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). Rs
provide 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 7th
Edition, 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.
