Experiment 1 Gas Chromatoghraphy Optimization Of Flow Rate And Column Temperature

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CHM510
ANALYTICAL SEPARATION METHOD



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




ABSTRACT

The separation of the compounds will travel faster through the column when column temperature is high, volatility of compound is low, increasing the carrier gas flow rate and length of the column is increase.

OBJECTIVE
To study the volatility of compound, the effect of length of the column, the effects of column temperature and flow rate by carrier gas through the column.



INTRODUCTION
Efficient separation of compounds in GC is dependent on the compounds travelling through the column at different rates. Method development always starts with a general consideration of the sample. Method development is the process of determining what conditions are adequate or ideal for analysis required. There are several factors that affect GC separation such as volatility of compound, column temperature, the flow rate of gas through the column, length of the column, column polarity and polarity of compounds. However, this experiment focuses on the first four factors. The details for each factor are below:

1.      Volatility of Compound: Low boiling components (High Volatility) will travel faster through the column than high boiling point components. Boiling points of the different components is the main factor in GC separation.

2.      Column Temperature: Raising the column temperature speeds up the elution of all the compounds in a mixture. Separation based on isothermal temperature or temperature programming.
3.      Flow Rate of The Gas through the Column: Speeding up the carrier gas flows will increases the speed with which all compounds move through the column.

Length of The Column: The longer the column, the longer it will take all compounds to elute. Longer columns are employed to obtain better separation (Higher N).
The resolution of two chromatographic peaks is defined by:
                             Rs= [2(Tr2-Tr1)/ W1+W2]
Where Tr1 and  are Tr2 retention times of the two peaks (peak 1 elutes first) and w2 is the baseline width of the peaks. The Rs value indicates the quality of separation between two adjacent peaks (Analytical Chemistry 7th Edition, p.615). Rsprovide a quantitative measure of the ability of the column to separate 2 analytes. Rs must be calculated to explore gas chromatography, including retention time and resolution using a mixture of analyte so the all four factors will be investigated well.


REAGENT AND SOLUTIONS
There are 5 individual methyl esters compounds such as methyl laurate, methyl myristate, methyl palmitate, methyl stearate and methyl linoleate. Standard mixture of methyl laurate (0.20 mg mL-1), methyl myristate (0.20 mg mL-1), methyl palmitate (1.0 mg mL-1), methyl stearate (0.70 mg mL-1) and methyl linoleate(0.35 mg mL-1).

INSTRUMENT
Gas chromatography (Agilent Technologies 6890N) equipped with flame ionization detector (FID) and 30 m x 250 µm HP5-MS capillary column.



PROCEDURE

a)      The instrument is set up like below:
Injection port: Split (40:1)
Injection port temperature: 250oC
Column Temperature: 210oC
Carrier gas flow rate: 30 cm sec-1
Detector temperature: 250oC

b)     Effect of carrier gas flow rate on isothermal GC separation of methyl esters.
0.4 µL standard mixtures are injected isothermally at 210oC at carrier gas flow rate of 30 cm sec-1. The flow rate is increased to 50 cm sec-1. The system is allowed to equilibrate for a few minutes before injecting the standard again. The procedure is repeated at flow rate 70 cm sec-1. The optimize flow rate is chose to continue with the next steps.

c)      Effect of column temperature on the isothermal GC separation of methyl esters.
0.4 µL of standard mixture is injected isothermally at 170oC, followed by 190oC at optimal carrier gas flow rate. The effects of column temperature on separation, resolution and analysis time are evaluated.

d)     Identification of components in methyl esters mixture.
Each of methyl ester is injected individually to identify the various compounds in the standard mixture using optimized GC conditions.



RESULTS
*Calculation Of Resolution based on Peak 2 and Peak 3 as references.

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


Condition
Injection
Retention Time of Peak 2 & Peak 3
Peak Width of Peak 2 & Peak 3
Resolution
Average Resolution
30 m s-1, 210oC
1
4.992, 6.814
0.0784, 0.1308
17.4187
17.41
2
4.992, 6.814
0.0781, 0.1312
17.4104
50 m s-1, 210oC
1
2.994, 4.091
0.0495, 0.0940
15.2891
15.81
2
2.994, 4.091
0.0496, 0.0847
16.3366
70 m s-1, 210oC
1
2.162, 2.953
0.0456, 0.0833
12.2731
12.70
2
2.162, 2.953
0.0469, 0.0736
13.1286
Table shows that the effect of the variation of gas flow on resolution

From the table (1), the optimized separation time of methyl ester is 70 m s-1.

2.      Effects on the variation of column temperature at optimized column temperature on the resolution:

1.        Condition
Injection
Retention Time of Peak 2 & Peak 3
Peak Width of Peak 2 & Peak 3
Resolution
Average Resolution
70 m s-1, 170oC
1
4.423, 8.245
0.1208, 0.2709
19.5149
19.37
2
4.426, 8.247
0.1258, 0.2717
19.2252
70 m s-1, 190oC
1
2.828, 4.461
0.0692, 0.1364
15.8852
15.95
2
2.821, 4.464
0.0690, 0.1362
16.0136
70 m s-1, 210oC
1
2.162, 2.953
0.0456, 0.0833
12.2731
12.70
2
2.162, 2.953
0.0469, 0.0736
13.1286
Table (2) shows the effect of the variation of column temperature at optimized column temperature on resolution.

The optimized column temperature at 70 m s-1 of gas flow rate is 210oC column temperature because it is produce the resolution nearest to the ideal resolution value that is 1.5 and also shorter analysis time.



2.      Retention time of standard compounds of methyl esters:
Standard Compound
Retention Time (min)
Methyl Laurate
1.744
Methyl Myristate
2.165
Methyl Palmitate
2.963
Methyl Linoleate
4.483
Methyl Stearate
5.095


3.      Sample calculation: *Condition of 70 m s-1 gas flow rate at 210oC:















DISCUSSION

The variation of the mobile phase flow rate affect the retention time of the compounds which is slow mobile phase flow rate give better separation but very long time taken. It means, high flow rate will shorten the analysis time but will cause broadening due to mass transfer (C-term) in Van Deemter Plat because the solute does not fully interact with the stationary phase. To reduce the analysis time and produce better separation, the optimum gas flow rate must be used. In this experiment, the optimum mobile phase flow rate is 70 m s-1 that give good resolution of 12.70 compared to others that are far from the ideal resolution value which is 1.5.


The column temperature also affects the separation resolution and the analysis time. High column temperature will give short analysis time but some of the earlier peaks may be overlapped while low column temperature produces better separation but will take very long analysis time. The optimum column temperature must be used in analysis time, the optimum column temperature must be used in order to separate each compounds adequately. 210oC is the best column temperature to separate each of the compounds. Based on this experiment, the best condition to separate the methyl ester mixture is by using 70 m s-1 gas flow rate at 210oC column temperature that will give adequate separation between compounds and shorter analysis time.


Optimum gas flow rate and optimum column temperature produce better separation, high efficiency, good resolution and short analysis time for the separation. Because the separation of gas chromatography is based on the boiling point of the compound, it can be concluded that methyl laurate has the lowest boiling point and followed by methyl myristate. The highest boiling point is methyl palmitate.



CONCLUSION
The optimum condition for the separation of the methyl esters is 70 m s-1  gas flow rate and 210°C of column temperature. The first peak after the solvent peak is corresponds to methyl laurate followed by methyl myristate and then methyl palmitate.



REFERENCE
1.      Christian, Dasgupta & Schug (2014),  Analytical Chemistry 7thEdition, p. 614
2.      Mardiana Saaid, Gas Chromatography Lecture Notes
3.      Nor’ashikin S., Ruziyati T., Mardiana S. (2012), Analytical Separation Methods Laboratory Guide (2nd edition).
4.      Gas Chromatography, 4/10/2014, http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography/Gas_Chromatography.

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