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EXPERIMENT 1

TITLE
Gas Chromatography (GC): Optimization of flow rate and column temperature

OBJECTIVE
To determine the optimum flow rate and column temperature to give optimum peaks of
chromatogram and to understand the concept of retention time in gas chromatography
To determine the optimum temperature programming for the analysis of standard mixture.

INTRODUCTION
Gas chromatography is a term used to describe the group of analytical separation techniques used
to analyze volatile substances in the gas phase. In gas chromatography, the components of a
sample are dissolved in a solvent and vaporized in order to separate the analytes by distributing
the sample between two phases: a stationary phase and a mobile phase. The mobile phase is a
chemically inert gas that serves to carry the molecules of the analyte through the heated column.
Gas chromatography is one of the sole forms of chromatography that does not utilize the mobile
phase for interacting with the analyte. The stationary phase is either a solid adsorbent, termed
gas-solid chromatography (GSC), or a liquid on an inert support, termed gas-liquid
chromatography (GLC).
The efficient separation of compounds in GC is greatly depends on the compounds that travel
through the column at different rate. The rate of compounds that travel through a particular GC
system is depends on several factors which are volatility of compounds, column temperature,
carrier gas flow rate and length of the column. Component that has low boiling points will elute
faster than the high boiling point components. Next, raising the column temperature and the
carrier gas flow rate will increase the speed which all the compounds move through the column.

Length of column also affects the efficient separation in which the longer the column, the longer
it will take for the column to elute but it is important to get better separation.
For this experiment, the detector used in GC is flame ionization detector. It is one of the most
common detectors used in GC due to its insensitivity towards noncombustible gases such as
carbon dioxide and noble gases. Using air-hydrogen flame, it pyrolyzes organic compounds into
ions and electrons. The charges are then detected by the detector.
We will look further on how to measure how good the separation is. It can be measured by
resolution, Rs. The Resolution Rs of a column tells us how far apart two bands are relatives to
their widths. The resolution provides a quantitative measure of the ability of the column to
separate two analyte. It can be deduced in formulas below:

Rs =

2Z
WA +WB

t R)A - (tR)B]

=
2

(tR)A = retention time for component that elute later


(tR)B = retention time for component that elute first
WA = peak width for component that elute later
WB

= peak width for component that elute first

Note that if RS is > 1.5, it gives an essential complete separation of two neighboring solutes
which provide baseline resolution. Whereas a RS = 0.75 does not provide a good resolution which
show overlap of the two peaks.

ANALYTICAL PROCEDURE
a. Instrument set-up
Injection port
Injection port temperature
Column temperature
Carrier gas flow rate
Detector temperature

: Split (40:1)
: 250 0 C
: 210 0 C
: 30 cm sec-1
: 250 0 C

b. Effect of carrier gas flow rate on isothermal GC separation of methyl esters


i.

0.4L of standard mixture was injected isothermally at 2100C at carrier gas flow

ii.
iii.

rate of 30 cm sec-1.
The flow rate was increased to 50 cm sec-1.
The system was allowed to equilibrate for a few minutes and the standard

iv.

mixture was injected again.


The same procedure was repeated at flow rate of 70 cm sec-1.

c. Effect of column temperature on the isothermal GC separation of methyl esters.


i.

Using the optimum flow rate from part (b), then injected 0.4m standard mixture

ii.
iii.

isothermally but change the oven temperature at 1700C


Repeated the step by change the column temperature at 1900C.
The effect of column temperature on the separation, resolution and analysis time
was evaluated.

d. Separation of methyl esters using column temperature programming


i.
ii.
iii.

The standard mixture was injected at the optimal carrier gas flow rate using used
linear temperature ramp from 1000C to 2900C at optimal flow rate.
The separation of the compounds was observed
Modification of the temperature programming was made to improve the
resolution of the compounds

e. Identification each methyl ester individually to identify the various compounds in the
standard mixture using the optimized GC condition.

i.

The standard individual methyl ester compounds were injected with 0.4Lvolume

ii.

using the optimized GC conditions.


The standard individual methyl ester compounds are methyl laurate, methyl myristate,
methyl palmitate, methyl stearate and methyl linoleate.

RESULTS AND DATA


*Calculation of resolution is based on peak 2 and 3 as references.
A. Effects of the variation of the gas flow rate on the resolution:

Condition

Injection

Retention

Peak width

time for peak

for peak 2

2 and 3
2.242

and 3
0.0763

3.581

0.1451

2.742

0.0763

3.851

0.1452

Resolution

Average
resolution

8.910

30 cm/s
2100C
2

10.018

9.46

2.334

0.1000

10.459

50 cm/s

3.735

0.1679

2100C

2.332
3.742

0.1028
0.1710

10.2995

1.181

0.0434

7.7032

1.669

0.0833

1.178

0.0436

1.666

0.0833

1
70 cm/s

210 C

10.38

7.6549
7.68

From the above table, we can figure out that the optimized separation time for methyl ester
mixture is at 70 m/s of gas flow rate.

B. Effects of the variation of column temperature at optimized column temperature on the


resolution
Condition

Injection

Retention

Peak width

time for peak

for peak 2

2 and 3
2.210

and 3
0.1099

4.398

0.2877

2.205

0.1119

4.377

0.2253

3.430

0.1179

Resolution

Average
resolution

11.000

70 cm/s
1700C

12.883

9.46

70 cm/s

5.636

0.1994

1900C

3.432
5.649

0.1219
0.2216

13.918
12.908
10.38

1
70 cm/s
0

210 C

1.181

0.0434

1.669

0.0833

1.178

0.0436

1.666

0.0833

7.7032

7.6549
7.68

The optimized column temperature at 70m/s of gas flow rate is 210C column temperature
because it produced the resolution nearest to the ideal resolution value that is 1.5 and also shorter
analysis time.

C. Retention time of standard compounds of the methyl esters:

Standard compounds
Methyl laurate
Methyl myristate
Methyl palmitate

Retention time
1st injection

2nd injection

Average Rt

1.175
1.653
2.630

1.178
1.648
2.660

1.1765
1.6505
2.645

DISCUSSION

In part A of this experiment was to determine the optimum flow rate for being used which give
the optimum peaks of chromatogram to be run in GC analysis of standard mixture 1. It was done
by using three different flow rates in isothermal conditions which are 70 cm sec-1, 50 cm sec-1, 30
cm sec-1. These variations of mobile phase flow rates will affect the retention time of the
compounds. The higher the mobile phase flow rate, the shorter the retention time for the standard
mixture compared to the lower mobile phase flow rate which give longer retention time. But,

longer retention time will cause higher resolutions thus, lower the quality of separations of
chromatogram in this GC analysis. As the data shown above, we choose mobile phase flow rate
of 70 cm sec-1 as the most suitable flow rates for this experiment. It is because it has the lowest
resolution which is 7.68 nearer to 1.5 compared to the others. As 70 cm sec-1 was the highest
flow rates, it causes the analytes to be retained in the stationary phase shorter. Thus, it decrease
retention time and give good separation in the chromatogram.
Next, part B was discussed in determining the most suitable temperature for being used in this
experiment. We ran three different temperatures which are 210C, 190C and 170C. Based on
the data above it show that high column temperature gave short analysis time but some of the
earlier peaks may be overlapped while low column temperature produce better separations but
gave long analysis time. So, the most optimum oven temperature to be used in this experiment
was 210C. Usually, the oven temperature should be 50C higher than the average boiling points
of analytes in the mixture.Based on both testing, we found that the optimum condition in running
GC analysis for standard mixture 1 is 210C: 70 cm sec-1.
In part C, used the optimum condition, we ran individual standards of methyl esters in GC
analysis which are methyl laurate, methyl myristate, methyl palmitate, methyl linolate and
methyl stearate. By comparing the individual standards with the standard mixture 1, we can
conclude that peaks 1 represents solvent peaks, peaks 2 represents methyl laurate, peaks 3
represents methyl myristate while the last peaks represents methyl palmitate.
Temperature programming was an alternative in GC methods which being used in GC analysis of
standard mixture 2. It is used in overcoming problems that are connected with isothermal
conditions which can lower the retention time thus produce better separations. The best
resolution was 1.5 and above meanwhile if the resolution is lower than 1.5, it signifies that the
separation is poor and the peaks are overlapping to each other. Somehow, the retention time and
the quality of separation are much lower in temperature programming than in isothermal
condition of 210C: 70 cm sec-1. This might due to the usage of different GC instruments as for
temperature programming, we used auto samplers instead of normal injection.
Factor that determines the separations in GC analysis was the boiling points. The higher the
boiling points of the analytes, the faster it elutes which provide shorter retention time. Based on

the experiments, we can show that methyl laurate has the lowest boiling points whereas methyl
striate has highest boiling point. Another factor that determines separations in GC was polarity of
analytes. Since all the analytes in this experiment were all methyl esters which under ester group,
they consist about the same polarity which is semi-polar.
There are some precaution that we have to give attentions which are there should be no bubble in
the syringe during an injections. The bubble will cause shoulder in the peaks of chromatogram
and affect the separations of peaks. We also should focus on the technique during injections
which do not let the syringe expose at the surrounding too long. It will cause the analyte to
evaporate hence loss of sample.

CONCLUSION
The most optimum condition to be used in isothermal GC analysis for methyl esters is 210C: 70
cm sec-1. We found that in standard mixture 1, peak 1 represents methyl laurate, second peak
represents methyl myristate and the last peak represents methyl palmitate. In standard mixture 2,
the identified peaks are as follows; peak 1 represents methyl laurate, peak 2 represents methyl
myristate, peak 3 represents methyl palmitate, peak 4 represents methyl linoleate and the last
peak represents methyl stearate.

REFERENCES
Holler, F.J., Skoog, D.A., & Crouch, S.R. (2007). Principles of Instrumental Analysis. Belmont,
USA: Brooks/Cole.
Norashikin Saim, Ruziyati Tajudin, & Mardiana Saaid. (2012). Analytical Separation Methods
Laboratory Guide. Selangor, Malaysia : Penerbit Press Universiti Teknologi MARA.