BACHELOR OF SCIENCE (HONOR) APPLIED CHEMISTRY (AS245)

ANALYTICAL SEPARATION METHOD

CHM510

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

AND COLUMN TEMPERATURE

NAME NURANISAH NADIAH BINTI MOHD NIZAM

STUDENT ID 2022855638

GROUP MEMBERS
1) NUR AMIRAH BALQIS BINTI MOHD KOSIM
(2022800388)
2) AINUL SYARAFANA BINTI SHAMSUL HAYAT
(20224942820)
3) NURATHIRAH NABIHAH BINTI SAIRI (20224942820)
4) NUR HIDAYAH MOHD NAJIB (2022

CLASS AS2452S1

NAME OF LECTURER DR WAN NAZIHAH WAN IBRAHIM

a. Introduction

Gas chromatography is a method of separating chemical compounds that uses variations in the
partitioning behaviour between a flowing mobile phase and a stationary phase to do so. For
hydrocarbons like butane or hexane, the flame ionisation detector (FID) is the most sensitive
gas chromatographic detector.

The sample solution is injected into the machine and then enters a gas stream that carries it into
a separation tube called a "column," according to the gas chromatography principle. The so-
called carrier gas is either nitrogen or helium. Inside the column, the different parts are divided.
The detector counts the components as they leave the column. An injection of a standard sample
with a known concentration into the instrument is necessary to measure a sample with an
unknown concentration. To determine the concentration, the test sample's peak retention time
and area are compared to the standard samples.

Compounds have to pass through the column at various rates in order for the GC to separate
them effectively. Considering the sample broadly is always the first step in developing a
method. Determining whether conditions are suitable or appropriate for the required analysis
is the process of developing a method. The volatility of the compound, the column temperature,
the velocity of gas flow through the column, the length of the column, column polarity, and the
polarity of the compound are some of the elements that affect GC separation. However, the
first four elements are the main focus of this experiment. Below are specifics for every aspect.
1. Volatility of compound

2. Column temperature

3. Flow rate of the gas through the column

4. Length of the column

The resolution of the two chromatographic peaks is calculated using equation:

Rs = [ 2(tr2 – tr1) / W2 + W1)

b. Objectives

To study the volatility of compound, the effect of the length of the column, the effect of column
temperature and flow rate by carrier gas through the column

c. Apparatus

Reagents and Solutions

a. Individual methyl esters compounds: methyl laurate, methyl

myristate, methyl palmitate, methyl stearate and methyl linoleate.

b. Standard mixture of methyl laurate (0.20 mg mL-1), methyl myristate (0.20 mg mL-1) and
methyl palmitate (1.0 mg mL-1), methyl stearate (0.70 mg mL-1) and methyl linoleate (0.35
mg mL-1).
Instrument

Gas chromatograph (Agilent Technologies 6890N) equipped with flame ionization detector
(FID) and 30 m x 250 µm x 0.25 µm HP5- MS capillary column

Analytical Procedure

a. Instrument set-up (may vary depending on instrument):

Injection port: Split (40:1)

Injection port temperature: 250 oC

Column temperature: 210˚C

Carrier gas flow rate: 30 cm sec-1

Detector temperature: 250 ˚C

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

methyl esters.

1. 0.4 μL standard mixture was injected isothermally at 210˚C at carrier gas flow rate of
30 cm sec-1.
2. The flow rate was increased to 50 cm sec-1.
3. A few minutes for the system was allowed to equilibrate and the standard mixture was
injected again.
4. The same procedure was repeated at flow rate of 70 cm sec-1. Which flow rate is most
suitable for this separation?

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

1. 0.4 μL standard mixture was injected isothermally at 170˚C, followed by

190˚C, at the optimal carrier gas flow rate. The effect of column temperature was evaluated on
the separation, resolution, and analysis time.

2.. Methyl esters was separated using column temperature programming. Standard mixture was
injected at the optimal carrier gas flow rate using a linear temperature ramp from 100˚C to
290˚C at optimal flow rate. Comment on the separation of the compounds. If the resolution is
not adequate for your needs, attempt a modification to the temperature programming in order
to improve the resolution of the compounds.

3. Components in methyl esters mixture were identified. Each methyl ester was injected
individually to identify the various compounds in the standard mixture using the optimized GC
condition
D. Data and results

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

Condition Injection Retention Time Peak Width Resolution Average

of Peak 2 & of Peak 2 & Resolution
Peak 3 Peak 3
30 cm sec-1, 1 4.385 0.0683 21.7828 21.5528
2100C 6.120 0.0910
2 4.401 0.0738 21.3227
6.142 0.0895
50 cm sec-1, 1 2.631 0.0531 17.6014 17.6735
2100C 3.673 0.0653
2 2.631 0.0513 17.7455
3.670 0.0658
70 cm sec-1, 1 1.886 0.0428 14.2718 14.7491
2100C 2.621 0.0602
2 1.871 0.0410 15.2263
2.611 0.0562

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

Condition Injection Retention Peak Width Resolution Average
Time of of Peak 2 & Resolution
Peak 2 & Peak 3
Peak 3
70cm sec-1, 1 5.841 0.1603 18.9428 26.2226
1700C 10.016 0.2805
2 5.647 0.0888 33.5023
9.612 0.1479
70cm sec-1, 1 3.096 0.0735 19.2845 18.8005
1900C 4.794 0.1026
2 3.112 0.0757 18.3164
4.820 0.1108
70cm sec-1, 1 1.931 0.0521 9.8074 9.8907
2100C 2.695 0.1037
2 1.931 0.0531 9.9739
2.695 0.1001
3. Retention time of standard compounds of methyl esters
Standard Compound Retention Time (min)
Methyl Laurate 1.173
Methyl Myristate 1.901
Methyl Palmitate 2.662
Methyl Linoleate 4.204
Methyl Stearate 4.667

Calculation
2 (t R2 − t R1 )
Rs =
W1 + W2
Condition of 30cm sec-1 gas flow rate at 2100C
2 (6.120−4.385)
First injection: 𝑅𝑅𝑅𝑅 = = 21.7828
0.0683+0.0910

2 (6.142−4.401)
Second injection: 𝑅𝑅𝑅𝑅 = = 21.3227
0.0738+0.0895
21.7828−21.3227
Average 𝑅𝑅𝑅𝑅 = 2
= 21.5528

e. Discussion
As shown by the findings above, this can be used to show how changes in the mobile phase
flow rate at constant column temperature will impact the analytes' retention times in the
standard combination. Due to the carrier gas's diffusion, slower flow rates will result in better
analyte separation but relatively lengthy analysis times. While the analysis will be shortened at
larger flow rates, a broad peak will result from mass transfer as predicted by Van Deemter
theory. This occurs as a result of the analytes' incomplete interaction with the stationary phase.

In this experiment, the best separation is achieved at a flow rate of 70 m/s, which results in a
resolution of 21.5528. However, this resolution needs to be improved more till it is nearly at
the 1.5 acceptable resolution.

The retention period of the analytes in the standard mixture will also change depending on the
column temperature. Even if the mixture's analytes are better separated at lower temperatures,
analysis takes longer. The experiment's analysis time will be shortened and the peaks'
resolution will be closer to 1.5 if a higher temperature is employed. The best separation and
quickest analysis times in this experiment were achieved at a column temperature of 210oC.
Additionally, if the temperature is higher than the analytes' boiling points, the resolution will
be too low, which will result in overlapped analyte peaks in the mixture.

Based on the results of this experiment, a flow rate of 70 m/s and a column temperature of
210˚C were found to be the optimal conditions for running the analysis on the standard mixture.
It is known as optimum condition

f. Conclusion

A carrier gas flow rate of 70 m/s and a column temperature of 210˚C are ideal for the separation
of methyl esters. Methyl laurate is the first peak on the chromatogram, followed by methyl
myristate and methyl palmitate.

g. Reference
• Gas Chromatography- Definition, Principle, Parts, Steps, Uses
https://microbenotes.com/gas-chromatography/
• Gas Chromatography
https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Module
s_(Analytical_Chemistry)/Instrumentation_and_Analysis/Chromatography/Gas_Chro
matography