EXPERIMENT 1:

Gas Chromatography (GC) : Op miza on of Flow Rate

and Column Temperature

NAME GEORGE ENGGONG AK VALENTINE

STUDENT ID 2022899586
LECTURER DR NORAINI KASIM
GROUP AS2453M1
OBJECTIVES :

1. To determine the op mum flow rate on isothermal GC separa on of methyl esters.

2. To determine the op mum column temperature on isothermal GC separa on of
methyl esters.

INTRODUCTION :

A gas chromatography (GC) instrument is used to separate analytes from their

compounds baed on vola lity. More vola le compounds elute faster and spend less me in
the column, resul ng in a shorter reten on me. The injec on technique is cri cal for avoiding
peak shoulder and broadening separa on. Because the samples are concentrated , split mode
injec on is used as an injec on method in this experiment. The flow rate and temperature are
the most important factors to consider in gas chromatography to achieve good resolu on and
sharp peak separa on of the analytes.

The efficiency of compound separa on by GC is determined by the rate at which the

compounds pass through the column, four factors influence GC systems performance, one of
which is compound vola lity, which mean that vola le compounds elute faster than
compounds with high boiling points. The temperature of the GC column is the next factor to
consider, as the higher the temperature of the column, the faster the compound separates in
the sta onary phase. The carrier gas flow rate, which increases as the carrier gas level rises,
also influences the rate of compound mobilisa on through the column. Finally, the longer the
column, the longer the analyte in it will migrate, requiring more me to be eluted. However,
the longer column, on the other hand will provide more separa on

Resolu on is the measured of how well separa on in the column. The formula of Resolu on
is :
PROCEDURES :

For this experiment, gas chromatography GC was used to determine the op mum flow
rate and temperature of methyl esters to achieve the best separa on and resolu on
condi ons. This experiment was also used to determine the various components of the
standard methyl ester mixture. A standard methyl ester is made up of three components:
methyl laurate, methyl myristate, and methyl palmitate. Split injec on with ra o of 40:1 was
used to set up the GC instrument. The flame ionisa on detector (FID) was used in this
experiment because it is suitable for methyl esters due to their vola lity.

Firstly, the effect carrier gas flow rate on isothermal GC separa on of methyl esters were
inves gated. 0.4 L of standard mixture was injected at 210 ℃ at a carrier gas flow rate of 30
cm sec-1 isothermally. A er the reading, the flow rate raised to 50 cm sec-1. Allowed the
system to equilibrate for a few minutes before injec ng the standard mixture again. The
procedure repeated at a flow rate of 70 cm sec-1. Then, the most suitable flow rate was
decided to be used in the second part of the experiment.

For the second part of the experiment, effect of column temperature on the isothermal
GC separa on of methyl esters were inves gated. Firstly, 0.4 µL standard mixture inject was
isothermally at 170℃, followed by 190℃ at the op mal carrier gas flow rate. A er the reading,
this factor was determined by evalua ng peaks, resolu on, and analysis me for each
temperature 170 ℃, 190 ℃ and 210 ℃.

Next, the third part of this experiment, separa on of methyl esters using column
temperature programming. Temperature programming refers to changing the temperature of
the column during chromatography analysis which provides a stable thermal environment for
the analysis. Standard mixture was injected at the op mal carrier gas flow rate and op mal
flow rate using a linear temperature ramp from 100 ℃ to 290 ℃.

Then, the resolu on was calculated by using the resolu on formula. If the resolu on was
not adequate, the temperature programming should be modified by increasing the
temperature to permits the higher resolu on of lighter compounds and sharp peaks for
heavier compounds, reducing the long run mes generated by heavier compounds. For the
last part of this experiment, iden fica on of components in methyl esters mixture. Each
methyl ester was injected individually by turn in the op mized temperature and flow rate. The
reading was compared to each other to iden fy the compound in the standard mixture.

Figure 1 Chromatogram of Standard Mixture, Temp 210,Flow 30 1st Injec on

Figure 2 Chromatogram of Standard Mixture, Temp 210, Flow 30, 4th Injec on
A. Effect of the varia on of gas flow rate on the resolu on

Peak 2 and 3 as reference

Condi on Injec on Reten on Time of Peak Width of Resolu on Average

Peak 2 and Peak 3 Peak 2 and Peak Resolu on
(min) 3 (min)

30 m/s 1st 2.674 4.127 0.0396 0.0712 26.227 27.008

210℃ 4th 2.671 4.123 0.0407 0.0638 27.789
50 m/s 3rd 1.574 2.423 0.0257 0.0458 23.748 23.920
210℃ 4th 1.571 2.419 0.0226 0.0478 24.091

70 m/s 2nd 1.115 1.716 0.0192 0.0372 21.312 21.164

210℃ 3rd 1.112 1.712 0.0200 0.0371 21.016

Peak 3 and 4 as reference

Condi on Injec on Reten on Time of Peak Width of Resolu on Average

Peak 3 and Peak 4 Peak 3 and Peak Resolu on
(min) 4 (min)

30 m/s 1st 4.127 5.573 0.0712 0.1131 15.69 17.87

210℃ 4th 4.123 5.566 0.0638 0.0802 20.04
50 m/s 3rd 2.423 3.268 0.0458 0.0727 14.26 14.41
210℃ 4th 2.419 3.265 0.0478 0.0684 14.56

70 m/s 2nd 1.716 2.313 0.0372 0.0605 12.22 12.34

210℃ 3rd 1.712 2.310 0.0371 0.0589 12.46
B. Effects on the varia on of column temperature at op mized column temperature on
the resolu on :
Peak 2 and 3 as reference

Condi on Injec on Reten on Time of Peak Width of Resolu on Average

Peak 2 and Peak 3 Peak 2 and Peak Resolu on
(min) 3 (min)

70 m/s 1st 1.093 1.670 0.0135 0.0277 28.00 28.01

210℃ 2nd 1.088 1.668 0.0146 0.0268 28.02
70 m/s 1st 1.378 2.574 0.0150 0.0319 51.00 45.86
190℃ 3rd 1.370 2.567 0.0191 0.0397 40.71

70 m/s 1st 1.964 4.677 0.0287 0.0711 54.37 56.24

170℃ 3rd 1.964 4.675 0.0240 0.0693 58.11

Peak 3 and 4 as reference

Condi on Injec on Reten on Time of Peak Width of Resolu on Average

Peak 2 and Peak 3 Peak 2 and Peak Resolu on
(min) 3 (min)

70 m/s 1st 1.670 2.237 0.0227 0.0321 18.963 19.41

210℃ 2nd 1.668 2.230 0.0268 0.0298 19.859
70 m/s 1st 2.574 3.820 0.0319 0.0469 31.624 28.413
190℃ 3rd 2.567 3.812 0.0397 0.0591 25.202

70 m/s 1st 4.677 7.713 0.0711 0.1348 29.490 29.35

170℃ 3rd 4.675 7.724 0.0693 0.1394 29.219
Effects on the Reten on me of standard compounds of methyl esters :

Standard Compound Reten on Time (min)

Methyl laurete 1.174
Methyl myristate 1.910
Methyl palmitate 2.658
Methyl linoleate 4.207
Methyl stearate 4.674

Sample calcula on :

a. To calculate resolu on
At 30ms-1 ,210 C

( . . )
𝑅𝑠 = =26.227
( . . )

b. To calculate average Rs
At 30ms-1 ,210 C
. .
=27.008

DISCUSSION
Reten on me (TR ) is the amount of me that elapses between the introduc on of
sample and the highest signal of a specific compound at the detector. Resolu on refer to the
ability to dis nguish between two signals. In chromatography, this is the ability to dis nguish
between two peaks.
A er obtaining the results, we proceed to calculate the resolu on to determine the
op mal condi ons which include the temperature and flow rate of the mobile phase. We
begin analyzing the op mum flow rate, using 30 ms-1 , 50 ms-1 and 70 ms-1 with an isothermal
temperature 210°C. We observed that at 70 ms-1, 210°C has the lowest resolu on for both
peak 2 and peak 3, and peak 3 and peak 4, which is the best for our me analysis. Therefore ,
we choose 70 ms-1 as our op mum flow rate. For temperature, we use 70 ms-1 as constant
flow rate and vary the temperature between 170 , 190 and 210 . We observe that at 210 has
the lowest resolu on for both peak 2 and 3, and peak 3 and peak 4, therefore we choose 210
as our op mum temperature.
Hence at 70 ms-1, 210 C is the op mum condi on for our analysis to be done prac cally.
Comparison of our sample injec on with each standard methyl ester shows that at 70 ms-
1,210 C chromatogram has the closest results with the standard methyl ester’s reten on me.
However , there are poten al errors in this experiment such as slow injec on which could lead
to bigger dead me (Tm) and cause the analysis to be done slower . addi onally, it is important
to choose the appropriate injec on mode when analysing concentrated methyl esters, as we
have used split mode to ensure the analysis is accurate for only the ra o taken from the
sample.

CONCLUSION
In a nutshell, the op mum gas carrier flow rate and op mum column temperature were
obtained which are 70 ms-1 and 210 °C respec vely. 70ms-1 and 210 °C are also the most
suitable methyl esters to obtain an ideal resolu on separa on. Overall the experiment was
successful in achieving its objec ves.

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3. Venton, D. B. (2018). JoVE Science Educa on. Retrieved from Gas Chromatography
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