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Experiment 3

Fatty Acid Determination Using Gas Chromatography (GC)


Name: Maxvicklye Rayner Abid
Matrix number: 2014586975
Group members:
1. Norfatin Zawan Binti Isa
2. Nur Izzati Binti Sumail
3. Muhammad Asyraf Bin Isyak
4. Suhaili Binti Sairi

Group: AS2453D1
Lecturer name: Dr.Rozita Binti Osman
Date of experiment: 07/10/2015
Date Submit: 17/12/2015

Introduction:

Gas chromatography (GC) separate usually volatile and chemically stable compound.
Fatty acid are not volatile enough to be inject on GC, therefore it was chemically
modified the compound to become more volatile and chemically stable compound. In this
experiment, fatty acid was changed into fatty acid methyl ester (FAME) that are more
volatile than fatty acid by undergoes esterification with metholic solution with catalyst
esterification reagent. Fatty acid methyl esters (FAME) are a type of fatty acid ester that
is derived by esterification of fats with methanol. The molecules in biodiesel are
primarily fame, usually obtained from vegetable oils by esterification.
Esterification is the name given to a reaction in which a Carboxylic acid combines
with an alcohol in the presence of concentrated sulphuric acid to form an ester. For
example: Ethanoic acid reacts with carbonates and hydrogen carbonates to give rise to a
salt, carbon dioxide and water
The aim of the experiment are to introduced derivation of non-volatile fatty acid that
normally used in fat analysis in GC converted into more volatile and chemically stable
fatty acid methyl ester(FAME) and determine the amount of fatty acid methyl
ester(FAME) in derivative sample.
The amount of FAME determine by response factor (RF) formula.
Respone factor (RF) =

amount of sample(std)
peak area

Amount of unknown = peak area of unknown x RF(std)

Experimental:

A. Preparation of fatty acid methyl ester sample from fat samples


1. The fat sample has been weighed 2,0529g, 2.0065g, 2.0231g respectively.
2. Sample has been transferred into 50 mL flask equipped with condenser.
3. 5 mL of 0.5 M methanolic solution was added into flask and reflux for 34minutes.
4. 15mL of esterification reagent was added into flask and continue reflux for
3minute.
5. The mixture was transferred into separating funnel with 50 mL of saturated NaCl
and 25mL diethyl ether. It was shaking vigorously for 2 minutes and the aqueous
layer has been discarding.
6. Step 2-5 was repeated for other potion of fat sample.
7. The organic layer has been transferred into screw cap vial.

B. Instrument set-up
Injection port

: Split (40:1)

Injection port temperature

: 250c

Column temperature

: 100C to 290C at 40C min-1

Carrier gas flow rate

: 30mLs-1

Detector temperature

: 250C

C. Quantitative analysis of FAME.


1. Each of derivative fat samples was injected onto GC column.
2. The injection was repeated until get reproducible peak area.
3. By using data obtained from standard esters, amount of fatty acid in sample has
been calculated.

RESULTS AND DISCUSSION


A. Peak area for analytes in standard FAME.

Amount of FAME

Peak area (pA*s)

Response factor

Peak 2

in standard(ppm)
100

360.38354

0.2775

Peak 3
Peak 4
Peak 5
Peak 6

100
100
100
100

376.79562
3612.25244
615.52899
3081.19360

0.2654
0.0277
0.1625
0.0325

B. Comparison of retention time for standard and sample.


Retention time

Retention time

Retention time

Retention time

for standard

for sample 1

for sample

for sample 3

Peak 2

(min)
1.046

(min)
0.842

2(min)
1.046

(min)
1.046

Peak 3

1.314

1.046

1.313

1.311

Peak 4

2.032

1.131

2.026

2.024

Peak 5

3.396

2.031

3.425

3.432

Peak 6

3.578

3.427

C. Amount of FAME in sample.

Sample 1

Sample 2

Sample 3

Rf corresponding

Peak area

Amount of

peak
0.2775
0.2654
0.0277
0.1625
0.0325
0.2775
0.2654
0.0277
0.1625
0.0325
0.2775
0.2654
0.0277
0.1625
0.0325

(pA*s)
31.52422
481.01089
205.28319
3009.74390
3114.29810
281.60815
120.72981
1827.53076
1930.30139
23.74359
9.48938
17.95598
175.03383
-

FAME
8.7479710
127.6602
5.686344
489.08338
101.214
78.14626
32.04169
50.62260
313.6739
6.588846
2.518481
0.497380
28.44299

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

SAMPLE CALCULATION.

Response factor of peak 2 in standard= 100/360.38534


= 0.2775
Amount of FAME in peak 2 (sample 1) = 0.2775 x 31.54222
= 8.74797

DISCUSSION
The component in the sample compared with the standard components by the
retention time. From the retention time of standard and sample. It is proven that
components 5, peaks is not present in all 3 sample because of the differences of the
retention time between standard and sample are too big. Peak 5 in each sample give very
large different in the amount of FAME, this may be due to un-complete separation
process during shaking process or the discarding process. From table above presents the
experimental response factor values obtain from FAME standard mixture and the other 3
sample. All of sample, we run two times to choose either one is the best analysis from this
experiment .The retention time for first samples presence is 0.842, 1.046, 1.131, 2.031,
3.427. For the second and the third sample retention time are shown on the table above.
The recommendation for this experiment is clean the flask properly before we using to
prevents from contaminants affected and the measurements of the solution must be
accurate before adding or transfer to another container

CONCLUSION
The derivatization technique used in this experiment is esterification to convert nonvolatile fatty acid to more volatile fatty acid methyl ester (FAME). There are 5
components in the standard mixture while the 3 sample 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. www.ncbi.nlm.nih.gov/pmc/articles
2. Moffat AC, Osselton MD, Widdop B. Clarkes Analysis of Drugs and Poisons.
London: Pharmaceutical Press; 2004
3. http://www.en.wikipedia.org/wiki/fatty-acidmethylester