1.

0 INTRODUCTION

The separation of analytes from its compound by using Gas Chromatography (GC) instrument
is based on volatility. The more volatile compound will elute faster and spend less time in the
column which means have small retention time. The injection technique is very important
to prevent peak shoulder and broadening. There are two modes of injection which are split and
splitless. In this experiment, the split mode is chosen because the sample is concentrated. The
flow rate and temperature are the most important things in gas chromatography in order to
separate the analyte at good resolution and sharp peak.

Efficiency separation of compound by gas chromatography is dependent on the compounds

travelling through the column at different rate. There are four factors that influence the
performance of GC system. One of the factors is volatility of compound, the volatile compound
will elute faster than compound that have high boiling points. Next is column temperature of GC.
The higher the temperature of column the faster the separation of the compound in the stationary
phase. Carrier gas flow rate also give impact to the separation where increasing the carrier gas
flow will increase the speed of compound mobilization through the column. The longer the
column, the longer the migration time of the analyte in it and take longer time to be eluted. But
the longer column will give better separation.

Resolution is the measure of how well the separation of compound in the columns. This
definition can be proven by this formula :

2(tR 2tR 1)
Rs=
Wb 1+Wb 2

Where tR : retention time of analytes

Wb: peak base width

Rs >1.5 is ideal resolution that indicate good separation or baseline separation between species.
Rs<1.5 is when the two species is not completely separated while too high resolution will need
longer analysis time. The objective of this experiment is to explore gas chromatography
including the concepts of retention time and resolution using a mixture of methyl esters, methyl
laurate, methyl myristate, methyl palmitate, methyl stearate and methyl linoleate. The effects of
column temperature and flow rate on the separation of these compounds will be investigated.
2.0 OBJECTIVE

To explore gas chromatography including the concepts of retention time and resolution using a
mixture of methyl ester like methyl laurate, methyl myristate, methyl palmitate, methyl stearate
and methyl lionate. To study the effects of column temperature and flow rate on the separation of
these compound.

REAGENTS AND SOLUTION

a. Individual methyl ester compound : methyl laurate, methyl myristate, methyl palmitate,
methyl stearate and methyl linoleate.
b. Standard mixture of methyl laurate (0.20mg/mL), methyl myristate (0.20mg/mL), methyl
palmitate (1.0mg/mL), methyl stearate (0.70mg/mL) and methyl linoleate (0.35mg/mL).

INSTRUMENT

Gas chromatograph (Agilent Technologies 6890N) equipped with flame ionization

detector (FID) and 30m x 250m x 0.25m HP5-MS capillary column.
3.0 METHODOLOGY

a. Isothermal elution was set up with condition of:

Injection port: split (40:1)

Injection port temperature: 250C.
Column temperature: varies (170C, 190C, 210C).
Carrier gas flow rate: varies (30cm/s, 50cm/s, 70cm/s)
Detector temperature: 250C.

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

1. The standard mixture was injected with 0.4L at 210C isothermally and 30cm/s of gas flow
rate.

2. For the second injection with 0.4L volume of standard mixture, the gas flow rate was
increased to 50cm/s with same temperature of 210C.

3. The same procedure was repeated with 70cm/s gas flow rate.

4. The resolution of 3 different flow rates at the same temperature was calculated and compared
to determine which gas flow rate give the better separation of the compounds in the
standard mixture.

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

1. By using the best gas flow rate from the above procedure, the same standard mixture with
0.4L was injected into the column isothermally at 170C and followed by 190C.

2. The resolution of each chromatogram was calculated and was compared based on the
resolution and the analysis time.

d. Identification of components in methyl esters mixture:

1. The standard individual methyl ester compounds were injected with 0.4L volume using the
optimized GC conditions.

2. The standard individual methyl ester compounds are methyl laurate, methyl myristate, methyl
palmitate, methyl stearate and methyl linoleate.
4.0 RESULT AND DISCUSSION

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

Table 1 : Flow Rate 30cm/s and Column Temperature 210oC.

Repeatabilit Peak Retention Width, Rs1 peak Average Rs2 peak Average
y time, tR Wb 2&3 3&4
1 2 4.032 0.0436 18.07 24.91
3 4.989 0.0587 18.71 25.41
4 6.840 0.0870
2 2 4.025 0.0502 17.43 24.41
3 4.982 0.0596
4 6.788 0.0884
Table 2 : Flow Rate 50cm/s and Column Temperature 210oC.

Repeatabilit Peak Retention Width, Rs1 peak Average Rs2 peak Average
y time, tR Wb 2&3 3&4
1 2 2.391 0.0398 13.88 20.78
3 2.971 0.0438 13.88 20.78
4 4.069 0.0619
2 2 2.391 0.0398 13.88 20.78
3 2.971 0.0438
4 4.069 0.0619
Table 3 : Flow Rate 70cm/s and Column Temperature 210oC

Repeatabilit Peak Retention Width, Rs1 peak Average Rs2 peak Average
y time, tR Wb 2&3 3&4
1 2 1.718 0.0307 12.36 12.43 17.45 17.51
3 2.137 0.0371
4 2.944 0.0554
2 2 1.711 0.0304 12.49 17.56
3 2.128 0.0364
4 2.918 0.0536
B. Effects of the variation of column temperature at optimized gas flow rate on the resolution:

Table 4 : Flow Rate 70cm/s and Column Temperature 190oC.

Repeatabilit Peak Retention Width, Rs1 peak Average Rs2 peak Average
y time, tR Wb 2&3 3&4
1 2 2.056 0.0376 18.37 24.27
3 2.874 0.0488 18.94 25.07
4 4.540 0.0841
2 2 2.048 0.0384 17.79 23.47
3 2.867 0.0537
4 4.530 0.0880
Table 5 : Flow Rate 70cm/s and Column Temperature 170oC.

Repeatabilit Peak Retention Width, Rs1 peak Average Rs2 peak Average
y time, tR Wb 2&3 3&4
1 2 2.725 0.0461 25.6 29.81
3 4.461 0.0820 27.10 30.35
4 8.311 0.1717
2 2 2.720 0.0492 24.10 29.27
3 4.441 0.0885
4 8.249 0.1717
Table 6 : Comparison of Resolution in Different Flow Rate and Temperature.

Flow Rate and Temperature Resolution, Rs1 Resolution, Rs2

Flow rate 30cm/s 18.07 24.91
Temperature 210oC
Flow rate 50cm/s 13.88 20.78
Temperature 210oC
Flow rate 70cm/s 12.43 17.51
Temperature 210oC
Flow rate 70cm/s 18.37 24.27
Temperature 190oC
Flow rate 70cm/s 25.6 29.81
Temperature 170oC

The optimized column condition is at 70cm/s of gas flow rate and 210oC column temperature
because it is produced the resolution nearest to the ideal resolution value that is 1.5 and also
shorter analysis time.

C. Retention time of standard compound of the methyl ester at optimum condition.

Standard Compound Retention time, tR

Methyl laurate 1.727
Methyl myristate 2.138
Methyl palmitate 2.924
Methyl stearate 4.423
Methyl linoleate 5.075

Table 7 : Comparison of Average Retention Time of Unknown Peak and Individual Standards at
Temperature 210oC, Flow Rate 70cm/s.

Peak Standard mixture, Standard mixture,

tR1 tR2
2 1.718 1.711
3 2.137 2.128
4 2.944 2.918
Average Methyl laurate, tR Methyl myristate, Methyl palmitate,
tR tR
1.715 1.727 2.138 2.924
2.133
2.931
Example of resolution calculation: Resolution value at 70cm/s and 210oC.

2 ( tR 2tR 1 )
Rs(2,3 ) Wb 1+ Wb 2

0.0307+0.0371

= 2 ( 2.1371.718 )

= 12.36

2 ( tR 2tR 1 )
Rs(2,3 )
Wb 1+ Wb 2

0.0554+0.0371

= 2 ( 2.9442.137 )

= 17.45

DISCUSSION

In this experiment, gas chromatography was used to identify the various components in the
standard mixture of methyl ester using the optimized GC conditions. The standard methyl ester
contains three individual components, methyl laurate, methyl myristate and methyl palmitate.
The instrument set to use split injection because only small amount of sample introduced into the
column. For optimum column efficiency, the sample for the injection should not too large and
introduced onto the column as a plug of vapor because slow injection of large sample will cause
band broadening and loss resolution.

The effects of carrier gas flow rate and column temperature on gas chromatography separation of
compounds mixture were investigated in this experiment. The optimum condition for this
experiment is determined by injection of sample at different temperature and flow rates. The
standard mixture injected at flow rate of 30, 50 and 70 cm/s and temperature of 170oC, 190oC,
and 210oC in order to determine the suitable flow rate and temperature for the separation. The
resolution value at different temperature and flow rate is compared in order to determine the best
condition for better separation. Based on the chromatograms of standard mixture, the optimum
condition of this experiment achieved at temperature 210oC and at flow rate 70cm/s. The
injection of sample at temperature 210oC and flow rate 70cm/s gives the lowest resolution value
compared to other temperature and flow rate. The ideal resolution value for chromatography
separation is around 1 to 20. If the resolution value between two peaks calculated is greater than
20, the separation takes longer time to complete.

Then the individual methyl esters are identifying by comparing the chromatograms of individual
compounds and the standard mixture. The average retention times of individual peaks of methyl
laurate, methyl myristate and methyl palmitate at optimum GC condition is 1.727, 2.138, and
2.924. The average retention time of the standard mixture at the same condition is 1.715, 2.133,
and 2.93. So, the individual components of methyl ester can be identified. Based on the
comparison of retention time of standard mixture and individual components of methyl ester,
methyl laurate elute first followed by methyl myristate and methyl palmitate.

5.0 CONCLUSION

The optimized condition for the separation of the methyl ester is at 70cm/s of gas flow rate and
210oC of column temperature. The elution order of compound are methyl laurate followed by
methyl myristate and methyl palmitate.

6.0 REFERENCES

Jennies, W. Mittlefehldt, E. & Stremple, P. (1997). Analytical Gas Chromatography.

Massachusetts: Academic Press.

Mardiana Saaid, Gas Chromatography Lecture Notes, 3/5/2017.

Norashikin S., Ruziyati T., Mardiana S. (2012), Analytical Separation Methods Laboratory
Guide (2nd edition), 3/5/2017.

Skoog, D. A; Holler, F. J; & Crouch, S. R (2007). Principle of Intrumental Analysis. Canada:

Cengage Learning.