1.

0 INTRODUCTION

Gas chromatography is a technique generally used to separate gases in a gaseous solution.

The more common technique is gas-liquid chromatography in which the stationary phase is a
porous solid covered with an absorbing liquid. Gas-liquid chromatography or GLC is used to
separate a wide variety of organic compounds. The basic requirements for GLC are that the
sample be volatile and that it not decompose in the vaporization process. Some factors that
affect the rate of which a compound travels through the GC column are:

 Flow rate of carrier gas

 Temperature of column
 Length of column
 Volatility of compound

Resolution

Resolution is the degree of separation of 2 species expressed in terms of retention time and
peak widths. The Rs of two species, A and B is defined as:

2Z 2[(t R )B − (t R )A ]
Rs = =
WA + WB WA + WB

Where Z is the separation between peaks A and B; and W A and W B are the widths at
the base of peaks A ad B, respectively. Fitting resolution is on the order of 1.0<Rs<1.5, and
baseline resolution between two peaks requires Rs>1.5.

The objective of this experiment is to discover gas chromatography, including the

concepts of retention time and resolution using a mixture of methyl esters; methyl laurate,
methyl myristate, methyl palmitate, methyl stearate, methyl oleate and methyl linoleate. The
outcomes of column temperature and flow rate on the separation of these compounds will be
examined.
2.0 METHODOLOGY

There were two types of samples injected into the column. The first sample set was individual
methyl esters compound or individual standard which were methyl laurate, methyl myristate,
methyl palmitate, methyl stearate and methyl linoleate. The second sample was a standard
mixture 1 consisted of first three individual standard; methyl laurate, methyl myristate and
methyl palmitate. Standard mixture 1 was used for the isothermal method. The instrument
used in the experiment was Gas Chromatography (Agilent Technologies 6890) equipped with
flame ionisation detector (FID) and 30 cm × 250 m × 0.25 m HP5 – MS capillary column.
Prior to sample injection, the instrument was set up accordingly. The injection port was made
sure in split mode with the ratio of 40:1 with the temperature of 250C. 250C was chosen to
avoid volatilization of liquid stationary phase. The column temperature was set at 210C and
the carrier gas flow rate was at 70 cm per second. The carrier gas which was also the mobile
phase used was nitrogen gas. The temperature of the detector was made sure to be at 250C.

Before sample was injected into the injector port, the GC was let to prep run for
approximately one to two minutes until it reached ‘ready to run’ status. 0.4 L standard mixture
1 was injected into the injection port efficiently by using a syringe rinsed with ether. The method
was run isothermally at 210C with a carrier gas flow rate of 70 cm per second. The analysis
was done twice to get the optimum resolution. Afterwards, another two flow rates, 50 and 30
cm per second were proceeded at the same temperature with another wo reproducible results
for each flow rate. By calculating the resolution between the two reproducible results for each
flow rate, the most suitable flow rate could be found and considered as Optimal Flowrate. After
optimal flowrate was obtained, the experiment as continued with another two temperature by
using the optimum flowrate. By calculating the resolution again, the optimal temperature was
obtained. Then, optimize condition was obtained so that the best resolution and the best
separation will be able to be chosen. Subsequently, injection on each five individual methyl
esters compounds was applied for once for each compounds for identification of components.
3.0 RESULTS AND DISCUSSION

a) Effect of flow rate

In this experiment, the effect of flow rate on the separation of methyl ester compounds was
learned by injecting the standard mixture at constant temperature (210C) using different flow
rate (30, 50 and 70 cm per second). Separation using flowrate 70 cm/s had shorter retention
times compared to other flowrates. From this, we can conclude that the higher the flowrate of
carrier gas, the shorter the retention time and the faster the component elute from the column.
By comparing the resolution between the first injection and second injection using all
the flowrates, the most optimum flowrate was 70 cm per second. Based on the Figure 1 and
2, the peaks are well separated compared to chromatograms of flowrate 50, Figure 3 and 4
and 30 cm/s, Figure 5 and 6.

2
3

Figure 1: First chromatogram of methyl ester mixtures. 1=methyl laurate, 2=methyl myristate and 3=methyl
palmitate separated at temperature of 210 C and flowrate at 70 cm per second.

2
3

Figure 2 Second chromatogram of methyl ester mixtures. 1=methyl laurate, 2=methyl myristate and 3=methyl
palmitate separated at temperature of 210 C and flowrate at 70 cm per second.
 1

2
3

Figure 3: First chromatogram of methyl ester mixtures. 1=methyl laurate, 2=methyl myristate and 3=methyl
palmitate separated at temperature of 210 C and flowrate at 50 cm per second.

2
3

Figure 4: Second chromatogram of methyl ester mixtures. 1=methyl laurate, 2=methyl myristate and 3=methyl
palmitate separated at temperature of 210 C and flowrate at 50 cm per second

2
3

Figure 5: First chromatogram of methyl ester mixtures1=methyl laurate, 2=methyl myristate and 3=methyl
palmitate separated at temperature of 210 C and flowrate at 30 cm per second
 1

2 3

Figure 6: Second chromatogram of methyl ester mixtures. 1=methyl laurate, 2=methyl myristate and 3=methyl
palmitate separated at temperature of 210 C and flowrate at 30 cm per second.

Table 1: Resolution (Rs) of standard methyl ester mixture separated at temperature of 210C
with different flowrate.

Flow 70 cm/s 50 cm/s 30 cm/s

Rs 1st 2nd Avg 1st 2nd Avg 1st 2nd Avg

1 25.11 23.05 24.08 30.78 19.22 25.00 37.51 38.47 37.99

2 19.35 19.95 19.65 21.27 18.82 20.05 29.73 25.46 27.60

3 27.60 28.59 28.10 29.39 30.57 29.98 37.78 31.95 34.87

Avg: Average

During the experiment, there were many trials and errors. The first error was not
cleaning the syringe with ether. The second error was the injection was too slow. In order to
obtain better peak height and avoid peak accumulation at one place, inject quickly and
efficiently using the correct method of manual injection.
b) Effect of Temperature

In this experiment, the effect of temperature on the separation of methyl ester compounds was
learned by injecting the standard mixture at constant flowrate (70 cm per second) using
different temperature (210, 190 and 130C). Separation using temperature 210C had shorter
retention times compared to other temperature. From this, we can conclude that the higher
the temperature of column, the shorter the retention time and the faster the component elute
from the column. By comparing the resolution between the first injection and second injection
using all the temperature, the most optimum temperature was 210C. Based on the Figure 7
and 8 temperature 210C, the peaks are well separated compared to chromatograms of
temperature 190C, Figure 9 and 10, and 170C, Figure 11 and 12.

2
3

Figure 7: First chromatogram of methyl ester mixtures1=methyl laurate, 2=methyl myristate and 3=methyl
palmitate separated at temperature of 210 C and flowrate at 70 cm per second

2
3

Figure 8: Second chromatogram of methyl ester mixtures1=methyl laurate, 2=methyl myristate and 3=methyl
palmitate separated at temperature of 210 C and flowrate at 70 cm per second

The peaks from both chromatograms are well separated but not that efficient because the
average resolution calculated for peak 2 and 3 is 30.18.
 1

2
3

Figure 9: First chromatogram of methyl ester mixtures1=methyl laurate, 2=methyl myristate and 3=methyl
palmitate separated at temperature of 190 C and flowrate at 70 cm per second

2
3

Figure 10: Second chromatogram of methyl ester mixtures. 1=methyl laurate, 2=methyl myristate and 3=methyl
palmitate separated at temperature of 190 C and flowrate at 70 cm per second.

1
2 3

Figure 11: First chromatogram of methyl ester mixtures. 1=methyl laurate, 2=methyl myristate and 3=methyl
palmitate separated at temperature of 170 C and flowrate at 70 cm per second.
 1
2 3

Figure 12: Second chromatogram of methyl ester mixtures. 1=methyl laurate, 2=methyl myristate and 3=methyl
palmitate separated at temperature of 170 C and flowrate at 70 cm per second.

Table 2: Resolution (Rs) of standard methyl ester mixture separated at flowrate 70 cm per
second with different temperature.

Temp
210C 190C 170C

Rs
1st 2nd Avg 1st 2nd Avg 1st 2nd Avg

1 N/A 12.52 12.52 N/A 76.44 76.44 100.72 102.21 101.47

2 30.36 30.00 30.18 42.87 42.96 42.92 52.73 53.85 53.29

3 22.43 22.86 22.65 30.53 30.15 30.34 39.80 39.88 39.84

Based on the results obtained, the optimal temperature is 210C because it has the least
resolution of all temperature. The peaks are also well separated. The retention time also
decrease for all compounds.
4.0 CONCLUSION
In conclusion, we were able to discover gas chromatography and obtained the optimal
temperature and flowrate for the use in separation of methyl esters compounds.

5.0 REFERENCES

1. Tripathi, R. B. (n.d.). STEPS TO BE CONSIDERED DURING METHOD

DEVELOPMENT AND VALIDATION FOR ANALYSIS OF RESIDUAL SOLVENTS
BY GAS CHROMATOGRAPHY. Retrieved October 2, 2019, from
https://www.academia.edu/5081032/STEPS_TO_BE_CONSIDERED_DURING_MET
HOD_DEVELOPMENT_AND_VALIDATION_FOR_ANALYSIS_OF_RESIDUAL_SOL
VENTS_BY_GAS_CHROMATOGRAPHY.
2. GC Method Development. (n.d.). Retrieved October 2, 2019, from
https://www.crawfordscientific.com/training-consultancy/gc-training/gc-method-
development.
3. Libretexts. (2019, September 5). Gas Chromatography. Retrieved October 2, 2019,
from
https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules
_(Analytical_Chemistry)/Instrumental_Analysis/Chromatography/Gas_Chromatograp
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