CHM 510 : ANALYTICAL SEPARATION METHODS

EXPERIMENT 1
GAS CHROMATOGRAPHY (GC) : OPTIMIZATION OF FLOW
RATE AND COLUMN TEMPERATURE

NAME : NURUL AISHAH BINTI AMRAN

MATRIX NO : 2017761847
GROUP : AS2533B1
GROUP MEMBERS :
1. MUHAMMAD FARID BIN EMJAH (2017420642)
2. RADZIATUL NABIELA
3. ASFARIZAH BINTI ADI (2017540919)
DATE OF EXPERIMENT : 24TH SEPTEMBER 2018
DATE OF SUBMISSION : 8TH OCTOBER 2018
LECTURER’S NAME : MADAM HALIZA KASSIM
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 techninue 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 has 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(𝑡𝑅2 − 𝑡𝑅1)
𝑅𝑠 =
𝑊𝑏1 + 𝑊𝑏2

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, ethyl palmitate, methyl
stearate and methyl linoleate. Other than that, to study the effect of column temperature and
flow rate on the separation of these compounds.

REAGENT AND SOLUTION

a. Individual methyl ester compound : methyl laurate, methyl myristate, methyl

palmitate, methyl stearate and methyl linoleate.
b. Standard mixture of methyl laurate x0.20 mg/mL), methyl myristate x0.20 mg/mL),
methyl palmitate x1.0 mg/mL), methyl stearate x0.70 mg/mL) and methyl linoleate
x0.35 mg/mL).

INSTRUMENT

Gas chromatograph xAgilent Technologies 6890N) equipped with flame ionization detector
xFID) and 30m x 250µm HP5-MS capillary column.

3.0 METHODOLOGY

a. Isothermal elution was set up with condition of :

Injection port : split x40:1)

Injection port temperature: 250 °C

Column temperature: varies x170 °C, 190 °C, 210 °C)

Carrier gas flow rate: varies x30cm/s, 50cm/s,70cm/s)

Detector temperature: 250 °C

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

1. The standard mixture was injected with 0.4 µL at 210 °C isothermally and
30cm/s of gas flow rate.
2. For the second injection with 0.4 µL volume of standard mixture, the gas flow
rate was increased to 50 cm/s with same temperature of 210 °C.
3. The same procedure was repeated with 70cn/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 0.4µL was injected into the column isothermally at 170 °C and
followed by 190 °C.
 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 0.4µL
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:

Retention time Peak width of

Resolution, Average
Condition Injection of peak 2 and 3, peak 2 & 3,
RS resolution
tS (min) Wb (min)
30 m/s, 1 4.487, 6.248 0.0420, 0.0549 36.35
37.13
210°C 2 4.486, 6.247 0.0418, 0.0511 37.91
50 m/s, 1 2.693, 3.748 0.0282, 0.0312 35.52
35.46
210°C 2 2.690, 3.743 0.0251, 0.0344 35.39
70 m/s, 1 1.928, 2.683 0.0264, 0.0283 27.61
27.69
210°C 2 1.929, 2.684 0.0266, 0.0278 27.76

B. Effects of the variation of column temperature at optimized gas flow rate on the
resolution:

Retention time Peak width of

Resolution, Average
Condition Injection of peak 2 and 3, peak 2 & 3,
RS resolution
tS (min) Wb (min)
70 m/s, 1 3.603, 6.977 0.0310, 0.0567 76.94
75.24
170°C 2 3.606, 6.985 00346, 0.0573 73.54
70 m/s, 1 2.569, 4.159 0.0291, 0.0386 46.97
48.60
190°C 2 2.566, 4.153 0.0260, 0.0372 50.22
70 m/s, 1 1.928, 2.683 0.0264, 0.0283 27.61
27.69
210°C 2 1.929, 2.684 0.0266, 0.0278 27.76

The optimized column condition is at 70cm/s of gas flow rate and 210 °C column
temperature because it produced the resolution which is nearest to the ideal resolution value
which is 1.5 other than having shorter analysis time.
 C. Retention time of standard compound of the methyl ester at optimum condition.

Standard Compound Retention time , tR

Methyl laurate 1.930
Methyl myristate 2.686
Methyl palmitate 4.212
Methyl stearate 7.257
Methyl linoleate 6.699

Calculation: Resolution value at 70cm/s and 210°C.

2 (2.683−1.928)
RS (2,3) =
0.0283+0.0264

= 27.61 (first trial)

2 (2.684−1.929)
RS (3,4) =
0.0278+0.0266

= 27.76 (2nd trial)

27.61+27.76
Average resolution =
2

= 27.69

DISCUSSION

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

As the effects of carrier gas flow rate and column temperature on gas chromatography
separation of compounds mixture were studied in this experiment, the optimum condition
for this experiment is determined by injection of sample at different temperature and flow
rates. The standard mixture was injected at flow rate of 30, 50 and 70cm/s and temperature
of 170°C, 190°C, and 210°C in order to determine the suitable flow rate and temperature for
the separation. The resolutions have different value at different temperature and flow rate has
been compared in order to determine the best condition for the better separation.

Based on the chromatograms of standard mixture, the optimum condition of this experiment
was at temperature 210°C and at flow rate of 70cm/s. The injection of sample at temperature
210°C 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 1.930, 2.686 and 4.212. The average retention time of the standard
mixture at the same condition is 2.306. 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 210°C of column temperature. The elution orders of compound are methyl laurate
followed by methyl myristate and methyl palmitate.