TITLE: Gas Chromatography(GC): Optimization of Flow Rate and Column Temperature

ABSTRACT:
The separation of the compounds will travel faster through the column when column temperature
is high, volatility of compound is low, increasing the carrier gas flow rate and length of the
column is increase.

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

INTRODUCTION:
Efficient separation of compounds in GC is dependent on the compounds travelling through the
column at different rates. Method development always starts with a general consideration of the
sample. Method development is the process of determining what conditions are adequate or ideal
for analysis required. There are several factors that affect GC separation such as volatility of
compound, column temperature, the flow rate of gas through the column, length of the column,
column polarity and polarity of compounds. However, this experiment focuses on the first four
factors. The details for each factor are below:

1. Volatility of Compound: Low boiling components (High Volatility) will travel faster through the
column than high boiling point components. Boiling points of the different components is the
main factor in GC separation.

2. Column Temperature: Raising the column temperature speeds up the elution of all the
compounds in a mixture. Separation based on isothermal temperature or temperature
programming.
3. Flow Rate of The Gas through the Column: Speeding up the carrier gas flows will increases the
speed with which all compounds move through the column.

Length of The Column: The longer the column, the longer it will take all compounds to elute.
Longer columns are employed to obtain better separation (Higher N).
 The resolution of two chromatographic peaks is defined by:

Rs= [2(Tr2-Tr1)/ W1+W2]

Where Tr1 and are Tr2 retention times of the two peaks (peak 1 elutes first) and w2 is the
baseline width of the peaks. The Rs value indicates the quality of separation between two
adjacent peaks (Analytical Chemistry 7th Edition, p.615). Rs provide a quantitative measure of
the ability of the column to separate 2 analytes. Rs must be calculated to explore gas
chromatography, including retention time and resolution using a mixture of analyte so the all
four factors will be investigated well.

REAGENT AND SOLUTIONS:

There are 5 individual methyl esters compounds such as methyl laurate, methyl myristate, methyl
palmitate, methyl stearate and methyl linoleate. Standard mixture of methyl laurate (0.20 mg mL-
1
), methyl myristate (0.20 mg mL-1), methyl palmitate (1.0 mg mL-1), methyl stearate (0.70 mg
mL-1) and methyl linoleate(0.35 mg mL-1).

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

PROCEDURE:
a) The instrument is set up like below:
Injection port: Split (40:1)
Injection port temperature: 250oC
Column Temperature: 210oC
Carrier gas flow rate: 30 cm sec-1
Detector temperature: 250oC
b) Effect of carrier gas flow rate on isothermal GC separation of methyl esters.
0.4 µL standard mixtures are injected isothermally at 210oC 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 column temperature on the isothermal GC separation of methyl esters.

0.4 µL of standard mixture is injected isothermally at 170oC, followed by 190oC 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.

RESULTS:
*Calculation Of Resolution based on Peak 2 and Peak 3 as references.

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

Condition 1 Condition 2 Condition 3
Gas flow rate 30cm/s 50cm/s 70cm/s
Column 210oC 210oC 210oC
Temperature
Retention Peak area Retention Peak area Retention Peak area
time (pA*s) time (min) (pA*s) time (min) (pA*s)
(min)
Methyl laurate 1.927 41.52623 1.349 45.45727 0.976 43.42664
Methyl myristate 2.420 128.46342 1.811 98.73732 1.652 79.96885
Methyl palmitate 3.911 265.74643 2.898 129.83837 2.743 168.38438
Methyl stearate - 4.819 167.18466 3.381 111.75437
2. Effects on the variation of column temperature at optimized column temperature on the
resolution:
Condition 4 Condition 5
Gas flow rate 50cm/s 50cm/s
Column temperature 170oC 190oC
Retention time Peak area Retention time Peak area
(min) (pA*s) (min) (pA*s)
Methyl laurate 4.523 41.52531 2.796 46.43243
Methyl myristate - - 5.477 102.73898
Methyl palmitate - - 7.989 175.84429
Methyl stearate - - - -

3. Retention time of methyl esters using column temperature programming:

Column 6
Gas flow rate 50cm/s
Column temperature programming 100oC to 290oC
Retention time (min) Peak area (pA*s)
Methyl laurate 3.536 43.64355
Methyl myristate 4.785 122.76672
Methyl palmitate 6.563 173.84346
Methyl stearate 7.642 167.93526

Sample Calculation:

*Liquid form
Methyl Laurate = 0.20g/mol
Molecular weight = 214.5 g/mol
Density = 0.87g/mL
0.87𝑔 1𝑚𝐿 1𝑚𝑜𝑙
M1 = 𝑚𝑜𝑙 x1𝑋10−3 𝐿x214.35𝑔 = 4.059 mol/L
0.2𝑚𝑔 1𝑚𝐿 1𝑚𝑜𝑙 1𝑋10−3 𝑔
M2 = x1𝑋10−3 𝐿x214.35𝑔x = 9.331x10-4mol/L
𝑚𝐿 𝑚𝑔
M1V1= M2V2
(4.059 mol/L)V1 = (9.331x10-4 mol/L)(0.025 L)
V1 = 5.747x10-6L
= 5.747 𝜇L (dilute in diethyl diether)

*Solid form
Methyl palmitate = 1.0 mg/mL x 100mL = 100 mg
= 0.1 g (dilute in diethyl ether)

DISCUSSION:
The variation of the mobile phase flow rate affect the retention time of the compounds
which was slow mobile phase flow rate give better separation but very long time taken (Falwell,
1997). 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, gas chromatography including the concepts of
retention time and resolution using a mixture of methyl esters; methyl laurate, methyl myristate,
methyl palmitate and methyl stearate were investigated. The effects of column temperature and
flow rate on the separation of these compounds were observed. It was necessary to prepare all
the samples by diluting them with the solvent of diethyl ether and mixed them as a mixture. Six
different methods of various conditions were conducted on four samples. Three early methods
were run due to various gas flow rate followed by two differents column temperature and ended
with column temperature programming. According to mass transfer (C-term) of Van Deemter
plot, the higher the velocity of mobile phase, the worse the broadening becomes (Falwell, 1997).
From the experimental data, the increasing of flow rate of the gas (30m/s,50m/s and 70 m/s)
affect the analyte to elute faster but cause band broadening. Overall resolution in the mixture of
each methods were much higher than baseline resolution (1.5) which was well separated between
two peaks (Driscoll, 1985). Thus, all of the analytes were well separated but to make sure that
they are fully separated, a comparison of the analytes was made based on retention time and peak
area in the mixture. The most suitable flow rate was 50cm/s because the peak area for methyl
esters were much higher than peak area in 70cm/s. Although the retention time should be lower
which indicates faster elution but we considered the peak area of the analytes for all separation.

The column temperature also affects the separation resolution and the analysis time. High
column temperature will give short analysis time but some of the earlier peaks may be
overlapped while low column temperature produces better separation but will take very long
analysis time (Falwell, 1997). The optimum column temperature must be used in analysis time,
the optimum column temperature must be used in order to separate each compounds adequately.
The optimum gas flow rate for method four and five was 50cm/s. The elution time of the
analytes were much higher at low column temperature. In this case, 210oC of column
temperature was the best temperature to separate each of the compounds. Based on this
experiment, the best condition to separate methyl esters mixture was by using 50cm/s gas flow
rate at 210oC column temperature resulting adequate separation between compounds. Optimum
column temperature will produce better separation, high efficiency, good resolution and short
analysis time for the separation (Driscoll, 1985).

The column that was used in the GC was HP-5 capillary column. This column consist of
5% of phenyl group and this column slightly less non-polar because in order to separate the
standard methyl esters mixture which was slightly less non-polar compound. This stationary
phase will alow the mixture to retain in the column and to separate at their retention time. The
most non-polar analyte will less retain in column and elute first and vice versa to the slightly less
non-polar compounds (Falwell, 1997). The methyl laurate was first compound eluted followed
by methyl myristate, methyl palmitate and methyl stearate at optimum condition. According to
polarity of the compounds, methyl stearate is the most retain by the column and high less non-
polar compared with others.
Overall, optimum gas flow rate and optimum column temperature produce better
separation, high efficiency, good resolution and short analysis time for the separation. Because
the separation of gas chromatography is based on the boiling point of the compound, it can be
concluded that methyl laurate has the lowest boiling point and followed by methyl myristate. The
highest boiling point is methyl palmitate. Experimental error that should be avoid is during the
preparation of the samples because dilution is important to lowering the concentration and avoid
contamination of the samples.

CONCLUSION:
In conclusion, The optimum condition for the separation of the methyl esters was 70
m/s gas flow rate and 210°C of column temperature. The first peak after the solvent peak was
corresponds to methyl laurate followed by methyl myristate and then methyl palmitate.

REFERENCE:
Driscoll, J.N. REview of Photoionization Detection in Gas Chromatography: The first Decade.
Journal of CHromatographic Science , Vol 23. November 1985. 488-492.
Falwell, S.O. (1997) Modern gas chromatographic instrumentation, in Analytical
Instrumentation Handbook (ed. G.W. Ewing), Marcel Dekker, New York, Chapter 23.
Nor’ashikin S., Ruziyati T., Mardiana S. (2012), Analytical Separation Methods Laboratory
Guide (2nd edition).