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CHM 510

ANALYTICAL SEPARATION METHODS


EXPERIMENT 3
FATTY ACID DETERMINATION USING GAS
CHROMATOGRAPHY

NAME: NABILAH BINTI ABD RAHMAN


STUDENT ID: 2015484718
LAB PARTNERS: 1. ANIZA BINTI ABDULLAH (2015827038)
2. NIK NURFARAHAIN RIFHAN BINTI NIK
AZMAN
(2015896328)
GROUP: AS2453D1
LECTERURS NAME: DR. MARDIANA BINTI SAAID
DATE PERFORMED: 6TH APRIL 2016

DATE OF SUBMISSION: 27TH MAY 2016


TITLE
Experiment

Fatty

Acid

Determination

Using

Gas

Chromatography
OBJECTIVE
This experiment introduces a procedure that is used routinely for fat
analysis in which nonvolatile fatty acids are chemically converted to
the corresponding volatile methyl esters. The resulting volatile
mixture can be analyzed by gas chromatography.

INTRODUCTION
Fats consist of glycerol esters and long chain aliphatic acids
(fatty acids). The backbone of these compounds contains from 4 to
more than 20 carbon atoms. Most natural sources of these
compounds have an even number of carbon atoms because the
biosynthetic pathway builds the backbone two carbons at a time.
Fatty acid chains may contain one or more double bonds at specific
positions (unsaturated and polyunsaturated), or they may be fully
saturated. The physical and chemical properties of a fat depend on
the composition of the fatty acid mixture. Animal fats tend to have a
larger proportion of long chain-saturated acids and are solids at
room temperature. Fats from plant sources contain a higher
proportion of unsaturated acids and are often liquids at room
temperature due to hydrogen bonding. Polyunsaturated fats are
usually of vegetable origin. Crisco is an example of a vegetablederived, unsaturated fatty acid that has been hydrogenated to form
a solid material. Fats are used in cooking because they are very high
boiling compounds. Their high boiling points therefore make this
class of compounds ill suited for analysis by gas chromatography.

However, the glycerol esters can be chemically decomposed into


methyl esters of each individual fatty acid.
Gas chromatography separates the analytes that is volatile
and chemically stable. Fatty acids are not sufficiently volatile for GC
analysis, so that it needs to be modified chemically to produce a
new compound, which has properties that are suitable for analysis.
If the unsuitable sample is introduced into GC analysis, it tends to
cause peak tailing due to the adsorption and non-specific interaction
with the column. In this experiment, the fatty acid is changed to
fatty acid methyl ester (FAME) that is more volatile, suitable for GC
analysis by using esterification reaction that used metholic solution
with catalyst of esterification reagent. The objective for this
experiment is to introduce a derivatization procedure routinely used
for fat analysis in which non-volatile fatty acids are chemically
converted to the corresponding volatile methyl ester (FAME) and to
determine the amount of FAME in the derivatized samples.

EXPERIMENTAL PROCEDURE
a.

Preparation of fatty acid methyl ester samples from fat

samples
1.

3 samples of approximately 2 g of fat (butter) were weighed


out and the exact weight were recorded.

2.

The samples were then transferred into a 50mL flask equipped


with air condenser.

3.

5mL of 0.5 M methanolic solution was added into the flask and
was refluxed for about 3 to 4 minutes until the mixture turned
to golden brown colour.

4.

15mL of esterification reagent was added and continued to


reflux again for another 3 minutes.

5.

The mixture was then transferred into a separatory flask and


50mL of saturated sodium chloride (NaCl) and 25mL of diethyl
ether were added into the flask. The mixture was shaken
vigorously and vented to release the pressure formed for
about 2 minutes. The aqueous layer was discarded.

6.

Step 5 was repeated with another 25mL of saturated NaCl and


the aqueous layer was discarded again.

7.

The organic layer was transferred into a screw cap vial and be
made sure that only the organic layer was transferred.

b.

Instrument set up:


Injector port: split (40:1)
Injection port temperature: 250C
Column temperature: 100C to 290C at 40C/min
Carrier gas flow rate: 30mL/s
Detector temperature: 250C

c.

Quantitative analysis of FAME:

1.

Each of the derivatized samples was injected into GC column


by using automated injector.

2.

FAME standard mixture was injected into the GC column.

3.

The amount of fatty acid in each sample was calculated.

EXPERIMENTAL RESULTS, DATA AND CALCULATIONS


A.

Response Factor (RF) for Analytes in Standard FAME

Peaks

Retention
Time of
Peaks
(min)

Base Peak
Width of
Peaks
(min)

Area of
Peaks
(pA*s)

Peak 2
Peak 3

2.084
2.765

0.0160
0.0217

107.40754
117.88110

Resolutio
n of 2
peaks
(peak 3 &
peak 4)

Response
Factor (Rf)
of Peaks
0.93103
0.8483

Peak 4
Peak 5
Peak 6

3.921
5.899
6.865

0.0299
0.0496
0.0432

1022.17914
698.57605
276.47214

45

0.09783
0.1431
0.3617

Amount of Standard Sample (ppm)


= 100 ppm

Calculation of resolution of 2 peaks:


Rs(2,3) = 2( Rt,3- Rt,2 )
(Wb,2 + Wb,3)
Rs(2,3) = 2( 3.921 2.765 )
(0.0299 + 0.0217)

= 45 #

Response Factor (Rf) of each peak (FAME)


1.

Peak 2
Response Factor (RF) = amount
peak area
= 100 (ppm)
107.40754

2.

= 0.93103 #

Peak 3
Response Factor (RF) = amount
peak area
= 100 (ppm)
117.88110

3.

Peak 4

= 0.8483 #

Response Factor (RF) = amount


peak area
= 100 (ppm)
1022.17914
4.

= 0.09783 #

Peak 5
Response Factor (RF) = 100 (ppm)
698.57605

1.

= 0.1431 #

Peak 6
Response Factor (RF) = amount
peak area
= 100 (ppm)
276.47214

B.

Peak 2

Peak 3

Peak 4

= 0.3617 #

Comparison of Retention Time in Standard and Samples


Retention

Retention

Retention

Retention

Time, Rt for

Time, Rt for

Time, Rt for

Time, Rt for

Standard

Sample 1

Sample 2

Sample 3

(min)
2.084

(min)
= 2.105 +

(min)
= 2.106 +

(min)
= 2.106 +

2.106

2.106

2.106

= 2.106
= 2.811 +

= 2.106
= 2.814 +

= 2.106
= 2.812 +

2.812

2.812

2.813

= 2.812
= 4.004 +

= 2.813
= 4.003 +

= 2.813
= 4.006 +

4.004

4.004

4.005

2.765

3.921

Peak 5

Peak 6

C.

5.899

6.865

= 4.004
= 6.360 +

= 4.004
= 6.363 +

= 4.006
= 6.371 +

6.369

6.365

6.365

= 6.365
= 7.005 +

= 6.364

= 6.368
= 7.023 =

7.004

7.015

= 7.005

= 7.019

Resolution of Each Sample of the FAME


Retention Time

Base Width of

Resolution of

(Rt) of Peak 3 &

Peak 3 & 4

Peak 3 & 4

Sample 1

5 (min)
2.812, 4.004

(min)
0.0494,

18

Sample 2
Sample 3

2.813, 4.004
2.813. 4.006

0.08495
0.0493, 0.0857
0.0493, 0.0838

18
18

Calculation of resolution of 2 peaks:


1.

Sample 1
Rs(2,3) = 2( Rt,3- Rt,2 )
(Wb,2 + Wb,3)
Rs(2,3) = 2( 4.004 2.812 )
(0.0494 + 0.08495)
= 18 #

2.

Sample 2
Rs(2,3) = 2( Rt,3- Rt,2 )
(Wb,2 + Wb,3)
Rs(2,3) = 2( 4.004 2.813)
(0.0493 + 0.0857)
= 18 #

3.

Sample 3
Rs(2,3) = 2( Rt,3- Rt,2 )
(Wb,2 + Wb,3)
Rs(2,3) = 2( 4.006 2.813 )
(0.0493 + 0.0838)
= 18 #

D.

Amount of FAME individuals in Samples


Response
Factor, Rf of

Peak Area
(pA*s)

Correspond

Sample
1

Sample
2

Sample
3

Amount of
FAME (Fatty
Acid),

Peak
Peak
Peak
Peak
Peak

2
3
4
5
6

ing peak
0.93103
0.8483
0.09783
0.1431
0.3617

35.89295
16.54252
234.95041
18.673855
6.16273

(ppm)
33.417
14.033
22.958
2.6729
2.2291

Peak
Peak
Peak
Peak
Peak

2
3
4
5
6

0.93103
0.8483
0.09783
0.1431
-

11.16741
3.72386
44.85535
33.72593
-

10.397
3.159
4.388
4.8261
-

Peak
Peak
Peak
Peak
Peak

2
3
4
5
6

0.93103
0.8483
0.09783
0.1431
0.3617

14.295845
5.26132
68.67184
44.42255
10.71028

13.310
4.4632
6.718
6.357
3.874

Calculation of Amount of Sample (ppm)

= Response Factor of Standard X Area (pA*s)

DISCUSSION
Fatty acid is an important component of lipids (fat-soluble
components of living cells) in plants, animals, and microorganisms.
Generally, a fatty acid consists of a straight chain of an even
number of carbon atoms, with hydrogen atoms along the length of
the chain and at one end of the chain and a carboxyl group
(COOH) at the other end. It is that carboxyl group that makes it an
acid (carboxylic acid). If the carbon-to-carbon bonds are all single,
the acid is saturated; if any of the bonds is double or triple, the acid
is unsaturated and is more reactive.
GC can be used to analyze fatty acids either as free fatty acids
or as fatty acid methyl esters. The primary reasons to analyze fatty
acids as fatty acid methyl esters include; in their free, underivatized
form, fatty acids may be difficult to analyze because these highly
polar compounds tend to form hydrogen bonds, leading to
adsorption issues. Reducing their polarity may make them more
amenable for analysis and to distinguish between the very slight
differences exhibited by unsaturated fatty acids, the polar carboxyl
functional groups must first be neutralized. This then allows column
chemistry to perform separations by boiling point elution, and also
by degree of unsaturation, position of unsaturation, and even the cis
vs. trans configuration of unsaturation.
The esterification of fatty acids to fatty acid methyl esters is
performed using an alkylation derivatization reagent. Methyl esters

offer excellent stability, and provide quick and quantitative samples


for

GC

analysis.

The

esterification

reaction

involves

the

condensation of the carboxyl group of an acid and the hydroxyl


group of an alcohol. Esterification is best done in the presence of a
catalyst (such as boron trichloride). The catalyst protonates an
oxygen atom of the carboxyl group, making the acid much more
reactive. An alcohol then combines with the protonated acid to yield
an ester with the loss of water. The catalyst is removed with the
water. The alcohol that is used determines the alkyl chain length of
the resulting esters (the use of methanol will result in the formation
of methyl esters whereas the use of ethanol will result in ethyl
esters).
In this experiment, a gas chromatography technique is used to
determine the amount of fatty acid present in the butter or
margarine. Butter commonly contains a lot of fatty acid types, and
fatty acids are derivatized from the butter using the techniques
mentioned in the experimental procedure. Generlly, 5 types of fatty
acids are contained and that is methyl laurate, methyl myristate,
methyl palmitate, methyl stearate and methyl linoleate and they are
commonly known as Fatty Acid Methyl Esters (FAME). Fatty Acid
Methyl Esters (FAME) are esters of fatty acids. The physical
characteristics of fatty acid esters are closer to those of fossil diesel
fuels than pure vegetable oils, but properties depend on the type of
vegetable oil. A mixture of different fatty acid methyl esters is
commonly referred to as biodiesel, which is a renewable alternative
fuel. FAME has physical properties similar to those of conventional
diesel. It is also non-toxic and biodegradable.
Since methyl laurate is least retained by the stationary phase,
it eluted out first, at the 2.0th minute followed by methyl myristate at
the 2.8th minute, methyl palmitate at 4.0th minute, methyl stearate
at 6th minute and methyl linoleate at 7 th minute. By comparing the
retention time of standard and the 3 samples, it can be told that the

retention time are almost the same, which means that the 5
compounds of samples elute out at almost the same time as the
standard. Methyl laurate is represented by peak 2, and at the
standard it eluted out at 2.084th minute and the samples it eluted
out at 2.1 minute. Peak 3 is methyl myristate, and in the standard
the compound elute out at 2.765th minute and the samples at 2.8th
minute. The 4th peak is methyl palmitate, and in the standard it
elute out at 3.921 minute and the in the samples methyl palmitate
elute out at the 4th minute. Methyl stearate is represented by peak 5
and in the standard it elute out at the 5.899 th minute but in the
samples peak 5 elute out at 6.3 rd minute, which differs slightly. And
lastly at peak 6, is methyl linoleate and it elute out at 6.865 th minute
and in the sample it elute out at 7th minute which also differs
slightly.
By the response factor calculated in the standard, we can tell
the amount of each methyl esters in the sample. And the amount of
fatty acids in each sample are calculated and stated in the results
and data section.
CONCLUSION
The derivatization technique used in this experiment is esterification
to convert non-volatile fatty acids to more volatile fatty acid methyl
ester (FAME). There are 5 components in the standard mixture that
is the methyl esters. The concentration of each component is
calculated by using the response factor of the standard.
REFERENCES
1.

http://global.britannica.com/science/fatty-acid

2.

http://www.sigmaaldrich.com/analyticalchromatography/analytical-products.html?
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