CHM 510

ANALYTICAL SEPARATION METHODS

GAS CHROMATOGRAPHY
(GC):
1. OPTIMIZATION OF FLOW RATE
AND COLUMN TEMPERATURE
2. DETERMINATION OF FATTY ACID

NAME : NUR ADAWIYAH BINTI MANSOR (2012663606)

LAB PARTNER : NUR ANITH BINTI MOHD SAHARUDIN
(2012441998)
GROUP : AS2253B
LECTURER : MRS. HALIZA KASSIM
DATE OF EXPERIMENT : 12th NOVEMBER 2013

DATE OF SUBMISSION: 26th DECEMBER 2013

OBJECTIVE :
1. To study the effect of carrier gas flow rate and column temperature on
isothermal GC separation of methyl esters.
2. To perform separation of methyl esters using temperature
programming.
3. To identify each component in methyl ester mixture.
4. To determine concentration of fatty acid by using gas chromatography.

ABSTRACT :
Gas chromatography(GC) is an analytical technique for separating
components/ solutes based primarily on their volatilities which means that
the separation is based on differences in boiling points of the solutes. It
may also based on the solutes interaction with the stationary phase.This
experiment was carried out to optimize the flow rate and column
temperature of methyl esters by using Gas chromatography(Agilent
Technologies 6890N) equip with flame ionization detector(FID) ABD 30m x
250m x 0.25m HP5-MS capillary column. The effect of carrier gas flow
rate and column temperature on isothermal GC separation of methyl
esters can be observed by varying the flow rate and the temperature of
column. The suitable condition for separation of methyl ester is at column
temperature 2100C and flow rate 70cm3/sec.
For identification of each component in methyl ester mixture, we
must compare the retention time of individual sample with standard
mixture. The first eluted is methyl laurate, followed by methyl myristate
and last eluted is methyl palmitate. For separation of methyl esters,
standard mixture two using column temperature programming the
resolution is smaller compare to resolution for standard mixture one, that
using isothermal method.
For determination of fatty acid, this analysis is to determine type of
fatty acid which contain in a majerin product but this analysis was done
through the use of gas chromatography technique. This creates a problem

because fatty acid is not volatile enough, reactive and too polar for the
column to separate them. To overcome this problem, that fatty acid was
converted into their corresponding volatile methyl esters first by
esterification process. . Fatty acids are converted to esters by reaction
with excess alcohol, using acid catalyst or a lipase. In preparing methyl
esters for gas chromatography analysis, boron trifluoride, sulfuric acid, or
anhydrous hydrogen chloride in methanol are commonly used. The
reaction is completed by refluxing. This involves the condensation of
carboxyl group of an acid and hydroxyl group of alcohol, with water
elimination. After we managed to prepare the methyl esters and we must
keep it in refrigerator to keep it cold and inject it into gas chromatography
machine under temperature programming condition. From the
chromatogram GC of fatty acid, we can calculate the concentration of
methyl ester and standard mixture two as our response factor (RF).
Table A : Type of metyhl esters that present in this experiment.
M.Myristate

M. Laurate

Formula: C15H30O2
Melting point: 18C
Boiling point: 323C
Molar mass: 242.4 g/mol
Density: 0.855 g/cm
Classification : Ester

Formula: C13H26O2
Melting point: 5 C
Boiling point: 261-262 C
Molar mass: 214.34 g/mol
Density: 0.87 g/cm
Classification : Ester

M.Palmitate

M.Linoleate

Formula: C17H34O2
Melting point: 28-34C
Boiling point: 135-137 C
Molar mass: 270.45g/mol
Density: 0.853 g/cm
Classification : Ester

Formula: C19H34O2
Melting point: 35 C
Boiling point: 192C
Molar mass: 294.47g/mol
Density: 0.889g/cm
Classification : Ester

M.Stearate

Formula: C19H38O2
Melting point: 37-41 C
Boiling point: 215C
Molar mass: 298.50 g/mol
Density: 0.84g/cm
Classification : Ester

INTRODUCTION:
Gas chromatography is a chromatographic technique of analytical
chemistry that is used in separation of volatile organic compounds. The
technique is applied for testing the purity of a substance, separating the
different components of a mixture, and identification of a compound. A
gas chromatograph consists of a flowing mobile phase, an injection port, a
separation column containing the stationary phase, a detector, and a data
recording system.

Diagram A: Component of Gas Chromatogram

Carrier gas acts as mobile phase for gas chromatography. It
functions to transport the analyte through the column without interacting
with the molecules of the analyte. The characteristic of carrier gas is that
it must be chemically inert, dry, oxygen free and high purity. To achieve

high purity level, molecular sieve acts to filter and remove any
contaminants. The common type of gasses used include nitrogen,
hydrogen, helium, argon, and carbon dioxide, depending on the type of
detector used. The carrier gas system also contains a molecular sieve to
remove water and other impurities. The flow rate of carrier gas are
controlled the flow controller.
There are two types of column, the packed column and the open
tubular (capillary) column. Packed columns are typically a glass or
stainless steel coil (typically 1-5 m total length and 2-6 mm inner
diameter) that is filled with the stationary phase, or a packing coated with
the stationary phase. Capillary columns have a very small inner diameter
(0.1-0.5 mm) with length of 5-100m long and have 0.1-5m thick
stationary phase coated on inner walls. They provide much higher
separation efficiency than packed columns (broader peak, longer retention
time, less resolution) but are more easily overloaded by too much sample.
There are three types of capillary column. Wall-coated open tubular
(WCOT) having the inner wall directly coated with liquid stationary phase
without support material. Support-coated open tubular (SCOT) have the
liquid stationary phase coated on solid support (thin film of support,
around 30m) attached to the inside wall of column. As for porous-layered
open tubular (PLOT) the stationary phase is a solid substance that is
coated to the column wall. Column temperature is set slightly above the
average boiling point of the sample. For various mixtures having different
boiling point, temperature programing is needed.
Column selection is based on stationary phase, column diameter
and length, and the thickness of stationary phase. Long and narrow
column and also a suitable stationary phase can yield good resolution. The
stationary phase of gas chromatography is a non-volatile liquid coated on
inside of column or on a fine solid support. It mus have low volatility,
thermal stability, chemical inertness and low viscosity. The type are
chosen by their polarity, thus it depends on whether analyte and mobile
phase are polar or not.

Injector port act as introducer of sample to column. The inlet is a piece of

hardware attached to the column head. Sample is injected through the
inlet using a microsyringe, then instantly evaporated to gas carried by
carrier gas into the column. To ensure fast and complete vaporization, the
injector port temperature is set 50oC higher than column temperature.
Three types of injectors are the split injector, splitless injector, and oncolumn injector. Split injection used for high analyte concentrations.
Splitting only allow only a small amount of sample into the column.
Excessive part of sample will be vented out, then the split outlet is closed.
The reason of splitting are to prevent overloading of the column and
produces narrow and sharp peaks. While splitless injection is used for low
concentration samples or trace analysis of high boiling compounds in low
boiling solvents. Split vent are opened for 30 to 90 seconds after injection,
removing the bulk of the solvent to avoid large solvent peak overlaping
with the analyte peak, but leaves most of the sample condensed at the
top of the column. This will give higher peak, making it suitable for trace
samples. As for on-column injection, it is applied for samples that
decompose above their boiling point or thermally labile compounds. This
type of injection can handle dilute or concentrated solutions and relatively
large or small volumes.
There are four categories of detectors; specific detector that responds to a
single chemical compound, selective detector responds to a range of
compounds having a common physical or chemical property, a nonselective or universal detector responds to all compounds except the
carrier gas and a non-destructive detector does not destroy the sample
used. The most commonly used detectors are the flame ionization
detector (FID) and the thermal conductivity detector (TCD). Recorder will
then record the signal from the detector as the analyte elute from column.
In this experiment, we are trying to determine the concept of gas
chromatography retention time and resolution by using mixture of methyl
esters, methyl laurate, methyl myristate, methyl palmitate, methyl
stearate and methyl linoleate. We are also trying to see the effects of
column temperature and flow rate on the compounds separation

Fatty acid is a carboxylic acid having a long carbon chain. They exist as
saturated or unsaturated form. Fatty acids bearing carbon-carbon double bonds
are known as unsaturated fatty acid, while those that did not have double bond
are saturated fatty acid. Most fatty acids contain an even number of carbon
atoms in the hydrocarbon chain. The chain lengths may vary, but most natural
fatty acids are have 4 to 22 carbons, with 18 carbons the most common one.
Those with odd number of carbon mostly exist in bacteria and lower plants or
animals. Fatty acids are the main constituents of oils and fats. Unsaturated fatty
acids posseses one or more double bonds on the carbon chains. The double
bonds can be broken by the addition of hydrogen atoms, converting it to
saturated fatty acid, hence naming it unsaturated.
The most reactive sites in fatty acids are the carboxyl group and double
bonds, as the carbon chain rarely show reactivity. Conversion of acids to esters
and vice versa, also the exchange of ester groups are one of the most widely
done in industry and chemistry. Fatty acids are converted to esters by reaction
with excess alcohol, using acid catalyst or a lipase. In preparing methyl esters for
gas chromatography analysis, boron trifluoride, sulfuric acid, or anhydrous
hydrogen chloride in methanol are commonly used. The reaction is completed by
refluxing. This involves the condensation of carboxyl group of an acid and
hydroxyl group of alcohol, with water elimination.
The hydrocarbon chains of carboxylic acid plays important role in
determining polarity. It is non-polar, thus counter balancing the polar acid
functional group. In acids with only a few carbons, the acid functional group
dominates, giving polar character to the molecule. However, in fatty acids, the
non-polar hydrocarbon chain gives the molecule a non- polar character.
Margarine is a semi-solid emulsion mostly contains vegetable fats and
water composition. The making of margarine involves emulsifying hydrogenated
vegetable oils with skimmed milk, then chilling it to solidify the mixture.
Vegetable and animal fats are similar compounds but different in melting points,
due to the difference in carbon-carbon double bonds in the fatty acids
components. The higher number of double bonds will result in lower melting
point. Margarines contain saturated and unsaturated fats. Vegetable fats contain
around 7% to 86% saturated fatty acids margarines contain more saturated fat.
In firmer margarines, the amount of saturated fats is higher. Unsaturated oils
exist in two variants, mono- and poly-unsaturated fats. In margarine

development, some of the unsaturated fats are converted into hydrogenated fats
or trans fats, making them have higher melting point so that they are solid at
room temperatures. In this experiment, we used margarines fatty acid to
convert them to methyl esters, then test their separation in gas chromatography.

ANALYTICAL PROCEDURE :
1. OPTIMIZATION OF FLOW RATE AND COLUMN TEMPERATURE
a) Instrument set-up
Injection port
Injection port temperature
Column temperature
Carrier gas flowrate
Detector temperature

: Split (40:1)
: 250 oC
: 210 oC
: 30 cm sec -1
: 250 oC

b) Effect of carier gas flow rate on isothermal GC separation of

methyl esters.
0.4L standard mixture injected isothermally at 210 oC at carrier gas
flowrate of 30 cm sec-1 . then the flowrate increased to 50 cm sec -1 . the
system was allowed to equilibratefor a few minutes and the standard
mixture injected again. the same procedure repeated at flowrate of 70 cm
sec -1. The most suitable flowrate was determined.
c) Effect of column temperature on the isothermal GC separation
of methyl esters.
0.4L standard mixture injected isothermally at 170 oC, followed by 190 oC
, at the optimal carrier gas flowrate . the effect of column temperature on
the separation, resolution, and the analysis time were evaluated.
d) Separation
programming.

of

methyl

esters

using

column

temperature

The standard mixture injected at the optimal carrier gas flow rate using a
linear temperature ramp from 100 oC to 290 oC at the optimal flow rate.
e) Identification of components in methyl esters mixture.
Each mehyl esters were injected individually to identify the various
compounds in the standard mixture using the optimized GC conditions.

Methyl
Methyl
Methyl
Methyl
Methyl

laurate
myristate
palmitate
stearate
linolate

2. DETERMINATION OF FATTY ACID

a) Preparation of fatty acid methyl ester samples
i.

2g of margerine is weighed and the reading is recorded.

ii.

Then, the sample is transferred into a 50 mL flask that equipped

with air condenser

iii.

5 ml of 0.5 M methanolic solution is added into the flask and

refluxed for 3-4 minutes.

iv.

15 ml of esterification reagent is added into the flask and

refluxed again for another 3 minutes.

v.

Next, the mixture is transferred into a separatory flask. 50 ml of

saturated NaCl and 25ml of diethyl ether is added together in the
separatory flask. The mixture is shaken vigorously for 2 minutes
and the aqueous layer is discarded.

vi.

Step (v) is repeated with another 25ml of saturated NaCl and the
aqueous layer is discarded once again.

vii.

The organic layer is transferred into a screw cap vial. Make sure
that only the organic layer is injected into the GC as water can
ruin the GC column.

b) Instrument set-up
Injection port
Injection port temperature
Column temperature
Carrier gas flow rate
Detector temperature

: Split (40:1)
: 250oC
: 100oC to 290oC at 40oC /min
: 30 mL/s
: 250oC

c) Quantitative analysis of FAME

i.

0.4L of standard esters are injected to the column. The

injection is repeated in order to get reproducible peak areas.

ii.

0.4L of derivatized samples are injected to the column. The

injection is repeated in order to get reproducible peak areas.

iii.

The amount of each fatty acid in the sample were calculated

by using the data from the standard esters.

DATA AND CALCULATIONS :

Table 1: Analysis for temperature: 170 oC and flow rate: 70 cm/s
Injection
s

Peaks

Retention
time, (tR)

Width,
(min)

2.272

0.1111

4.540

0.2181

10.072

0.6920

2.270

0.1127

4.530

0.2227

10.069

0.7117

2.275

0.1124

4.545

0.2187

10.051

0.7068

Average Resolution time , RS,

Resolution time , RS,

peak 2 & 3

Peak 3&4

2(4.540-2.272)
0.2181+0.1111

2(10.072-4.540)
0.6920+0.2181

= 13.78
2(4.530-2.270)
0.2227+0.1127
= 13.48
2(4.545-2.275)
0.2187+0.1123
= 13.72
13.78+13.48+13.
72
3
= 13.66

= 12.16
2(10.069-4.530)
0.7117+0.2227
= 11.86
2(10.051-4.545)
0.7068+0.2187
= 11.90
12.16+11.86+11
.90
3
= 11.97

Table 2: Analysis for temperature: 190 oC and flow rate: 70 cm/s

Injection
s

Peaks

Retention
time, (tR)

Width,
(min)

1.540

0.0657

2.561

0.1366

4.853

0.2594

Resolution time , RS,

peak 2 & 3
Peak 3&4
2(2.561-1.540)
0.0657+0.1366
= 10.09

2(4.853-2.561)
0.2594+0.1366
= 11.58

1.542

0.0658

2.565

0.1397

4.849

0.2530

1.542

0.0665

2.565

0.1387

4.863

0.2607

Average Resolution time , RS,

2(2.565-1.542)
0.0658+0.1397
= 9.96
2(2.565-1.542)
0.0665+0.1387
= 9.97
10.09+9.96+9.97
3
= 10.00

2(4.849-2.565)
0.2530+0.1397
= 11.63
2(4.863-2.565)
0.2607+0.1387
= 11.51
11.58+11.63+11
.51
3
= 11.57

Table 3: Analysis for temperature: 210 oC and flow rate: 30 cm/s

Injectio
ns

Peak
s

Retention
time, (tR)

Width,
(min)

2.770

0.0702

3.915

0.1336

6.309

0.2209

2.766

0.0731

3.911

0.1345

6.248

0.2142

2.767

0.0722

3.911

0.1293

6.291

0.2472

Average Resolution time , RS,

Resolution time , RS,

peak 2 & 3
Peak 3&4
2(3.915-2.770)
0.0702+0.1336
= 11.24
2(3.911-2.766)
0.0731+0.1345
= 11.03
2(3.911-2.767)
0.0722+0.1293
= 11.35
11.24+11.03+1
1.35
3
= 11.21

2(6.306-3.915)
0.2209-0.1336
= 13.49
2(6.284-3.911)
0.2142+0.1345
= 13.61
2(6.291-3.911)
0.2472+0.1293
= 12.64
13.49+13.61+
12.64
3
= 13.25

Table 4 : Analysis for temperature: 210 oC and flow rate: 50 cm/s

Injectio
ns

Peak
s

Retention
time, (tR)

Width,
(min)

1.663

0.0540

Resolution time , RS,

peak 2 & 3
Peak 3&4

2.356

0.0965

3.803

0.1782

2(2.356-1.663)
0.0965+0.0540
= 9.21

1.664

0.0492

2.359

0.0970

3.806

0.1769

1.666

0.0485

2.361

0.0949

3.804

0.1869

Average Resolution time , RS,

2(2.359-1.664)
0.0970+0.0492
= 9.51
2(2.361-1.666)
0.0485+0.0949
= 9.69

2(3.803-2.356)
0.1782+0.0965
= 10.54
2(3.806-2.359)
0.1769+0.0970
= 10.57
2(3.804-2.361)
0.1869+0.0949
= 10.27

10.54+10.57+
10.27
3
= 10.46
Table 5 : Analysis for temperature: 210 oC and flow rate: 70 cm/s
Injectio
ns

Peak
s

Retention
time, (tR)

Width,
(min)

1.186

0.0443

1.687

0.0780

2.729

0.1629

1.188

0.0461

1.686

0.0788

2.726

0.1530

1.189

0.0478

1.688

0.0804

2.730

0.1610

Average Resolution time , RS,

9.21+9.51+9.69
3
= 9.47

Resolution time , RS,

peak 2 & 3
Peak 3&4
2(1.687-1.186)
0.0443+0.0780
= 8.19
2(1.686-1.188)
0.0461+0.0788
= 7.97
2(1.688-1.189)
0.0804+0.0478
= 7.78
8.19+7.97+7.78
3
= 7.98

2(2.729-1.687)
0.0780+0.1629
= 8.65
2(2.726-1.686)
0.1530+0.0788
= 8.97
2(2.730-1.688)
0.0804+0.1610
= 8.63
8.65+8.97+8.6
3
3
= 8.75

RESULTS:
1. OPTIMIZATION OF FLOW RATE AND COLUMN
TEMPERATURE
Table 6 : The resolution for optimization of flow rate and
temperature
TEMPERATURE(OC
)

170

190

210

FLOW
RATE(cm/s)

Rs 2,3
=11.21
Rs 2,3
=9.47

30
50

Rs2,3
Rs 3,4
Rs 2,3
Rs 3,4
Rs 2,3
=13.66 =11.97 =10.00 =11.57 = 7.98

70

Rs 3,4
=13.25
Rs
3,4=10.4
6
Rs 3,4
=8.75

Table 7 : The average retention time, tR for optimization of flow

rate and temperature.
170 oC

T
FR
30cm
/s
50cm
/s
70cm
/s

tR,1=
2.272

tR,2=
4.538

T = temperature

tR,3=
tR,1=
10.06 1.541
4
RF = Flow rate

190 oC

tR,2=
2.563

210 oC

tR,3=
4.855

tR,1=
4.152
tR,1=
1.664
tR,1=
1.188

tR,2=
3.912
tR,2=
2.419
tR,2=
1.687

tR,3=
6.283
tR,3=
3.804
tR,3=
2.728

Table 8 : Analysis for temperature programming of standard

mixture 2.
Injectio
ns

Pea
k

Retenti
on
time,
(tR)

Widt
h,
(min)

0.080
2
3
3.099
0.144
4
4
5.393
0.225
6
5
7.744
0.182
2
6
7.983
0.105
0
2
2
1.988
0.078
4
3
3.103
0.142
3
4
5.407
0.232
1
5
7.537
0.183
3
6
7.979
0.102
0
Average Resolution time , RS,

Resolution time , RS,

Peak
2&3

Peak
3&4

Peak
4&5

Peak 5&6

9.89

12.4

11.53

1.66

10.10

12.31

10.26

3.098

10.00

12.36

10.90

2.37

1.988

Table 9 : Analysis for isothermal of standard mixture 2.

Injectio
ns

Pea
k

Retenti
on
time,
(tR)
1.188

1.685

2.713

Widt
h,
(min)
0.039
9
0.075
6
0.141

Resolution time , RS,

Peak
2&3

Peak
3&4

Peak
4&5

Peak 5&6

8.61

9.47

10.60

9.96

6
5

4.469

4.825

0.189
7
0.261
2

Table 10 : The Retention time, tR of standard mixture

2(temperature programming).
Peak
st

1 Injection
1.988

3.099

5.393

7.544

7.983

Retention time, tR (min)

2nd Injection
Average
1.988
1.988+1.988
2
= 1.988
3.103
3.099+3.103
2
= 3.101
5.407
5.393+5.407
2
= 5.400
7.537
7.537+7.544
2
= 7.5405
7.979
7.983+7.979
2
= 7.981

Table 11 : Comparison of Resolution,RS, between temperature

programming and isothermal for standard mixture 2.
Peaks

Resolution, RS,
Isothermal

Peak 2&3

Temperature
programming
10.00

Peak 3&4

12.36

9.47

Peak 4&5

10.90

10.60

Peak 5&6

10.90

9.96

8.61

Table 12 : Comparison of retention time between temperature

programming and isothermal for standard mixture 2.
Peaks

Average Retention time, (tR)

Temperature

Isothermal

Peak 2

programming
1.988

1.188

Peak 3

3.101

1.685

Peak 4

5.400

2.713

Peak 5

7.540

4.469

Peak 6

7.981

4.825

Table 13: The Retention time, tR of standard individual sample.

Methyl
Esters

Retention time, tR (min)

M.Laurate

1st Injection
1.190

2nd Injection
1.185

Average
1.1875

M.Myristate

1.685

1.690

1.6875

M.Palmitate

2.722

2.715

2.7185

M.Linolate

4.458

4.435

4.4465

M.Stearate

4.838

4.837

4.8375

Table 14 : Comparison of retention time for individual samples

with standard mixture 1.
Methyl Esters

Retention time, tR (min)

M.Laurate

Average tR for individual

sample
1.1875

Average tR for std mixture

1
1.1877

M.Myristate

1.6875

1.687

M.Palmitate

2.7185

2.7283

Table 15 : Comparison of standard mixture 2 with individual

sample in terms of retention time.
Methyl Esters

Retention time, tR (min)

M.Laurate

Average tR for individual

sample
1.1875

Average tR for std

mixture 2
1.188

M.Myristate

1.6875

1.685

M.Palmitate

2.7185

2.713

M.Linolate

4.4465

4.469

M.Stearate

4.8375

4.825

2. DETERMINATION OF FATTY ACID

Standard mixture 2
Response factor,RF =
peak area
sample amount
Table 16 : Response factor,RF of fatty acid.
Response factor,RF
Methyl Esters
M.Laurate

M.Myristate

M.Palmitate

M.Linolate

M.Stearate

1st Injection

2nd Injection

1141.59460
250
= 4.5664
769.81403
220
= 3.4992
96.87212
1010
= 0.0959
35.14044
781
= 0.04499
20.71200
350
= 0.0592

1067.89673
250
= 4.2716
711.45239
220
= 3.2339
91.98003
1010
= 0.0911
33.44002
781
= 0.0428
17.95727
350
= 0.0513

Sample 1 : FATTY ACID

Average
4.4190

3.3666

0.0935

0.0439

0.0553

Concentration of Methyl Esters =

peak area
response factor
Table 17 : Average concentration of fatty acid for sample 1
Methyl Esters

M.Laurate

M.Myristate

M.Palmitate

M.Linolate

M.Stearate

Concentration (ppm)
1st Injection

2nd Injection

3rd Injection

27.75001
4.4190
= 6.2797
16.41573
3.3666
= 4.8761
224.15385
0.0935
= 2397.3674
14.89066
0.0439
= 339.1950

29.62523
4.4190
= 6.7041
13.97977
3.3666
= 4.1525
228.10072
0.0935
= 2439.5799
8.47154
0.0439
= 192.9736

27.18356
4.4190
= 6.1515
11.44884
3.3666
= 3.4007
211.08675
0.0935
= 2257.6123
1.11003e-1
0.0439
= 2.5285

377.13971
0.0553
= 6819.8863

239.02115
0.0553
= 4322.2631

221.57201
0.0553
= 4006.7271

Average
6.3784

4.1431

2364.8532

266.0843

4164.4951

Sample 2 : FATTY ACID

Table 18 : Average concentration of fatty acid for sample 2.
Methyl Esters
M.Laurate

M.Myristate

M.Palmitate

M.Linolate

M.Stearate

Concentration (ppm)
1st Injection

2nd Injection

3rd Injection

Average

55.56129
4.4190
= 12.5733
22.25484
3.3666
= 6.6105
265.55234
0.0935
= 2840.1320
2.27972e-1
0.0439
= 5.1930
285.07483
0.0553
= 5155.0602

53.82813
4.4190
= 12.1811
28.23556
3.3666
= 8.3870
301.04279
0.0935
= 3219.7090
8.37783
0.0439
= 190.8390
293.88065
0.0553
= 5314.2975

53.69234
4.4190
= 12.1503
23.02673
3.3666
= 6.8398
304.28159
0.0935
= 3254.3486
22.29779
0.0439
= 507.9223
314.11569
0.0553
= 5680.2114

12.3016

6.7251

3237.0288

234.6514

5383.1897

Sample 3 : FATTY ACID

Table 19 : Average concentration of fatty acid for sample 3.
Methyl Esters
st

M.Laurate

M.Myristate

M.Palmitate

M.Linolate

M.Stearate

1 Injection
11.73461
4.4190
= 2.6555
14.36507
3.3666
= 4.2669
60.38026
0.0935
= 645.7782
30.02446
0.0439
= 683.9285
54.99415
0.0553
= 1037.6255

Concentration (ppm)
2 Injection
3rd Injection
9.03226
11.95307
4.4190
4.4190
= 2.0440
= 2.7049
3.99646
14.17661
3.3666
3.3666
= 1.1871
= 4.2110
63.39573
76.86593
0.0935
0.0935
= 678.0292
= 822.0955
28.72328
1.74857
0.0439
0.0439
= 654.2889
= 39.8308
59.57137
3.21798
0.0553
0.0553
= 1123.9881
= 60.7166
nd

Average
2.6802

4.2389

661.9037

664.6087

1080.8068

Table 20 : Average concentration for Sample 1, Sample 2 and

Sample 3.
Methyl Esters
M.Laurate
M.Myristate
M.Palmitate
M.Linolate
M.Stearate

Sample 1
6.3784
4.1431
2364.8532
266.0843
4164.4951

Average Concentration (ppm)

Sample 2
Sample 3
12.3016
2.6802
6.7251
4.2389
3237.0288
661.9037
234.6514
664.6087
5383.1897
1080.8068

DISCUSSION :
Gas chromatography (GC), is a common type
of chromatography used in analytical chemistry for separating and
analyzing compounds that can be vaporized without decomposition.
Typical uses of GC include testing the purity of a particular substance, or
separating the different components of a mixture. In some situations, GC

may help in identifying a compound. In preparative chromatography, GC

can be used to prepare pure compounds from a mixture. In gas
chromatography, the mobile phase is a carrier gas, usually an inert gas
such as helium or an unreactive gas such as nitrogen. The stationary
phase is a microscopic layer of liquid or polymer on an inert solid support,
inside a piece of glass or metal tubing called a column. The instrument
used to perform gas chromatography is called a gas chromatograph. The
gaseous compounds being analyzed interact with the walls of the column,
which is coated with a stationary phase. This causes each compound
to elute at a different time, known as the retention time of the compound.
The comparison of retention times is what gives GC its analytical
usefulness.
In this experiment, type of gas chromatogram used is Gas
chromatography(Agilent Technologies 6890N) equip with flame ionization
detector(FID) ABD 30m x 250m x 0.25m HP5-MS capillary column.
Flame ionization detector(FID) consist of a hydrogen / air flame and a
collector plate. The effluent from the GC column passes through the
flame, which breaks down organic molecules and produces ions. The ions
are collected on a biased electrode and produce an electric signal. The
column used is capillary column.
The two major factors that control elution time in gas
chromatography is flow rate and column temperature. Although solute
elution rate increases linearly with flow rate, elution rate increases
approximately exponentially with column temperature and, thus, is far
more effective in eluting strongly retained solutes. Firstly we will, analyse
the effect of carrier gas flow rate on isothermal GC separation of methyl
esters. Three difference flow rate which is 30 cm/s , 50 cm/s and 70cm/s
been analyse with temperature 210oC. Based on resolution flow rate of 30
cm/s , 50 cm/s and 70cm/s , the best resolution is resolution flow rate at
70 cm/s because for peak2,3 the resolution is only 7.98and for peak3,4 is
only 8.75 it is the lowest resolution compare to flow rate at 30 cm/s and
50 cm/s which is 9.47 and 10.46 for flow rate at 30 cm/s and, 11.21

and13.25 for flow rate at 50 cm/s. Based on retention time, the most
shortest analysis time also for flow rate at 70cm/s with retention time for
first peak 1.188min, second peak with 1.687min and 2.728min for the last
peak. Compare to other retention time, flow rate at 30 cm/s the retention
time is 4.152,3.912 and 6.283 for first, second and third peak respectively
and flow rate at 50 cm/s the retention time is 1.664, 2.419 and 3.804 for
first, second and third peak respectively. We can make a conclusion here
that the shortest retention time is when at flow rate 70cm/s and the best
flow rate for gradient elution of chromatography.
The second factors that control elution time in gas chromatography
are temperature. We will analyse the effect of carrier gas on isothermal
GC separation of methyl esters. Three difference temperature which is
170 oC, 190 oC and 210oC are been analyse at flow rate 70cm/s. The best
temperature based on resolution, is 210oC because the resolution for
temperature 210oC is only 7.98 and 8.75 which is the shortest resolution
compare to resolution for temperature 170 oC which is 13.66 and 11.97
and resolution for temperature 190 oC is 10.00 and 11.57. Based on
retention time, the retention time for temperature 210 oC is the shortest
compare to other. The retention time for temperature at 210 oC are 1.188,
1.687 and 2.728 for first, second and last peak respectively. For
temperature 170 oC the retention time is 2.272, 4.538 and 10.064,
meanwhile for temperature at 109 oC the retention time is 1.541, 2.563
and 4.855 for first, second and last peak respectively. We can conclude
that the best temperature for elution gradient is 210 oC by analysis of
resolution and retention time at difference temperature. Based on the
chromatograms of standard mixture, the optimum condition of this
experiment achieved at temperature 210C at flow rate of 70 cm3/sec.
The injection of sample at temperature 210C and flow rate of 70 cm3/sec
gives the lowest resolution value compared to other temperature and flow
rate.
Before this we used method isothermal in order to know the effect of
carrier gas flow rate and column temperature for separation of methyl

esters. Now we going to used column temperature programming for

separation

of

chromatography

methyl

esters.

development

Temperature
technique

programming

used

largely

is
in

a
gas

chromatography to accelerate the elution rate of late peaks that,

otherwise, would take a very long time to elute. It is achieved by
continuously raising the column temperature, usually as a linear function
of time, during the elution process. Column temperature is increased
either continuously or in steps as the analysis proceeds. But practically,
we did not get the result as we expected, based on Table 11 and Table 12
we can see that the best retention time is when using
technique

not

temperature

programming.

The

isothermal

analysis

time

for

temperature programming is longer than isothermal technique, to elute all

samples, isothermal technique only take 4.469min but for temperature
programming it take 7.981min. In term of resolution, the resolution for
isothermal

technique

is

better

than

resolution

for

temperature

programming .
In this experiment, gas chromatography was used to identify the
various components in the standard mixture of methyl ester using the
optimized GC conditions at temperature 210C and at flow rate of 70
cm3/sec. For standard mixture 1, from the chromatogram we can see 4
peak appear first peak is solvent peak and to know the other three peaks,
we must compare the retention time between individual sample with
standard mixture 1. Based on Table 14 we can predict the component that
will be eluted first is methyl laurate, follow by methyl myristate and lastly
methyl palmitate. For standard mixture 2, we also compare the retention
time between standard mixture and individual sample. Based on Table 15
we can see that the order of elution, component that will be eluted first is
methyl laurate, follow by methyl myristate, then methyl palmitate, methyl
linolate and lastly, methyl stearate.
For determination of fatty acid using gas chromatography(GC), the
order of elution of compound same with order of elution for standard
mixture 2 when using temperature programming. The experiment was

done using margarine to obtain fatty acid. Since fatty acid was not
volatile to be analyzed in gas chromatography, the sample was modified
into methyl ester. Short fatty acids, having fewer than six carbon chains
tend to be volatile. The longer chained are much difficult to be tested with
gas chromatography due to little volatility, plus the highly polar
compounds tend to form hydrogen bonds, leading to adsorption problems.
This leads to the esterification process of the acid, thus reducing their
polarity. Esterification reaction involves the condensation of the carboxyl
group of an acid and the hydroxyl group of an alcohol, resulting in a type
of fatty acid ester named fatty acid methyl esters (FAME). Base-catalyzed
methanolysis proceeds faster under mild temperature conditions
compared to acid-catalyzed reactions. BF3 is a commonly used acid
catalyst for methylation and methanolysis. However, it is hazardous and
has a limited shelf life. Another effective acid catalyst for FAME synthesis
is H2SO4, but another downfall is it has a very corrosive property and must
be handled with care. 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 along with the water. Methyl esters offer excellent
stability, and provide quick and quantitative samples for gas
chromatography analysis Moisture must be removed prior to analysis, as
to prevent any damage towards the GC. The sample was also neutralized
from any form of acidity by adding the NaCl solution.
To calculate the concentration of the component we must calculate
the response factor first. The result of response factor tabulated in Table
16. After that, we can calculate the concentration of each component for
sample 1, sample 2 and sample3.Based on Table 20, we can see that
methyl stearate has the highest concentration among the other, second
highest in term of concentration is methyl palmitate, than methyl linolate,
methyl myristate and lastly methyl laurate.

CONCLUSION :

As a conclusion, the separation of analytes mixture using gas

chromatography affected by the column temperature and carrier gas flow
rate. Compounds separated better at temperature, 210oC and flow rate,
70cm/s based on the value of resolution calculated. The elution order of
standard mixture, first is methyl laurate, follow by methyl myristate, then
methyl palmitate, methyl linolate and lastly, methyl stearate.
The derivatization procedure is routinely used for fat analysis in
which nonvolatile fatty acids are chemically converted to the
corresponding volatile methyl esters (FAME) so that it can be analyzed by
gas chromatography.From the experiment, we can conclude that of methyl
stearate was the largest compund present in margarine, and methyl
laurate being the least.

REFERENCES :
1. Holler, Skoog, and Crouch, Principles of Instrument bAnalysis 6 th
Edition(2007)
2. 124-10-7 CAS MSDS (METHYL MYRISTATE) Melting Point Boiling Point
Density CAS Chemical Properties.
3. https://www.google.com.my/webhp?
hl=en&tab=ww#hl=en&q=ester+Methyl+Myristate+properties
4. https://www.google.com.my/webhp?
hl=en&tab=ww#hl=en&q=ester+Myristate+wiki
5. http://en.wikipedia.org/wiki/Prefix
6. http://www.telecomabc.com/p/prefix.html
7. https://www.google.com.my/webhp?hl=en&tab=ww#hl=en&q=C14H28O2

APPENDIX :

Conc. of Methyl Esters =

area

peak
response

factor
Response factor,RF =
Resolution, Rs =
2[(tR)A (tR)B]

amount

WA
WB

peak area
sample

Formula
of plate
height, H =
L
N