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GAS CHROMATOGRAPHY
(GC):
1. OPTIMIZATION OF FLOW RATE
AND COLUMN TEMPERATURE
2. DETERMINATION OF FATTY ACID
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.
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.
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
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
ii.
iii.
iv.
v.
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
ii.
iii.
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
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
Peaks
Retention
time, (tR)
Width,
(min)
1.540
0.0657
2.561
0.1366
4.853
0.2594
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
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
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
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
Peak
s
Retention
time, (tR)
Width,
(min)
1.663
0.0540
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
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
9.21+9.51+9.69
3
= 9.47
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
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
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,
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
Pea
k
Retenti
on
time,
(tR)
1.188
1.685
2.713
Widt
h,
(min)
0.039
9
0.075
6
0.141
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
1 Injection
1.988
3.099
5.393
7.544
7.983
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
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
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
M.Laurate
M.Myristate
1.6875
1.687
M.Palmitate
2.7185
2.7283
M.Laurate
M.Myristate
1.6875
1.685
M.Palmitate
2.7185
2.713
M.Linolate
4.4465
4.469
M.Stearate
4.8375
4.825
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
Average
4.4190
3.3666
0.0935
0.0439
0.0553
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
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
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
Sample 1
6.3784
4.1431
2364.8532
266.0843
4164.4951
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
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
of
chromatography
methyl
esters.
development
Temperature
technique
programming
used
largely
is
in
a
gas
not
temperature
programming.
The
isothermal
analysis
time
for
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 :
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 :
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