Isolation of xylans from bleached Eucaliptus kraft pulp by

precipitation with antisolvents

Ana Isabel da Fonseca Marques
ana.f.marques@tecnico.ulisboa.pt

Instituto Superior Tcnico, Lisbon, Portugal

December 2014
Chemical Engineering Department, Instituto Superior Tcnico, Avenida Rovisco Pais, 1, 1049-001 Lisbon, Portugal.

Abstract
In the last decades, more attention has focused on lignocellulosic materials, for being sustainable
resources with high potential to create new value-added biomaterials. Among these resources main
highlight has been given to hemicelluloses, in particular xylans, since they have, together with their
derivatives, a wide range of applications in industry, namely, pulp, food, pharmaceutical and biofuels.
The main purpose of the present work was focused on alkaline extraction of bleached Kraft pulp of
Eucalyptus Globulus, and in the isolation of xylans present in alkaline extract, with high purity and yield. To
optimize the extractions, the effect of two parameters was evaluated, the stirring speed and the extraction
time. With the speed increase there was an increase in yield, however the second parameter does not
show any influence. The extraction yield, for the various tests, varied between 65 -85%.
The isolation of xylans was performed by two different methods: neutralization with dilute acid and
precipitation with alcohol (ethanol and methanol). Due to the presence of impurities in the precipitate, the
washing importance was studied by different ways. Water washing yielded samples with purity values up to
99%, while with methanol those values range from 36-84%. The characterization of xylan indicated that the
average molecular weights were 16.8 to 22.7 kDa and the xylan yields varied between 43 and 97%.
It was concluded that nitric acid is the solvent that yielded more pure xylan and the use of alcohol
requires the subsequent washing with water to neutralize the sample, resulting in low yields due to the
losses in wash process. It was also found that the alcohol content appears to influence the diversity of
molecular weights of precipitate.
Keywords: Eucalyptus globulus, Bleached Kraft pulp, Alkaline Extraction, Precipitation and Xylans.
sunflower hulls, wheat, rye and oat bran or sugarbeet
pulp (Srossy, Zsuzsa, 2011).
Portugal, a country with a large forest area, 38% of
the national territory, where the industrial sector of
pulp and paper contributes a lot to the Portuguese
economy, shows great potential to obtain xylans.
Eucalypt wood is the increasingly dominating source
of short fibers for the pulp and paper industry in our
country. Particularly, Eucalyptus globulus is the
dominant raw material, rich in hemicelluloses namely
xylan (about 16%) for bleached hardwood kraft pulp
production (Lisboa, S., et al., 2005; Shatalov, A., et
al., 1999).
The structural diversity and complexity of xylans are
shown to depend on the botanic source and are also
changed by the way they are extracted and purified.
Various suitable extraction procedures for the isolaton
of xylans from different plant sources are described in

1. Introduction
Considerable attention has been focused on
alternative and sustainable resources to produce fuels
and chemicals that are currently derived from fossil
oils.
One example of a promising biomaterial is xylan, a
hemicellulose largely found in nature, being
considered the second most abundant polysaccharide
after cellulose. They occur in close association with
cellulose and lignin in plant cell wall in lignified
tissues.
The most potential sources of xylans include forest
and pulping waste products from hardwoods and
softwoods and many agricultural crops such as straw,
sugar cane, corn stalks and cobs, hulls and husks
from starch production (Ebringerova & Heinze, 2000),

the literature (Deutschmann, R., et al., 2012; Peng,

Feng, et al., 2012; Zsuzsa, S., 2011).
Traditionally, hemicelluloses can be isolated from
materials by extraction with alkaline solutions (NaOH,
KOH,etc). The main advantages of this method is that
it is simple to implement and inexpensive (Peng, F., et
al., 2010).
The recovery of the extracted xylan can be done by
acid neutralization (sulfuric, acetic, hydrochloric) or
with alcohol precipitation (ethanol, methanol,..).
Besides these, there are a variety of other
fractionation and purification procedures to obtain the
desired purity of xylans, namely, ammonium sulfate
precipitation,
iodine-complex
precipitation,
supercritical anti-solvent precipitation and membranes
(ultrafiltration, microfiltration, nanofiltration and
reverse osmosis) (Peng, Feng, et al., 2012).
Afterward, several washing steps were performed
including washing with water and methanol.
There is an interest in the application of the xylan
polymer in the paper, pharmaceutical, cosmetic,
biofuel and food industries.
The current uses of xylans on an industrial scale
involve their conversion to xylose, xylitol and furfural
(Peng, F., et al., 2010).
Xylan has drawn considerable interest due to its
potential for packaging films and coating food, as well
as for its use in biomedical products (Li et al., 2011).
Xylan has also been used as an adhesive, thickener
and additive to plastics. It increases their stretch and
breaking resistance as well as their susceptibility to
degradation (nlu et al., 2009). Another interesting
application for xylan may be found in the food industry
as an emulsifier and protein foam stabilizer during
heating (Ebringerova et al., 1995).
In addition, the beneficial effect of some xylans in
papermaking was confirmed in the case of
hemicelluloses that might be used as a beater additive
(Peng, F., et al., 2009).
Since the early 1990s attention has focused on the
oligomers of xylose that can be produced from
hemicellulosic xylans, xylo-oligosaccharides (XOS).
They can be applied as prebiotics, as an agent to
control the level of ammonia in blood, to reduce
complications associated with diabetes (glucosuria,
proteinuria). They can also be used antioxidants and a
preventive agent against anemia, arteriosclerosis,
osteoporosis
and
certain types
of cancer
(Deutschmann, R., et al. 2012).
The purpose of the present work is to obtain xylans
from bleached E.Globulus kraft pulp after an alkaline
extraction and a depth study of acid and alcoholic
precipitation from filtrate.
In this paper, the results obtained from xylans isolated
from E.Globulus pulp are presented.

The bleached kraft pulp from Eucalyptus globulus

used in this work, Table 1, was supplied by RAIZ
(Paper and Forest Research Institute, Aveiro,
Portugal).
Tab. 1 Ash content, pentosans, humidity and SR of

2. Materials and Methods

The following procedures were carried out:

In the case of acid addition,
a- Washed with methanol (LMT);

bleached Kraft pulps.

Sample

Pulp 1

Pulp 2

Ash (%)

0,139

0,194

Pentosans (%)

16,89

16,44

SR

64

66

Humidity (%)

58,0

70,6

2.2 Alkaline extraction of pulp xylans

The xylans were extracted from the Kraft pulp by
alkaline extraction with NaOH according to the
method indicated by RAIZ. Previous studies have
optimized this step by alkaline extraction setting the
NaOH content in 10%, the temperature in 25C and
5% consistency. In these experiments, extractions
were performed with a total time of 1 and 2 hours.
Taking into account the extreme importance of stirring
speed on the extraction yield, the influence of this
parameter was studied, ranging between 120 and
1100 rpm.
All tests were started with the weighing of a certain
amount of pulp, and calculation of the amount of total
suspension, using the values of pulp weight, its
humidity and consistency value. After these
calculations, sodium hydroxide (10%) and sodium
borohydride (10mg/100g suspension) were weighed.
Finally the quantity of water to make up the total value
of mass of suspension was weighed. After the
stipulated time of extraction, the suspension was
filtered off, and the alkaline extract separated from the
extracted pulp. This one was washed with distilled
water till pH 7 by vacuum filtration. After this
procedure, the extracted pulp was dried in an oven at
105C.

2.3 Isolation of alkaline extract xylans

The alkaline extract was treated in two ways:
- adjusted to pH 7 with dilute acids at the same
concentration (7,5 N): sulfuric (SA), hydrochloric (HA),
acetic (AA), nitric (NA), phosphoric (PA) and formic
(FA) acid. The solution was neutralized under
constant agitation (300 rpm) in a water bath, and a
solid phase is formed.
-precipitated with alcohols, ethanol (ET) or methanol
(MT) (30, 50 and 70%) in the same conditions referred
before.
-The supernatant of both treatments was removed off.

2.4 Purification of xylans

2.1 Materials
2

b- Without washing (NL);

c- Washed several times with water until constant
conductivity (H2O).
In the case of alcohol addition:
d- Only washed with methanol (remaining alkaline)
(LM);
e- Neutralized with acetic acid in a support liquid
(filtrate or supernatant) (AALF and AASO,
respectively) and with sulfuric acid in supernatant as
support liquid (ASSO);
f- Washed several times with water until pH=7 (H2O).
All precipitates obtained were dried at room
temperature.
The ash content was quantified for each xylan sample
(Tappi T211 om 02).

in a test tube and vortexed for 30s. The temperature

of the mixture rises rapidly within 10-15 s after
addition of sulfuric acid. Then, the solution was cooled
in ice for 2 min to bring it to room temperature. Finally,
UV light absorption at 315 nm is read using UV
spectrophotometer
(Hitachi
U-2000
UV-VIS).
Reference solution of xylose is prepared following the
same procedure as above, except that the
carbohydrate aliquot is replaced with distilled water
(Albalasmeh, A., et al., 2013). The concentration of
samples was determined from the calibration graph of
standard xylose (Fluka, 99% (assay HPLC)) at 30,
40, 50, 0, 80, 80 and 100 mg/L.

2.8

Solubility of xylans obtained from alkaline extract and

from supernatant precipitation with ethanol were
tested in a wide range of solvents, namely, water,
aqueous solutions: NaOH 0,1 M (Hakala, T., et al.,
2013), NaCl 0,02 N with 0,005M phosphate buffer
(Luo, Q., et al., 2012; Peng, F. et al., 2009),
DMSO:H2O (90:10) + 0,05M LiCl e DMSO:H2O
(20:80) + 0,1M NaNO3 (Saake, B., et al., 2001) and
organic solvents: Toluene, DMF(Saake, B., et al.,
2001), DMAc (Evtuguin, D., et al., 2003; Evtuguin, D.,
et al., 2007; Pinto, P.C., et al., 2005; Shatalov, A., et
al., 1999), DMSO (Saake, B., et al., 2001) and THF
(Gabrielii, I., et al., 2000).
Xylan samples weighing approximately 30 mg were
dispersed in 15 mL of solvent, in a closed container,
under constant agitation (300 rpm) at room
temperature overnight (Silva, S., et al., 1998). The
solubility of xylan samples in those solvents were
classified as good or bad depending on the aspect of
the solution: clear or cloudy, respectively. The
reagents used in the solutions preparations were
sodium hydroxide (98% , Panreac), sodium chloride
(99,9%, AnalaR NORMAPUR VWR), (pro-analysis
>99%, Merck), anhydrous sodium phosphate (98102%, Panreac), Lithium chloride extra pure (>98%,
Riedel-de Han), sodium nitrate (>98%, Scharlau),
DMSO (99,9%, Sigma-Aldrich), toluene (pro-analysis
>99,5%, Merck), DMF (100%, AnalaR NORMAPUR
VWR), THF (99,99%, Fisher Scientific) and DMAc
(99%, Sigma-Aldrich).

2.5 Fractionation of hemicelluloses by graded

Ethanol precipitation (Supernatant)
Hemicellulosic fractions of supernatant were obtained
by fractionations. Supernatant (from sulfuric,
hydrochloric and nitric acid experiments) was
precipitated with alcohol contents of 25, 30, 45, 60
and 80% (in solution) under constant agitation at 300
rpm. The precipitate obtained was dried at room
temperature. The ash content was quantified for each
hemicellulosic fraction (Tappi T211 om 02).

2.6

Pentosan Analysis

Pentosan analysis of xylans and pulps was performed

according to the procedure described in Tappi T223
cm-84 and PE-02-011 RAIZ (2014).

2.7

Carbohydrate analysis

2.7.1

Acid Hydrolysis

The isolated xylans were subjected to treatment with

72% H2SO4 at 20C for 3 h followed by dilution with
4,4 mL distilled water at 100C for 2,5 h (Seaman
hydrolysis) (Shatalov, A., et al., 1999). In order to
prevent degradation of the samples, they were stored
in a refrigerator.

2.7.2

HPAEC-PAD

The constituent neutral sugar was determined by

high-performance anion exchange chromatography
(HPAEC). After hydrolysis, the sample was diluted,
filtered and injected into the HPAEC system (Dionex
DX500) with an amperometric detector (Dionex ED50)
and a Carbopac PA1 column (4x250 mm, Dionex).
Neutral sugars were separated in 2 mM NaOH
(carbonate free and purged with helium) at a rate of 1
mL/min. Run time was 40 min, followed by a 20 min
elution with 0,3 M NaOH to re-equilibrate the column.
Calibration was performed with standard solutions of
arabinose, glucose, xylose, mannose and galactose.

2.7.3

Solubility of xylan

2.9

Size-exclusion chromatography (GPC)

The isolated xylans were dissolved in 50 L of 8%

LiCL (w/v) solution in N,N-dimethylacetamide (DMAc)
HPLC grade at 105C and further diluted with 600 L
DMAc.
The SEC analysis was carried out on two PLgel 10m
Mixed D 300 x 7,5 mm columns protected by a PLgel
10m pre-column (Polymer Laboratories, UK) using a
PL-GPC 110 system (Polymer Laboratories). The
columns, injector system and the detector (RI) were
maintained at 70C during the analysis. The eluent
(0,1 M LiCl solution in DMAc) was pumped at a flow
rate of 0,9 mL/min. The analytical columns were

UV-H2SO4 Method

Initially, 2 mL aliquot of carbohydrate solution is

rapidly mixed with 6 mL of concentrated sulfuric acid
3

calibrated
with
pullulan
standard
Laboratories) in the range of 5,8-48 kDa.

2.10

(Polymer

variation in pentosan content than in ash content

(Figure 2).

Spectroscopic Characterization (FTIR)

FT-IR spectra of hemicellulosic samples were

obtained on a FTIR spectrophotometer ABB, FTLA
-1
2000 Analyzer in the range of 4000 to 500 cm and
64 scans per sample.

2.11

Extracted Pulp (g) vs initial pulp (g)

Thermogravimetic Analysis (TGA)

Thermal behavior of the xylans and pulps were

performed using thermogravimetric analysis (TGA)
and differential thermal analysis (DTA) on a thermal
analyzer NETZSCH STA 409 PC Luxx. The
apparatus was continually flushed with an air flow of
150 mL/min. The sample weighed between 30 and
100 mg was heated from room temperature to 1100C
at a rate of 25C/min. A standard xylan sample from
Beechwood (>90% of xylan residues) (Sigma
Aldrich) was used

2.12

Fig. 1 Variation of pulp extracted dry weight with the

mass of starting pulp, for various tests.
Pentosan content
Ash content

Conductivity of wash water samples

In order to dissolve the impurity contents, some xylan

precipitates were washed with water and the
concentration of the liquid followed by water
conductivity measurements (Crison GLP 32). The
measured values were adjusted to 25C Before
measurements, water samples were vacuum filtrated.
Calibration curves were obtained beforehand for
calculating the concentration of salt (g / L) (and their
ions) existing in each water sample. The calibration
was carried out with aqueous solutions of sodium
sulfate (pro-analysis > 99% Merck), sodium nitrate
(>98%, Scharlau), anhydrous sodium phosphate (proanalysis > 99%, Merck) and also of sodium hydroxide
(98%, Panreac) depending on the compound present
in the wash water sample.

Tests

Fig. 2 Pentosan and ash content for each extracted

pulps.
The representation of the previous contents
(Pentosan and ash) for the two different series of
initial pulp (12,5 g Fig.3 and 25 g Fig.4) denotes
the influence of the stirring speed on extraction of
xylans. In the low speed region (green circle)
pentosan content is higher, when compared with high
speed region (orange circle) (Figure 3 and 4). This
means that with higher stirring speed, more xylan was
extracted and goes to alkaline filtrate.

2.13
Washed water analysis by Laboratrio
de Anlises of IST
In parallel with the previously method, some wash
water samples were analysed, by ion chromatography
at the Laboratrio de Anlises of IST, for ion
concentration determinations.

Pentosan content
Ash content

3. Results and Discussion

3.1 Alkaline Extraction
Tests

In alkaline extraction, three important parameters

were analyzed: initial dry mass pulp (12, 25 or 50 g),
stirring speed (120 to 1100 rpm) and extraction time
(1 and 2 hours).
As shown the dry mass of extracted pulp presents
little variability in tests carried out for the same initial
amount of pulp (Figure 1). This indicates the
reproducibility of the various experiments.
Ash and pentosan content were determined for both
initial and extracted pulps. Comparing these two
parameters, is noted that there is a more pronounced

Fig. 3 Pentosan and ash content for extracted pulp

obtained for 12,5 g of initial dry pulp.

Yield of extraction (%)

Pentosan content
Ash content

Yield of extraction

Tests
Stirring speed (rpm)

Fig. 4 Pentosan and ash content for extracted pulp

obtained for 25 g of initial dry pulp.

Fig. 8 Variation of extracted xylan yield for 25 g

initial dry pulp tests with stirring speed.

With mass and pentosan content of initial and

extracted pulp is possible to determine the mass of
extracted xylan and therefore the extraction yield.

(%) =
100
( )

Relatively to ash content, this parameter show, the

inorganic matter that exists in initial bleached Kraft
pulps and in extracted pulps and the difference is very
small, concluding that water washing of extracted
pulps were efficient.
It was concluded that the extraction time does not
influence the yield of extraction and 1 hour of
extraction is enough to extract xylans.
In the extraction process, in addition to the extracted
pulp, the alkaline filtrate was also obtained. At the end
of the alkaline extraction, the pulp was separated from
solution by vacuum filtration. This solution is
constituted with NaOH and also with some organic
matter including xylans.

Extracted xylan and extraction yield shows the same

variation behavior. For an initial dry mass pulp of 12,5
g extracted xylan varies between 1,4-1,8 g (Figure 5)
and for 25 g the range is from 3-3,5 g (Figure 6).
Extracted xylan (g)

Yield of extraction (%)

3.2 Xylan precipitation from alkaline filtrate

Tests

Fig. 5 Extracted xylan and yield for 12,5 g initial dry

pulp tests.

Extracted xylan (g)

Yield of extraction (%)

The isolation of xylans from alkaline filtrate was

performed by two methods: acid neutralization and
alcohol precipitation (ethanol and methanol) followed
by centrifugation. From this process result a
precipitate and a supernatant. The precipitate consists
of organic matter (xylans), salts or NaOH (inorganic
matter) and humidity. To reduce / remove the
inorganic matter of the precipitate several washing
procedures
were
performed
(item
2.4).The
supernatant was also studied and the results are
showed in section 3.3. For dry precipitate, purity and
yield, two important parameters were determined
through ash and pentosan contents:

Tests

Fig. 6 Extracted xylan and yield for 25 g initial dry

pulp tests.
The extraction yields vary between 65-84% and 7085% for two series of initial pulp, respectively. This
variation shows the influence of stirring speed (Figure
7 and 8). It was concluded that in alkaline extraction
experiments the higher the stirring speed, greater the
yield of extraction.

(%) =
(%) =

()
100
()

()
100
()

Yield of extraction (%)

3.2.1 Diluted acid neutralization

Acid neutralization consists in addition of an acid to
alkaline filtrate until reach pH=7. Several diluted acids
are studied: sulfuric, hydrochloric, acetic, nitric,
phosphoric and formic acids. Figure 9 shows the
behavior of neutralization process for different tests.

Yield of extraction

Stirring speed (rpm)

Fig. 7 Variation of extracted xylan yield for 12,5 g

initial dry pulp tests with stirring speed.
5

Tests

3.2.2 Alcohol precipitation

In this step, the purpose was to isolate xylans by
addition of various amounts of alcohol: 30, 50 and
70%, namely ethanol and methanol. After alcohol
addition the precipitate remains alkaline being
necessary to neutralize. Some neutralization studies
were performed (section 2.4). After alcohol addition
and precipitate washing, purity of solids was
determined (Figure 11 and 12).

Ethanol

Acid/Filtrate (mL/mL)

Fig.9 pH variation along acid neutralization.

It was observed that the three experiments (6, 12 and
15 all with SA neutralization) which showed a lower
ratio are related to a more concentrated acid used
indicating that the more concentrated acid, the smaller
amount of acid is needed to reach pH 7. The other
experiments (with the same acid concentration-7,5 N)
have the same behavior with a ratio acid/filtrate
(mL/mL) of about 0,3. Phosphoric acid (7.5 N) was
different because as a polyprotic acid it produces
various salts.
The acid addition induces the formation of a solid
phase and several washing procedures were used to
evaluate the purity of precipitates (Figure 10).

AALF-acetic acid-liquid filtrate; AASO-acetic acid-supernatant; ASSOsulfuric acid-supernatant; H2O washed with water until pH=7; LMMethanol washing only (alkaline pp)

Fig.11 Purity (%) of precipitated samples obtained by

alkaline filtrate ethanol precipitation (ET).

Methanol

AALF-acetic acid-liquid filtrate; AASO-acetic acid-supernatant; ASSOsulfuric acid-supernatant; H2O washed with water until pH=7; LMMethanol washing only (alkaline pp)
LMT-Washed with methanol; NL-Not washed; H2O-washed with water
(to remove salts);

Fig.12 Purity (%) of precipitated samples obtained by

alkaline filtrate methanol precipitation (MT).

Fig.10 Purity (%) of precipitated samples obtained by

alkaline filtrate acid neutralization.

From these experiments was concluded that washing

the precipitate with water is the more efficient method,
with xylan purity of about 99%. Neutralization with
acetic acid gives xylan with a purity of 60-81% and
with sulfuric acid the precipitate has the lowest purity
observed (3-28%) and almost no xylans in precipitate.
The xylans yield obtained from alcohol precipitation
ranged from 43-97%, and the best yield was obtained
from 30% ethanol.

The results indicate that the most suitable acid to the

neutralization of the alkaline filtrate is what produces
the most soluble salts.Nitric acid (HN) was the best
acid with a xylan purity of 87-88%. In the washing
processes, methanol proved to be very important to
drag impurities and remove the excess water present
in precipitates, reducing the ash content between 48% compared to unwashed samples. On the other
hand, water washing was very efficient because the
resulting precipitates have purity between 97-99%.
Although it originated some precipitate losses during
washing and xylan yields vary between 50-60%.
The yield of xylans in the precipitated derived from
acid neutralization ranged from 51-97% and the best
xylan yield was obtained for formic acid experiments.

3.3 Fractionation of hemicelluloses by graded

Ethanol precipitation (Supernatant)
As was previously referred, from alkaline filtrate
process always result a precipitate and a supernatant.
In this section, a brief reference to xylan precipitation
of the supernatant is mentioned. This study was
performed through the ash content for each dry
sample obtained by ethanol precipitation (Figure 13).

Fig.13Ash content of hemicellulosic

obtained by graded ethanol precipitation.

Xylose is the predominant sugar suggesting the

presence of a high proportion of xylan. Glucose is the
second major sugar component. Minor quantities of
galactose, arabinose and mannose were also
identified. It is thought that some samples (AA LMT,
NA LMT, and MT70 ASSO) presents some
contamination and analyzes needed to be repeated. It
is possible to verify that samples washed with water
(H2O), have the highest xylose content. In order to get
more detailed conclusions would be necessary to
perform further analysis with a complementary study
1
of morphology of xylan by H NMR, for example.

samples

These tests showed that the supernatant has almost

no xylan, because it was mostly isolated by
precipitation with acid/alcohol. Supernatant is mainly a
mixture of salt (from acid precipitation) or NaOH and
alcohol (in alcohol precipitation).

3.2.2 UV-H2SO4 Method

From this method was possible to determine the
xylose content of some precipitated samples. This
method gives the total sugar composition of samples
and as xylan is the main polysaccharide existing in
precipitate, xylose was considered the major sugar in
samples (Figures 16 and 17).

3.2 Carbohydrate Analysis

Precipitated xylans from alkaline filtrate were
classified about their monosaccharide content by two
methods: ion chromatography (HPAEC-PAD) and
spectrophotometric UV-VIS (UV-H2SO4).

3.2.1 HPAEC-PAD
The hemicelluloses are a mixture of different
polysaccharides, in which the sugar composition can
vary depending on its source and method of isolation.
The sugar composition of hemicellulosic samples is
showed in Figure 14 for samples obtained by alkaline
filtrate neutralization with acid and in Figure 15 for
samples obtained by alkaline filtrate precipitation with
alcohol.
Fig.16 Xylose composition of xylan samples obtained
by alkaline filtrate neutralization with acid.

Ara-Arabinose;
Mannose

Gal-Galactose;

Glc-Glucose;

Xyl-Xylose;

Man-

Fig.14 Sugar composition of xylan samples obtained

by alkaline filtrate neutralization with acid.

Fig.17 Xylose composition of xylan samples

obtained by alkaline filtrate precipitation with alcohol.
HPAEC-PAD and UV-H2SO4 can only be compared in
terms of xylose content. As can be seen, UV method
agrees with HPAEC, showing that xylose content is
higher for samples washed with water (H2O). In this
method HA LMT obtained by hydrochloric acid
neutralization shows major xylan content than in
previous method. The deviations may have occurred
due to the possible degradation of the samples with
time, even being stored in a refrigerator. In this
method other possible error is considering all sugar
content as xylose. The main advantage of this method
compared to the HPAEC-PAD is that it is faster and
more accessible. In literature, Deutschmann, R., et al.

Fig.15 Sugar composition of xylan samples obtained

by alkaline filtrate precipitation with alcohol.

(2012) refers that the spectrophotometric method

allows to obtain good results and this is also proved
here. So, this is a method to consider for this type of
analysis.

The weight-average (Mw) and number average (Mn)

molecular weight were 16,828-22,715 and 8,69014,810 kDa for the hemicellulosic samples analyzed.
The highest molecular weight was obtained for the
sample produced from neutralization with hydrochloric
acid. The lower molecular weight was related to the
sample obtained by neutralization with phosphoric
acid. Possibly, due to these acid properties, samples
have a wide range of molecular weights, causing a
smaller average molecular weight.

3.3 Solubility Tests

Solubility tests in xylan samples showed that DMSO
and DMSO:H2O (90:10)+0,05M LiCl, toluene and
DMF are the best solvents to solubilize xylans. On the
other hand, DMAc partially dissolves xylan, only at
high temperatures, 105C, at about one hour. It was
proved also that aqueous solutions do not dissolve
xylans from E.Globulus.

3.5 Spectroscopic Characterization (FTIR)

Spectroscopic Characterization was used to identify
xylans in samples and conclude about the existence
of other compounds present in the sample (salts,
NaOH, and others) through the bands that
characterizes them.
Figure A and B shows two examples of the FTIR
spectra of the alkali hemicellulosic fractions 15 a, b, c
(obtained with sulfuric acid) and 30, 50 and 70%
ethanol, neutralized with H2O (collected from 4000 to
-1
-1
500 cm ). The absorbance at about 3450 cm was
attributed to the stretching of OH-groups. The C-H
-1
stretching vibration gave a signal at 2933 cm . The
-1
absorbances at 1414, 1327, 1043, 950 and 901 cm
were associated with hemicelluloses. The bands
-1
between 1130 and 1000 cm were typical of xylans.
-1
The predominant absorbance at 1043 cm was
attributed to C-O-H bending modes (Luo, Q., et al.,
2012). Moreover, the most important band
-1
observed at about 901 cm corresponded to the
glycosidic C1-H deformation mode with ring
vibration contribution for the -anomer form of the
pyranoid ring, which is characteristic of glycosidic linkages between the sugar units (Peng,
F., et al., 2010).
The results confirmed the presence of xylan on
samples. However, the spectra also show the
impurities of xylan (Figure A). In this figure at 1100
-1
1
cm a sodium sulfate characteristic band (the salt
formed in sulfuric acid neutralization) is observed. On
the other hand, this band is not present in red spectra,
because it belongs to the sample washed with water.
In Figure B the results obtained for the precipitates
with the three contents of ethanol and washed with
water, indicate that there are no impurities and
spectra are similar to the standard xylan.

3.4 Size-exclusion chromatography (GPC)

In the present work, gel permeation chromatography
(GPC) was employed. The gel permeation
chromatograms of seven hemicellulosic samples
analyzed (Figure 18) showed the elution peaks
between 10 and 13,5 min.
Internal
standard:
glucose

RI

Xylans

Salt: LiCl

Fig. 18 GPC elution curves of xylan isolated from alkaline

Kraft pulp by acid/alcohol precipitation.
By observing the elution curves, can expect that, the
larger the curve, the grater the molecular weight
distribution and the higher the retention time, the
lower the molecular weight.
The determination of samples weight-average (Mw)
and number-average (Mn) molecular weights and
polydispersivity (Mw/Mn) are listed in Table 2.
Table 2 Average molecular weight (Da) of xylans
isolated from kraft pulp.
Sample

Mw

Mn

PD

HA LM
AA LM
SA H2O
ET50 H2O
NA H2O
PA H2O
MT30 ASSO

22715
21773
21233
18658
19938
16828
20326

14810
14339
14688
12317
13712
8690
13722

1,53
1,51
1,45
1,51
1,45
1,93
1,48

3.6 Thermogravimetic Analysis (TG)

The thermal properties of the hemicelluloses were
investigated using the thermogravimetric analysis and
derivative thermogravimetry (TG-DTG) technique in
the temperature range of 25C-1100C. These
analyses are a valuable analytical method to
investigate the physicochemical properties of

HA- neutralization w/ hydrocloric acid; AA- w/ acetic acid; SA-w/ sulfuric acid; NAw/ nitric acid; PA w/phosphoric acid; ET50-precipitation w/50%Ethanol; MT30
precipitation w/ 30%Methanol.

http://webbook.nist.gov/cgi/cbook.cgi?ID=B6000542&Units=
SI&Mask=80#IR-Spec

hemicelluloses. Thermal degradation of biomass, and

in particular decomposition of carbohydrates, is a
complex process and it includes many different
reactions occurring simultaneously, e.g. dehydration
(<190C),
depolymerization
(186
to
330C),
fragmentation,
rearrangement,
repolymerization,
condensation and carbonization (>330C). The
pyrolysis process results in a char residue (fixed
carbon and ash) and gaseous products (Luo, Q., et
al., 2012). The TG-DTG curves for one of the
samples analyzed (PA LMT obtained by
phosphoric acid neutralization), compared to
standard xylan are shown in Figure C. From
thermal degradation of the samples is possible to
know its composition and with the derivative curve
(DTG) is possible to quantify. Another important
task is the use of thermogravimetric analysis to
qualitatively compare (between thermograms) the
difference in molecular weights of samples by the
displacement of thermogram with temperature
(higher molecular weights show more degradation
resistance with temperature and weight loss is
observed at higher temperatures). In Figure C, the
split into two peaks (200-400C for standard xylan)
indicates a wide range of molecular weights and
sample PA LMT shows molecular weights between
this range but very next to each other.

- The xylans yield of precipitated obtained from acid

neutralization ranged from 51-97% and the best yield
was obtained for formic acid experiment (97,3%);
In alkaline filtrate alcohol precipitation was concluded
that:
-The formed precipitated remains alkaline and xylans
may depolymerize over time and was necessary to
neutralize them;
- Washing the precipitate with water was the more
efficient neutralization with xylan purity of 99%;
- Precipitate neutralization with acetic acid gave xylan
with a purity of 60-81%;
-Sulfuric acid neutralization has the low purity
observed (3-28%) and there are not almost xylans in
precipitate;
-The xylans yield of precipitated obtained from alcohol
precipitation ranged from 43-97% and the best yield
was obtained from 30% ethanol;
The supernatant has almost no xylan and these were
isolated mostly by adding acid/alcohol to the filtrate;
Infrared analysis of precipitated samples confirmed
the existence of xylan;
The UV-H2SO4 method has proved to be an effective
method for the analysis of carbohydrates in the
alternative chromatographic method (HPAEC-PAD);
Solubility tests in xylan samples showed that DMSO
and DMSO:H2O (90:10)+0,05M LiCl, toluene and
DMF are the best solvents to solubilize xylans. It was
proved also that aqueous solutions do not dissolve
xylans from E.Globulus;
The weight-average (Mw) and number average (Mn)
molecular weight were 16,828-22,715 and 8,69-14,81
kDa, respectively, for the hemicellulosic samples
analyzed.
Finally, it was concluded that thermogravimetric
analysis
was
a
very
complete
technique,
complementing the previous analyzes.

Conclusions

It was concluded that in alkaline extraction

experiments:
-The higher the stirring speed, the greater is the
efficiency of extraction;
-The extraction time does not influence the yield of
extraction;
- Pentosan and ash content are important parameters,
because from them it is possible to determine xylans
and organic matter mass, respectively;
- Pentosan content is a parameter that varies widely
throughout the tests while the pulp extracted mass
proves to be a more regular value;
-Pulp alkaline extraction yields vary from 65-85%;
In alkaline filtrate acid neutralization was concluded
that:
-The more soluble salt from acid used (in water), more
suitable is the acid to the neutralization of the alkaline
filtrate. Nitric acid was the best acid with a xylan purity
of 87-88%;
-Methanol washing of precipitates proved to be very
important to drag impurities and remove the excess
water present in samples, reducing the ash content
between 4-8% compared to unwashed samples;
-Water washing was very efficient because water
dissolve salts from neutralization reaction resulting in
precipitated samples with purity between 97-99%;
-This procedure originated some losses of precipitate
during washing and xylan yields vary between 5060%;

4. Acknowledgements
This work was supported by the grants from Prof. Dr.
Lurdes Serrano (Thesis coordinator - IST) and Prof.
Dr. Ana Paula Dias (IST) for the collaboration in FTIR
and thermogravimetric analysis, to RAIZ for the
collaboration in HPAEC analysis and pentosan
determination and to Aveiro University for the
collaboration in GPC analysis.

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APPENDIX

Fig. A FTIR spectra of xylan obtained from alkaline filtrate by sulfuric acid neutralization and washed with methanol (LM blue
spectra), not washed (NL- green spectra), washed with water (H2O- red spectra) and standard xylan (black spectra).

Fig. B FTIR spectra of xylan obtained from alkaline filtrate ethanol precipitation (30% red spectra, 50%-blue spectra and
70%- green spectra, in solution), washed with water (H2O) and standard xylan (black spectra).

Fig. C TG-DTG curves of xylan obtained from alkaline filtrate by phosphoric acid neutralization and washed with methanol (PA
LMT green thermogram) and standard xylan (blue thermogram).

11