. (6)

(ΔH = –65,7 kcal/mol)

On the basis of presented values of thermodynamic parameters we can do some


conclusions. The reaction of radical’s recombination (5) is a slightly endothermic and exist
the possibility of its realization. On the other hand the value of ΔF of reaction of
disproportionation (6) is less for 27,7 kcal/mol. That is this reaction is more prefer from the
point of view of thermodynamic.
The final product (oxygen) is obtained by the recombination of biradicals (O•• + O•• –>
O2). This reaction characterize by a great exothermic effect (–268 kcal/mol).
Let us consider the thermodynamics of the reaction (4). Thermodynamic parameters ΔH,
ΔS and ΔF are equal to –130,7 kcal/mol, –44,2 cal/mol K and –117,4 kcal/mol
correspondingly. The results of calculations show that the exothermic effect of the reaction is
extremely great but the value of entropy is negative. The result of the negative entropy is
decreasing of the free energy of reaction.
Thus the reaction of catalytic interaction between catalase and hydrogen peroxide can be
presented as follow.

H O
[Catalase × H2O]+ OH– ⎯⎯2 ⎯
2
→ [Catalase×H2O2]+ OH– –> Catalase (OH)2– + 2 OH● –
>

H O+
⎯⎯3 ⎯→ [Catalase × H2O]+ OH– (7)

Every presented stages and possibility of its proceeding are stated above. It is necessary
to note that the same results of calculations were obtained in the case of changing of molecule
H2O at the complex [Catalase × H2O]+ OH– to the fragment of polypeptide chain. The
interactions and the results of calculations are presented below (eqs. (8) – (10)).
Thermodynamics for Catalase and Hydrogen Peroxide Interaction 235

[Catalase × C3O2H4N]+ OH– + H2O2 –>[ Catalase ×H2O2]+OH– + C3O2H4N (8)


(ΔF = –43,4 kcal/mol)

[Catalase ×H2O2]+ OH– –> Catalase (OH)2– + OH● (9)

(ΔF = –66,4 kcal/mol)

Catalase (OH)2– + H3O+ + C3O2H4N –> [Catalase ×C3O2H4N]+ OH– + 2H2O (10)
(ΔF = –118,4 kcal/mol)

As we can see the all stages are thermodynamically profitable.


The space pattern of the complex [Catalase × C3O2H4N]+ OH– presented at the Fig. 4.
It is permitted that peptide fragment possess some energetic barrier of rotating around the
Fe–N bond and the interaction between Fe and N is minimal at the some conformation states
of macromolecule. This assumption need the more detailed investigations but it can not be
declined because the new nuances of biocatalysis may be explained in case of its
confirmation.

Figure 4. Space pattern of the complex [Catalase × C3O2H4N]+ OH–.

Authors [5] propose the next scheme of interaction between catalase and hydrogen
peroxide.

Fe(III)–E –> O=Fe(IV)–E(+) –> Fe(III)–E (11)


236 A. A. Turovsky, A. R. Kytsya, L. I. Bazylyak et al.

But catalase is an electronically delocalized system and the term “valency” is ill-posed in
accordance with the modern conception of matter’s structure.
The thermodynamic calculations of presented scheme where done (eqs. (12), (13)). The
values of thermodynamic parameters for all substances were obtained (see Table 2). Space
pattern of the complexes are presented at Figures 5 and 6.

Figure 5. Space pattern of the complex Fe(III)–E

Figure 6. Space pattern of the complex O=Fe(IV)–E(+)


Thermodynamics for Catalase and Hydrogen Peroxide Interaction 237

Let us consider the process stage-by-stage.

Fe(III)–E + H2O2 –> O=Fe(IV)–E(+) + H2O, (ΔF = –84,3 kcal/mol) (12)

O=Fe(IV)–E(+) + H2O2 –> Fe(III)–E + H2O, (ΔF = +28,9 kcal/mol) (13)

The values of free energies are calculated by the data presented at Table 2.
Taking into account obtained results we can do the next conclusion. From the point of
view of thermodynamics the first state of proposed process is virtual but the second is
absolutely forbidden. It is means that proposed scheme (11) [5] is beneath criticism.

3.2. Interaction of Catalase and Organic Hydroperoxides

As a rule enzymes are characterized by such peculiarity as stereospecificity. That is why


we considered the interaction of catalase and organic hydroperoxides from the
thermodynamic point of view.
The reaction of complexation of catalase and methyl hydroperoxide (structure of the
complex is presented at Fig. 7) is characterized by the next thermodynamic parameters:
ΔH = –3,1 kcal/mol, ΔS = 40,0 cal/mol K, ΔF = –14,8 kcal/mol.

Figure 7. Space pattern of the complex [Catalase×CH3OОH]+ OH–.

On the basis of results of calculations we can do the next conclusion. As we can see from
the presented results the values of ΔH and ΔF of reaction are too smaller than the same for
238 A. A. Turovsky, A. R. Kytsya, L. I. Bazylyak et al.

reaction of complexing of catalase and hydrogen peroxide. This fact means that the
interaction between catalase and methyl hydroperoxide is worse than hydrogen peroxide one
and taking into account the errors of calculations may be impossible.
At the carrying out of the calculations for the reaction of catalase and
tertbutylhydroperoxide complex formation (Fig. 8) it has been determined that such reaction
is not neutralized.

Figure 8. Space pattern of the complex [Catalase ×(CH3)3COОH]+ OH–.

Let us consider the catalase and organic hydroperoxides complex formation. Under
interaction of catalase and hydrogen peroxide, methyl and tertbutyl peroxides the numerical
values of electron affinity are equal to 0,115 eV, 0,304 eV and 0,523 eV respectively. Hence,
we can calculate the values of orbital energies. There are equal to 0,00151*const eV,
0,00155*const eV, 0,0016*const eV. Presented data shows that the values of orbital energies
at the series “H2O2 – CH3OOH – (CH3)C3OOH” increasing. It denotes that power inputs via
complexation between catalase and hydroperoxides increase. But the predominant cause in
such processes performs the volume of substrate’s molecule.

3.3. Influence of Solvation Upon the Catalytic Decomposition of


Hydrogen Peroxide

With the aim of determination of influence of solvation sphere upon the catalytic
decomposition of hydrogen peroxide the thermodynamic of the following reaction was
considered.

[Catalase × H2O]+ OH– + H2O2×2 H2O –> [Catalase × H2O2×2 H2O]+ OH– + H2O (14)
Thermodynamics for Catalase and Hydrogen Peroxide Interaction 239

(ΔH = –31,7 kcal/mol, ΔS = 23,0 cal/mol K, ΔF = –38,6 kcal/mol)

Figure 9. Space pattern of the complex [Catalase × H2O2×2 H2O]+ OH–.

Process of solvation of hydrogen peroxide by water is thermodynamically efficient.

H2O2 + 2 H2O –> H2O2×2 H2O (15)


(ΔH = –22,2 kcal/mol, ΔS = –49,3 cal/mol K, ΔF = –8,5 kcal/mol)

The results of calculations show that the solvation acts to decreasing of entropy. It is
mean that organization of complex H2O2×2 H2O (Fig. 10) is more than one of initial system.
It is necessary to mark that the electron affinities of solvated and non-solvated molecules
H2O2 are quite different. It is the reason of different values of orbital energies of
complexation.

Figure 10. Space pattern of the complex H2O2×2 H2O.


240 A. A. Turovsky, A. R. Kytsya, L. I. Bazylyak et al.

Let us consider the solvation of H2O2 by the molecule of rhamnolipid in accordance with
the presented scheme with the aim of compare to the above mentioned process (space patterns
of the rhamnolipid molecule and its complex with H2O2 are presented at Figs. 11 and 12).

H2O2 + Rhamnolipid –> H2O2 × Rhamnolipid (16)

(ΔH = –7,9 kcal/mol, ΔS = 28,9 cal/mol K, ΔF = –16,4 kcal/mol)

Figure 11. Space pattern of the rhamnolipid molecule.

Figure 12. Space pattern of the complex H2O2 × Rhamnolipid.


Thermodynamics for Catalase and Hydrogen Peroxide Interaction 241

This process is more thermodynamically prefer than the solvation of hydrogen peroxide
by water. The electron affinity of the complex H2O2 × Rhamnolipid is rather greater than the
one of H2O2×2 H2O. It is mean that electron transfer from the atom of iron at the catalase
must be easier.
Let us consider the complaxation of catalase and solvated by rhamnolipid hydrogen
peroxide.

[Catalase × H2O]+ OH– + H2O2 × Rhamnolipid –> [Catalase × H2O2 × Rhamnolipid]+



OH + H2O

(ΔH = 16,5 kcal/mol, ΔS = –95,7 cal/mol K, ΔF = 45,0 kcal/mol) (17)

Presented results of calculations show that such reaction is impossible from the point of
view of thermodynamics.
From the another hand the obtained experimental data show that the reaction of catalytic
decomposition of hydrogen peroxide is strongly accelerated in the presence of rhamnolipid
and its rate depend on the concentrations of the last. Unfortunately this fact can not be explain
on the basis of presented data and needs the more detailed investigations.

Figure 13. Space pattern of the complex [Catalase × H2O2 × Rhamnolipid]+ OH–.

It is interesting to consider the process of interaction of H2O2 and Fe (II) at the acid
medium. In accordance with the Chorner’s mechanism this reaction can be written as follow.

Fe2+ + H2O2 –> Fe3+ + HO– + HO• (18)


It is means that reaction proceed as a radical reaction. Formation of radicals is proven by
[6]. However in water this reaction has to proceed in two stages and can be written as follow.
Stage 1:
242 A. A. Turovsky, A. R. Kytsya, L. I. Bazylyak et al.

[Fe2+ × 6 H2O] + H2O2 –> [Fe2+ × 5 H2O × H2O2] + H2O (19)

(ΔH = –42,3 kcal/mol, ΔS = 2,9 cal/mol K, ΔF = –43,2 kcal/mol)

Stage 2:

[Fe2+ × 5 H2O × H2O2] –> [Fe3+ × 5 H2O] OH– + OH•

(ΔH = –12,0 kcal/mol, ΔS = 20,8 cal/mol K, ΔF = 5,8 kcal/mol)

[Fe3+ × 5 H2O] OH– + H3O+ –> [Fe3+ × 6 H2O] (20)

(ΔH = 185,4 kcal/mol, ΔS = –45,5 cal/mol K, ΔF = 199,0 kcal/mol)

In case of reaction of catalase and hydrogen peroxide the intermediate product


Catalase (OH)2– forms as a result of reaction between complex “Catalase+ – OH–” and OH–
anion which is the product of H2O2 decomposition. Hydroxyl posses a small value of
ionization potential (2,309 eV) and can easily give back the electron to the atom of iron.
However this phenomenon is not observed in the case of ionic reaction between Fe2+ and
H2O2. Intermediate reaction “[Fe3+ × 5 H2O] + OH– + OH•” is not realize or proceeds very
weakly with forming the product [Fe2+ × 5 H2O] OH– which turns back to the parent
substance in accordance with the next scheme.

[Fe2+ × 5 H2O] OH– + H3O+ –> [Fe2+ × 6 H2O] + H2O (21)

In a case of such reaction proceeding we would observed the catalytic process of the
hydrogen peroxide decomposition. But at the acid medium reducing of iron Fe3+ using the
OH– group not realize because the competitive reaction OH– + H+ –> H2O which proceeds
very rapidly. At the alkaline medium the stable substance Fe(OH)3↓ forms. That is why the
data concerning the investigations of this reaction at the alkaline and neutral mediums are
absent.
It is necessary to note that the reaction of oxidation of ions Fe2+ by ions Fe3+ is not
excepted. This reaction characterized by the values of activation energy and preexponential
factor (lgA) equal to 9 – 11 kcal/mol and 7 – 9 correspondingly.
The rate of intermediate reaction (22) can depend on the concentration of ions of
hydrogen and ions Fe3+ × 5 H2O may exist in form Feaq3+ ⇔ Fe(OH)2+ + H+.

Fe3+ × 5 H2O + OH– –> Fe2+ × 5 H2O + OH• (22)

CONCLUSIONS
Firstly, it is necessary to mark that the all presented thermodynamic quantum-chemical
calculations are approximate and were done in accordance with the accepted scheme of
interaction “catalase – hydrogen peroxide”. According to the presented scheme we neglected
Thermodynamics for Catalase and Hydrogen Peroxide Interaction 243

the protein part of the enzyme and only haem of iron was used in calculations. However, the
protein part of enzyme (especially, the nearest fragments) can influences on the process of
catalysis. Today's such influence was not investigated. But the next assumption can be done.
It is known, that the protein fragments are characterized by some mobility (or rotation) which
is realized with some energetic barrier. This rotation can results to the “key effect” that is the
value of free energy when the formation of complex “catalase – hydrogen peroxide” will be
energy-efficient. Taking into account the presented results and considerations we can do the
next conclusions.
The mechanism of enzyme catalytic decomposition of hydrogen peroxide by catalase
based on the Chorner’s scheme is proposed. Thermodynamic analysis of elementary reactions
of proposed scheme is done. It was formed hypotheses about Michaelis’s complex [catalase +
H2O2] forming which further decay conditioned by the low ionization potential of iron atom
in the catalase.
A similar reaction of interaction between Fe2+ and H2O2 proceeds more slowly because
the elevated ionization potential of reaction Fe2+ – 1 e– → Fe3+.
It is shown that bioSAA (ramnolipid) forms the thermodynamically efficient complexes
with the hydrogen peroxide. But forming of these complexes did not influence onto the free
energy of interaction between catalase and hydrogen peroxide. It is probably that rhamnolipid
molecule plays some role in the processes of orientation and adsorption of hydrophobic part
of substrate and enzyme. Due to these processes the rate of catalytic decomposition of
hydrogen peroxide in the presence of rhamnolipid sharply (up to 5 times) increase and depend
on the concentration of the last. From the other hand rhamnolipid can play role of
concentrating agent for H2O2 and in this way accelerate the catalytic reaction of hydrogen
peroxide decomposition.
It is shown that the catalase’s catalytic specificity causes by the dimensions of
hydroperoxides which plays the main role in Michaelis’s complex forming.

REFERENCES
[1] Jiali Gao, Kyoungrim Lee Byun, Ronald Kluger. Catalysis by Enzyme Conformational
Change // In Topics in Current Chemistry v. 238, 2004, PP. 113–136.
[2] Berezin I.V. Osnovy phisicheskoy chimii fermentativnogo kataliza. Мoscow, Vysshaya
shkola, 1977, 275 p.
[3] V.L. Antonovski, М. М. Buzlanova. Analiticheskaya himija organicheskikh peroksidov.
Мoscow, Chimija, 1978, 307 p.
[4] Reakcionnaja sposobnost i puti reakcii. / Ed. by Klopman G.M., Мoscow, Мir, 1977 383 p.
[5] Chelikani P, Fita I, Loewen P. C. Diversity of structures and properties among catalases
// Cell. Mol. Life Sci. 61 (2): 2004, p. 192–208.
[6] Haber, F. and Weiss, J. Uber die Katalyse des Hydroperoxydes. Naturwissenschaften
20, (1932). – 948–950.
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.245-247 ©2010 Nova Science Publishers, Inc.

Chapter 18

DR. RER. NAT. WOLFGANG FRITSCHE –


SCIENTIST AND ORGANIZER OF
INTERNATIONAL SCIENCE
(SECRETARY GENERAL RTD. OF
GESELLSCHAFT DEUTSCHER CHEMIKER,
HONORARY PRESIDENT OF FEDERATION OF
EUROPEAN CHEMICAL SOCIETIES)

G. E. Zaikov*
N.M.Emanuel Institute of Biochemical Physics,
Russian Academy of Sciences, Moscow, Russia

Dr. Fritsche is a well-known world organizer of Chemical Science within the framework
of the German Chemical Society and International Union of Pure and Applied Chemistry
(IUPAC). He was born on 11 March 1928 at Dortmund (Germany). His career has several
important steps: doctorate in Inorganic Chemistry (University of Bonn) – 1954; Assistant to
Technical Director Vereinigte Ultramarinfabriken Marienberg (Germany). After that he was
Deputy Secretary of Gesellschaft Deutscher Chemiker (GDC) – 1960-1967; Secretary of
GDC – 1968-1971 and Secretary General of GDC – 1972-1991. Wolfgang was retired in
1991.
As a scientist he has had good success in the field of inorganic chemistry.
His activity in the Federation of European Chemical Societies (FECS) included several
very important steps: member of the Steering Committee (before 1970) and Founder member
(after 1970). He was also member of Executive Committee (1970-1976), Secretary General
(1976-1988), Chairman of the Council (1989-1992). He has position as Lifelong Honorary
President from 1993.

* N.M.Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygin st., Moscow 119991,
Russia, Chembio@sky.chph.ras.ru
246 G. E Zaikov

Dr. Fritsche was very active as a member of the Finance Committee as well as
ChemRAWN (Chemical Research Applied to World Needs) of the IUPAC 1982-1989
(together with prof. G.E. Zaikov from USSR), member of European Community Chemistry
Committee (1973-1991). Wolfgang was nominated by UNESCO as Founding Member of the
International Organization for Chemistry in Development (IOCD).
We should did indication about his honorary activities in German bodies:

1972-1996 Member of the Supervisory Board of VCH-Publishers;


1981-1991 Member of the Supervisory Board of Chemical Information Center in
Berlin;
1972-1992 Secretary of Deutscher Zentralausschuss fur Chemie (National Adhering
Organization of UPAC);
1972-1992 Secretary of the Association of Professors of Chemistry at German
Universities;
1972-1992 Member of the Board of the German Association of Technical and
Scientific Societies;
1972-1992 Member of the German National Committee of the World Energy Council

Dr. Wolfgang Fritsche has many medals and orders for his scientific activity as well as
for activity as organizer.
We should first of all remember about Honorary medal of FECS “for outstanding
services to the Federation and for furthering of international cooperation in the field of
chemistry” (!985), medal of the Societe Royale de Chimie of the occasion of the 100th
Anniversary of the Belgian Society (1987), Lavoisier-Medal of the Societe Francaise de
Chimie “pour l’ensemble des eminents services qu’il rend a la Communaute Europeenne des
Chimistes en sa qualite de Secretaire General de la Federation Europeenne des Societes
Chmiques” (1987), nomination as Life-Long Fellow of the Institute of the Chemistry of
Ireland (FICI) – 1987, Honorary Document of the Societa Chimica Italiana “ comme
espressione di profonda stima e del piu vivo ringraziamento per l’opera svolta, originale e
richissima, nella promozione della cultura chimica e nella collaborazione internazionale, …”
(1988), Gold Medal of the Fedaration of Technical and Scientific Societies of Bulgaria for the
promotion of international cooperation in Europe (1990), Nomination as Honorary Fellow of
the Royal Society of Chemistry on the occasion of the 150th Anniversary of the Royal Society
of Chemistry – UK (1991), Honorary Document of the Chinese Chemical Society ‘for his
great contributions in promoting friendship between Chinese and German chemical
communities in the past decades” (1991), Carl-Duisberg-Medal of GDC for his merits for the
benefit of Chemistry and the GDC (1991), Honorary Document of the Gesellschaft
Osterreichischer Chemiker (Austrian Chemical Society) ‘in acknowledgement of his great
merits for the Society” (1991), Hanus-Medal of Czechoslovak Chemical Society “for his
merits in promoting cooperation in Europe and the furthering of the Czechoslovak Chemical
Society effected thereby (1992), Thilo-Medal of Chemical Society of Germany “in
acknowledgement of his contributions leading to the unification of the two Chemical
Societies after the re-unification of Germany” (1992), Nomination as Life-Long Honorary
President of the FECS (1993).
Dr. Rer. Nat. Wolfgang Fritsche 247

Today Dr. Wolfgang Fritsche is 82 years old but he is full energy, force and ideas in the
field of inorganic chemistry as well as in the field of organization and cooperation in
chemistry.
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.249-250 ©2010 Nova Science Publishers, Inc.

Chapter 19

PROFESSOR VICTOR MANUEL DE MATOS LOBO ON


HIS 70TH ANNIVERSARY

Gennady E. Zaikov*1 and Artur J. M. Valente±2


1
N.M. Emanuel Institute of Biochemical physics,
Russian Academy of Sciences, Moscow, Russia
2
Coimbra University, Chemical faculty, Coimbra, Portugal

Prof. Victor Lobo graduated in Physics and Chemistry from University of Coimbra in
1963. At the end of his first undergraduate year he was invited as an “assistant demonstrator”
and as such helped in laboratory classes up to his final year. He had a University scholarship
and a research grant from “National Institute of High Culture”. While an undergraduate
student did research in radiochemistry.
Invited to join the teaching staff of the University of Coimbra in 1963, he was asked to go
“on leave” with his Professor to the University of Mozambique for three years, where he did
research in flame photometry in parallel with his teaching activity.

* N.M. Emanuel Institute of Biochemical physics, Russian Academy of Sciences, 4 Kosygin Street, 119334
Moscow, Russia, Chembio@sky.chph.ras.ru
± Coimbra University, Chemical faculty, Coimbra 3004-535, Portugal, AValente@ci.uc.pt
250 Gennady E. Zaikov and Artur J. M. Valente

He has got a Ph.D. degree from the Cambridge University (U.K.), under the supervision
of Dr. J. Agar, in 1971. During Ph.D. studies he developed a new isothermal diffusion cell,
with which he received a Gold Medal at the “15th International Exhibition of Inventions and
New Techniques”, Geneve, Switzerland in 1987.
After completing his Ph.D. studies, he returned to Mozambique for about one year,
finally to return to his post, as a lecturer, at the University of Coimbra in 1972. He was
“Associate” Professor of Electrochemistry in 1975 and Full Professor in 1981. During his
academic career he has been lecturing Electrochemistry and Corrosion, Electrolyte Solutions,
Physical Chemistry, Chemical Thermodynamics, Chemical Analysis, Medical Chemistry and
General Chemistry.
Prof. Victor Lobo has dedicated much of his life to the research of thermodynamic and
transport properties (in particular on diffusion) of electrolyte solutions, and through polymers
as well, and electrochemistry, particularly metallic corrosion. In recent years, his interests
have focused on the diffusion of multicomponent systems and diffusion of electrolytes
through polymer membranes.
He was invited researcher or visiting professor in several universities, such as University
of East Anglia (U.K.), University of Canberra (Australia), University of Gottingen (Germany)
and University of Regensburg (Germany).
Prof. Lobo has been the main responsible for the organization (1984-1989) of a databank
with data on density, viscosity, equivalent conductance, transport number, diffusion
coefficients and activity coefficients of electrolyte solutions, for the Scientific Engineering
Research Council, SERC, U.K..
He has given a considerable number of invited lectures in Universities, and conferences
on issues related to transport properties. He has been titular member of IUPAC, and
Portuguese representative in technical commissions of CEN and ISO. He was president of
Portuguese Electrochemistry Society, President of General Assembly of Portuguese Chemical
Society, Member of National Bureau of Education, and Head of the Chemistry Department.
He published more than 150 scientific articles in international and national refereed journals.
Moreover, he has published some 40 articles in the Portuguese daily press on matters
concerning education.
He has written and edited more than 10 books, of which considerable prominence must
be given to the widely used “Handbook of Electrolyte Solutions” and “Self-diffusion of
Electrolyte Solutions”, the latter written with R. Mills, published by Elsevier.
He is the Editor of Portugaliae Electrochimica Acta – the journal of the Portuguese
Electrochemical Society – since September 2002.
Besides his professional skills he has a lovely character.
On the occasion of his 70th birthday, it is with a great pleasure that we wish to Prof.
Victor Lobo, who has dedicated many years to Academy and Science, good health and further
achievements.
Prof. Victor Manuel de Matos Lobo is full of energy and new ideas for research in
electrochemistry, physical chemistry as well as in chemistry of highmolecular compounds.
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.251-261 ©2010 Nova Science Publishers, Inc.

Chapter 20

THE SCIENTIST WHO OUTSTRIPPED HIS TIME

Revaz Skhiladze and Tengiz Tsivtsivadze


Georgian Technical University, Tblisi, Georgia

ABSTRACT
This article is dedicated to the 100 year anniversary of the birth of the Georgian
prominent scientist, Professor Akaki Gakhokidze. He was one of the outstanding
representatives of the Georgian school of chemistry. High theoretical preparation,
mastery of experiments, unusual scientific flair and intuition allow him to leave a great
and light footstep for posterity on the way of scientific research and pedagogical activity.
The fundamental investigation of A. Gakhokidze won international recognition.
His works were published and broadly considered in the special literature, monographs
and manuals of chemistry. There were created the specific terms - “Synthesis of
Gakhokidze”, the “method of Danilow-Gakhokidze” etc.

A. Gakhokidze was born on 14 August 1909 in the village Zeda Khuntsi, Martvili district
of Georgia. Martvili belongs to the region of Mingrelia which is the native land of many
remarkable persons in the past. They are the pride of Georgian people today. One of them on
which we want to draw your attention to was a bright representative of the scientific elite of
Georgia Akaki Gakhokidze, the person world renowned, scientist of international scale, who
made a huge contribution in the development of chemical sciences.
His initial education was received at home, in his village. At first he attended the Khuntsi
elementary school and then proceeded to the seven year school. During school study he was a
very diligent and industrious pupil.
From 1924 A. Gakhokidze continued studies in Tbilisi Melikishvili college, which he
graduated with high academic success after what he was sent for practice passing in Baku in
1927 where A. Gakhokidze begin work in the oil purification plant. There turned out that only
theoretical preparation without practice is quite insufficient for him. He recalled later “I
thought that I know very well oil technology but soon I understood that an engineer without
practice is like to the unarmed warrior. I started to study everything, beginning from the
252 Revaz Skhiladze and Tengiz Tsivtsivadze

simple worker, finishing the engineer. It is clear that all became easy for me and I have soon
connected the theory with practice”.
In 1928 A. Gakhokidze graduated the college very successfully and he begin work in the
soap plant (next the chemical industrial complex) of Tbilisi. At the same time he begin
preparation for examinations of higher school. On August of 1928 he entered in the
polytechnic faculty of Tbilisi University. Parallel studying A. Gakhokidze interested the
problem of sugar production in Georgia.
In this time there were functioned two institute in the former USSR – in Kiev (one –
educational, second - scientific research) where the sugar production problems were being
solved. Student A. Gakhokidze was their frequent guest.
For A. Gakhokidze the task to build the sugar production plant in Georgia was the one of
great importance. He elaborated in Kiev the sugar production plant for Georgia. There is the
fragment of recollection of A.Gakhokidze: “I composed and wrote the plant project right
away, made up 30 design drawing, the explanatory letter took 400 page, in Tbilisi it was not
required corrections. After it consideration professor I.Burjanadze signed the project on the it
public presentation. This project drawing up gave me important knowledge”. When sugar
production plant director in Kiev acquainted this project, he told to project author with
admiration: “the building of sugar plant in Georgia must be realized by your project.” In June
1929 A. Gakhokidze successfully defended the sugar plant project and he became an engineer
technologist. Since 1932 year A. Gakhokidze continued post-graduated study in Leningrad
(St.Peterburg). He works there in the field of carbohydrate chemistry under direction of
professor Danilow. S.Danilow was the student of Russian outstanding chemist A.Favorski,
whose scientific activity goes back to the founder of organic chemistry Butlerow. A.Favorski
was the head of Leningrad Chemical Society where in general meeting two times in month
was being made the report about new discovery in chemistry. At 18 December 1935 professor
Danilow in the next meeting made the brief information about Gakhokidze’s work. He noted
that like simple aldehydes the sugars are proved analogous isomerization and there are
obtained saccharinic acids. “One of our students represented dissertation about this problem”,
told Danilow. Favorski asked him who was this young scientist and the last pointed to
Gakhokidze. Later one jester chemist explained to Gakhokidze the reason of Favorski
interest: “you are now his grandchild and he wanted to see you.” The joker meant following
sequence: Zinin → Butlerow → Favorski → Danilow → Gakhokidze.
At November of 1935 A. Gakhokidze successfully defends dissertation “Isomerization of
glucose in saccharinic acid”. Scientific council by a solid vote appropriates to A. Gakhokidze
the candidate‫ۥ‬s degree. In the same year was published his large experimental work in the
German journal “Berichte der Deutschen Chemischen Gasellschaft”. This work had large
comments between chemists. Method elaborated in it was included in the manual for students
of higher school as the “Danilow-Gakhokidze method”.
In 1939 A. Gakhokidze comes back in Georgia and begins work in the Institute of
processing of a wood material as a docent. Since 1937 he works in Tbilisi Pedagogical State
Institute as Dean and next as a Head of faculty of chemistry till his death. In 1934 A.
Gakhokidze was sent again in Leningrad by recommendation of Georgian Science Academy
as a person working for doctor's degree. Doctor dissertation was defended in 1948 very
successfully. In this time he was a first doctor chemist in organic chemistry in Georgia.
1940-1947 years A. Gakhokidze worked (parallel of pedagogical activity) in the institute
of chemistry of Georgia Science Academy and then in 1947-1950 – in the Scientific-Research
The Scientist Who has Outstripped His Time 253

Chemical- Pharmaceutical Institute in Tbilisi, where he founded the department of medicine


technology.
Professor A. Gakhokidse is most outstanding representative of Georgian Chemists
School. His life credo is very many-sided. He was a kind and tactful person. There were not
for him large or small problems. He with identical attention concerned them. All students for
him were young men which should be become not only good specialists but also worthy
citizens of our country.
The fundamental investigations of prof. A. Gakhokidze have an international recognition.
The goal of this article is to touch more detail to activity of prof. A. Gakhokidze in
carbohydrate chemistry. He has elaborated original method of disaccharides synthesis by
which there appeared a possibility to receive unknown (until then) type of disaccharides
synthesis. This type compound caused a great interest because of foreigner scientists working
on the same problem possibility of existence of new type (1,2- and 1,3- bond consisted)
disaccharides except of trehalose, maltose and gentiobiose type (1,1-, 1,4- and 1,6-bond
consisted) disaccharides. For example we can bring the schemes of synthesis of glucosyl- 2-
glucose (sophorose) and glucosyl-3-glucose (laminaribiose). From 2,3,4,6,-tetra-0-acetyl-α-
D-glucopyranosyl bromide(I) and 1,3,4,6–tetra-0-acetyl-β-D-gluco-pyranose (II) he obtained
2-0-β-glucopyranosyl-D-glucopyranose (sophorose, III):

CH2OAc CH2OAc CH2OAc


O O OAc O OAc
ZnCl2, P2O5 CH3ONa
OAc + OAc OAc
AcO Br AcO AcO
OAc OH
CH2OAc
I II O
O
Ac = COCH3
OAc
AcO
OAc

CH2OH
O OH

OH
HO

CH2OH
O O

OH
HO
OH
III
254 Revaz Skhiladze and Tengiz Tsivtsivadze

Structure of new disaccharide were determined by author by following way: by action of


hydroxylamine on the disaccharide was obtained corresponding oxyme (IV) and by
interaction of last with acetic anhydride is obtained acetylated nitrile of glucosyl -2-gluconic
acid (V). Author transferred this substance into glucosyl-1-arabinose, via of saponification
and nitrile group splitting, which does not act with phenylhydrasine:

CH2OH CH2OAc
OH OAc

OH CH NOH OAc C N
HO AcO
III CH2OH CH2OAc
O O O O

OH OAc
HO AcO
OH OAc
IV V

HO
HO
CH2OH
OH
O O
OH
HO
OH
VI

By oxidation of disaccharide (III) (with brome water) the author received glucosil-2-
glucone acid (VII) and it’s derivatives, but by hydrolyze of acid – glucose(VIII) and glucone
acid (IX):

CH2OH
O

HO OH
O OH
CH2OH
O
OH
HO
OH
XII
The Scientist Who has Outstripped His Time 255

CH2OH
OH

OH COOH COOH
CH2OH
HO
O
OH
III CH2OH
OH OH + HO
O O
HO OH
OH OH
OH
HO CH2OH
OH VIII IX
VII

Analogously, disaccharides with different monosaccharide residues (glucosyl-2-


galactose, galactosyl-2-galactose, galactosyl-2glucose, manosyl-2glucose, manosyl-2-
manose) has been obtained.
From 2,3,4,6,-tetra-0-acetyl-α-D-glucopyranose (X) and 1,2-0-isopropyliden-4,6-0-
benziliden-α-D-glucopyranose (XI) A. Gakhokidze obtained 3-0-β-D-glucopyranosyl-D-
glucopyranose (laminaribiose,XII):

CH2OAc OCH2
O H5C6
O
OAc + OH ZnCl2, P2O5

AcO OH O O
OAc O CH3

X XI
CH3
OCH2
H5C6
O

CH3ONa
O O
CH2OAc O O CH3
O
CH3
OAc
AcO
OAc

By similar procedures, A. Gakhokidze synthesized disaccharides with different


monosaccharide residues.After some tens years there were corroborated once again the
structure of substances obtained by A. Gakhokidze by several scientists with application of
modern physical and chemical research methods.
Lately, it was proved that new type compounds discovered by A. Gakhokidze are
widespread in nature and they have high physiologic (immunologic, antitumoral and others)
activity. Methods of synthesis of those class carbohydrates in the world scientific literature
are known under name of “Gakhokidze’s syntheses”.
256 Revaz Skhiladze and Tengiz Tsivtsivadze

The central problems of A. Gakhokidze’s scientific researches were the investigation of


physiological active compounds, their study and application. The theory of A. Gakhokidze
was the foundation of application of carbohydrate as a transporter of medicine preparations.
The principle of glycosylation of medicine means elaborated by A.Gakhokidze, which is
based on the active transport of carbohydrate fragments in the cell membranes, represents a
new approach of a creation of medicine preparations of task-oriented action.
Application of these preparations in clinical pharmacology which are not solved in water
has very important disadvantage which consists that their obtaining is possible only internal
way or with external influence. Mentioned circumstance obviously restricts to the possibility
of their usage in medical practice. A. Gakhokidze provided for great importance of
conversion of medicine means in water soluble form in which was being given the possibility
to introduce parenterally these medicines. In result there would be changed not only
absorption but accelerated physiologic effectiveness and sharply decreased the preparation
toxicity. All above spoken pointed out to actuality of synthesis development of water soluble
medical preparations. It was the question of very great significance solution of which had a
great importance in the provision of population by medical preparations.
Clinical medicine in this period widely used sulfa drugs which not being solved in water.
A. Gakhokidze envisaging the actuality of problem laid down the aim to converse just
sulfanilamide in solved form in water.
After studying the question in detail, A. Gakhokidze solved the problem by following
way. He realized the sulfanilamide condensation with glucose for obtaining of soluble one
and determined the conditions of reaction. In the solution of diluted ethanol at the interaction
of streptocide with glucose, at presence of calcium chloride gives monoglucosulfanilamide
(XIII), but if repeat this reaction in the absolute ethanol mediom, we obtain
diglucosulfanilamide (XIV).

What about the question of obtaining of soluble aspirin, A. Gakhokidze obtained chlorine
anhydride of aspirin by interaction of sodium salt of aspirin and thionyl chloride, then he
realized the condensation of glucose and last component (with presence of quinoline) and
The Scientist Who has Outstripped His Time 257

partially saponification by sodium acetate of obtained pentaaspiringlucose (XV) he prepared


pentasalicylglucose (XVI). Obtained product is the water soluble preparation what gives the
possibility to prepare injection form of medicine for parenteral application.

Prof. A. Gakhokidze obtained also water soluble monoaspiringlucose (XVII) by


following scheme:

OAc OAc
OH
O O OCOC 6 H 4 OH

AcO
OAc
Br
+ H4 C 6
Pb
AcO
OAc
COO
OAc 2 OAc

OH OH
O OCOC 6 H 4 OH O OCOC 6 H 4OAc
OH OH
HO HO
OH OH

XVII

As a conducted researches showed insoluble preparations solve in the water by


“fastening” of carbohydrate molecules and absorb easy by organism. There is significantly
increased the physiologic effectiveness of medical preparations and decreased their toxicity.
Clinical medicine today widely uses such an approach “to ennoble” the preparations for
treatment of malignant tumors.
For example interaction product of glucose and nitrogen yperit is used successfully in
oncology:
258 Revaz Skhiladze and Tengiz Tsivtsivadze

Cl
OH
O O CO CH2 N
OH n
HO
OH
Cl

n=1,2,3

In comparison of live cells with neoplasm ones, the tumor cells are distinguished by
intensive glycolise. This reason is considered by scientists to be unusual penetration of
glucose in the shell of neoplasm cells. Therefore in such compound the carbohydrate residue
is the carrier, which provides more selected concentration in the neoplasm cells. The same
idea has laid down for obtaining of chlorethylamides of aldaric and aldonic acids.
It is known that natural compounds are easy subjected to decomposition, therefore they
can not perform the role of ideal medicines. This noted circumstance force the researchers
find the way of natural compounds modification, for obtaining of stable medicine means of
long duration pharmacologic influence. The light confirmation of this is the elaboration of
carbohydrate condensation methods with ascorbic acid and other physiologic active
substances.
In today world the searching of new medical means is most expensive, long and less
effective process. This was mentioned on the one of meetings of National Academe of USA
in New York.
Medicine service market today need not increase of new, synthesis means potential but
the correction of known, approved by clinical medicine drugs i.e. increase of their specificity,
acceleration of their participation in the pathologic region, reversal of toxic influence from
organism what cardinally changes the pharmacokinetics and pharmacodynamics of
preparations.
Prof. A. Gakhokidze obtained also the soluble starch. There has a great importance of
A.Gakhokidze‫ۥ‬s achievement in veterinary practice, in the obtaining of helminthologic means.
There must be noted the obtaining new preparations between which substances consisted
manganese and arsenic obtained by mining of natural deposits of Chiatura and Racha.
One of basic purposes of A. Gakhokidze was the study and application of natural
resources. By him was stated the structure of some glucosides and pigments and elaborated
the methods of their synthesis. For example he isolated new flavonides – akrammerin
(XVIII), olmelin (XIX) and 3-D-glycosylepicatechin (XX) with different acids and glycosides
from plant gleditschia triacanthos to which the clinicians appropriate an important value in
phitotherapy as a means of cardiovascular and malignant diseases.
The Scientist Who has Outstripped His Time 259

OH
O
OH
OH O HO

OH

HO OH O
OCH 3
H 3CO O

XVIII
akramerini XIX
olmelini

OH
HO
O
OH
OH
O

OH O
HO OH
OH

XX
3-D-glukozilepikateqini

Prof. A. Gakhokidze synthesized akrammerin and olmelin according to the following


scheme:

OH OCH 2 C 6 H 5
HO OH HNO3
C6H5CHCl2
H 5 C 6 H 2 CO OCH 2 C 6 H 5 H 5 C 6 H 2 CO OCH 2 C 6 H 5

OH OCH 3
OCH 3
(CH3)2SO4
COCH 3

H 5 C 6 H 2 CO OCH 2 C 6 H 5 H 5 C 6 H 2 CO OCH 2 C 6 H 5
H 5 C 6 H 2 CO OCH 2 C 6 H 5
OH OCH 3
OCH 3

OCH 3
H 3 CO OCH 3 OCH 3 OH
OCH 3 OH
H 3 CO O OH O
COOK
OCH 3 HI
OH

HO HO
H 3 CO O OH O

dimeTilirebuli
Demethylated
akramerini akrammerin
OCH 3
OCH 3
H 3 CO O

OCH 3

H 3 CO
H 3 CO O

meTilirebuli
Methylated
akrameriniakrammerin
260 Revaz Skhiladze and Tengiz Tsivtsivadze

HO OH
NC HCl HO OH
H2O
+ OCH 3 +
NH2 Cl
-

OH
OH
fluroglucini
Phloroglucinol
OCH 3

HO OH
O
O HCOOC2H5
HO

OH

OCH 3 OH O
OCH 3

Prof. A. Gakhokidze elaborated method of hydroxicarbonic acids synthesis which is


based on the condensation of ketones and esters of carbonic acids:

R R'' R R''
CO + CH2 C(OH) CH
R' COOR''' R' COOR'''

R R''
C(OH) CH
R' COOH

On the base of these investigations by A. Gakhokidze was arisen an original theory of


organic acid formation in the plants. In accordance with this theory, for example, citric acid is
formed from glucose according to the following scheme:

COOH COOH
COOH COOH
CHO COOH H OH
H OH CH 2 CH 2
H OH H OH HO H HCOOH
HO H CO C(OH)COOH
HO H HO H H OH
H OH CH 2 CH 2
H OH H OH COOH
CO COOH COOH
H OH H OH
CH2OH
CH 2OH CH 2OH
+
HCOOH

This scheme was proved as chemical so biochemical synthesis. Splitting between 4 and 5
carbon atoms of 5-keto-gluconic acid gives rise to other organic acids.
A. Gakhokidze paid a great attention to the application of agricultural wastes. He
obtained from maize waste xylotrihydroxyglutaric acid. It can change the tartaric acid in the
techniques. Yield of this acid from maize waste consisted 6%.
A. Gakhokidze separated citric acid from tobacco waste, from different region of
Georgia.There was stated that waste from Lagodekhi consisted 6.37% of citric acid, from
Gagra region – 4.71%.
By processing of shell of tung-tree A. Gakhokidze separated the dye and stated it‫ۥ‬s
empirical formula.
The Scientist Who has Outstripped His Time 261

Prof. A. Gakhokidze has founded chemical investigation of the oil in Georgia, which is of
great theoretical and practical meanings. He studied oil deposits of Supsa, Mirzaani and
Shirakhi.
In 1942 A. Gakhokidze came out with an original hypothesis about oil genesis. He
showed that transformation of carbohydrates in nature besides biochemical conversion is
possible also by geologic metamorphosis. For proving his opinion, he obtained 2-
methylheptane (isooctane) from glucose by method elaborated by him (Gakhokidze was the
first investigator who used metalorganic synthesis in carbohydrate chemistry):

CH2OH CH2OH CH2OAc


O O O
Br2, CaCO3 (CH3CO)2O
OH 1. CH3MgI
OH O O
OAc
HO OH HO 2. H2O
AcO
OH OH OAc

CH3 CH3 CH3

H 3C COH H3C CI H3C CH

HCOH CH2 CH2


HI, P Mg
HOCH CH2 CH2
HCOH CH2 CH2
HCOH CH2 CH2
CH2OH CH3 CH3

This research is very important for power engineering in future. A. Gakhokidze‫ۥ‬s


considerations were proved after 40 years by Canadian scientists. There were discovered
microorganisms which transform the carbohydrates into oil (oil fermentation).
Prof. A. Gakhokidze wrote many courses of chemistry for students; there were published
course of organic chemistry, manual of organic and biological chemistry, inorganic chemistry
and many other books.
The life of prof. A. Gakhokidze interrupted very unexpectedly at the age of 55 years in
1964. He could not realize completely many new scientific ideas, new scientific beginnings,
but what he has created that remains forever in a treasury of a world science.
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.263-265 ©2010 Nova Science Publishers, Inc.

Chapter 21

PROF. DR. RYSZARD MICHAL KOZLOWSKI:


HALF A CENTURY IN SCIENCE AND TECHNOLOGY

Gennady Zaikov*
N.M.Emanuel Institute of Biochemical Physics,
Russian Academy of Sciences, Moscow, Russia

Prof. Ryszard Michal Kozlowski retired in 2009 from the position of the director of
Institute of Natural Fibres and Medicinal Plants (Poznan, Poland). It is now the right time to
make a short review of his activity in pure and applied science as well as in the organization
of science and technology.
Ryszard (Richard) Kozlowski was born on July 28, 1938 in Uniejów (pronounce:
Oonyeyoov), Poland. He graduated from the Adam Mickiewicz University in Poznan in 1961,
receiving the degree of Master of Applied Chemistry. In 1970 he received Ph.D. degree in
chemical technology from the same University. After his graduation he worked for the
Institute of Bast Fibers, later renamed to the Institute of Natural Fibers. In 1973 he completed
postgraduate studies in the field of research organization. Three years later, he became deputy
director of the Institute of Natural Fibers, and at the same time he was the head of the
Department of Lignocellulosic Boards and By-Products. In 1979 he went through a special
training in the field of the manufacture and upgrading of particle boards and preservation of
wood at Technical Research Center of Finland. On August 1, 1987, he became General
Director of the Institute of Natural Fibers in Poznan and did this duty until December 31,
2008. In 1990, President of the Republic of Poland made him a professor of technical
sciences.
Professor Richard Kozlowski carried out innovative research in the field of
lignocellulosic fibrous raw materials, wool and silk, processing of bast fibers, environmental
protection, utilization of by-products and waste materials from textile industry and the
manufacture of agro- fine chemicals. He was also involved in research on the application of
natural polymers and composites to industry, including modern composites reinforced with
natural fibers. Other areas of his research work were reclamation of soils polluted with heavy

* N.M.Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygin st., Moscow 119991,
Russia, Chembio@sky.chph.ras.ru
264 Gennady Zaikov

metals and the application of such soils to the cultivation of raw materials for textile industry.
The above studies were financially supported by the European Framework Projects V, VI and
VII, as well as by the Polish Ministry of Science and Ministry of National Economy, and by
international companies such s Airbus, Lenzig, and others.
In 1989, professor Kozlowski was appointed to a post of coordinator of the Flax Research
Group, operating under the auspices of Food and Agriculture Organization (FAO) of the
United Nations in Rome. In 1993, the Institute of Natural Fibers headed by Professor
Kozlowski became the Coordination Center of FAO’s European Cooperative Research
Network on Flax and other Bast Fibers. Activities of Professor Kozlowski in the field of
projects carried out under the auspices of FAO, which lasted for over 20 years, significantly
contributed to the intensification of the exchange of scientific ideas and technology transfer in
the field of natural fibers between research centers in Europe and in the world. From 2008,
Professor Kozlowski is a coordinator of Focal Point ESCORENA, which works for the good
of many important areas of European agriculture.
Professor Kozlowski represents the Institute of Natural Fibers and Polish science in a
number of scientific and professional organizations in the world. He gave invited lectures in
Australia, Brazil, China, Romania, Russia, Ecuador, Columbia, Republic of South Africa and
other countries. As an international consultant for cellulosic and protein fibers he is an author
of analyses, forecasts and economic plans. Professor Kozlowski in an author of the treatise
“Hemp – an alternative plant”, which was prepared for the European Office of FAO in Rome.
He is a consultant of UNIDO in Vienna and an expert of the Polish Ministry of Culture and
Arts on safety of public collections – the protection of materials from fire. Professor Richard
Kozlowski is an author and co-author of 300 original research papers, 7 books, 25 patents and
24 production technologies that were implemented to industry. He is the author of production
technologies for fiber degumming with the use of osmosis, enzymes and ultrasounds,
production technologies for waterproof and fireproof composites. New generations of fire
retardant, fungi-resistant and insect resistant agents for the protection of lignocellulosic
materials were developed by Professor Kozlowski and implemented to industry. These
technologies (including intumescent coatings of Expander type) make over 90 % of modern
protection agents manufactured in Poland.
Professor Kozlowski also carried out pioneering research in the field of therapeutic and
dietetic properties of flax-seed and the application of biotechnology to the cultivation of
fibrous plants and biosynthesis of cellulose.
Results of Professor Kozlowski’s studies are of importance to developing countries,
particularly as concerns reactivation and intensification of production and processing of
natural fibrous raw materials, which is very important for the development of rural areas.
For his outstanding research achievements, Richard Kozlowski received the title of
Professor Honoris Causa (Honorary Professor) of the Pontifical Catholic University in
Ibarra, Ecuador. He received many Polish honors and distinctions, among the order “Polonia
Restituta” (he is the knight of the order “Polonia Restituta”), Golden Cross of Merit, awards
of Ministry of Science, Higher Education and Technology. Moreover he was “Man of the
Year” in 1992/93 and 1995/96 and his biography was included in “International Who’s Who
of Intellectuals” “Dictionary of International Biography”, “Five Thousand Personalities of the
World”.
Professor Kozlowski is an honorary member of The Textile Institute in Manchester,
charter-member and vice-president of the Polish Section of the The Textile Institute, member
Prof. Dr. Ryszard Michal Kozlowski, Half a Century in Science and Technology 265

of Polish National Committee of ICOMOS (International Council of Monuments and Sites)


and a number of other organizations.
Professor Richard Kozlowski is an editor-in-chief of “Journal of Natural Fibers”
(published by Taylor & Francis, USA), EUROFLAX Newsletter (Information bulletin of the
FAO European Cooperative Research Network on Flax and other Bast Fibers). He is also a
member of editorial boards of such journals as “Colourage”, “Polymer Research Journal”
(Nova Science Publishers, USA), “Vlasberichten”, “Bioresources Technology Journal”,
“Fibres & Textiles in Eastern Europe”.
Professor Kozlowski significantly contributed to a new approach to natural fibers and
polymers and to the decision of the United Nations and FAO that the year of 2009 was
proclaimed the “International Year of Natural Fibers”.
Today Ryszrad Michal is full energy, force and ideas in the field of pure and applied
chemistry. No doubt, we will see his new success in chemistry, biology and medicine.
In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0
Editors: G. E. Zaikov, R. M. Kozlowski, pp.267-269 ©2010 Nova Science Publishers, Inc.

Chapter 22

THE SECOND INTERNATIONAL CONFERENCE ON


BIODEGRADABLE POLYMERS AND
SUSTAINABLE COMPOSITES
(BIOPOL-2009)

G. E. Zaikov, L. L. Madyuskina and M. I. Artsis


N.M.Emanuel Institute of Biochemical Physics,
Russian Academy of Sciences, Moscow, Russia

The Second International Conference on Biodegradable Polymers and Sustainable


Composites (BIOPOL-2009) was held in the period of September, 30 – October, 2 2009 at the
University of Alicante, Spain. Scientists from both academic and industrial laboratories who
are interested in biodegradable polymers and biocomposites were encouraged to participate
with the aim to exchange up-to-date ideas on current research and new applications.
150 scientists from 16 countries (Spain, Italy, Portugal, France, UK, Holland, Belgium,
Georgia, Russia, Sweden, USA, Canada, Argentina, Hungary, Greece, Czech Republic ) took
part in this conference and represented 65 research centers of universities, institutes and
companies.
World known scientists in the field of polymer chemistry were members of Scientific
committee of this conference: Luc Averous (Université Louis Pasteur, Strasbourg, France),
Lars Berglund (KTH, Stockholm, Sweden), Norman Billingham (University of Sussex,
Brighton, UK), Giovanni Camino (Politecnico di Torino, Italy), Philippe Dubois (Université
Mons-Hainaut, Belgium), Alain Dufresne (Institut National Polytechnique Grenoble, France),
Alessandro Gandini (University of Aveiro, Portugal), Sigbritt Karlsson (KTH, Stockholm,
Sweden), José M. Kenny (Universitá di Perugia, Terni, Italy), José M. Lagaron (IATA-CSIC,
Valencia, Spain), Carmen Mijangos (ICTP-CSIC, Madrid, Spain), Iñaki Mondragón
(Universidad del País Vasco, San Sebastián, Spain), Carlos Pascoal Neto (University of
Aveiro, Portugal), Kristiina Oksman (Lulea University of Technology, Sweden), Andrea
Pipino (Centro Ricerche Fiat, Orbassano, Italy), David Plackett (RISØ-DTU, Roskilde,
Denmark), Amparo Ribes (Universidad Politécnica de Valencia, Spain), Roxana Ruseckaite
268 G. E. Zaikov, L. L.Madyuskina and M. I. Artsis

(Universidad Nacional de Mar del Plata, Argentina), Julio San Román (ICTP-CSIC, Madrid,
Spain), Sabu Thomas (Mahatma Gandhi University, India).
The chairman of Conference was Prof. Alfonso Jiménez from the University of Alicante.
Carmen Bueno, Nuria Burgos, M. Carmen Garrigós, Verónica Martino, Olimpia Mas,
Eduardo Paredes, Mercedes Peltzer, Marina Ramos, Raquel Sánchez, Amanda Terol, José
Luis Todolí were members of Organizing Committee of this conference.
Scientific programme of the conference was divided into 8 sessions. The first session
included 2 invited lectures. Alessandro Gandini spoke about furan monomers and furan
chemistry at the service of polymer science. The second lecture was done by Sigbritt Karlsson
and had the title “Resource and environmental aspects of sustainable biocomposites”.
The second session included 7 oral presentations which were devoted to the next
problems: evaluation of thermal degradation and dynamic mechanical properties of PP / hemp
fibres composites: fibre plasma modification; electrospun cross-linked collagen nanofibers as
novel bone tissue interfaces; on the plastifying effect of β-carotene in biopolyester matrices;
synthesis of graft copolymer of ethylmethacrylate onto polydichlorophosphazene and its
ultrasonic degradation; citric acid/starch catalysed sterification.
The third session included 1 invited lecture and 2 oral presentations. Lars Berglund in the
invited lecture spoke about nanocelluloses as unique polymeric building blocks for
nanostructured polymer systems. The information about PLA and PCL nanocomposites
preparation and biodegradation and the information about thermal analysis applied to the
characterization of degradation in soil of polylactide were discussed in the oral presentations.
The fourth session included 2 invited lectures and 2 oral presentations. The invited
lectures were devoted to nano-biocomposites: agropolymer/nanoclay systems (Luc Averous),
and processing and characterization of PLGA-carbon nanotube nanocomposites for bone
tissue engineering (Jose M. Kenny). The oral presentations included the information about
development and characterization of nanobiocomposites based on plasticized poly(lactic acid)
and organomodified montmorillonite and the information how to shift toughness of PLA into
non break area and create high impact flax fibre reinforcements.
The fifth session included 5 oral presentations which were devoted to the next problems:
influence of plasticization on barrier properties of poly(lactic acid); influence of the
recrystallization conditions on the crystallinity and barrier properties of polylactide acid
(PLA) food packaging films; development of new biodegradable nanocomposites based on
polylactic acid / natural rubber blends for packaging applications; nano-biocomposites based
on poly(lactic acid)-poly(hydroxybutyrate) blends; polyesters from renewable resources based
on furan monomers: alternative materials to aromatic counterparts.
The sixth session included 1 invited lecture and 3 oral presentations. The invited lecture
was done by Gennady E. Zaikov (Institute of Biochemical Physics Russian Academy of
Sciences, Russia) and was about biodegradation and medical application of microbial poly(3-
hydroxybutyrate). The problems of new ways for blending biodegradable polymers with
poly(vinyl chloride), of secondary plasticizers for PVC obtained from cardanol and of
improved properties of formol-phenolic adhesive by adding natural-based fillers were
discussed in the oral presentations.
The seventh session of this conference included 2 invited lectures and 2 oral
presentations. David Plackett in the first invited lecture spoke about nanocellulose-reinforced
bioplastics – prospects for further development. Jean M. Raquez. gave information about
recent development of novel (bio)polymers and related composites implemented by reactive
The Second International Conference on Biodegradable Polymers … 269

extrusion. The oral presentations were devoted to high barrier nanocomposites of


biopolyesters, proteins and polysacharides for coating and packaging applications and to
starch nanoparticles for eco-efficientpackaging: influence of botanic origin.
The last eighth session included 4 oral presentations, in which there were discussed the
next problems: olive stone as a renewable source of biopolyols; simple and quick method to
prepare highly hydrophobic cellulosic materials by vapor-phase reaction with chlorosilanes;
development of a method for biodegradability evaluation on leather used in the footwear
industry; chitosan as an antimicrobial agent for footwear leather components.
The two poster sessions of this conference included 70 presentations in which were
discussed some particular tasks in the field of biodegradable polymers and sustainable
composites. Particularly there were some posters of Russian scientists: stabilization of
polymers from the influence of biological media, kinetic method of biocide efficiency
estimation; diagnostics of quality and prognosing of potatoes safe storage duration; effects of
lead diacetate on structure of neurotropic drug (piracetam): conformational polymorphism;
application of poly-HEMA embolic agent for target delivery cytostatic drug – doxorubicin.
The next 3rd International Conference on Biodegradable Polymers and Sustainable
Composites will be held in Strasbourg (France) in last week of August 2011. Prof. Luc
Averous is responsible for organization of this conference.
INDEX

aliphatic polymers, 45
A  alkyl macroradicals, 146, 148
allergic reaction, 171
absorption, 13, 16, 57, 58, 93, 125, 133, 141, 166,
alpha-tocopherol, 42
206, 211, 212, 215, 256
alternatives, 92
abstraction, 8, 129, 131, 138
aluminum, 92, 111, 179, 180
access, 51, 156, 208
amines, 143, 144, 168, 172
acclimatization, 77
amino acids, 171
acetone, 168
ammonia, 168, 178, 188
acetonitrile, 130, 131
ammonium, 171, 177
acetylcholinesterase, 24
amorphous polymers, 57, 191, 195, 199, 210
acid, 7, 13, 22, 31, 32, 38, 40, 63, 67, 75, 76, 77, 78,
amplitude, 31
79, 80, 81, 83, 143, 149, 156, 158, 159, 161, 168,
anatomy, 97
169, 173, 178, 179, 187, 211, 212, 213, 215, 217,
anchoring, 173
222, 227, 241, 242, 252, 254, 260, 268
aniline, 168, 188
acidity, 221, 222, 225
anisotropy, 119
acrosome, 80
annealing, 197, 198, 201, 203
acrylate, 189
ANOVA, 103, 104
activation energy, 3, 242
antibacterial soap, 171
active additives, 12
antioxidant, ix, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22,
active centers, 126
24, 27, 29, 66, 67, 70, 71, 72, 73, 81, 83, 84, 85,
active oxygen, 172
86, 87, 89, 90, 159, 207, 217
active radicals, 124, 133
antioxidative activity, 72, 89
active transport, 256
aorta, 23
adaptation, 81
applications, x, 62, 123, 133, 134, 136, 165, 166,
additives, 63, 148, 168, 205, 208, 209, 210, 211, 212,
178, 180, 219, 267, 268, 269
214
aqueous solutions, 31
adenine, 81
Arabidopsis thaliana, 78, 81
adenosine, 76
aromatic polyimide, 62, 206
adhesion, 109, 116
aromatic rings, 129, 136, 138
adsorption, 243
Arrhenius equation, 6
AFM, 106, 107
ARs, 123, 124, 125, 127, 128, 130, 131, 132, 133,
agar, 183, 184, 185
134, 136, 137, 146, 148
aggregation, 182
arsenic, 258
aging process, 12
arthropods, 119
agriculture, 27, 264
articular cartilage, 93
alcohols, 3, 31, 67, 71, 86, 89, 107, 130, 131, 167
ascorbic acid, 167, 170, 258
aldehydes, 67, 85, 88, 107, 171, 252
272 Index

atomic force, 107 carbon, x, 1, 5, 9, 45, 46, 51, 53, 61, 62, 63, 75, 76,
atoms, 50, 57, 78, 80, 125, 127, 130, 135, 136, 138, 78, 80, 83, 128, 132, 133, 137, 139, 206, 216,
166, 213, 215, 223, 224, 225, 230, 231 260, 268
autooxidation, 11, 12, 13, 14, 15, 19, 65, 66, 67, 83, carbon atoms, 75, 78, 80, 137, 260
84, 85, 86, 87 carbon dioxide, x, 9, 45, 46, 51, 53, 61, 62, 63
Avogadro number, 192 carbon nanotube nanocomposites, 268
carbon tetrachloride, 133
carbonic acids, 260
B  carbonyl groups, 144, 211
carboxylic acids, 168
bacteria, 166, 170, 171, 172, 174, 175, 177, 178, carboxylic groups, 217
183, 184, 185, 186, 188 carboxymethyl cellulose, 169, 188
bacteriostatic, 176, 178, 183, 184, 185 carotene, 268
bacterium, 188 carrier, 67, 77, 85, 258
benzene, 136, 148 cartilage, 92, 93
binding, 38, 40, 125, 150, 173, 215, 228 cast, 46, 58, 61
biochemistry, 10, 130 catalysis, 133, 206, 227, 228, 243
biodegradability, 269 catalyst, 180, 192
biodegradation, 268 catalytic properties, 229
biological activity, 65, 84 catalytic reaction, 228, 230, 243
biological media, 269 cell, 13, 22, 30, 35, 38, 51, 76, 78, 81, 92, 93, 95,
biological systems, 22, 24, 92, 93 108, 109, 110, 111, 112, 114, 115, 116, 117, 166,
biomass, 22, 23 171, 172, 173, 174, 177, 185, 250, 256
biopolymer, 134 cell death, 172, 174
biosynthesis, 264 cell membranes, 30, 78, 171, 185, 256
biotechnology, 80, 82, 264 cell surface, 173
bisphenol, 206, 211 cellulose, x, 21, 22, 23, 27, 92, 107, 108, 178, 184,
black tea, 73, 90 188, 264
bleaching, 178 chain branching, 207
blends, 176, 268 chain mobility, 228
blocks, 158, 162, 172, 216 chain propagation, 14
body fluid, 176 chain scission, 4, 5, 7, 8, 218
bonding, 7, 19, 216 character, 32, 125, 135, 136, 161, 193, 201, 250
bonds, 5, 8, 9, 57, 60, 131, 132, 148, 178, 212, 213, chemical interaction, 211, 227, 228
228, 229, 230, 233 chemical kinetics, 133
bone, 92, 93, 268 chemical properties, 46, 53, 119, 123, 129, 133, 136,
branching, 12, 207, 212, 221, 222 139, 161
bromine, 130, 131 chemical reactions, ix, x, 131, 133, 139, 227
buffer, 23, 98 chemical stability, 139
building blocks, 165, 268 chitin, 92, 94, 95, 99, 100, 103, 105, 106, 107, 120
bulk materials, 166 chlorine, 2, 3, 130, 131, 178, 256
Burkina Faso, 73, 90 chlorobenzene, 13, 18
butyl methacrylate, 62, 63 chloroform, 97, 214, 215
by-products, 157, 263 cholesterol, 38, 40
chromatography, 66, 67, 77, 84, 85
C  chromatography analysis, 77, 85
classification, ix, 1, 107, 119
cadmium, 172 cleaning, 166, 176, 186
calcium, 158, 159, 256 cluster model, 191, 192, 194, 196, 197, 199, 200,
capillary, 65, 66, 67, 77, 83, 84, 85 201
carbohydrate, 252, 253, 255, 256, 257, 258, 261 clusters, 168, 191, 201
CO2, 8, 9, 46, 51, 52, 53, 55, 56, 57, 58, 59, 60, 61,
63
Index 273

coatings, 118, 159, 167, 175, 176, 178, 185, 264 covering, 108, 110, 112
cobalt, 159 cross-linked polymers, 203
collagen, 268 crystal polymers, 210
color, 169, 170, 180, 183, 211, 213, 214 crystal structure, 112
combustion, 211, 217 crystalline, 7, 59, 194
communication, ix, 225 crystallinity, 268
compatibility, 134, 176 crystallites, 7
competitive process, 60 crystallization, 159
compilation, 93 crystals, 91, 93, 107, 108, 109, 111, 112, 113, 115,
complexity, 22, 136 117, 180
components, 22, 23, 27, 65, 66, 67, 68, 69, 70, 71, cultivation, 22, 264
72, 73, 76, 77, 84, 85, 86, 87, 89, 90, 92, 95, 103, culture, 23, 30, 173, 185
105, 106, 107, 124, 127, 134, 158, 173, 175, 198, curing, 179, 192, 193
208, 269 curing process, 179
composites, 92, 121, 263, 264, 268, 269 cuticle, 91, 92, 93, 94, 95, 96, 98, 99, 100, 102, 103,
composition, 9, 22, 65, 66, 67, 69, 70, 71, 72, 73, 76, 105, 106, 107, 108, 109, 112, 114, 116, 117, 118,
78, 79, 80, 82, 83, 84, 85, 86, 87, 88, 89, 90, 93, 119, 120, 121
107, 138, 155, 192, 229 cutin, 107, 121
compounds, 3, 7, 11, 20, 68, 86, 88, 107, 129, 130, cytokines, 77
132, 135, 136, 139, 146, 156, 171, 177, 188, 207, cytoplasm, 171
214, 215, 217, 250, 255, 256, 258
compression, 192, 193, 198
computer technology, 51 D 
concentration, x, 1, 2, 6, 8, 12, 13, 14, 15, 18, 21, 23,
24, 25, 27, 30, 32, 33, 34, 38, 39, 40, 46, 66, 67, Dagestan, 191, 197
68, 70, 71, 76, 77, 83, 85, 86, 87, 89, 134, 157, decay, 16, 125, 128, 129, 131, 133, 138, 199, 201,
158, 159, 168, 169, 173, 181, 182, 183, 242, 243, 203, 227, 243
258 decomposition, 5, 9, 133, 138, 157, 159, 161, 163,
condensation, 215, 256, 258, 260 211, 228, 238, 241, 242, 243, 258
conformational analysis, 61 defects, 134, 198, 208
conformity, 164 deformation, 92, 106, 107, 117, 198
conjugation, 14, 125, 141, 216 degenerate, 12, 208
constant rate, 3, 4, 12, 206 degradation, x, 2, 4, 5, 6, 7, 8, 9, 22, 40, 80, 134,
consumption, 4, 8, 9, 13, 14, 15, 16, 17 175, 205, 206, 207, 211, 213, 215, 216, 218, 268
contact time, 185 degradation process, 4, 7
contamination, 91, 103, 109, 115, 116, 117, 118, 141 degradation rate, 4, 8
contour, 49, 165, 166 degumming, 264
control, 23, 24, 27, 30, 33, 34, 35, 36, 37, 40, 51, 66, density, 49, 51, 55, 56, 57, 58, 61, 92, 94, 105, 108,
70, 75, 76, 77, 84, 85, 141, 156, 157, 165, 166, 112, 125, 127, 129, 132, 139, 157, 158, 192, 194,
167, 168, 175, 176, 178, 184, 185, 209 195, 198, 201, 202, 203, 215, 250
control group, 75 density functional theory, 125, 132, 139
conversion, 12, 17, 207, 256, 261 density values, 55, 203
cooling, 148 deposition, 179, 180, 186
coordination, 215, 229, 230 deposits, 258, 261
copolymers, 142, 144 derivatives, 68, 85, 86, 129, 178, 254
copper, 172, 177 desiccation, 94, 96, 99, 100, 103, 107, 114, 117
correlation, 11, 14, 19, 32, 33, 35, 38, 39, 53, 57, 58, desorption, 51, 52, 55, 60
59, 93, 127, 128, 142, 183, 193, 195 DFT, 132
correlation coefficient, 14, 33, 53, 57, 58, 59 DGEBA, 192
corrosion, 250 diallyldimethylammonium chloride, 179, 180
cotton, 177, 182, 186, 187, 188, 189 diaminodiphenylmethane, 192
coupling, 139 dianhydrides, 46
covalent bond, 177, 178, 230 differential scanning calorimetry, 49, 53
diffraction, 210
274 Index

diffusion, 1, 7, 49, 51, 52, 58, 60, 63, 128, 177, 184, elytra, 119
185, 208, 210, 216, 250 emulsions, 170
diffusion time, 49 encapsulation, 177
diglycidyl ether of bisphenol, 192 endothermic, 5, 234
dimerization, 125, 129, 171 energy, 22, 57, 95, 125, 132, 174, 225, 228, 233,
diseases, 175, 258 243, 247, 250, 265
disinfection, 178, 189 engineering, 91, 92, 106, 107, 158, 160, 161, 192,
displacement, 102, 103, 105, 112, 113, 114, 116, 120 261, 268
dissociation, 125, 129, 132, 230 entropy, 234, 239
distillation, 13, 85 environment, 31, 32, 76, 94, 107, 109, 176
distilled water, 31, 184 environmental conditions, 108
distribution, 38, 134, 161, 168, 170, 213, 215 environmental protection, 263
diversity, 107 enzymes, 22, 24, 30, 171, 172, 184, 227, 228, 237,
division, 173 264
DNA, 172, 173 EP-1, 192, 193, 195
double bonds, 3, 5, 6, 66, 68, 149 EP-2, 192, 193, 195
drawing, 9, 163, 252 epidermis, 95, 114, 118
dressings, 175 epoxy polymer, x, 191, 192, 193, 194, 195, 197, 198,
Drosophila, 106, 120 203
drought, 76, 77, 78, 80, 81, 82 equilibrium, 52, 53, 56, 60
drug delivery, 166 equipment, 49, 157, 158, 161, 163
drugs, 12, 258 erythrocyte membranes, 42
drying, 58, 61, 96, 103, 117, 170, 184 ESR, 9, 29, 30, 31, 32, 38, 72, 89, 123, 125, 126,
DSC, 49, 53, 57, 58 127, 128, 133, 135, 136, 139, 142, 145, 148, 149,
DSC method, 53, 57, 58 208, 215, 219
durability, 134, 175, 176, 178, 182, 185 ESR spectra, 9, 30, 32, 125, 126, 127, 133, 135, 136,
duration, 13, 73, 90, 258, 269 139, 149, 208
dynamics, 33, 51, 68, 134 ESR spectroscopy, 123, 139
ester, 212, 213, 217
ethanol, 13, 49, 98, 169, 179, 182, 183, 256
E  ethers, 131, 217
ethyl alcohol, ix
elasticity, 120, 191, 192, 193, 196, 199, 200, 201, ethylene, 142, 167, 168, 183
203 ethylene glycol, 167, 168
elasticity modulus, 191, 193, 196, 199, 200, 201, 203 ethylene oxide, 142, 183
elastin, 92 eucalyptus, 108, 114
elastomers, 2, 5 Euclidean space, 194
electric current, 166 evolution, 119, 120
electrical conductivity, 166 exocytosis, 35
electrochemistry, 250 exoskeleton, 94
electrolysis, 19 experimental condition, 15, 168
electrolyte, 250 exposure, 1, 4, 5, 7, 8, 9, 132, 179, 183, 198
electromagnetic, 170 extraction, 30, 33, 155, 159, 215
electron, 98, 118, 123, 125, 126, 127, 129, 135, 136, extrapolation, 2
137, 172, 173, 174, 175, 179, 180, 181, 182, 211, extrusion, 198, 200, 202, 203, 269
212, 215, 216, 229, 230, 233, 234, 238, 239, 241,
242
electron density distribution, 215 F 
electron microscopy, 118, 173, 179, 180, 181, 182
electron pairs, 125, 229, 230 fermentation, 261
electron state, 216 fibers, 92, 94, 95, 99, 100, 134, 165, 167, 175, 176,
electronic structure, 125, 221, 222 178, 179, 180, 182, 185, 186, 187, 263, 264, 265
electrons, 57, 130, 131, 132, 141, 143, 166, 171, 215 filament, 107
electroplating, 159 filled polymers, 134
Index 275

film thickness, 52 glutamic acid, 169, 187


films, 1, 2, 4, 7, 8, 46, 49, 52, 58, 61, 105, 120, 134, glycol, 8, 139, 168
140, 146, 148, 149, 203, 207, 208, 209, 210, 268 glycosylation, 256
filters, 158, 159, 178 granules, 107
filtration, 159, 161 groups, 2, 3, 4, 5, 7, 9, 16, 35, 45, 46, 57, 58, 59, 60,
financial support, 62 61, 62, 84, 95, 125, 128, 129, 133, 136, 137, 138,
flame, 66, 77, 85, 211, 218, 249 139, 140, 143, 144, 145, 146, 147, 149, 150, 161,
flame retardants, 218 166, 172, 178, 187, 211, 213, 217, 228, 229
flavonoids, 11, 12, 14, 15, 16, 18, 19, 107 growth, x, 15, 22, 67, 75, 76, 77, 80, 82, 86, 166,
flexibility, 59, 62, 161, 194 168, 171, 172, 173, 175, 178, 180, 181, 183, 185,
flexible manufacturing, x, 155, 156 198, 199, 201
fluid, 23, 114, 115, 117 growth rate, 171
fluorescence, 77, 78, 215 growth time, 168
fluorine, 3, 148 guidelines, 24
fluorine atoms, 3
food, 12, 22, 65, 84, 169, 268
food products, 12 H 
formaldehyde, 145
formamide, 167 halogens, 130, 171
formula, 32, 50, 51, 194, 198, 222, 260 Hamiltonian, 126
fractal dimension, 194 hardness, 92, 93, 96, 102, 103, 104, 105, 112, 113,
fragments, 76, 124, 125, 130, 135, 141, 145, 228, 114, 115, 117, 118, 119, 120
229, 243, 256 harmful effects, 176
free energy, 227, 228, 229, 234, 243 health, ix, 157, 175, 177, 178, 250
free radicals, 9, 12, 16, 72, 89, 123, 132, 136, 137, health problems, 177
139 heat, 178, 205, 206, 207, 209, 210, 211, 213, 215,
free rotation, 60 216, 218
free volume, 45, 46, 50, 51, 55, 56, 57, 58, 59, 61, heat aging, 209
62, 203 heating, 49, 57, 60, 140, 141, 168, 211
friction, 91, 93, 95, 96, 116, 118, 119, 120 heavy metals, 166, 264
fuel, 22, 27 height, 100, 192, 198
functional approach, 118 helium, 67, 85
functionalization, 132, 149, 165 hemp, 268
fungi, 166, 171, 175, 178, 264 heterogeneous systems, 134
fungus, 22, 187 hexane, 65, 66, 67, 68, 69, 70, 71, 77, 83, 84, 85, 86,
furan, 268 87
fusion, 35 HIV, 186
HIV-1, 186
homeostasis, 81
G  hospitals, 171, 175
human subjects, 42
gases, 1, 2, 6, 107 humidity, 103, 108, 184
gel, 8, 188 hybrid, 230
gene, 76, 118 hybridization, 125
gene expression, 76 hydrazine, 167, 168, 170
generation, 75, 77, 78, 80, 81, 82, 123, 124, 128, 135 hydrocarbons, 66, 67, 68, 70, 71, 85, 86, 89, 130,
genome, 177 138, 170, 211, 218
geometrical parameters, 227 hydrogen, 2, 3, 4, 5, 7, 8, 19, 38, 57, 58, 59, 61, 76,
glass transition temperature, 45, 46, 49, 53, 54, 55, 80, 81, 84, 129, 131, 132, 135, 136, 138, 158,
56, 57, 58, 61, 192, 199, 201 159, 178, 222, 225, 227, 228, 230, 231, 232, 233,
glassy polymers, 45, 63, 201 234, 235, 238, 239, 241, 242, 243
glucose, 23, 24, 25, 26, 27, 168, 178, 252, 253, 254, hydrogen abstraction, 3
256, 257, 258, 260, 261 hydrogen atoms, 2, 4, 5, 8, 57, 131, 132, 135, 136
glucose oxidase, 23 hydrogen bonds, 57, 58, 59, 61, 129
276 Index

hydrogen peroxide, 80, 81, 178, 227, 228, 230, 231, iron, 178, 214, 215, 227, 228, 229, 230, 231, 232,
232, 233, 234, 235, 238, 239, 241, 242, 243 234, 241, 242, 243
hydrolysis, x, 21, 22, 23, 24, 26, 27, 211, 212, 213 irradiation, 126, 133, 135
hydroperoxides, 5, 13, 135, 227, 237, 238, 243 IR-spectroscopy, 218
hydroxide, 158 isobutylene, 4, 5, 148
hydroxyl, 4, 38, 131, 139, 149 isomerization, 252
hydroxyl groups, 4, 139 isoprene, 5, 6, 148
hygiene, 175 isotactic polypropylene, 146
hyperfine interaction, 31, 126 isotope, 131
hypothesis, 57, 116, 261



ketones, 107, 137, 260
images, 96, 97, 98, 99, 101, 105, 106, 107, 110, 111, kinetic curves, 13, 14, 16, 17, 18, 206
112, 180, 181, 182, 183 kinetics, ix, 1, 11, 13, 14, 123, 134, 168, 206, 212,
imide rings, 59 216
imidization, 60, 63 KINS program, 13
immobilization, 165, 178, 180
impacts, 40, 206
impregnation, 51, 63 L 
in vitro, x, 29, 30, 35, 36, 37, 39, 40, 41
in vivo, 106, 120, 166 laboratory tests, 185
indication, 138, 230, 246 lactate dehydrogenase, 24
indices, 67, 77, 81, 213 lactic acid, 268
induction, 11, 12, 13, 14, 15, 16, 19, 81, 134 leaching, 177
induction period, 11, 12, 13, 14, 15, 16, 19, 134 leakage, 171
industrial sectors, 165, 178 life cycle, 156
industry, x, 27, 67, 85, 155, 156, 159, 177, 178, 263, lifetime, 38
264, 269 ligand, 168
infection, 167, 171, 175, 176 light transmittance, 213, 214
inhibition, x, 11, 16, 18, 19, 38, 39, 65, 66, 72, 83, line, 13, 17, 30, 33, 53, 55, 57, 58, 128, 163
84, 86, 89, 132, 172, 173, 206, 211, 212, 216 linear chain termination, 15
inhibitor, 13, 21, 22 linear defects, 194
initial reagents, 161 linear dependence, 2, 6
initiation, ix, 9, 12, 13, 14, 18, 23, 38, 215 linearity, 12
inoculation, 183 linkage, 172
inoculum, 185 links, 192
insects, 91, 94, 95, 98, 104, 106, 109, 115, 116, 117, lipid metabolism, 81
120 lipid peroxidation, 12, 14, 18, 72, 75, 76, 84, 89
integument, 95, 106, 119, 120 lipids, 22, 24, 30, 31, 33, 34, 38, 39, 40, 75, 78, 81,
interface, 94, 120 82, 95, 96, 105, 106, 107, 118, 172
interference, 100, 101 liquid chromatography, 65, 83
international standards, 156 liquids, 127
interval, 13, 32, 201 lithium, 142, 147, 148
intrinsic viscosity, 1, 4 liver, 29, 30, 32, 37, 40
intuition, 251 liver cells, 29, 30, 32
invertebrates, 92, 95 livestock, 22, 27
iodine, 130 living conditions, 96, 177
ionization, 66, 77, 85, 125, 130, 215, 227, 242, 243 living radical polymerization, 132
ionization potentials, 130 local order, 191, 200
ions, 149, 168, 170, 172, 242 low density polyethylene, 217
IR spectra, 2, 5, 8, 60, 125, 211 low-molecular substances, 216
IR spectroscopy, 2, 125 LPO intensity, 22
Index 277

microorganism, 172
M  microscope, 98, 184
microscopy, 100, 101, 107, 118
macromolecular chains, 45, 57
microviscosity, 30, 33, 34, 40, 78, 228
macromolecules, 2, 4, 5, 6, 7, 8, 9, 51, 123, 128, 132,
middle lamella, 108
134, 139, 140, 141, 145, 147, 148, 149, 150, 191,
migration, 179
200, 211
mining, 258
macroradicals, 9, 141, 145, 148
mitochondria, 77, 80, 81
magnetic properties, 134
mixing, 23, 148, 159, 163, 170
magnetization, 134
MNDO, 221, 222
majority, ix, 70, 93, 136
mobility, 38, 91, 96, 116, 133, 208, 228, 243
malignant tumors, 257
model, 11, 12, 13, 16, 50, 63, 66, 67, 68, 71, 72, 76,
maltose, 253
77, 83, 84, 85, 86, 87, 89, 98, 115, 128, 132, 141,
manganese, 258
188, 194, 197, 207, 211, 212, 215, 218, 222, 230
manpower, 161
model system, 13, 66, 67, 68, 72, 83, 84, 85, 86, 87,
manufacturer, 176, 178
89, 207
manufacturing, 164, 166, 186
models, 63, 105, 156, 216
market, 258
modulus, 92, 94, 96, 102, 103, 104, 105, 106, 112,
marketing, 159
113, 114, 115, 116, 117, 120, 193, 199
mastery, 251
moisture, 76, 77, 79, 159, 170, 175, 176
materials science, 91, 93
molds, 171, 198
mathematics, 63
molecular biology, 118, 130, 133, 134
matrix, 57, 58, 92, 94, 103, 107, 149, 177, 191, 200,
molecular dynamics, 134
201, 203, 208
molecular mass, 142
measurement, 49, 72, 89, 105, 117
molecular mobility, 134, 208, 210
mechanical degradation, 134
molecular orientation, 201, 203
mechanical properties, x, 45, 91, 92, 93, 95, 96, 102,
molecular structure, 14
103, 105, 106, 107, 108, 109, 112, 113, 117, 118,
molecular weight, 9, 51, 53, 107, 192
121, 197, 201, 268
molecules, 4, 12, 16, 31, 38, 39, 40, 57, 76, 127, 129,
mechanical stress, 201
133, 137, 166, 172, 174, 208, 221, 222, 225, 228,
media, 135, 139, 168
239, 257
melt, 210
monomers, 136, 141, 143, 144, 177, 211, 268
membrane permeability, 35, 175
monosaccharide, 255
membranes, 12, 29, 30, 33, 35, 38, 75, 76, 77, 78, 79,
Monte Carlo method, 50, 63
80, 81, 119, 121, 172, 174, 250
morphology, 7, 108, 118, 168, 172, 174
men, ix, 253
motion, 23, 31, 93, 208
Mendeleev, 21, 23
MTS, 117
mercury, 1, 172, 198
multilayered structure, 91, 95, 114, 116
metabolism, 76, 166, 172, 177
mutant, 115
metabolites, 171
mutation, 109
metal oxides, 166
metal salts, 168
metals, 159, 172, 187 N 
metamorphosis, 261
methacrylic acid, 179, 180 Na+, 169, 187
methanol, 77, 97 NaCl, 181
methyl groups, 136 nanocomposites, 187, 268, 269
methyl methacrylate, 62, 63 nanocrystals, 168, 170, 180, 188
methyl oleate oxidation, ix, 16, 19 nanofibers, 268
methylation, 77 nanoindentation, 93, 96, 106, 115, 119
MFI, 213 nanomaterials, 166
mice, 29, 30, 32 nanometer, 91, 92, 108, 115, 165, 166, 191
microcrystalline cellulose, 21, 23 nanometer scale, 91, 92
microemulsion, 188
278 Index

nanoparticles, 165, 166, 167, 168, 169, 170, 172, oxidation, x, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
173, 174, 175, 178, 179, 180, 181, 182, 185, 186, 22, 24, 30, 65, 66, 67, 68, 69, 70, 71, 75, 76, 80,
187, 188, 191, 269 83, 85, 86, 87, 88, 126, 130, 131, 134, 138, 143,
nanostructures, x, 197, 198, 201 144, 149, 206, 207, 211, 212, 215, 216, 217, 218,
nanotechnology, 165, 166, 178, 186 242, 254
narcotics, ix oxidation products, 24, 67, 85, 206
natural polymers, 263 oxidation rate, 13, 14, 18, 85
natural resources, 258 oxide nanoparticles, 174
neoplasm, 258 oxides, 67, 86, 138, 206, 216
nerve, 35 oxygen, 1, 12, 13, 14, 16, 17, 18, 38, 57, 61, 72, 75,
network, 192, 202, 208 76, 77, 80, 84, 85, 89, 125, 128, 130, 136, 139,
nickel, 159 164, 172, 175, 178, 206, 207, 208, 210, 213, 215,
nicotinamide, 81 216, 230, 231, 234
nitrates, 3, 161 oxygen absorption, 13, 206
nitric oxide, 132 oxygen consumption, 72, 89
nitrogen, ix, 1, 2, 4, 5, 9, 49, 123, 125, 128, 129, 136, ozone, 2, 130
139, 230, 231, 257
nitrogen dioxide, 2, 4, 5, 9
nitrogen oxides, ix, 1, 123 P 
nitron, 137, 138, 145
nitroso compounds, 124, 128, 132, 134, 135, 136, palladium, 98
137, 146, 148 paradigm, 121
nitroxyl radicals, 29 parallel, 24, 33, 68, 95, 100, 157, 158, 185, 249, 252
NMR, 211, 213, 215, 218, 219 parameter, 15, 16, 18, 19, 29, 31, 32, 33, 35, 36, 38,
nodes, 192 49, 55, 100, 128, 195, 215, 225
nonequilibrium, 191, 196 parameters, 12, 18, 27, 29, 31, 32, 33, 34, 39, 41, 46,
non-inhibited oxidation, 206 50, 52, 60, 62, 100, 103, 134, 157, 158, 191, 194,
nuclei, 212 195, 199, 201, 215, 221, 222, 232, 234
nucleic acid, 134, 171 particles, 109, 134, 148, 165, 166, 167, 168, 170,
nucleus, 81, 127, 212 172, 173, 178, 179, 182, 183, 185, 186, 187, 188,
nutrients, 175 189
pathogens, 178
PCA, 205
O  peptides, 173
percolation, 195
oil, 22, 67, 68, 69, 70, 71, 72, 73, 83, 85, 86, 87, 88, performance, 30, 62, 93, 96, 158, 169, 171, 185
89, 90, 170, 251, 261 permeability, 172, 174
oil samples, 67, 85 peroxidation, 22, 76
oils, x, 65, 66, 67, 68, 70, 71, 72, 73, 83, 84, 85, 86, peroxide, 14, 30, 75, 80, 131, 138, 217, 227, 233,
87, 88, 89, 90 238, 243
olefins, x, 221, 222 peroxide radical, 131, 138, 217
oligomers, 141 PET, 218
operator, 126 PETF, 218
optimization, 161, 222 petroleum, 169
oral presentations, 268, 269 pH, 23, 30, 98, 139, 168, 171, 180
order, 14, 29, 31, 36, 38, 49, 53, 56, 61, 91, 96, 97, pharmaceuticals, 84
105, 116, 131, 170, 176, 180, 191, 195, 199, 206, pharmacokinetics, 258
210, 264 pharmacology, 256
organelles, 76 phase transitions, 35
organic compounds, 123, 131, 215 phenol, 23, 66, 68, 84, 85, 86, 171, 192, 215
organic solvents, 139 phenoxyl radicals, 137
organism, 30, 33, 171, 183, 257, 258 phosphates, 161
orientation, 31, 92, 99, 100, 108, 201, 203, 243 phospholipids, 24, 38, 40, 42
osmosis, 264 phosphorous, 156, 158, 161, 206
Index 279

phosphorus, x, 155, 156, 157, 158, 159, 161, 163, polymer matrix, 57, 58, 61, 149, 178
164, 173, 205, 211, 212, 214, 215, 217, 218 polymer oxidation, 134
phosphorylation, 173, 211, 216 polymer properties, 198, 203, 207
photochemical transformations, 133 polymer solutions, 1, 61
photolysis, 132, 133, 145, 146, 148 polymer structure, 206, 207, 208
photooxidation, 145 polymer swelling, 52
photostabilizers, 134 polymer systems, 191, 197, 268
physical aging, 46 polymeric materials, 1, 2, 124, 133, 194
physical chemistry, 250 polymerization, x, 4, 134, 141, 142, 143, 147, 149,
physical properties, 45, 46, 62, 123 169, 177, 211, 221, 222
physicochemical properties, 78 polymethylmethacrylate, 3, 148
physics, 1, 123, 133, 249 polymorphism, 269
physiology, 120 polyolefins, 211
plants, 65, 72, 76, 77, 80, 81, 83, 90, 104, 107, 108, polypeptide, 169, 227, 228, 229, 234
109, 111, 112, 116, 118, 120, 260, 264 polypropylene, 2, 134, 146, 148
plasma membrane, x, 30, 32, 33, 34, 37, 38, 40, 76, polystyrene, 4, 63, 140, 142, 145, 146, 147, 218
172, 174 polyurethanes, 8, 9
plasticity, 203 polyvinylacetate, 139, 140
plasticization, 203, 268 polyvinylchloride, 2, 3
platform, 179, 180 potassium, 22, 77, 167
PMMA, 3 power, 161, 184, 222, 238, 261
poison, 177, 178 pressure, 1, 2, 46, 51, 114, 115, 117, 179, 181, 182,
polar groups, 4 183, 192, 198, 212
polarity, 32, 213 prevention, 91, 93, 116, 158, 179
polarization, 218 probability, 33, 40, 58, 141, 212
pollutants, 1, 2, 157 probe, 29, 31, 32, 208
pollution, 165, 178 process control, 156
poly(3-hydroxybutyrate), 268 product life cycle, 156, 164
poly(ethylene terephthalate), 218 production, 22, 27, 28, 65, 81, 155, 157, 158, 159,
poly(methyl methacrylate), 218 160, 161, 162, 163, 164, 188, 252, 264
poly(vinyl chloride), 268 production technology, 157
poly(vinylpyrrolidone), 168 productivity, 27, 80
polyamides, 1, 62, 205, 206, 218 program, 229
polyamidoacid, 207 programming, 85
polybutadiene, 1, 2, 148 project, 156, 157, 158, 159, 160, 163, 252
polycondensation, 143 proliferation, 81
polydispersity, 142 promoter, 228
polyesters, 268 propane, 22
polyheteroarylenes, x, 45, 46, 49, 52, 53, 56, 60, 61, protein kinase C, 23, 24, 38
62, 63 protein kinases, 76
polyimides, 46, 57, 58, 60, 61, 62, 205, 206 protein structure, 95
polyisoprene, 5, 148 proteins, 22, 24, 38, 76, 80, 94, 105, 106, 172, 173,
polymer, x, 1, 4, 7, 9, 20, 46, 51, 52, 57, 58, 59, 60, 175, 227, 269
61, 92, 94, 123, 124, 133, 134, 139, 140, 143, prothorax, 96, 98, 99
145, 146, 147, 148, 149, 150, 171, 178, 191, 193, protons, 136, 149
194, 195, 197, 199, 201, 203, 205, 206, 207, 208, Pseudomonas aeruginosa, 171
210, 211, 215, 216, 250, 267, 268 purification, 13, 31, 157, 251
polymer amorphous state, 197 purity, 213
polymer amorphous state structure, 197 PVC, 159, 268
polymer blends, 134 pyrolysis, 211, 218
polymer chains, 9, 58, 61, 124 pyromellitic dianhydride, 55
polymer films, 46, 148
polymer materials, 20, 197
280 Index

regulations, 158
Q  regulators, 75, 76, 134
relationship, 14, 194, 195
quantitative estimation, 191
relaxation, 106, 107
quantum-chemical calculations, 127, 221, 227, 228,
relevance, 88
242
reliability, 33
quartz, 30, 77
replacement, 92
quaternary ammonium, 178, 189
residuals, 169, 227
quinones, 132
residues, 23, 214, 255
resins, 159, 205, 216
R  resistance, 80, 81, 95, 184
resolution, 98, 107, 211
radiation, 150, 167 resonator, 30
radical formation, 16 resources, 22, 161, 268
radical mechanism, 213 respect, 2, 213, 215
radical polymerization, 132, 142, 148 respiration, 172
radical reactions, 123, 207, 211 respiratory, 171
radicals, ix, 5, 9, 11, 12, 16, 17, 18, 19, 84, 123, 124, respiratory disorders, 171
125, 127, 128, 130, 131, 132, 133, 134, 135, 136, retention, 61, 67, 77, 81, 85, 130
137, 138, 139, 140, 143, 149, 207, 234, 241 reticulum, 76
radius, 50, 127 rhamnolipid, 228, 240, 241, 243
Raman spectra, 125 rights, iv
range, x, 4, 13, 15, 21, 22, 23, 29, 30, 33, 35, 38, 40, rings, 14, 145, 146
46, 51, 53, 92, 103, 105, 107, 108, 113, 115, 116, risk, 2, 171
125, 128, 133, 139, 165, 166, 169, 171, 177, 178, robotics, 92
188, 192, 193, 201, 205, 215 rolling, 80, 178
raw materials, 22, 263, 264 Romania, 45, 62, 264
reactants, 146 ROOH, 4, 6, 13, 14, 15
reaction mechanism, 3, 8 room temperature, 3, 15, 66, 84, 110, 131, 136, 137,
reaction rate, 13, 27 169, 170, 179, 198, 215
reaction temperature, 168, 169 roughness, 91, 100, 103, 109
reactions, 3, 4, 9, 12, 15, 19, 123, 124, 125, 128, 129, roughness measurements, 100
130, 131, 132, 133, 134, 136, 138, 139, 141, 145, Royal Society, 119, 120, 246
146, 147, 148, 206, 214, 217, 218, 227, 228, 234, rubber, 1, 2, 5, 23, 95, 188, 199, 201, 268
243 rubbers, 1, 148, 149
reactive groups, 141, 192 rural areas, 264
reactive oxygen, 12, 75, 77, 78, 80, 81 RUS, 81
reactivity, ix, 1, 2, 9, 18, 132, 213, 216 Russia, x, 1, 11, 21, 22, 29, 45, 65, 66, 75, 77, 83,
reagents, 77, 168 85, 123, 155, 205, 221, 222, 245, 249, 263, 264,
reason, 12, 13, 29, 30, 87, 100, 109, 125, 178, 196, 267, 268
239, 252, 258
receptors, 77, 79

recognition, 251, 253
recollection, 252
salts, 22, 76, 130, 131, 159, 161, 164, 168, 177, 187,
recombination, 9, 16, 132, 213, 234
189, 214, 256
recommendations, iv, 157
saturated fat, 38, 40, 79
recovery, 106, 107, 156, 164
saturated fatty acids, 79
recrystallization, 268
scanning electron microscopy, 96, 98, 107, 109, 111,
recycling, 159
179, 180
reflection, 107
secretion, 81, 109
reflexes, 211
seed, 264
region, 4, 95, 108, 125, 192, 221, 251, 258, 260
senescence, 80
regulation, 30, 77, 80, 81, 198, 210
sensing, 120, 188
Index 281

sensitivity, 136 stable radicals, 123, 124


sensors, 92 starch, 187, 258, 268, 269
seta, 115 stereospecificity, 237
shade, 233 stoichiometry, 38
shape, 94, 97, 98, 99, 106, 188, 211 storage, 65, 67, 68, 70, 73, 83, 84, 85, 86, 87, 88, 90,
shock, 93 118, 158, 164, 269
signal transduction, 38, 80, 81 strain, 129, 176, 192, 198, 199, 200, 202
signaling pathway, 173 stratification, 158, 228
signals, 38, 211 stress, 65, 76, 78, 80, 81, 176, 191, 192, 193, 194,
signs, 142, 212 195, 196, 198, 199, 200, 201, 203
silica, 66, 85, 187 stress-strain curves, 193
silk, 179, 180, 181, 187, 263 stretching, 125
silver, 146, 166, 167, 168, 169, 170, 172, 173, 174, structural changes, 100, 103, 201
175, 178, 179, 180, 181, 182, 183, 185, 186, 187, structural characteristics, 30, 81
188 structural transformations, 35, 38
SiO2, 187 structural transitions, 30, 31, 33, 34, 37
sludge, 155, 156, 157, 158, 159, 161, 163, 164 structuring, 156
smooth muscle cells, 23 students, 252, 253, 261
sodium, 142, 147, 148, 155, 156, 157, 158, 159, 160, styrene, 40, 140, 142, 147, 149
161, 162, 163, 164, 167, 169, 170, 180, 181, 188, suberin, 121
256 substrates, 22, 24, 131, 173, 175, 177
sodium hydroxide, 158 sugar, 252
soil, 167, 176, 268 sulfa drugs, 256
solid matrix, 208 sulfur, 131, 173, 179, 182
solid phase, 135 sulphur, 173, 179
solid polymers, 134 surface area, 31, 33, 35, 40, 172, 175
solid state, x, 136 surface layer, 95, 105, 107, 228
solid surfaces, 118 surface modification, 178
solid waste, 157 surface structure, 99, 107, 109, 111, 112, 177
solid-state extrusion, 197 surfactant, 168, 170
solubility, 46, 136 swelling, x, 45, 46, 49, 51, 52, 53, 55, 56, 57, 58, 59,
solvation, 238, 239, 240, 241 60, 61, 62, 148
solvents, 19, 46, 60, 61, 125, 128, 129, 132, 136, swelling process, 46, 61
137, 138, 168, 170, 203 synergistic effect, 88
sorption, 49, 61, 228 synthesis, x, 3, 46, 60, 61, 76, 123, 124, 130, 134,
space, 164, 228, 233, 235, 240 146, 147, 148, 149, 155, 156, 159, 160, 161, 163,
species, 9, 12, 73, 75, 76, 77, 78, 80, 81, 86, 88, 90, 164, 167, 168, 169, 170, 186, 187, 188, 253, 255,
107, 108, 112, 113, 116, 119, 128, 139, 171, 177 256, 258, 260, 261, 268
specific surface, 170 synthetic fiber, 176
spectroscopy, 218 synthetic polymers, 141
spectrum, 4, 11, 13, 26, 31, 32, 92, 95, 125, 127, synthetic rubbers, 211
128, 149, 178, 186, 208, 215 system analysis, 161, 164
speed, 31, 51, 77, 84, 85, 215
spin, x, 29, 30, 31, 32, 72, 89, 123, 126, 127, 128,
129, 130, 132, 133, 134, 135, 136, 137, 138, 139, T 
140, 141, 142, 143, 146, 147, 148, 149, 208
spin labels, 123, 130, 133, 134, 149 T cell, 81
stability, 9, 65, 68, 71, 76, 81, 83, 86, 87, 89, 94, tanks, 158
108, 113, 125, 128, 134, 136, 168, 209, 213, 217, taxonomy, 118
218, 229 TEM, 174, 182, 183
stabilization, 12, 40, 125, 134, 161, 205, 206, 207, temperature, 4, 19, 23, 29, 30, 33, 34, 35, 36, 38, 40,
208, 210, 211, 212, 213, 216, 217, 218, 269 46, 49, 51, 58, 61, 66, 77, 85, 108, 133, 134, 158,
stabilizers, 12, 20, 134, 215, 217 159, 168, 169, 175, 187, 192, 195, 198, 199, 201,
206, 207, 211, 212, 213, 217
282 Index

tensile strength, 95
terpenes, 66 U 
textiles, x, 165, 166, 167, 171, 175, 176, 178, 179,
ultrastructure, 119
183, 184, 185, 186, 187, 189
UNESCO, 246
thermal activation, 215
universal gas constant, 192
thermal aging, 208
urethane, 7
thermal analysis, 268
UV, 1, 2, 3, 5, 7, 132, 136, 166, 167, 179, 214, 215
thermal decomposition, 218
UV light, 1, 2, 5, 7, 132, 167, 179
thermal degradation, 148, 218, 268
UV radiation, 3
thermal destruction, 148
thermal oxidation, 205, 206, 207, 209, 211, 212, 215,
216 V 
thermal stability, 45, 139, 205, 207, 216
thermal treatment, 141, 203 vacuum, 13, 159, 198, 211
thermodynamic calculations, 236 valence, 223, 224, 225
thermodynamic equilibrium, 227 Van der Waals radius, 50
thermodynamic parameters, 231, 234, 236, 237 vapor, 269
thermodynamically nonequilibrium solids, 195 vector, 194
thermodynamics, 227, 233, 234, 237, 241 Vickers hardness, 92, 105
thermoregulation, 35 vinylchloride, 211
thin films, 4, 62, 118, 120 viscosity, 29, 31, 32, 38, 127, 210, 250
titanium, 187, 217
tobacco, ix, 81, 260
toluene, 132, 148 W 
toxicity, 256, 257
TPA, 216 waste, x, 155, 156, 171, 186, 260, 263
transformation, 156, 161, 215, 261 water absorption, 212
transformation product, 215
transformations, 134, 207, 218

transition, 29, 35, 38, 40, 53, 55, 57, 58, 125, 211,
214, 215, 228
X-ray, 42, 174, 209, 215
transition metal, 211, 214, 215
transition temperature, 53, 55, 57, 58
transitions, 35, 53, 57, 58, 133 Z 
transmission, 40, 175
transport, 172, 174, 176, 216, 250 zinc, 187
tumor cells, 258 zinc oxide (ZnO), 166, 187
tyrosine, 80, 173