i

CHARATERIZATION OF ANNATTO (Bixa orellana L.)SEED OIL AND COMPOUNDS

BY
NAME REG NO:
1. NJANE PETER NJOGU CPE/21/06
2. ELIAS RIAMAN CPE/29/06
3. CYRIL M. JUMA CPE/05/06
4. GIBSON N. IRERI CPE/1010/04

AN EXPERIMENTAL PROJECT SUBMITTED IN PARTIAL FULFILLMENT OF
REQUIREMENTS FOR THE DEGREE OF BACHELOR OF ENGINEERING IN
CHEMICAL AND PROCESS ENGINEERING


SUPERVISORS NAME: DR. AMBROSE KIPROP
MR. ANIL KUMAR




DATE: 31

MARCH, 2011


ii

DEDICATION
We dedicate this work to our dear loving parents and friends who have selflessly supported us
throughout our studying period. God bless them.


















iii

ABSTRACT
Annatto extract which is a natural food colouring is obtained from the seeds of the Annatto tree,
Bixa orellana L. The study on annatto seeds dates back to ancient times. The annatto seed oil
has particularly found application in the cosmetic industry as it is used as an ingredient in
suntan formulation. Extraction was done and the samples were analysed to characterise the oil.
Various analytical techniques such as: Fourier transform Infra red spectroscopy (FT-IR)
spectroscopy was used to analyse the extract. For the characterization of the oil saponification
value, acid value, free fatty acids, specific gravity and refractive index were determined. The
report has five chapters: introduction, literature review, material and methods, results and
discussion, and conclusion and recommendation in that order.














iv

TABLE OF CONTENTS
DEDICATION .............................................................................................................................................. ii
ABSTRACT................................................................................................................................................ iii
TABLE OF CONTENTS .......................................................................................................................... iv
LIST OF FIGURES ................................................................................................................................... vi
LIST OF TABLES .................................................................................................................................... vii
ACKNOWLEDGEMENT ........................................................................................................................ viii
CHAPTER ONE ......................................................................................................................................... 1
1.1 INTRODUCTION ........................................................................................................................ 1
1.2 JUSTIFICATION ........................................................................................................................ 1
1.3 OBJECTIVE ................................................................................................................................ 1
CHAPTER TWO ......................................................................................................................................... 2
2.0 LITERATURE REVIEW ........................................................................................................... 2
2.1 ANNATTO PLANT PROFILE .................................................................................................. 2
2.2 COMPOSITION AND PROPERTIES OF THE ANNATTO PERICARP EXTRACTS ..... 2
2.3 Uses of Annatto ........................................................................................................................ 5
2.3.1 Traditional uses of annatto ................................................................................................ 5
2.3.2 Modern Uses ....................................................................................................................... 6
2.4 Annatto Leaves ......................................................................................................................... 7
2.5 Extraction of Annatto Pigment ............................................................................................. 7
2.6 Annatto Oil ................................................................................................................................. 8
CHAPTER THREE ................................................................................................................................... 10
3.0 MATERIALS AND METHODS ............................................................................................. 10
3.1 Sample Preparation ............................................................................................................... 10
3.2 Extraction of Oil ...................................................................................................................... 11
3.3 Solid Analysis (ATR-FTIR Spectra) ................................................................................... 11
3.4 CHEMICAL ANALYSIS OF THE OIL ................................................................................... 12
3.4.1 Determination of Acid Value ........................................................................................... 12
3.4.3 Determination of Saponification Value .......................................................................... 12
3.5 PHYSICAL ANALYSIS OF THE OIL .................................................................................... 13
3.5.1 Determination of Specific Gravity .................................................................................. 13
3.5.2 Determination of refractive index ................................................................................... 13
v

CHAPTER FOUR ..................................................................................................................................... 14
4.0 RESULTS AND DISCUSSIONS ................................................................................................ 14
4.1 Extraction of Oil ...................................................................................................................... 14
4.3 Saponification Value ............................................................................................................. 15
4.4 Acid value................................................................................................................................. 17
4.5 Free Fatty Acids (FFA) .......................................................................................................... 18
4.6 ATR-FTIR spectrum for Annatto hexane extract (solid sample) ................................ 18
4.7 Specific Gravity ...................................................................................................................... 20
4.8 Refractive Index (RI) .............................................................................................................. 20
CHAPTER FIVE ...................................................................................................................................... 22
5.1 CONCLUSION AND RECOMMENDATION ........................................................................ 22
5.1.1 Conclusion ......................................................................................................................... 22
5.1.2 Recommendation ............................................................................................................ 22
REFERENCES ......................................................................................................................................... 23


vi

LIST OF FIGURES
Figure 2-1 Annatto fruit .................................................................................................................... 2
Figure 2-2 Chemical structures of some bixin/norbixin isomers ................................................. 4
Figure 2-3 Steam Distillation ........................................................................................................... 7
Figure 4-1 A block diagram for the extraction process .............................................................. 15
Figure 4-2 ATR-FTIR spectrum for Annatto hexane extract (solid sample).....19
vii

LIST OF TABLES
Table 2-1 General properties and brief descriptions of annatto pericarp extracts ..................... 3
Table 2-2 Composition of matter suitable as suntan oil ................................................................. 9
Table 4-1 Results for percentage oil extracted .............................................................................. 14
Table 4-2 Results for saponification test ......................................................................................... 16
Table 4-3 Blank test results ............................................................................................................... 16
Table 4-4 Results for the acid test ................................................................................................... 17
Table 4-5 Results for refractive index test ...................................................................................... 20
















viii

ACKNOWLEDGEMENT
We would like to give our sincere gratitude to our supervisors Dr. Ambrose Kiprop and Mr. Anil
Kumar for their wonderful guidance and support during the project.

We are indebted to Moi University, Chemical and Process Engineering Department for having
provided as with facilities chemicals and reagents that were really needed for the eventual
success of the project.

We greatly express our sincere thanks to Nancy university in France for their facilities which
were used for the FTIR analysis.

Our fellow classmates have also demonstrated cooperation and ready to assist spirit during the
studies, may God bless them all.










1

CHAPTER ONE
1.1 INTRODUCTION
Annatto plant is a tropical plant which has brown fruits at maturity. These fruits have
dark red seeds. Major producers are Peru, Brazil and Kenya (Mercadante et al., 1997b).
Some of the useful compounds extracted from annatto seeds include; bixin which is oil
soluble and norbixin which is water soluble. Bixin has found wide application in food,
cosmetic, pharmaceutical, textile and ink industry as an effective colouring agent (Silva
et al., 2005; Silva et al., 2007).
Annatto oil is extracted from seeds of annatto plant (Bixa Orellana L.). This was
obtained by solvent extraction.
Other compounds extracted from annatto seeds are essential oil and apocarotenoids.

1.2 JUSTIFICATION
Annatto oil has found uses in the cosmetic industry where it is used in the formulation of
the suntan oil which is applied on human skin to treat sunburns.
In the tropical regions where sun is experienced throughout the year most people suffer
from sunburns. There is need, therefore, to characterize annatto compounds so as to
understand their contribution to sunburn treatment.

1.3 OBJECTIVE
The objective of this experimental project is to extract and characterize annatto
compounds in order to understand their properties.

2

CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 ANNATTO PLANT PROFILE
Annatto also known as (a.k.a) Roucou, is a derivative of the achiote trees. The english name is
Arnotta or Annatto plant while family is Bixaceae and species is Bixa orellana.
The heart-shaped fruits (figure 2.1 below) are brown or reddish brown at maturity, and are
covered with short, stiff hairs. When fully mature, the fruit splits open exposing the numerous
(10-60) dark red seeds (Mercadante et al., 1997b).

Figure 2- 1 Annatto fruit (courtesy of http://en.wikipedia.org/wiki/Annatto).

Achiote is a hardy tree and does not require very fertile soil for its cultivation. An average soil
which has moisture retaining capacity is suitable for the cultivation of this crop. Soil which is
poor in drainage and has rocks in the subsoil should be avoided. It can be grown successfully in
a warm dry climate. It cannot withstand frost and hence unsuitable for cultivation in the places
where frost occurs frequently. Achiote originated in South America and has spread in popularity
to many parts of Asia. It is commonly found in Latin America and Caribbean.
Major commercial producers of annatto seeds are Peru, Brazil and Kenya, and the largest
importers are Japan, U.S.A and England (Mercadante et al, 1997b).
2.2 COMPOSITION AND PROPERTIES OF THE ANNATTO PERICARP
EXTRACTS
The pericarp of annatto seeds is composed of up to 80% of the carotenoid cis-bixin (IUPAC:
methyl hydrogen 9-cis-6,6-diapocarotene-6,6-dioate) and the remaining 20% include:
trans- and cis-norbixin (6,6-diapocarotene-6,6-dioic acid and 9-cis-6,6-
diapocarotene-6,6-dioic acid),
several apocarotenoids e.g. methyl(9Z)-ap-8-lycopenoate, methyl (all-E)-apo-6-
lycopenoate, methyl (all-E)-apo-8-lycopenoate, etc.
3

volatile compounds and
Other uncharacterized substances (Presto and Rickard, 1998; Mercadante et al., 1997a,
1997b and 1999).
Table 2-1 below gives the general properties of annatto pericarps extracts with brief
descriptions.
Table 2-1 General properties and brief descriptions of annatto pericarp extracts (sources:
Scotter et al, 1998; Silva et al, 2007; Silva et al, 2005)

Properties of annatto
pigment
Description
Colour Orange yellow
Colour pigments Carotenoids bixins and norbixins
Solubility Water soluble and oil soluble
Stability Fades in strong or direct light and elevate temperatures
Stable in alkaline mediums
Degrades in acids
Degrades under oxidation
Resistant to microbial attack

As stated earlier, bixin (C
25
H
30
O
4
) is present in large concentration in the annatto seeds. It is
unstable and degrades when exposed to elevated temperature, light and oxygen. When treated
with alkali, all its isomeric forms are transformed to their water-soluble norbixin counterpart
(Silva et al, 2005; Silva et al, 2007).
Norbixin (C
24
H
28
O
4
) is obtained by alkaline hydrolysis of cis-bixin at (45-50C). It decomposes at
temperatures higher than 280C

and it is the major coloring component of commercial water-
soluble annatto extracts (Silva et al, 2007).

Figure 1.2 below shows the chemical structure of some bixin/norbixin isomers. For norbixin,
R=H while R=CH
3
for bixin.

4



Figure 2-2 Chemical structures of some bixin/norbixin isomers (Source: Scotter et al, 1998)


HOOC
COOR
9'
HOOC
9'
COOR
13
15
15'
9
13'
HOOC
13'
COOR
9'
9', 13'-di-cis-
trans-
9'-cis-
5



Figure 2-3 Chemical structure and
13
C NMR data for cis-norbixin.

2.3 Uses of Annatto
Annatto plant has been discovered and used for a long time by natives in tropical areas where it
naturally grew. Its uses therefore can be classified under traditional uses and modern use.
2.3.1 Traditional uses of annatto
In Latin America and Caribbean, they are used as both a coloring agent and for
flavoring.
Central and South American natives use the seeds to make body paint, and lipstick. For
this reason, the achiote is sometimes called the lipstick-tree.
In Venezuela, annatto is used in the preparation of hallacas, perico, and other
traditional dishes.
In Jamaica, annatto has had many uses over the centuries, including as a:
o food dye
o body paint
o treatment for heartburn and stomach distress sunscreen and
o insect repellant.
Annatto in the Philippines, is called atsuete and is used as:
o food coloring in traditional dishes and
o a major ingredient in the popular spice blends "Sazn" made by Goya Foods
(http://en.wikipedia.org/wiki/Annatto).
6

2.3.2 Modern Uses
Carotenoids are a family of natural fat/oil soluble pigments found principally in chloroplasts
where they function as accessory pigments to chlorophyll (Lotfi et al, 2008).
Bixin and norbixin are widely applied in food industry, condiments, cosmetics, textiles and inks
as an effective natural coloring agent (Silva et al., 2005). These pigments are sold commercially
as annatto.
Annatto formulations are generally found as oil- or water-soluble solutions, suspensions,
emulsions, encapsulated products and spray-dried powders (with carriers).
Other natural colourants (carotenoids) include; anthocyanins (flavonoids), curcuminoids and
betalains. Betalains are extracted from the roots of red beets (Beta vulgaris) and from some
species of the fungal genera amanita and hygrocybe (Cai et al., 2003; Castellanos-Santiago et
al., 2008).
Carotenoids play an essential role in human health due to their provitamin A activity and their
function as biological antioxidants thus protecting tissues and cells from the destructive effects
of free radicals and quenches the reactive oxygen species (Lotfi et al., 2008).
In general, these colourants exhibit antiflammatory, antioxidant, antibacterial (antiviral) activities
and anticarcinogenic and anti-tumor promoting effects (Cai et al., 2003).
Besides its uses in food as a natural colorant and spice, the medicinal uses of annatto (Giuliano
et al., 2003) include:
anecdotal treatment of diabetes
skin infections
burns
fever
measles,
gonorrhea
diarrhea and asthma
Commercial annatto extracts have biological activities against microorganisms of significance
to:
food fermentation
preservation and
Safety.

7

2.4 Annatto Leaves
Annatto leaves can also be used to colour food, but, in general, they will give only a modestly
green colour.
2.5 Extraction of Annatto Pigment
The pigment from the annatto pericarp is extracted by steam distillation. Steam distillation is a
special type of distillation (a separation process) for temperature sensitive materials like natural
aromatic compounds. Many organic compounds tend to decompose at high sustained
temperatures. Separation by normal distillation would then not be an option, so water or steam
is introduced into the distillation apparatus. By adding water or steam, the boiling points of the
compounds are depressed, allowing them to evaporate at lower temperatures, preferably below
the temperatures at which the deterioration of the material becomes appreciable. Figure 2-3
shows how extraction of annatto pigment by steam distillation is carried out.



Figure 2-3 Steam Distillation
After distillation the vapors are condensed as usual, mostly yielding a two phase system of
water and the organic compounds, allowing for simple separation. When a mixture of two
practically immiscible liquids is heated while being agitated to expose the surfaces of both the
liquids to the vapor phase, each constituent independently exerts its own vapor pressure as a
function of temperature as if the other constituent were not present. Consequently, the vapor
pressure of the whole system increases. Boiling begins when the sum of the partial pressures of
the two immiscible liquids just exceeds the atmospheric pressure.
8

There are three commercial processes used to extract the pigment from dried annatto seeds. To
obtain oil- or fat-based formulation of annatto, direct extraction into oil process is employed. Hot
oil facilitates isomerization of 9-cis-bixin to relatively more soluble trans-bixin. Direct aqueous
alkali extraction produces alkali metal or ammonium salt solutions of 9-cis-norbixin. Finally,
indirect extraction of annatto involves using solvents to extract the pigment which are then
evaporated leaving powder (Scotter et al, 1998).
After the extraction of the pigment, the samples can be characterized using the following
analytical techniques:
Thin layer chromatography (TLC)
High pressure liquid chromatography (HPLC)
pH tests
Gas chromatography mass spectrometry (GCMS)
UV-visible spectroscopy (UV-Vis)
FT-IR spectroscopy, among many other techniques.
2.6 Annatto Oil
Annatto oil is obtained from the seed by conventional expression and solvent extraction
techniques using a variety of solvents such as hexane and ether. Annatto oil is that fatty oil used
to make suntan formulation which is highly successful in enhancing the human skin tanning
process. When annatto is added to the formulation of suntan oil, preferably with other naturally
occurring oils, wearer receives a suntan that is commercially more desirable. Suntan oil is
applied to human skin before, during and after exposure to a natural or artificial ultra violet rays
source.
The composition of matter suitable as suntan oil is shown in Table 2-2 below.


9

Table 2-2 Composition of matter suitable as suntan oil
Percentage by weight
Ingredients Preferred Allowable range
White mineral oil
Sesame oil
Sanflower
Avocado oil
Sweet almond oil
Apricot kernel oil
Grape seed oil
Kukuui nut oil
Wheat germ oil
Cod liver oil
COVI-ox
Annatto
Mango fragrance
Antioxidant
54.93
15
10
3
3
5
6
0.1
2
0.5
0.01
0.25
0.2
0.01
52-57
13-17
8-12
2-4
2-4
4-6
5-7
0.8-0.12
1-3
0.4-0.6
0.08-0.012
0.1-1.0
0.1-0.4
0.001-0.02




10

CHAPTER THREE
3.0 MATERIALS AND METHODS
Plant material: Dried seeds of Bixa orellana were purchased from Coast Province; Kenya.
Chemicals: The reagents that were used include;
Potassium hydroxide pellets
Phenolphthalein indicator
Hydrochloric acid
Ethanol
Normal hexane
All these reagents were obtained from the Chemical and Process Engineering Laboratory.
0.1M ethanolic potassium hydroxide was prepared by dissolving 2.8g of potassium
hydroxide pellets in 500ml of ethanol.
Phenolphthalein indicator was prepared by mixing 50ml of distilled water with 50ml of
ethanol and 3g of phenolphthalein powder.
0.5M of HCl was prepared by mixing 210ml of water with 10ml of 11M HCl. The correlation
between molarity and volume that was used is:

Apparatus: The apparatus that were used include:
Refractometer
Rotor vacuum distiller
Mill
ATR-FTIR spectrophotometer
Vacuum filter
The refractometer, vacuum filter and rotor vacuum distiller were obtained from the Chemical and
Processing Laboratory while a mill from Cheboywa Trading Centre was used. ATR-FTIR
spectrophotometer in Nancy University, France was used.
3.1 Sample Preparation
The annatto seeds were ground before extraction. A mill was used to crush the seeds into very
small sizes.

11

3.2 Extraction of Oil
1.0 kg of ground annatto seeds were soaked in 1.0 litre of n-hexane for five days in a flat
bottomed flask. The soaked sample was then decanted and solid residue was discarded. The
filtrate was subjected to further filtration. Filter paper in vacuum filtration equipment was used.
The solid residue was preserved for analysis. The filtrate was subjected to vacuum distillation to
separate solvent from oil and recover the solvent. The oil substance then was left in the open for
7 days to allow evaporation of solvent remaining.
The percentage oil was calculated using the formula below;




This experiment was repeated for other two samples using the same procedure.
3.3 Solid Analysis (ATR-FTIR Spectra)
ATR-FTIR spectrum in the region between 4000-650 cm
-1
was obtained using PerkinElmer
Instruments, Spectrum One, FT-IR Spectrometer. The scanner velocity was 10 kHz, with the
resolutions of 4 cm
-1
. Reference is atmosphere for solids and pure water for aqueous solutions.
ATR spectra are shown with absorbance scale corresponding to log (R
reference
/R
sample
), where R
is the internal reflectance of the device.

12


3.4 CHEMICAL ANALYSIS OF THE OIL
3.4.1 Determination of Acid Value
0.1 M of KOH was prepared using ethanol. 1.0 g of oil sample was put into three 250 ml conical
flasks. 25 ml of ethanol was added into each of the three flasks. The mixture was then warmed.
Two drops of phenolphthalein indicator were added and the mixture was titrated with ethanolic
KOH till endpoint with consistent shaking for which a dark pink was observed and the volume of
0.1M ethanolic KOH (V
0
) was noted.
The acid value (AV) was calculated by the following formulae




Where;




3.4.2 Determination of Free Fatty Acid
The acid value was first obtained using the above procedure for acid value
Free Fatty Acid (FFA) was then calculated using the formulae below

3.4.3 Determination of Saponification Value
Into three 250ml conical flasks, 1.5g of sample was added in each. 50ml of 0.5M ethanolic KOH
was added into each of the flasks. Each of the mixture was refluxed for 30min. Then 1.0 ml of
phenolphthalein indicator was added to each flask and slowly titrated the hot soap solution with
0.5M HCl. The titre volume was noted (V
1
). The blank test was run upon the same quantity of
KOH solution at the same time and under the same conditions. The titre volume was noted as
V
2
.
The saponification value (SV) was calculated using the following formulae

(

13

Where;

3.5 PHYSICAL ANALYSIS OF THE OIL
3.5.1 Determination of Specific Gravity
The mass of a sample of the oil was obtained by a weighing balance and its volume was
determined by a measuring cylinder (10ml).
The density of the oil was obtained by the following formulae

The specific gravity was obtained by the following formulae

3.5.2 Determination of refractive index
Refractometer was used in this determination. Few drops of the sample were transferred into
the glass slide of the refractometer. Water at 30C was circulated round the glass slide to keep
its temperature uniform. Through the eyepiece of the refractometer, the dark portion viewed was
adjusted to be in line with the intersection of the cross. At no parallax error, the pointer on the
scale pointed to the refractive index. This was repeated and the mean value noted and recorded
as the refractive index.

14

CHAPTER FOUR
4.0 RESULTS AND DISCUSSIONS
4.1 Extraction of Oil
The results for extraction are shown in the table below

Table 4-1 Results for percentage oil extracted
Sample Mass of oil (g)
1 27.39
2 26.39
3 25.79
Average mass 26.50

The seeds were soaked for five days to give time for maximum extraction of the oil. Usually
annatto seeds contain oil up to a maximum of 5% oil. The percentage value of oil obtained in
this experiment lies within the range. However, in this case the percentage content of the oil
was lower than 5% since the annatto seeds provided had already had their pericarp removed
and this is attributed to loss of oil from the seeds during pericarp removal.
The following Figure 4-1 shows the block diagram for the extraction of annatto oil

15




Figure 4-1 A block diagram for the extraction process
4.3 Saponification Value
Saponification value represents the number of milligrams of potassium hydroxide or sodium
hydroxide required to saponify 1g of oil under the conditions specified. It is a measure of the
average molecular weight (or chain length) of all the fatty acids present. As most of the mass of
a fat/triester is in the 3 fatty acids, it allows for comparison of the average fatty acid chain length.
Titration results for the sample are shown in the table 4-2 below:

16

Table 4-2 Results for saponification test
1
st
2
nd
3
rd

Initial burette reading(ml) 0.0 3.5 7.0
Final reading(ml) 3.4 6.9 10.5
Titre volume(ml) 3.4 3.4 3.5
Average titre volume(ml) 3.4

For the blank test the results are shown in the table below;

Table 4-3 Blank test results
Initial burette reading(ml) 10.40
Final reading(ml) 15.80
Titre volume(ml) 5.4


( )

From the result above, annatto oil it has a soap value of 37.4. This can be interpreted to mean
that one needs 37.4 milligrams of potassium hydroxide in order to saponify 1000milligrams of
annatto oil. This depicts less number of carboxylic functional groups and hence a long chain
fatty acid.
Determination of the Average Molecular Weight of the Oil
The saponification equation for the annatto oil is as shown below.

0.0374g of KOH saponified 1.0g of the oil. Using the above equation, the average molecular
weight of the oil can be determined.

17

4.4 Acid value
The results of titration are as shown in the table below.
Table 4-4 Results for the acid test







Acid value is the mass of potassium hydroxide (KOH) in milligrams that is required to neutralize
one gram of fatty acid in the oil. The acid number is a measure of the amount of carboxylic acid
groups in a chemical compound, such as a fatty acid, or in a mixture of compounds.
As oil rancidity, triglycerides are converted into fatty acids and glycerol, causing an increase in
acid number. Rancidification results from the reaction of oil with oxygen in the air. It occurs at
the double bond or points of unsaturation (http://www.soonsoonoil.com.my/FAQ.htm)
The acid value obtained in this experiment indicates that rancidity is progressing. The factors
that contributed to the oxidative oils deterioration are
oxygen in air
Large surface of the oil that was exposed to air. This occurred in placing the oil in a
beaker to provide large surface area for solvent evaporation.
Raised temperature. This occurred during the solvent recovery process.
The rancidity reaction is:
Test 1
st
2
nd
3
rd
Average Value
Mass of oil used (g) 1.00 1.02 1.00 1.01
Volume of titrant (ml) 1.8 1.9 1.8 1.83
18



4.5 Free Fatty Acids (FFA)
The free fatty acid is determined from the acid value obtained.

The %FFA indicates the care and control exercised during extraction. It is an indication of fresh
oil quality. FFA is the result of reaction of water and fats at high temperature. In well refined oil
which doesnt contain an emulsifier, the typical FFA level should be less than 0.05%. The high
value obtained in the above experiment can be attributed to the high temperature (60-80C) for
which the oil was exposed to during the recovery of the solvent.

4.6 ATR-FTIR spectrum for Annatto hexane extract (solid sample)
The ATR-FTIR spectrum of the hexane crude extract (solid) is presented in Figure below.
This spectrum is characterized by four groups of absorption bands which appear in the following
wave number ranges: 3700 2350 cm
-1
, 1800 900 cm
-1
and 900 650 cm
-1
.
3700 2350 cm
-1
: this wave number range contains five distinct bands:
a OH stretching band of an alcohol OH functional group (3700 3300 cm
-1
)
a OH stretching band (wide band) of the associated OH group in a carboxylate
functional group (3400 2500 cm
-1
)
a CH stretching band of an aromatic CH bond (3075 3030 cm
-1
)

a
CH
3
and
a
CH
2
antisymmetric stretching bands of alkanes (2970 2915 cm
-1
)

s
CH
3
and
s
CH
2
symmetric stretching bands of alkanes (2880 2845 cm
-1
)
The broadband in the region 2350-1800 cm
-1
corresponds to the strong absorption of diamond
used in the ATR-FTIR instrument.
The observed band at 1715 cm
1
indicates the presence of C=O carbonyl band.
The broadband in the region 1719 1736 cm
-1
could be assigned to the C=O stretching
mode of four different functional groups: ketones, aldehydes, aliphatic or aromatic
esters, or dimers of carboxylic acids (Parker, 1971; Wojtkowiak and Chabanel, 1977).
19

The bands at 1550 and 1630 cm
-1
are assigned to the C=C of the aromatic ring.
The weak broadband in the 950 1017 cm
-1
region represents the in-plane CH
bending mode of aromatics.
Absorption bands in the low frequency region 710 895 cm
-1
result from the out-of-plane
CH bending of the aromatic ring CH bonds.
The spectrum is not smooth since we have not isolated into pure compounds and the
analyses carried herein are therefore for the n compounds in the crude extract.





Figure 4-2 ATR-FTIR spectrum for Annatto hexane extract (solid sample).


4000 3500 3000 2500 2000 1500 1000 500
0.002
0.004
0.006
0.008
0.010
1715
678
750
1008
1630
3700-2350
A
b
s
o
r
b
a
n
c
e
Wavenumber (cm
-1
)
Annatto hexane extract (solid)
20

4.7 Specific Gravity
The following results were obtained.

The specific gravity is then calculated as;

From (http://www.engineeringtoolbox.com/specific-gravity-liquids-d_336.html), Olive oil has a
specific density of 0.703, linseed oil has 0.932, cotton seed oil has 0.88, sesame seed oil 0.92.
So for the annatto seed oil a specific gravity of 0.83 is within that range.
4.8 Refractive Index (RI)

Refraction is the change of direction of light rays as it travels through one substance to another.
This occurs when the light travels at different speeds through materials with different densities.
The Table 4-5 below gives the results obtained for the refractive index.
Table 4-5 Results for refractive index test
Sample Refractive Index
1 1.4755
2 1.4725
3 1.4735
Average RI 1.4738

The refractive index of the oil is 1.4738.

21

From to (http://www.engineeringtoolbox.com/specific-gravity-liquids-d_336.html) most vegetable
oils have a refractive index of around 1.47 e.g. for corn oil-1.47, Clove Oil -1. 535,
Orange Oil -1.473, and Sunflower oil -1.466. So for the annatto oil it is well within that range.

22

CHAPTER FIVE
5.1 CONCLUSION AND RECOMMENDATION
5.1.1 Conclusion
The objective of this work was to extract and characterize annatto compounds in order to
understand their properties. Having carried out some of the main analysis on the annatto oil, we
can now confirm that the annatto seeds contain oil in the range of 1-5%. This oil can be used as
an ingredient in the suntan formulation which when applied on the skin acts as a remedy for sun
burns and after exposure to natural or artificial ultra violet rays. It can also be used in creams
and lotions. The oil is a long chain fatty acid as depicted by the low saponification value.
However, due to the limited number of tests carried out on the oil we encourage more research
on the contamination level of the annatto oil with other oils e.g. in iodine value test and the level
of its unsaturation i.e. in the peroxide value.
5.1.2 Recommendation
The application on to which annatto oil is used warrant further investigations on its effect on the
human skin on its application.
It is further recommended that:
Further research on the unsaturation and contamination levels of the annatto oil
and its effects be carried and field evaluation should be investigated.
Further characterization using more analytical techniques should be done.


23

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