EXPERIMENT 1

GAS CHROMATOGRAPHY (GC): Optimization of Flow Rate and Column Temperature

INTRODUCTION
By using Gas Chromatography instrument the separation of analytes from its compound is
based on volatility. The more volatile compound will elute faster and spend less time in the
column that will give small retention time. The injection technique is very important to prevent
peak shoulder and broadening. There 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.
Gas chromatography (GC) is an analytical method used for separating and analyzing
compounds that can be vaporized into a gaseous state. This method is used to analyze
compounds and samples that have low molecular weight and high volatility. Efficiency
separation of compound by gas chromatography is dependent on the compounds travelling
through the column at different rate. There are our 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.
i. Resolution
Resolution is the measure of how well the separation of compound in the column. This
definition can be proven by this formula:
2 2[( ) ( ) ]
= =
+ +
The formula equation of resolution.

Z is the separation between two peaks example peak A and B; W a and Wb are the widths at
the base of peak A and B respectively. The acceptable resolution is on Rs=1.0 and baseline
resolution between two peaks requires an Rs >1.5.
OBJECTIVE
1. To determine the retention time and resolution of a mixture of methyl esters; methyl
laurate, methyl myristate, methyl palmitate, methyl stearate, and methyl linoleate.
2. To analyse the effects of column temperature and flow rate on the separation of the
compounds.

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 isothermal GC separation of methyl esters.

0.4 micro litre of standard mixture is injected isothermally at 210 at carrier gas flow rate
of 30 cm/sec. The flow rate increased to 50 cm/sec. The system allowed to equilibrate
within minutes and the standard mixtures is injured again. The same procedure is applied
at flow rate 70 cm/sec.

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

0.4 micro litre of standard mixture is injected isothermally at 170, followed by 190 at the
optimal carrier gas flow rate. The effect of column temperature on the separation,
resolution, and analysis time is evaluated.

d. Separation of methyl esters using column temperature programming.

The standard mixture in injected at the optimal carrier gas flow rate using a linear
temperature ramp from 100 to 290 at optimal flow rate. The separation of the compounds
is evaluated. A bit modification is attempted to the temperature programming in order to
improve the resolution of the compounds.

e. Identification of components in methyl esters mixture.

Each of the methyl esters in injected individually to identify the various compounds in the
standard mixture by using the optimised GC conditions.
RESULT AND CALCULATIONS
*Calculation of resolution is based on peak 3 and 4 as references
The resolution calculated based on the formula;

A. Effect of the variation of gas flow on the resolution.

Condition Injection Retention Peak width of Resolution Average

time of peak 3 and 4 resolution
peak 3 and (min)
4 (min)
30 m/s, 1 5.217, 7.300 0.0721, 0.1181 21.903 22.145
210 C 2 5.217, 7.291 0.0721, 0.1131 22.397
50 m/s 1 3.139, 4.406 0.0479, 0.0808 19.689 19.782
210 C 2 3.139, 4.403 0.0475, 0.0797 19.874
70 m/s 1 2.252, 3.165 0.0448, 0.0716 15.687 11.551
210 C 2 2.243, 2.623 0.0445, 0.0580 7.414

Table a shows the effect of the variation of gas flow on resolution.

From table (a) the optimized separation time of methyl ester is at 70 m/s of gas flow
rate.
 B. Effects of the variation of column temperature at optimized column temperature
on resolution.

The resolution calculated based on the formula;

Condition Injection Retention Peak width of Resolution Average

time of peak 3 peak 3 and 4 resolution
and 4 (min) (min)
70 m/s 1 4.887, 9.348 0.1118, 0.2405 25.325 24.97
170C 2 4.886, 9.322 0.1115, 0.2489 24.617
70 m/s 1 3.085, 5.023 0.0682, 0.1253 20.031 20.539
190C 2 3.088, 5.017 0.0619, 0.1214 21.047
70 m/s 1 2.252, 3.165 0.0448, 0.0716 15.687 11.551
210C 2 2.243, 2.623 0.0445, 0.0580 7.414

Table b shows the effect of the variation of column temperature at optimized column
temperature on resolution.

The optimized column temperature at 70 m/s of gas flow rate is 210C 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.

The resolution calculated based on the formula;

Standard Compound Retention time (min)

Methyl laurate 1.776
Methyl myristate 2.253
Methyl palmitate 3.162
Methyl stearate 4.888
Methyl linoleate 5.583

Table c shows the retention time of individual standard of methyl ester.

Example of resolution calculation;

2(7.300 5.217)
(,) =
(0.0721 + 0.1181)
= . ( )

2(7.291 5.217)
(,) =
(0.1131 + 0.0721)
= . ( )

21.903 + 22.397
=
2
= .
DISCUSSION

Different flow rate of the mobile phase will affect the retention time of the compound for
example, the high flow rate of the gas will elute the analyte fast in shorter time but will cause
band broadening due to mass transfer (C-term) of Van Deemter plot, where the solute in the
column not completely interact and equilibrate. To reduce the band broadening and
overlapping peak of chromatogram, the optimum gas flow rate must be used for a better
separation in short retention time. Form the experimental results, the optimum flow rate of
gas is at 70 cm/s which at good resolution at 11.551 near to ideal resolution (1.5). Other than
flow rate of gas, the column temperature also the main reason for getting the good separation.
same goes like flow rate, the column with too high temperature will allow the analytes to
elute fast but for the volatile compound the chromatogram peaks will overlap and separated
well and vice versa of applying low temperature of column. The high temperature of column
also will lead to column bleeding. The optimum temperature for separation is at 210 C.
Based on the experiment, the optimum condition to separate the standard of methyl ester is
70 cm/s and 210 C.
The column that is used in the GC is HP-5 capillary column. This column consist of 5% of
phenyl group and we can assumed that this column is slightly less non-polar because in order
to separate the standard methyl ester mixture which is known as slightly less non-polar
compound. This stationary phase will allow 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.

From the standard mixture of methyl ester there are three type of individual standard which
are methyl laurate, methyl myristate and methyl palmitate. These three standards are eluted at
1.776, 2.253 min and 3.162 min respectively which the retention time of the standard mixture
closed to individual standard. The methyl laurate is first compound eluted and followed by
methyl myristate and methyl palmitate. According to polarity of the compounds the methyl
palmitate is most retain by the column and more less non-polar compared to methyl aurate
and methyl myristate. Other than that, the gas chromatography is also depends on the
temperature to separate. The methyl palmitate is less volatile and have high boiling point than
other two compounds, methyl laurate is more volatile than methyl myristate.
CONCLUSION

The optimized condition for the separation of the methyl ester is at 70 cm/s of gas flow rate
and 210C of column temperature. The first peak of the chromatogram is identified as methyl
laurate meanwhile the other two peaks are methyl myristate and methyl palmitate
respectively.

REFERENCES

Saim, N., Tajuddin, R., & Saaid, M. (2014). Analytical separation methods laboratory guide.
Selangor: UiTM Press.
 (EXPERIMENT 1)
GAS CHROMATOGRAPHY (GC):
OPTIMIZATION OF FLOW RATE AND COLUMN
TEMPERATURE

NAME: NURUL HAZIQAH BINTI HASAN

STUDENT ID: 2016666772
PARTNERS NAMES: 1. NUR AININA BINTI MOHAMED AINI
2. NURUL JUNAIDAH BINTI TERMIZI
3. RAHAYU BINTI ABDUL RAHMAN
DATE OF SUBMISSION: 09/06/2017