GAS LIQUID CHROMATOGRAPHY

INTRODUCTION:

 Gas liquid chromatography is one of the most versatile of all

the chromatography techniques.
 It was first discovered by JAMES and MARTIN in 1952

 This method involves the partitioning of the compounds

of the mixture in the vapor state between a mobile
stable gas phase and stationary non volatile liquid phase
dispersed on an inert support.

 Gas chromatography consists of gas liquid chromatography

and gas solid chromatography. In both the types gas is used
as mobile phase and either solid (or) liquid is used as
stationary phase.

Principle:
 The Basics for the separation of compounds in Gas liquid Chromatography in the
different in partition coefficient of the volatile sample components in a liquid
stationary phase.
 Gas liquid Chromatography is a form of column chromatography in which the
stationary phase is a non volatile liquid.
 This Liquid Stationary phase is dispersed on to the surface of the inert solid
support contain in the column.
 The solid support is inert and it doesn’t react with sample components.
 It only acts to hold the stationary liquid phase in a dispersed form to present a
large contact surface area.
 It is a liquid phase which interacts with the sample components.
 A Gas stream termed as carrier gas flows to the column at a rate which is
controlled, it act as a mobile phase.
 The volatile sample components introduced into the mobile phase are carried into
the column by this carrier gas.
 In the column the sample components become distributed between liquid
stationary phase and gas mobile phase.
  The sample components which interact more with the mobile phase will move
faster, and the sample components which interact more with the stationary phase
will move slowly.
 So the sample components will separate from another based on their partition
coefficients.
 The sample components eluting out of the column reach the detector which
measures the concentration of sample components present in the effluent and
converts the concentration into an equivalent electrical signal.
 Volume of effluent when plotted against time gives a characteristic pattern called
chromatogram.
 This is made up of a number of peaks. The area under each peak is proportional to
the concentration of sample components present in the effluent.
 The volume of effluent(sample components + carrier gas)which flows between
the time, the sample was introduced into the column and the time in which the
sample has eluted out of the column is termed as “Retention volume” vary which
is a characteristic useful for the analysis of sample components.
Instrumentation:
The Apparatus of GLC consists of
1. Pressure Regulators.
2. Detectors.
3. Columns.
4. Sample Injectors.
5. Recorder.
6. Oven.
 The carrier gas is stored in cylinders passes to a pressure regulator which controls
the carrier gas flow.
 Then it reaches the sample injection chamber in which the sample component can
be introduced into the carrier gas.
  From the sample injection chamber the carrier gas flow into column which is
coated with an inert solid support on which a liquid stationary phase is dispersed.
 The sample components distribute between the stationary liquid phase and the
mobile gas phase and get separated from each other on the basis of their
distribution coefficient.
 Then the sample component reaches the detector which measures the
concentration and converts it into an electrical signal which is recorded by
recorder.
 Since gas liquid chromatography is performed at high temperature. Sample
injection chamber, column and detector are thermostatically controlled by an
oven.

Columns:

Column is the heart of the chromatography.

 In the column ,the different solutes in the vaporized samples are

separated from each other by virtue of their different interaction with
Column packing.
 As the solutes emerge individually from the end of column, they enter
the detector.
 Column is one of the important part of gas chromatography which
decides the separation of efficiency.
 Columns are made up of glass (or) stainless steel.

Depending upon use, column is two types

1 .Analytical columns {lengths 1to 1.5mts and diameter ( 3

to6)nm}
2. Preparative columns {length 3to6and 6to9mm out side
diameter}

Depending on its nature, column is of two types

1. Capillary column
2. Packed column

1 Capillary columns:
 Capillary column is fabricated from capillary tubing the bore of which is coated
with a very thin film of the liquid phase.
  These columns {0.75 nm (or) less} are available in length up to 100mts because
of the fact that they have a very low pressure drop .

 These columns are of two types

[Link] coated open tubular:

 The stationary phase liquid is coated in the form of a thin film(0.5to 1)
 It is suitable for small volume of sample.

B. Solid coated open tubular:

 Solid coated open tubular is suitable for large scale sample separation.
 In these columns a mirror size porous layer of support material on the inner
wall of the capillary column and then coated with a thin film of liquid phase.
 These columns offer least resistance to the flow of carrier gas and hence they
are more efficient than packed columns.

2. Packed columns:


The column container for packed columns is usually stainless steel (or)copper
(or)glass tube packed with either a solid substrate (or)liquid coating on a inert
solid.

The internal diameter of the tubes is about 2-4mm and length varies from 2-50
feet


Ordinarily the tubes folded (or) coiled so that they can be conveniently fitted
into the gas chromatography oven.

Commonly used solid supports are diatomaceous earth, Teflon powder and gloss
beads .The stationary phase must be chosen on the basis of compound to be
analyzed.
Stationary Phase Nature Temperature

Poly Diethyl siloxane Non polar 60-320 0 c

Poly Diphenyl Diethyl Siloxane Bonded Polar 60-320 0c
 Poly ethylene Glycol Polar 50-280 0 c

Column Specifications:
[Link] column
Make:chramatopack.
Length:2 mts.
Diameter:1/8 inch.
Liquid:10%
Solid:ch-WIHP
Mesh Range:80/100.
Max Temperature: 275 0 c
[Link] column:
Make:Varian
Column type:WCOT fusid silica.
Stationary phase:CP-Sil-88.
Column length:50 mts.
Inside diameter:0.25mm.
Outside Diameter:0.39mm
Thickness:0.20mm.
Max Temperature:250 0c
It is used in separation of cis-trans fatty acids.

Solid support:
The main purpose of the solid phase is to provide support to the thin
uniform film of liquid phase. The ideal inert supporting solid of moderate porosity with
large porce,uniform particle size chemically inert with no catalytic or adsorption sites at
elevated temperature and structurally stable. The solid support should be readily wetted
by the liquid phase to give a uniform coating.
Diatomaceous earth, being a form of hydrated silica
containing many free hydroxyl groups on its surface. These can serve as sites at which
solute molecules can be absorbed. this effect can be reduced by treatment of the solid
with a sleazing agent such as hex methyl disilazane(HMDS)which converts the si-o-H
groups to si-o-si(CH3).
Detectors:
The Function of the detector is to detect and quantify the different components
of the sample as they emerge from the column. The choice of the detector depends on the
type of analysis being performed .
The most widely used detectors are
1. Flame ionization detector (FID).
2. Thermal conductivity detector (TCD).
3. Electron capture detector (ECD)
4. Flame potentiometer detector(FPD).
Flame ionization detectors are being used for both systems of JOCIL.
Flame Ionization Detector:
 Most organic compounds are readily pyrolysed when introduced into a hydrogen
oxygen flame. as a result of pyrolysis ions are produced .
 These ions provide a mechanism by which current can be carried through the the
flame.
 These ions can be collected at a changed electrode and the resulting current
measured by an electrometer amplifier.
 In FID the effluent from the gas chromatographic column is fed into an air
hydrogen flame.
 Two wire electrodes are also placed in the flame and an electric potential is
applied across them.
 When only the inert carrier gas enters the flame, the current across the electrodes
is small and constant.
  When the vapor of organic compounds from the end of the chromatographic
column enters the flame, the compound is broken up into fragments that are
highly conducting.
 These are easily detected by the change in the current flowing across the
electrodes.
 This detector is capable of detecting 10 to the power of -12gm of organic
materials.
 The ionization of carbon compounds in a flame is roughly proportional to the no.
of reduced carbon atoms in the flame.
 The FID has now become one of the most popular detectors since it is highly
sensitive and has a wide range of linear response.

Thermal Conductivity Detector:

 Thermal conductivity detectors are based upon change in thermal conductivity
of the gas stream .
 The thermal conductivity of gases are different, a change in composition of
gas causes the thermal conductivity to change.
Electron Capture Detector:
 The electron capture or electron affinity detector operates on the basis of
differential electron absorption by molecules or functional groups with the
molecule.
 The detector is composed of a radio active source which emits electrons.
 Chlorine has a high efficiency for the capture of electrons, the detector is
particularly sensitive to halogen compounds.
 The detector has a valuable applications in monitoring pesticides in the
environment.
 Flame Potentiometer Detector:
 The flame that is used in the FPD is similar to that of a flame ionization
detector and is operated hydrogen rich .
 This detector is highly selective for sulfur and phosphorous containing
molecules.
Carrier Gas :
 The main function of carrier gas is to carry the vaporized sample on the column
which contains the stationary phase material.
 The carrier gas is normally considered as it does not react chemically with the
sample or stationary phase.
 The flow rate carrier gas is carefully controlled by means of pressure regulators
and flow meters to keep the flow rate constant during analysis.
Sample Inlet Systems:
The sample inlet system called the injection port, is generally heated to
high temperature to vaporize quickly the sample to be analyzed. The most general
methods employed for liquids or solids is the syringe technique. Solids are dissolved in
suitable solvent and liquids are injected as such are diluted with a suitable solvent for
gases are gastight syringe of 0.5-10ml capacity or sample loop may be used.
 The carrier gas is conducted from the gas reservoir sample port injector.
since the samples to be chromatographic must be in the vapor state.
The injection port is heated to a temperature which will insure a rapid vaporization but
not thermal degradation of the solute. The porting designed for instantaneous injection
and vaporization of the sample so that the sample is injected immediately into the
column.
Samples of 0.5microleters of a 5 solution of fat in heptanes,hexane,diethyl ether
provide the optimum load(16)chloroform tends to strip the stationary phase from the
packing(17).As capillary columns posses a much load sample capacity compared to a
packed columns. The sample introduction is some what complicated. The sample load of
a capillary column is in the upper range, where as that of packed columns is about 200
ford higher. The sample up to 5 microlitre is injected directly onto the column,. thus
avoiding disturbance by the septum and the difference in size between injection port and
column inlet. however routine analysis needs automatic injection.

ANALYSIS-I
COCONUT SAMPLE FOR GLC:
PRINCIPLE:
Fats and oils are first saponified by treating the sample with methanolic
sodium hydroxide. the soaps are subsequently converted to the methyl esters of the fatty
acids by reaction with a methanolic boron triflouride.
Reagents:
[Link] sodium hydroxide-approximately 0.5 N
Dissolve 2gmsodium hydroxide in 100ml methanol containing not more than
0.5% w/w of water. store the solution in a closed vessel.
Eg:-A polythene bottle with screw cap in prolonged storage. a precipitate of
sodium carbonate may form; this doesn’t influence the methyl ester preparation.
2.10% methanolic boron triflouride solution.
[Link]-HPLC grade .
[Link] sulphate-anhydrous(LR grade)
[Link] chloride solution-saturated aqueous solution(LR grade)
Apparatus :
[Link] bottom flask -50ml with ground joint 15/23 or 19/26.
[Link] condenser-high efficiency helicalpasteure
[Link] condenser –length 20-30cms with ground joint fit flask
[Link] chips
[Link] capillary pipettes
[Link] pipette-10ml or higher volume
[Link] filter
[Link] stopper tubes.
Procedure::
Saponification of samples containing oils
 Heat the dry sample to just above its melting point if the fat is not entirely liquid .
 Transfer approximately 150mg of the sample to a round bottom flaslk with a
Pasteur capillary pipette. Add 4ml of methanolic sodium hydroxide solution and a
few boiling chips .
 Connect the round bottom flask to the reflex condenser and boil the mixture under
total reflux until all the fat globule have dissolved .i.e saponification is complete.
minimum boiling time is 5 minutes.
Conversion to Methyl Ester:
 Add 5 ml of methanolic Boron triflouride solution to the boiling mixture via the
condenser using the graduated pipette with a pipette filter. boil the mixture for 10
minutes and add about 5ml heptanes to the boiling mixture via the condenser and
boil for another 5 minutes.
 Remove the flask from heat source, remove the reflux condenser add some
saturated sodium chloride solution and stir the flask gently several times. add
more of the saturated sodium chloride solution to bring the liquid level of the
mixture into the neck of the round bottom flask.
 Transfer using a Pasteur pipette about 1ml of the upper (heptanes) layer into a
glass stopper test tube. add anhydrous sodium sulfate to the test to bind traces of
water. the solution of fatty acid methyl ester is now suitable for injection on a gas
chromatographic column.
 Store the sample if necessary under an inert gas in a refrigerator.
Fatty Acid Composition by GLC:
The following procedure is intended to give general guidance for the
application of gas liquid chromatography to the determination of the qualitative and
quantitative composition of a mixture of fatty acid methyl esters. the instructions given
relate to the ordinary equipment used for gas liquid chromatography, employing in a
packed column and a flame ionization detector.
Reagents:

 Carrier gas :inert gas(nitrogen ,helium)thoroughly dried.

 Auxillary gases : 1. hydrogen gas(not less than 99.9%pure)free from organic
impurities

[Link](or)oxygen

Reference standard: a mixture of methyl esters (or) the methyl ester of an oil of known
composition preferably similar to that of the fatty matter to be analyzed
Equipment:
Gas liquid chromatogram:
 Injection system should have the least dead space possible .preferably it should be
capable being heated to a high temperature than that of the column.
 Oven should be capable of heating the column to at least 220 and of maintaining the
desired temperature to with in 1
 If programmed heating is to be employed an apparatus with thin column is
recommended.

Test conditions:

Analysis parameters:
Injector temperature:250 0 c
Detector temperature:275 0 c
Gas flows :
Nitrogen 30ml/minute
Hydrogen:30ml/minute
Oxygen:300ml/minute
Temperature program:
Oven1: 150 0 c
Time1:2minutes
Rate1:5 0.c
Oven2:190 0 c
Time2:0 minutes.
Rate2:6 0 c/minute.
Oven3:225 0 c
Time3:5 minutes.
Report:
Inject the unknown sample under the certain conditions and for identification
inject reference standard also under the same conditions to identify the peaks from
retention times of the reference [Link] area percentage by the integrator is
considered as the relative concentration of the component in mixture.
Sample ID: Fatty acid methyl esters(CN oil)
C8(caprylic):8.733
C10(capric):5.449.
C12(Lauric):45.416.
C14(myristic):18.054
C16(Palmtic):9.069
C18(Stearic):3.465
C18:1(Oleic):7.607
C18:2(linoleic):1.768
C18:3(linolenic):0.058
 ANALYSIS-II
Sample ID:pkDFA sample
Procedure:
Introduce the PKDFA sample 0.15gm into the 50ml reaction flask .Then add 10ml of5%
BF3 methanol reagent through the condenser reflux 5-10 minutes add 2-5ml of N-
heptane through condenser and boil for 5 minutes remove and cool to oom temperature
and add saturated NaCl solution to float the heptane solution of methyl esters in to the
neck of the flask .trasfer about one ml of heptane solution into the test tube and a small
amount of anhydrous sodium [Link] dry heptane solution may be inject directly in
agas chromatography.
Test conditions:
Analysis Parameters:
Injector temperature:250 0 c
Detector temperature:275 0 c
Gas flows :
Nitrogen 30ml/minute
Hydrogen:30ml/minute
Oxygen:300ml/minute
Temperature program:
Oven1: 150 0 c
Time1:2minutes
Rate1:5 0 c
Oven2:190 0 c
Time2:0 minutes.
Rate2:6 0 c/minute.
Oven3:225 0 c
Time3:5 minutes.
Report: Inject the unknown sample under the certain conditions and for identification
inject reference standard also under the same conditions to identify the peaks from
retention times of the reference [Link] area percentage by the integrator is
considered as the relative concentration of the component in mixture.

Sample ID: PKDFA sample

C8(caprylic):0.172
C10(capric):0.804
C12(Lauric):52.426
C14(myristic):15.702
C16(palmtic):13.508
C18(stearic):5.310
C18:1(oleic):10.863
C18:2(linoleic):1.059
C18:3(linolenic):0.156
 ANALYSIS-III
Sample ID : Noodles sample
Procedure :
Weigh out an amount of sample which will be sufficient to yield about 5g of
fatty acids .dissolve in hot distilled water ,cool add 20% sulphuric acid to split the soap
by using methyl orange as indicator .cool ,dissolve fatty acids in diethyl ether .filter the
ether solution in to separating funnel. wash the ether phase with distilled water until it is
free from mineral acid .collect the fatty acid ,it is taken into RB flask add 5ml of
methanolic BF3 reagent then reflux 5-10 minutes under water condenser add 2-5ml of N-
heptane through the condenser and boil for 5 minutes ,remove and cool to room
temperature and add saturated NaCl solution to float the heptane solution of methyl
ester in to the neck of the flask transfer about 1ml of heptane solution into the test tube
and a small amount of anhydrous sodium [Link] dry heptane solution may be inject
directly in a gas chromatography.
Test conditions:
Analysis parameter:
Injector temperature:250 0
Detector temperature:275 0 c
Gas flows :
Nitrogen 30ml/minute
Hydrogen:30ml/minute
Oxygen:300ml/minute
Temperature program:
Oven1 :150 0 c
Time1 : 2 minutes
Rate1 : 5 0c /minute)
Oven2 : 190 0 c
Time2 : 0 minutes
Rate2 : 6 0 c /minute
Oven3 : 225 0 c
Time3 : 5 minutes

Report:
Inject the unknown sample under the certain conditions and for identification inject
reference standard also under the same conditions to identify the peaks from retention
times of the reference standard. The area percentage by the integrator is considered as the
relative concentration of the component in mixture.
Sample ID: Noodles sample
C12(Lauric):15.665
C14(myristic):0.467
C16(palmtic):42.608
C18(stearic):5.019
C18:1(oleic):30.071
C18:2(linoleic):6.068
C18:3(linolenic):0.102
 History of chilli seeds

Chili peppers have been a part of the human diet in the Americas since at least 7500 BC.
There is archaeological evidence at sites located in southwestern Ecuador that chili
peppers were domesticated more than 6000 years ago,and is one of the first cultivated
crops in the Americas that is self-pollinating.

Chili peppers were domesticated at least in different parts of South and Middle America.

Christopher Columbus was one of the first Europeans to encounter them (in the
Caribbean), and called them "peppers" because of their similarity in taste (though not in
appearance) with the Old World Black peppers of the Piper genus.

Chilies were cultivated around the globe after Columbus. Diego Álvarez Chanca, a
physician on Columbus' second voyage to the West Indies in 1493, brought the first chili
peppers to Spain, and first wrote about their medicinal effects in 1494.

From Mexico, at the time the Spanish colony that controlled commerce with Asia, chili
peppers spread rapidly into the Philippines and then to India, China, Korea and Japan.
They were quickly incorporated into the local cuisines.

An alternate sequence for chili peppers' spread has the Portuguese getting the pepper
from Spain, and thence to India, as described by Lizzie Collingham in her book Curry.
Collingham states in her book that the chili pepper figures heavily in the cuisine of the
Goan region of India, which was the site of a Portuguese colony (e.g. Vindaloo, an Indian
interpretation of a Portuguese dish). Collingham also describes the journey of chili
peppers from India, through Central Asia and Turkey, to Hungary, where it became the
national spice in the form of paprika.
Intensity

The substances that give chili peppers their intensity when ingested or applied topically
are capsaicin (8-methyl-N-vanillyl-6-nonenamide) and several related chemicals,
collectively called capsaicinoids. Capsaicin is the primary ingredient in pepper spray.

When consumed, capsaicinoids bind with pain receptors in the mouth and throat that are
normally responsible for sensing heat. Once activated by the capsaicinoids, these
receptors send a message to the brain that the person has consumed something hot. The
brain responds to the burning sensation by raising the heart rate, increasing perspiration
and release of endorphins. A 2008 study reports that capsaicin alters how the body's cells
use energy produced by hydrolysis of ATP. In the normal hydrolysis the SERCA protein
uses this energy to move calcium ions into the sarcoplasmic reticulum. When capsaicin is
present, it alters the conformation of the SERCA and thus reduces the ion movement; as a
result the ATP energy (which would have been used to pump the ions) is instead released
as heat.

The "heat" of chili peppers is measured in Scoville heat units (SHU), which is the number
of times a chili extract must be diluted in water for it to lose its heat. Bell peppers rank at
0 SHU, New Mexico green chilis at about 1,500 SHU, jalapeños at 3,000–6,000 SHU,
and habaneros at 300,000 SHU. The record for the hottest chili pepper was assigned by
the Guinness Book of Records to the Naga Jolokia, measuring over 1,000,000 SHU. Pure
capsaicin, which is a hydrophobic, colorless, odorless, and crystalline-to-waxy solid at
room temperature, measures 16,000,000 SHU.

Nutritional value
Peppers, hot chili, red, raw
Nutritional value per 100 g (3.5 oz)
Energy 40 kcal 170 kJ

Carbohydrates 8.8 g
- Sugars 5.3 g
- Dietary fiber 1.5 g
Fat 0.4 g
Protein 1.9 g
Water 88 g
Vitamin A equiv. 48 μg 5%
- β-carotene 534 μg 5%
Vitamin B6 0.51 mg 39%
Vitamin C 144 mg 240%
 Iron 1 mg 8%
Magnesium 23 mg 6%
Potassium 322 mg 7%
Percentages are relative to US
recommendations for adults.
Source: USDA Nutrient database

Red chilis contain high amounts of vitamin C and carotene ("provitamin A"). Yellow and
especially green chilis (which are essentially unripe fruit) contain a considerably lower
amount of both substances. In addition, peppers are a good source of most B vitamins,
and vitamin B6 in particular. They are very high in potassium and high in magnesium and
iron. Their high vitamin C content can also substantially increase the uptake of non-heme
iron from other ingredients in a meal, such as beans and grains.

Psychologist Paul Rozin suggests that eating chilis is an example of a "constrained risk"
like riding a roller coaster, in which extreme sensations like pain and fear can be enjoyed
because individuals know that these sensations are not actually harmful. This method lets
people experience extreme feelings without any risk of bodily harm.

Chili peppers drying in Kathmandu, Nepal

Evolutionary advantages

Birds do not have the same sensitivity to capsaicin because it targets a specific pain
receptor in mammals. Chili peppers are eaten by birds living in the chili peppers' natural
range. The seeds of the peppers are distributed by the birds that drop the seeds while
eating the pods, and the seeds pass through the digestive tract unharmed. This
relationship may have promoted the evolution of the protective [Link] based
on this substance have been sold to treat the seeds in bird feeders to deter squirrels and
other mammalian vermin without also deterring birds. Capsaicin is also a defense
mechanism against microbial fungi that invades through punctures made in the outer skin
by various insects.
Medicinal use

Capsaicin is a safe and effective analgesic agent in the management of arthritis pain,
herpes zoster-related pain, diabetic neuropathy, postmastectomy pain, and headaches.

Possible health benefits

All hot chili peppers contain phytochemicals known collectively as capsaicinoids.

 Capsaicin was shown, in laboratory settings, to cause cancer cell death in rats.
 Capsaicin in chilies has been found to inhibit chemically induced carcinogenesis
and mutagenesis in various animal models and cell culture systems.
 Recent research in mice shows that chili (capsaicin in particular) may offer some
hope of weight loss for people suffering from obesity.
 Researchers used capsaicin from chillies to kill nerve cells in the pancreases of
mice with Type 1 diabetes, thus allowing the insulin producing cells to start
producing insulin again.
 Research in humans found that "after adding chili to the diet, the LDL, or bad
cholesterol, actually resisted oxidation for a longer period of time, (delaying) the
development of a major risk for cardiovascular disease".
 Researchers found that the amount of insulin required to lower blood sugar after a
meal is reduced if the meal contains chili pepper.
 Chilli peppers are being probed as a treatment for alleviating chronic pain.
 Spices, including chilli, are theorized to control the microbial contamination
levels of food in countries with minimal or no refrigeration.
 Hot peppers are claimed to provide symptomatic relief from rhinitis, but a review
study found no effect.
 Several studies found that capsaicin could have an anti-ulcer protective effect on
stomachs infected with H. pylori by affecting the chemicals the stomach secretes
in response to infection.
 By combining an anesthetic with capsaicin, researchers can block pain in rat paws
without causing temporary paralysis. This anesthetic may one day allow patients
to be conscious during surgery and may also lead to the development of more
effective chronic pain treatments.

Possible health risks and precautions

 A high consumption of chili is associated with stomach cancer.
 Chili powders may sometimes be adulterated with Sudan I, II, III, IV, para-Red,
and other illegal carcinogenic dyes.
 Aflatoxins and N-nitroso compounds, which are carcinogenic, are frequently
found in chili powder.
 Chronic ingestion of chili products may induce gastroesophageal reflux (GER).
 Chili may increase the number of daily bowel movements and lower pain
thresholds for people with irritable bowel syndrome.
 Chilis should never be swallowed whole; there are cases where unchewed chilis
have caused bowel obstruction and perforation.
  Consumption of red chilis after anal fissure surgery should be forbidden to avoid
postoperative symptoms.

ANALYSIS-IV
Sample ID:Chilli oil sample
Procedure:
Chilli oil is extracted by taking 33.3gm of crushed chilli seeds and acts as
petroleum spirit as solvent.

And got 13.843% of chilli oil

The acid value of chilli oil is 2.6
The iodine value of chilli oil is 133
Saponification of samples containing oils
 Heat the dry sample to just above its melting point if the fat is not entirely
liquid .
 Transfer approximately 150mg of the sample to a round bottom flaslk with a
Pasteur capillary pipette. Add 4ml of methanolic sodium hydroxide solution and a
few boiling chips .
 Connect the round bottom flask to the reflex condenser and boil the mixture under
total reflux until all the fat globule have dissolved .i.e saponification is complete.
minimum boiling time is 5 minutes.

Conversion to Methyl Ester:
 Add 5 ml of methanolic Boron triflouride solution to the boiling mixture via the
condenser using the graduated pipette with a pipette filter. boil the mixture for 10
minutes and add about 5ml heptanes to the boiling mixture via the condenser and
boil for another 5 minutes.
 Remove the flask from heat source, remove the reflux condenser add some
saturated sodium chloride solution and stir the flask gently several times. add
more of the saturated sodium chloride solution to bring the liquid level of the
mixture into the neck of the round bottom flask.
 Transfer using a Pasteur pipette about 1ml of the upper (heptanes) layer into a
glass stopper test tube. add anhydrous sodium sulfate to the test to bind traces of
water. the solution of fatty acid methyl ester is now suitable for injection on a gas
chromatographic column.
 Store the sample if necessary under an inert gas in a refrigerator.
Fatty Acid Composition by GLC:
The following procedure is intended to give general guidance for the
application of gas liquid chromatography to the determination of the qualitative and
quantitative composition of a mixture of fatty acid methyl esters. the instructions given
relate to the ordinary equipment used for gas liquid chromatography, employing in a
packed column and a flame ionization detector.

Test conditions:
Analysis parameter:
Injector temperature:250 0 c
Detector temperature:275 0 c
Gas flows :
Nitrogen 30ml/minute
Hydrogen:30ml/minute
Oxygen:300ml/minute
Temperature program:
Oven1 :150 0 c
Time1 : 2 minutes
Rate1 : 5 0 c/minute
Oven2 : 190 0 c
Time2 : 0 minutes
Rate2 : 6 0 c/minute
Oven3 : 225 0 c
Time3 : 5 minutes

Report:
Inject the unknown sample under the certain conditions and for identification inject
reference standard also under the same conditions to identify the peaks from retention
times of the reference [Link] area percentage by the integrator is considered as the
relative concentration of the component in mixture.
Sample ID: chilli oil sample
C12(Lauric):0.046
C14(myristic):0.121
C16(palmtic):14.331
C18(stearic):3.404
C18:1(oleic):8.168
C18:2(linoleic):72.951
C18:3(linolenic):0.454
C20(arachidic):0.332