EXPERIMENT 3

FATTY ACID DETERMINATION USING GAS

CHROMATOGRAPHY
INTRODUCTION
In chemistry, and especially in biochemistry, a fatty acid is a carboxylic
acid with a long aliphatic tail (chain), which is
either saturated or unsaturated. Fatty acids are merely carboxylic acids with
long hydrocarbon chains. The hydrocarbon chain length may vary from 10-30
carbons (most usual is 12-18). The non-polar hydrocarbon alkane chain is an
important counter balance to the polar acid functional group. In acids with
only a few carbons, the acid functional group dominates and gives the whole
molecule a polar character. However, in fatty acids, the non-polar
hydrocarbon chain gives the molecule a non- polar character. The most
common fatty acids are listed. Note that there are two groups of fatty acids-saturated and unsaturated. Recall that the term unsaturated refers to the
presence of one or more double bonds between carbons as in alkenes.
A saturated fatty acid has all bonding positions between carbons occupied by
hydrogens. The melting points for the saturated fatty acids follow the boiling
point principle observed previously. Melting point principle: as the molecular
weight increases, the melting point increases. This observed in the series
lauric (C12), palmitic (C16), stearic (C18). Room temperature is 25oC, Lauric
acid which melts at 44o is still a solid, while arachidonic acid has long since
melted at -50o, so it is a liquid at room temperature. Fatty acids are
important sources of fuel because, when metabolized, they yield large
quantities of ATP. Many cell types can use either glucose or fatty acids for
this purpose. In particular, heart and skeletal muscle prefer fatty acids.
Despite long-standing assertions to the contrary, brain can use fatty acids as
a source of fuel in addition to glucose and ketone bodies.

OBJECTIVE

To prepare the fatty acid sample

To modify compound through derivation to procedure a new compound
which is suitable for gas chromatography

REAGENT AND SOLUTION

Methyl laurate (0.1 mg/ml)

Methyl myristate (0.1 mg/ml)
Methyl palmitate (1.50 mg/ml)
Methyl strearate (0.70 mg/ml)
Methanolic solution (0.5M) : NaOH in methanol
Esterification reagent : added 7.5 ml H2SO4 and 5g of NaCl in a 500ml
flask and reflux for 15 minutes.
Sample : oil or fat samples (margarine or butter) 10g

ANALYTICAL PROCEDURE
1. Preparation of fatty acid methyl ester samples from fat samples
a. Weighed 2g of fat and recorded the exact weight
b. Transferred the sample into a 50ml flask equipped with air
condenser
c. Added 5 ml of 0.5 M methanolic solution and refluxed for 3 to 4
minutes
d. Added 15 ml of esterification reagent and refluxed for 3 minutes
e. Transferred the mixture into a separatory flask. Added 50ml of
saturated NaCl and 25 ml of diethyl ether. Shake the mixture
vigorously for 2 minutes and discarded the aqueous layer.
f. Added another 25ml of saturated NaCl and again discarded the
aqueous layer.
g. Transferred the oraganic layer into vial
2. Instrument Set-up
Injection port : split(20:1)
Injection port temperature : 250C
Oven temperature : 100C to 290C at 40C/min
Column flow rate : 40 ml/s
Detector temperature : 250oC

PROCEDURE
1. Methyl strearate
Weighed 17.5 mg of methyl strearate
Then diluted in 25 ml diethyl ether in volumetric flask
2. Methyl laurate

Used the micro pipette, pipette 2.874L of methyl laurate

Then diluted in 25 ml diethyl ether in volumetric flask
3. Methyl myristate
Used the micro pipette, pipette 2.924L of methyl myristate
Then diluted in 25ml diethyl ether in volumetric flask
4. Methyl palmitate
Used the micro pipette, pipette 44.0L of methyl palmitate
Then diluted in 25ml diethyl ether in volumetric flask
5. Methanolic solution
Weighed 40 mg and then diluted in 50 ml water and 50 ml methanol

CALCULATION
Methyl Laurate
0.1mg
x
=
ml
25 ml
2.5 mg
x 1000=2.874 L
870 mg/cm3
Methyl myristate
0.1mg
x
=
ml
25 ml
2.5 mg
x 1000=2.924 L
855 mg/cm3
Methyl palmitate
1.5 mg
x
=
ml
25 ml
3.75 mg
x 1000=44.0 L
852mg/cm 3

Methyl strearate

0.7 mg
x
=
ml
25 ml
17.5 mg

RESOLUTION
peak 1peak 2=

2(3.6393.303)
0.0331+ 0.0264

11.294

peak 2peak 3=

2(4.5433.639)
0.0363+0.0264

28.836

peak 3 peak 4=

2( 4.6994.543)
0.0363+ 0.0319

4.575

RESULT
COMPOUND
RETENTION TIME (MIN)
Palmitate
4.543
Myristate
3.935
Laurate
3.276
Stearate
4.718
Table 1: Retention time for compound of Palmitate, Myristate, Laurate and
Stearate in standard solution.

PEAK

COMPOUND

RETENTION
RESOLUTION
TIME (MIN)
1
Laurate
3.303
Peak 1 & 2 Peak 2 &
Peak 3
2
Myristate
3.639
11.294
3
&4
3
Palmitate
4.543
28.836
4.575
4
Stearate
4.699
Table 2: Retention time of compound of Palmitate, Myristate,Laurate and
Stearate in sample of fatty acid.

DISCUSSION
Based on the result obtained, it shows that the fatty acid consist all the four
esters. This can be seen by comparing both compounds retention time in
standard and sample solutions. From the chromatogram, the four
components peak is determined to be the highest compare to other
components. This shows that they are the major compounds in the fatty acid.
Palmitate shows it peak at 4.543 min as same as the retention time in
standard solution. For Laurate and Stearate, the retention time in sample is
not the same as standard yet almost nearest. However, for Myristate the
retention time is a bit earlier in sample solution compare to standard. This
might be due to the condition applied in the sample is more suitable as it
elute faster. Based on the standard chromatogram for Stearate, the peak is
too small to recognize. This might be because of the samples temperature is
too broad compare to the setting temperature. Thus, the compound
remained in the column as it fully unable to vaporize completely.
From the table, the resolution for peak 1 & 2 and peak 3 & 4 shows a good
value, which is 11.294 and 4.575 respectively. However, for peak 2 & 3, the
resolution is above 20, which is 28.836, and this indicates a poor separation
between this two peaks. From the chromatogram, it can be seen that the
peak of Palmitate is not separated well. This might be due to the presence of
another compound in the sample that have the closest retention time with
the Palmitate. A new condition, for instance increase the temperature of the
column, need to be conducted to separate the overlapping peaks.
The peaks appear to be tailing. This is may be because the sample is too
soluble with column. Thus, it been retained in the column and elute lately as

a band to the detector. A large amount of compound arriving at the detector

causing the peak to occur as tailing.
In this experiment, derivization method was used. This is a method of
converting non-volatile method into volatile so that can be analyse using gas
chromatography. Derivatization will render highly polar materials to be
sufficiently volatile so that they can be eluted at reasonable temperatures
without thermal decomposition. For GC analysis, compounds containing
functional
groups with active hydrogens such as -SH, -OH, -NH and -COOH are of
primary concern
because of the tendency of these functional groups to form intermolecular
hydrogen bonds which affect the inherent volatility of compounds containing
them. Since GC is used to separate
volatile organic compounds, modification of the functional group of a
molecule by
derivatization enables the analysis to be conducted.

CONCLUSION
Derivization is a effective method in converting non-volatile samples into
volatile. The sample prepared using this method was successfully run using
gas chromatography. The result shows all the four esters found to be in the
sample fatty acid and they are the major components identified in this
analysis.

REFERENCES
1. Skoog, West, Holler, Crouch, Fundamentals of Analytical Chemistry,
Thomson Brooks/Cole Publishers,8th ed.,2004
2. Derivization Reaction and Reagents for Gas Chromatography Analysis,
https://www.google.com.my/url?
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%2F32817&ei=P0qGUYWLO8f7rAfvoYDYCw&usg=AFQjCNFHrAor4ES26
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3. Fatty acid, http://en.wikipedia.org/wiki/Fatty_acid, retrieved on 5 May

2013
4. Fatty acids,
http://www.elmhurst.edu/~chm/vchembook/551fattyacids.html,
retrieved on 5 May 2013