EXPERIMENT 1

Gas Chromatography (GC): Method

Development

NAME: DANG HUMAIRAH BT ANUAR

MATRIC NO: 2020989185

CLASS: RAS2453B

DATE OF SUBMISSION: 22/1/2021

LECTURER’S NAME: DR SHARIZAL HASAN

OBJECTIVE

To determine effects of separation using Gas Chromatography (GC) by changing their

volatility of compound, the effect of length of the column, the effects of column temperature
and flow rate of carrier gas.

INTRODUCTION

The creation of methodology is a mechanism used to show whether or not the

empirical procedure that has been conducted is suitable. The success of the separation of
compounds in Gas Chromatography (GC) depends on the compounds traveling at various
speeds across the column. There are factors that affect GC separation such as volatility of
compound, column temperature, flow rate of gas through the column, length of the column,
column polarity and polarity of the compounds. In this experiment, the only four variables
that have been based on are the volatility of the compound, the temperature of the column
and the flow rate of the gas through the column. Dependent on their properties, all variables
that influence the separation. For compound volatility, when the boiling point is low, the
compound volatility is high so they evaporate further, meaning that the compound travels
faster through the column than the high boiling point compound. Next, it can speed up the
compound elution process for the column temperature as the column temperature rises. Then,
for the flow rate of column. As the column flow rate increases, the gas can flow further into
the column such that it first elutes. A sharp and narrow peak with better separation will be
provided by compounds with shorter elution time. The last one is for the column's duration. If
the column is longer, eluting the compound will take some time. So, later than another
analyte, the substance is eluted. As for this experiment, it is determined that the resolution
determines whether or not the peak has stronger separation. If the resolution is 1.5, better
separation is available for the max. The peak has a higher resolution if the resolution is over
1.5, but the retention time is longer. The resolution is calculated as below:

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

Rs = resolution

Tr1 and Tr2 = retention times of two peaks

W1 and W2 = baseline width of the peaks

REAGENTS AND SOLUTIONS
a. Individual methyl esters compounds: methyl laurate, methyl myristate, methyl
palmitate, methyl stearate, methyl oleate, methyl linoleate.
b. Standard mixture: methyl laurate (0.20 mg m L−1 ¿, methyl myristate (0.20 mg m L−1 ¿,
methyl palmitate (1.0 mg m L−1 ¿, methyl stearate (0.70 mg m L−1 ¿, and methyl
linoleate (0.35 mg m L−1 ¿

INSTRUMENT
Gas chromatography (Agilent Technologies 6890N) equipped with flame ionisation detector
(FID) and 30m × 0.25µm HP5-MS capillary column.

ANALYTICAL PROCEDURE

1. The instrument set up as below:

Injection port: split (40:1)
Injection port temperature: 250℃
Column temperature: 210℃
Carrier gas flow rate: 30 cm sec-1
Detector temperature: 250℃

2. Effect of carrier gas flow rate on the isothermal GC separation of methyl esters.
The standard mixture of 0.40μL was isothermally injected at 210°C at a gas flow rate
of 30 cm sec-1. After that, the flow rate rose to 50 cm sec-1. Until the normal mixture
was injected again, the machine was allowed to balance for a few minutes. At a flow
rate of 70 cm sec-1, the same process was repeated. Based on the result, the optimum
was determined to be 70 cm sec-1.
3. Effect of column temperature on isothermal GC separation of methyl esters.
he normal mixture of 0.4μL was isothermally injected at 170 °C, followed by 190 °C
at the optimum rate of carrier gas flow. The influence of column temperature on the
time of separation, resolution, and analysis was assessed.
4. Standard mixtures were injected at the optimal flow rate using temperatures varying
from 100 °C to 290 °C. To boost the resolution of compounds, modify the
temperature programming. The methyl ester was individually injected using tailored
GC conditions to distinguish different components in the standard mixture.
RESULTS

Effect on resolution based on same temperature but different flow rate.

Condition Injection Retention Peak width Resolution Average

time (min) (min) Resolution
Temperature: 2 Peak 2: Peak 2: 34.7692 35.0642
210°C 4.570 0.0467
Peak 3: Peak 3:
Flow rate:
6.491 0.0638
30 m/s
3 Peak 2: Peak 2: 35.3591
4.568 0.0450

Peak 3: Peak 3:
6.488 0.0636
Temperature: 1 Peak 2: Peak 2: 30.1039 31.8834
210°C 2.740 0.0351
Peak 3: Peak 3:
Flow rate:
3.899 0.0419
50 m/s
2 Peak 2: Peak 2: 33.6628
2.738 0.0304
Peak 3: Peak 3:
3.896 0.0384
Temperature: 2 Peak 2: Peak 2: 25.0904 25.3061
210°C 1.956 0.0288
Peak 3: Peak 3:
Flow rate:
2.789 0.0376
70 m/s
4 Peak 2: Peak 2: 25.5218
1.959 0.0314
Peak 3: Peak 3:
2.791 0.0338
Table 1 shows effect on resolution based on constant temperature but different flow rate.

Condition Injection Retention Peak width Resolution Average

time (min) (min) Resolution
Column 1 Peak 2: Peak 2: 61.8101 60.8707
temperature: 3.644 0.0390
 170°C Peak 3: Peak 3:
Flow rate: 7.127 0.0737
70 m/s 2 Peak 2: Peak 2: 59.9312
3.631 0.0445
Peak 3: Peak 3:
7.116 0.0718
Temperature: 2 Peak 2: Peak 2: 36.9381 36.1247
190°C 2.578 0.0333
Peak 3: Peak 3:
Flow rate:
4.279 0.0588
70 m/s
3 Peak 2: Peak 2: 35.3112
2.579 0.0356
Peak 3: Peak 3:
4.281 0.0608
Temperature: 2 Peak 2: Peak 2: 25.0904 25.3061
210°C 1.956 0.0288
Peak 3: Peak 3:
Flow rate:
2.789 0.0376
70 m/s
4 Peak 2: Peak 2: 25.5218
1.959 0.0314
Peak 3: Peak 3:
2.791 0.0338
Table 2 shows effect on resolution based on constant flow rate different temperature

Standard mixture Retention time (min)

Methyl linoleate 4.002
Methyl myristate 1.938
Methyl laurate 1.559
Methyl palmitate 2.728
Methyl stearate 2.553
Table 3 shows retention of standard mixture of methyl ester.
DISCUSSION

The isolation technique of the molecule from its sample mixture is gas chromatography.
The flame ionization detector (FID) detector used for this experiment will detect the
components, whether they are present or not, and will display them in the form of different
peaks by chromatogram if they are present. When altering with different flow rate and
column temperature, this experiment conducted to know effect on peak separation and to
know optimum flow rate and temperature for better separation. Based on the resulting high
flow rate, lower retention time is given when adjusting the flow rate. But because of mass
transfer, which is the C-term in Van Deemter Plot, it causes the peak to broad because the
solute does not completely interact with the stationary phase. Where the peak is narrow and
sharp, and when the peak is not further apart, the peak has greater separation. In this
experiment, the optimum gas flow rate should be used to reduce retention time and to
generate better separation. The optimal gas flow rate in this experiment is 70 m/s with an
average resolution of 25.3061, which is 1.5 relative to the other flow rate, similar to the ideal
resolution.

Changing temperature also affects retention time and peak resolution. Retaining time is
inversely proportional to the temperature of the column. When the temperature rises, it will
minimize the time for the analyte to pass through the column, although certain peaks may
overlap with each other. The peak gives better separation when the temperature is lower, but
it has greater retention time. So, it might take some time for the analyte to move through the
column. Optimum temperatures must be used in order to minimize retention time and to
properly isolate the compounds. The findings suggest that the optimal temperature is 210 °C.
The experiment concludes that the ideal gas flow rate, 70 m/s and column temperature of 210
° C will provide the best resolution and better separation peak to isolate methyl. The retention
time was used from the latest data to understand the boiling point of the regular mixture. If
the boiling point is strong, the time from injection to detection is faster for the sample. That is
because when the sample has a lower boiling point, it is more volatile, so it increases the
sample speed to move through the column. The result shows the lowest boiling point is
methyl laurate while the highest boiling point is methyl linoleate.

CONCLUSION

Gas chromatography is type of chromatography used in analytical chemistry for separating

sample from its mixture. Mobile phase for gas chromatography is gas carrier while the
stationary phase is a chemical that can selectively attract components in a sample mixture.
From the experiment, it concludes that the best resolution and more efficient peak can be
achieve without worsen the quality of the peak by optimizes the gas flow rate and column
temperature. The optimum gas flow rate is 70 m/s while the optimum temperature is 210°C.
The first peak that out after the solvent peak is correspond to methyl laurate followed with
methyl myristate and end with methyl linoleate.

REFERENCES
Analytical Laboratories Applications GC with Electron Capture Detector (GC-ECD). (n.d.). Retrieved
from AIR PRODUCTS : http://www.airproducts.com.my/industries/Analytical-
Laboratories/analytical-lab-applications/product-list/gc-with-electron-capture-detector-gc-
ecd-analytical-laboratories.aspx?
itemId=2ED69212C574443C9354860ABEFCFE2B#:~:text=Gas%20Chromatography
%20%E2%80%9

Solid Phase Extraction (SPE). (2020, June 9). Retrieved from Chem.LibreTexts:
https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(An
alytical_Chemistry)/Analytical_Sciences_Digital_Library/Active_Learning/Contextual_Modul
es/Sample_Preparation/03_Solid-Phase_Extraction

Solid-Phase Extraction. (n.d.). Retrieved from ScienceDirect:

https://www.sciencedirect.com/topics/chemistry/solid-phase-extraction#:~:text=The
%20basic%20principle%20of%20SPE,greater%20affinity%20for%20the%20analytes.

What is Solid-Phase Extraction (SPE)? (n.d.). Retrieved from Waters:

https://www.waters.com/waters/en_US/Goals-and-Benefits-of-SPE/nav.htm?
cid=10083495&locale=en_US

APPENDIX

FIGURE 1 STANDARD MIXTURE OF METHYL ESTER

TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 30 2
 FIGURE 2 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 30 3
 FIGURE 3 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 50 1
 FIGURE 4 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 50 2
 FIGURE 5 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 70 2
 FIGURE 6 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 70 4
 FIGURE 7 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
170 70 1
 FIGURE 8 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
170 70 2
 FIGURE 9 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
190 70 2
 FIGURE 10 STANDARD MIXTURE OF METHYL ESTER
TEMPERATURE, °C FLOW RATE, m/s INJECTION
190 70 3
 FIGURE 11 STANDARD MIXTURE OF METHYL ESTER WITH
METHYL LINOLEATE
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 70 1
 FIGURE 12 STANDARD MIXTURE OF METHYL ESTER WITH
METHYL MYRISTATE
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 70 1
 FIGURE 13 STANDARD MIXTURE OF METHYL ESTER WITH
METHYL LAURATE
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 70 1
 FIGURE 14 STANDARD MIXTURE OF METHYL ESTER WITH
METHYL PALMITATE
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 70 1
 FIGURE 15 STANDARD MIXTURE OF METHYL ESTER WITH
METHYL STEARATE
TEMPERATURE, °C FLOW RATE, m/s INJECTION
210 70 1