UITM ARAU PERLIS

Experiment 1

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

Name : Nur Izzati Binti Othman Basri

Group : RAS2452C
Matrix No. : 2020991379
Date Of Experiment : 28 April 2020
Date of Submission : 5 May 2020
Lecturer’s Name : En Mohd Lias
Objectives
To explore gas chromatography, including the concepts of retention time and
resolution using a mixture of methyl esters such as methyl myristate, methyl laurate
and methyl palmitate. To investigate the effects of column temperature and flow rate
on the separation of these compound.

Abstract
The factors which contribute to the efficient separation of mixture of methyl esters are
examined. These factors included the affect of carrier gas flow rate on the isothermal
and temperature programming GC separation of methyl esters. The elution rate of a
compound depends on volatility of compound, column temperature, carrier gas flow
rate and length of the column of the particular GC system. This experiment is
examined gas chromatography, including the concepts of retention time and
resolution using a mixture of methyl esters which were methyl laurate (0.20mg/ml),
methyl myristate (0.20mg/ml) and methyl palmitate (1.0mg/ml). At the end of the
experiment the resolution (Rs) is measured to know of how well species are separated.

Introduction
The rate at which compound travels through a particular GC system depends on
several factors that include :
 Volatility of compound : Low boiling (volatile) compounds will travel faster
through the column than high boling compounds.
 Column temperature : Raising the column temperature speeds up all the
compounds.
 Carrier gas flow rate : Increasing the carrier gas flow increases the speed which
all compounds move through the column.
 Length of the column : the longer the column, the longer it will take for all
compounds to elute. Longer columns are employed to obtain better separation.
Resolution
Resolution (Rs) is a measure of how well species are separated. The Rs of two species,
A and B, is defined as :
Where ∆z is the separation between peaks A and B, and WA and WB are the widths at
the base of peaks A and B, respectively. Acceptable resolution is on the order of
1.0<Rs<1.5, and baseline resolution between two peaks requires an Rs>1.5.

Reagents and Solutions

a. Individuals methyl esters compounds : methyl laurate, methyl myristate, methyl
palmitate, methyl stearate, methyl oleate and methyl linoleate.
b. Standard mixture : methyl laurate (0.20mg/ml), methyl myristate (0.20mg/ml),
methyl palmitate (1.0mg/ml)

Instrument
Gas Chromatography (Agilent Technologies 6890 N ) equipped with flame ionisation
detector (FID) and 30m x 250 micrometer HP5-MS capillary column.

Analytical Procedure
a. Instrument set-up (may vary depending on instrument ):
Injection port : Split (40:1)
Injection port temperature : 250℃
Column temperature : 210℃
Carrier gas flow rate : 30 cm/sec
Detector temperature : 250℃

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

esters.
0.4 µL standard mixtures are injected isothermally at 210℃ at carrier gas flow rate of
30 cm sec-1. The flow rate is increased to 50 cm sec-1. The system is allowed to
equilibrate for a few minutes before injecting the standard again. The procedure is
repeated at flow rate 70 cm sec-1. The optimize flow rate is chose to continue with the
next steps.

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

esters.
 0.4 µL of standard mixture is injected isothermally at 170℃, followed by 190℃ at
optimal carrier gas flow rate. The effects of column temperature on separation,
resolution and analysis time are evaluated.

d. Identification of components in methyl esters mixture.

Each of methyl ester is injected individually to identify the various compounds in the
standard mixture using optimized GC conditions.

e. Separation of methyl esters using column temperature programming

Standard mixture is injected at the optimal carrier gas flow rate using temperature
programming, 190℃(1 min) to 210℃ at 30℃/min and 210℃ to 230℃ at 30℃/min.

Results

Table 1 : Effects on the variation of the gas flow rate on the resolution

Condition Injection Retention Time Peak Width Resolution Average

Flow rate/temperature Resolution
Peak 2 Peak 3 Peak 2 Peak 3
30ms /210℃
-1
1 4.570 6.491 0.0467 0.0638 34.769 35.064
2 4.568 6.488 0.0450 0.0636 35.359
50ms-1/210℃ 1 2.740 3.899 0.0351 0.0419 30.104 31.884
2 2.738 3.896 0.0304 0.0384 33.663
70ms-1/210℃ 1 1.956 2.789 0.0288 0.0376 25.090 25.306
2 1.959 2.791 0.0314 0.0338 25.521

Calculation :
For 30ms-1/210℃ :
1st Injection
Rs = 2 (6.491-4.570) / (0.0467 + 0.0638 ) = 34.769
2nd Injection
Rs = 2 (6.488-4.568) / (0.0450 + 0.0636 ) = 35.359
Average Resolution = ( 34.769 + 35.359 ) / 2 = 35.064

For 50ms-1/210℃ :
1st Injection
Rs = 2 (3.899-2.740) / (0.0351 + 0.0419 ) = 30.104
2nd Injection
 Rs = 2 (3.896-2.738) / (0.0304 + 0.0384 ) = 33.663
Average Resolution = ( 30.104 + 33.663 ) / 2 = 31.884

For 70ms-1/210℃ :
1st Injection
Rs = 2 (2.789-1.956) / (0.0288 + 0.0376 ) = 25.090
2nd Injection
Rs = 2 (2.791-1.959) / (0.0314 + 0.0338 ) = 25.521
Average Resolution = ( 25.090 + 25.521 ) / 2 = 25.306

Table 2 : Effects on the variation of column temperature at optimized column

temperature on the resolution

Condition Injection Retention Time Peak Width Resolution Average

Temperature/flow rate Resolution
Peak 2 Peak 3 Peak 2 Peak 3
170℃/70ms -1
1 3.644 7.127 0.0390 0.0737 61.810 60.871
2 3.631 7.116 0.0445 0.0718 59.931
190℃/70ms-1 1 2.578 4.279 0.0333 0.0558 38.182 36.747
2 2.579 4.281 0.0356 0.0608 35.311
210℃/70ms-1 1 1.956 2.789 0.0288 0.0376 25.090 25.306
2 1.959 2.791 0.0314 0.0338 25.521

Calculation :

For 170℃/70ms-1 :
1st Injection
Rs = 2 (7.127-3.644) / (0.0390 + 0.0737 ) = 61.810
2nd Injection
Rs = 2 (7.116-3.631) / (0.0445 + 0.0718 ) = 59.931
Average Resolution = ( 61.810 + 59.931 ) / 2 = 60.871

For 190℃/70ms-1 :
1st Injection
Rs = 2 (4.279-2.578) / (0.0333 + 0.0558 ) = 38.182
2nd Injection
Rs = 2 (4.281-2.579) / (0.0356 + 0.0608 ) = 35.311
Average Resolution = ( 38.182 + 35.311 ) / 2 = 36.747
For 210℃/70ms-1 :
1st Injection
Rs = 2 (2.789-1.956) / (0.0288 + 0.0376 ) = 25.090
2nd Injection
Rs = 2 (2.791-1.959) / (0.0314 + 0.0338 ) = 25.521
Average Resolution = ( 25.090 + 25.521 ) / 2 = 25.306

Table 3 : Retention time of standard compounds of methyl esters

Standard Compound Retention Time (min)

Methyl Laurate 1.916
Methyl Myristate 2.692
Methyl Palmitate 4.289
Methyl Linoleate 6.809
Methyl Stearate 7.495

Discussion

An analytical procedure is developed to test a defined characteristic of the

substance against established acceptance criteria for that characteristic. In the
development of a new analytical procedure, the choice of analytical instrumentation
and methodology should be based on the intended purpose and scope of the analytical
method.
Optimizing gas chromatography (GC) is crucial in improving laboratory
efficiencies and analytical performance. This experiment is examined gas
chromatography, including the concepts of retention time and resolution using a
mixture of methyl esters which were methyl laurate (0.20mg/ml), methyl myristate
(0.20mg/ml) and methyl palmitate (1.0mg/ml). In this experiment, the methyl ester
was injected individually to identified various components in standard mixture using
optimized GC conditions.
The variation of the mobile phase flow rate affect the retention time of the
compounds which is slow mobile phase flow rate give better separation but long time
taken. It means, high flow rate will shorten the analysis time but will cause
broadening due to mass transfer (C-term) in Van Deemter Plat because the solute does
not fully interact with the stationary phase. To reduce the analysis time and produce
better separation, the optimum gas flow rate must be used. In this experiment, the
optimum mobile phase flow rate is 70 m/s that give good resolution of 25.31
compared to others that are far from the ideal resolution value which is 1.5.
The column temperature also affects the separation resolution and the analysis
time. Increasing the temperature will send the vapors through the column faster,
which will lower the retention time and worsen the resolution. Lowering
the temperature or flow rate increases retention times and broadens the peaks. The
optimum column temperature must be used in order to separate each compounds
adequately. 210℃ is the best column temperature to separate each of the compounds.
Based on this experiment, the best condition to separate the methyl ester mixture is by
using 70 m/s gas flow rate at 210℃ column temperature thus give adequate separation
between compounds and shorter analysis time.
The most important benefit of temperature programming in gas chromatography
(GC) is the substantial reduction of analysis time compared to isothermal GC.
Temperature programming permits the higher resolution of lighter compounds and
sharp peaks for heavier compounds, reducing the long run times generated by heavier
compounds. Then, at optimum flow rate, standard mixture was injected at optimum
carrier flow rate using temperature range in 100℃ to 290℃ to improve resolution of
compounds.

Conclusion

Optimum gas flow rate and optimum column temperature produce better separation,
high efficiency, good resolution and short analysis time for the separation. The
optimum condition for the separation of the methyl esters is 70 m/s gas flow rate and
210°C of column temperature. The first peak after the solvent peak is corresponds to
methyl laurate followed by methyl myristate and then methyl palmitate.

References

1. Nor’ashikin S., Ruziyati T., Mardiana S. (2012), Analytical Separation Methods

Laboratory Guide (2nd edition).
2. Matt, (2011), Gas Chromatography (GC), Article of Organic Chemistry Practice
Problems and Problem Sets (Mendel Set).
3. Dr Santosh kumar, (2016), GC Method Development and validation, publication
of Jiwaji University.