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(EXPERIMENT 3)

FATTY ACID DETERMINATION USING GAS


CHROMATOGRAPHY (GC)
NAME:

MOHAMAD NOR AMIRUL AZHAR BIN KAMIS

STUDENT ID:

2014647344

PARTNERS NAMES: 1.

MOHAMAD HAMIZAN BIN MOHD ISA

2.

MOHAMAD SHAFIQ BIN PARMAN

3.

MOHAMAD AZMIZAM BIN MOHAMAD


NOOR

DATE OF EXPERIMENT:

30/10/2014

DATE OF SUBMISSION:

8/12/2014

INTRODUCTION:
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.
The amount of FAME is determined by using the response factor calculation.
Response Factor (RF) =
Amount of unknown compound = peak area of unknown compound RF (std)

EXPERIMENTAL:
a. Preparation of fatty acid methyl ester samples from fat samples:
1. 3 fat samples were prepared by weighing the mass of 2.0528g, 2.0894g
and 2.0327g respectively.
2. The samples were transferred into a 50mL flask equipped with air
condenser.
3. 5mL of 0.5M methanolic solution was added into the flask and was reflux
for about 3 to 4 minutes until the mixture turned to golden brown colour.
4. 15mL of esterification reagent was added and continue refluxing for
another 3 minutes.
5. The mixture then was 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 too.
7. The organic layer was transferred into a screw cap vial for easies the
analysis to be done with automated injector.
8. Note that, the preparation is the same for all 3 samples.

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.

RESULT AND DISCUSSION:


A. Response factor (RF) for analytes in standard FAME:
Amount of
Peak area (pA*s)
FAME in
standard (ppm)
Peak 2
100
65.90409
Peak 3
100
435.19760
Peak 4
100
1244.67700
Peak 5
100
125.80314
Peak 6
100
116.25892
B. Comparison of retention time for standard and samples:
Retention
Retention
Retention
time for
time for
time for
standard
sample 1
sample 2
(min)
(min)
(min)
Peak 2
1.269
1.275
1.276
Peak 3
1.671
1.684
1.684
Peak 4
2.366
2.390
2.390
Peak 5
3.537
3.613
3.605
Peak 6
3.739
7.822
-

Response
Factor (RF)
1.5174
0.2298
0.0803
0.7949
0.8601

Retention
time for
sample 3
(min)
1.278
1.686
2.391
3.620
-

C. Amount of FAME in samples:

Sample 1

Sample 2

Sample 3

Peak 2
Peak 3
Peak 4
Peak 5
Peak 6
Peak 2
Peak 3
Peak 4
Peak 5
Peak 6
Peak 2
Peak 3
Peak 4
Peak 5
Peak 6

RF of
corresponding
peak
1.5174
0.2298
0.0803
0.7949
0.8601
1.5174
0.2298
0.0803
0.7949
0.8601
1.5174
0.2298
0.0803
0.7949
0.8601

Peak area
(pA*s)

Amount of
FAME (ppm)

62.79572
34.40598
888.78308
975.41412
81.3408
49.48989
1123.19543
65.98615
86.24370
36.68810
1133.67981
1315.67725
-

95.29
7.91
71.37
775.36
123.43
11.37
90.19
52.45
130.87
8.43
91.03
1045.83
-

D. Sample calculation:
Response factor of peak 2 in standard =
= 1.5174
Amount of FAME in peak 2 (sample 1) = 1.5174 62.79572
= 95.29ppm
Amount of FAME in peak 2 (sample 2) = 1.5174 81.3408
= 123.43ppm
Amount of FAME in peak 2 (sample 3) = 1.5174 86.24370
= 130.87ppm

The components in the samples are compared with the standard components by the
retention time. From the retention time of standard and samples, it is proven that
component 5 (peak 6) is not present in all 3 samples because of the difference of the
retention time between standard and samples are too big. The amount of each
component is different in each samples may due to the different mass of the fat
initially. Peak 5 in each sample give very large different in the amount of FAME, this
may be due to the un-complete separation process during shaking process or the
discarding process. It is necessary to discard little organic layer to make sure that
there is no aqueous layer anymore to be injected onto GC. That condition also
affected by the contaminants in the flask that is not clean before using. The other
peaks that correspond to specific component show small difference that assumed to
be correct.
There is another way to derivatize or modified the low volatility fatty acid to more
volatile compound called silylation that BSTFA to yield trimethylsilyl (TMS) ester that
is more suitable to be analysed in GC.

CONCLUSION:
The derivatization technique used in this experiment is esterification to convert nonvolatile fatty acids to more volatile fatty acid methyl ester (FAME). There are 5
components in the standard mixture while the 3 samples only indicate 4 components
as shown in the standard mixture by comparison of the retention time. The
concentration of each component is calculated by using the response factor of the
standard.

REFERENCES:
1. Norashikin S., Ruziyati T., Mardiana S. (2012), Analytical Separation
Methods Laboratory Guide (2nd edition), 3/10/2014.
2. Mardiana Saaid, Sample Preparation Lecture Notes, 15/10/2014.