EXPERIMENT 1: GAS CHROMATOGRAPHY (GC): OPTIMIZATION OF FLOW

RATE AND COLUMN TEMPERATURE

OBJECTIVES

1. To study the effects of column temperature and flow rate by carrier gas through the
column on the resolution of the chromatography.
2. To study the retention time of standard compounds of the methyl esters.

INTRODUCTION

Gas chromatography (GC) is an analytical tool that is used to isolate and then detect the
chemical components of a sample mixture to determine their presence or absence and/or how
much is present. These chemical elements are typically compounds or gases that are organic.
In gas chromatography, the components of a sample are dissolved in a solvent and vaporized
by distributing the sample between two stages to isolate the analytes, which are the stationary
phase and the mobile phase. The mobile phase is a chemically inert gas that is used to move
the analyte's molecules through the heated column. Meanwhile, the stationary phase is either
a solid adsorbant, called gas-solid chromatography (GSC), or a liquid on an inert support,
named gas-liquid chromatography (GLC).

Efficient compound separation in GC depends on the compounds moving at different rates

through the column. Creation of methods often begins with a general sample consideration.
The creation of methods is the process of deciding what conditions are appropriate or optimal
for the necessary study. GC separation is influenced by many variables, such as compound
volatility, column temperature, gas flow rate through the column, column length, column
polarity, and compound polarity. However, this experiment focuses on these factors:

1. Column Temperature: Increasing the temperature of the column accelerates the

elution of all the compounds in a mixture. Separation is based on isothermal
temperature or temperature programming.

2. Flow Rate of The Gas through the Column: Speeding up the carrier gas flows
will increase the speed with which all compounds move through the column.
PROCEDURES

a. Isothermal elution was set up with condition of:

Injection port: split (40:1)
Injection port temperature: 250°C
Column temperature: varies (170°C, 190°C, 210°C)
Carrier gas flow rate: varies (30m/s, 50m/s, 70m/s)
Detector temperature: 250°C

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

1. The standard mixture was injected with 0.4μL at 210°C isothermally and 30m/s of
gas flow rate.
2. For the second injection with 0.4μL volume of standard mixture, the gas flow rate
was increased to 50m/s with same temperature of 210°C.
3. The same procedure was repeated with 70m/s gas flow rate.
4. The resolution of 3 different flow rates at the same temperature was calculated
and compared to determine which gas flow rate give the better separation of the
compounds in the standard mixture.

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

1. By using the best gas flow rate from the above procedure, the same standard
mixture with 0.4μL was injected into the column isothermally at 170°C and
followed by 190°C.
2. The resolution of each chromatogram was calculated and was compared based on
the resolution and the analysis time.

d. Identification of components in methyl esters mixture:

1. The standard individual methyl ester compounds were injected with 0.4μL volume
using the optimized GC conditions.
2. The standard individual methyl ester compounds are methyl laurate, methyl
myristate, methyl palmitate, methyl stearate and methyl linoleate.
RESULTS

A. Effects of the variation of the gas flow rate on the resolution

Condition Injection Retention time Peak width of Resolution Average

of peak 2 and peak 2 and 3 resolution
3 (min) (min)
1 4.570, 6.491 0.0467, 0.0638 34.769
30m/s, 35.064
2 4.568, 6.488 0.0450, 0.0636 35.359
210°C
1 2.740, 3.899 0.0351, 0.0419 30.104
50m/s, 31.884
2 2.738, 3.896 0.0304, 0.0384 33.663
210°C
1 1.956, 2.789 0.0288, 0.0376 25.090
70m/s, 25.306
210°C 2 1.959, 2.791 0.0314, 0.0338 25.521

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

the resolution.

Condition Injection Retention time Peak width of Resolution Average

of peak 2 and peak 2 and 3 resolution
3 (min) (min)
1 3.644, 7.127 0.0390, 0.0737 61.810
70m/s, 60.871
2 3.631, 7.116 0.0445, 0.0718 59.931
170°C
1 2.578, 4.279 0.0333, 0.0558 38.182
70m/s, 36.74
190°C 2 2.579, 4.281 0.0356, 0.0608 35.311
1 1.195, 6.809 0.0355, 0.0765 100.25
70m/s, 60.042
210°C 2 1.204, 1.919 0.0212, 0.0509 19.834

C. Retention time of standard compounds of the methyl esters

Standard compound Retention time (min)

Methyl laurate 1.201
Methyl myristate 1.204
Methyl palmitate 1.216
Methyl linoleate 1.195
Methyl stearate 1.203

DISCUSSION
Gas chromatography is a term used to describe the separation techniques used to analyse
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 the mobile phase and the stationary phase. The mobile phase is a chemically
inert gas that is used to move the analyte's molecules through the heated column. A glass
column filled with silica that is covered with a liquid goes through the mobile process.
Materials that are less soluble in the liquid can elute more easily with greater solubility than
the material. The rate of compound travel in the column depends on the compound volatility,
the temperature of the column, the mobile phase flow rate and the column length. The solvent
time in the column will be limited by high column temperatures and a high mobile phase flow
rate. Longer column length will take longer to elute all compounds, but due to a high number
of theoretical plates and smaller plate height, it will provide better separation. The liquid
stationary phase is adsorbed in a thin layer onto a solid inert package or immobilized on the
capillary tubing walls. In a capillary column, the tubing walls are coated or bonded with the
stationary phase liquid.

In this experiment, the technique proposed was used in isothermal gas

chromatography with split injection, because the analyte concentration is high. Isothermal gas
chromatography is an analysis in which the column temperature during the analysis is kept
constant. Resolution (Rs) is used to explain how well they distinguish the organisms in the
mixture. The ideal resolution is 1.5, which shows ample differentiation between species.
Lower than 1.5 resolutions are when the two species are not fully separated, whereas longer
analytical time would be needed for too high resolution.

As we can see, the higher the flow rate, the shorter time it takes to elute the
samples. The peak width of the samples also decreases as the flow rate of the
chromatography increases. This means that both the retention time and the peak width in
indirectly proportional to the flow rate. We can also see that as the flow rate increases, the
resolution number decreases as gets closer to 1.5. As the resolution is so high, we can assume
that there have been errors while conducting this part of the experiment. The error that has
occurred may be that the time taken to conduct the experiment is too short and that a longer
analytical time is needed.
 On the other part of the experiment, the column temperatures were being manipulated.
As the temperature was increased, the retention time decreases and so does the peak width.
But, on the condition where it is 210°C, only two peaks appeared. This may be caused by
some error that may have taken place when handling the equipment. Among the errors that
might the cause to this inefficiency are Incorrect gas flow rates to a flame ionization detector,
using the wrong syringe or heating a column without any carrier gas flow. If these were
avoided, the results may show more peaks than it did.

Furthermore, it is also established that methyl linoleate has the fastest retention time
followed by methyl laurate, methyl stearate and methyl myristate respectively. This means
that the slowest retention time was by methyl palmitate.
CONCLUSION

By the end of the experiment, the effects of column temperature and flow rate by carrier gas
through the column on the resolution of the chromatography were able to be studied. The
higher the column temperature, the lower the resolution. This is the same as the flow rate.
The higher the flow rate, the lower the resolution. Next, the retention time of standard
compounds of the methyl esters were also able to be investigated. The result shows that the
fastest retention time is methyl linoleate and the slowest retention time is for methyl
palmitate.