Exp 1 510.docx

TITLE: Gas Chromatography(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 mL1

), 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 sec1

. 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 1

Condition 2

Condition 3

Gas flow rate

30cm/s

50cm/s

70cm/s

Column

210oC

210oC

210oC

Temperature Retention Peak area Retention time

(pA*s)

Peak area Retention

time (min) (pA*s)

time (min)

Peak area (pA*s)

(min) Methyl laurate

1.927

41.52623 1.349

45.45727 0.976

43.42664

Methyl myristate

2.420

128.46342 1.811

98.73732 1.652

79.96885

Methyl palmitate

3.911

265.74643 2.898

129.83837 2.743

168.38438

Methyl stearate

-

4.819

167.18466 3.381

111.75437

2. Effects on the variation of column temperature at optimized column temperature on the resolution: Condition 4

Condition 5

Gas flow rate

50cm/s

50cm/s

Column temperature

170oC

190oC

Retention time Peak

Methyl laurate

area Retention time Peak

area

(min)

(pA*s)

(min)

(pA*s)

4.523

41.52531

2.796

46.43243

Methyl myristate

-

-

5.477

102.73898

Methyl palmitate

-

-

7.989

175.84429

Methyl stearate

-

-

-

-

3. Retention time of methyl esters using column temperature programming: Column 6 Gas flow rate

50cm/s 100oC to 290oC

Column temperature programming

Retention time (min)

Peak area (pA*s)

Methyl laurate

3.536

43.64355

Methyl myristate

4.785

122.76672

Methyl palmitate

6.563

173.84346

Methyl stearate

7.642

167.93526

Sample Calculation:

*Liquid form Methyl Laurate

= 0.20g/mol

Molecular weight

= 214.5 g/mol

Density

= 0.87g/mL 0.87𝑔

1𝑚𝐿

1𝑚𝑜𝑙

M1

= 𝑚𝑜𝑙 x1𝑋10−3 𝐿x214.35𝑔 = 4.059 mol/L

M2

=

0.2𝑚𝑔 𝑚𝐿

1𝑚𝐿

1𝑚𝑜𝑙

1𝑋10−3 𝑔

x1𝑋10−3 𝐿x214.35𝑔x

𝑚𝑔

= 9.331x10-4mol/L

M1V1= M2V2 (4.059 mol/L)V1 = (9.331x10-4 mol/L)(0.025 L) V1

= 5.747x10-6L = 5.747 𝜇L (dilute in diethyl diether)

*Solid form Methyl palmitate

= 1.0 mg/mL x 100mL = 100 mg = 0.1 g (dilute in diethyl ether)

DISCUSSION: The variation of the mobile phase flow rate affect the retention time of the compounds which was slow mobile phase flow rate give better separation but very long time taken (Falwell, 1997). 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, gas chromatography including the concepts of retention time and resolution using a mixture of methyl esters; methyl laurate, methyl myristate, methyl palmitate and methyl stearate were investigated. The effects of column temperature and flow rate on the separation of these compounds were observed. It was necessary to prepare all the samples by diluting them with the solvent of diethyl ether and mixed them as a mixture. Six different methods of various conditions were conducted on four samples. Three early methods were run due to various gas flow rate followed by two differents column temperature and ended with column temperature programming. According to mass transfer (C-term) of Van Deemter plot, the higher the velocity of mobile phase, the worse the broadening becomes (Falwell, 1997). From the experimental data, the increasing of flow rate of the gas (30m/s,50m/s and 70 m/s) affect the analyte to elute faster but cause band broadening. Overall resolution in the mixture of each methods were much higher than baseline resolution (1.5) which was well separated between two peaks (Driscoll, 1985). Thus, all of the analytes were well separated but to make sure that they are fully separated, a comparison of the analytes was made based on retention time and peak area in the mixture. The most suitable flow rate was 50cm/s because the peak area for methyl

esters were much higher than peak area in 70cm/s. Although the retention time should be lower which indicates faster elution but we considered the peak area of the analytes for all separation.

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 (Falwell, 1997). The optimum column temperature must be used in analysis time, the optimum column temperature must be used in order to separate each compounds adequately. The optimum gas flow rate for method four and five was 50cm/s. The elution time of the analytes were much higher at low column temperature. In this case, 210oC of column temperature was the best temperature to separate each of the compounds. Based on this experiment, the best condition to separate methyl esters mixture was by using 50cm/s gas flow rate at 210oC column temperature resulting adequate separation between compounds. Optimum column temperature will produce better separation, high efficiency, good resolution and short analysis time for the separation (Driscoll, 1985).

The column that was used in the GC was HP-5 capillary column. This column consist of 5% of phenyl group and this column slightly less non-polar because in order to separate the standard methyl esters mixture which was slightly less non-polar compound. This stationary phase will alow 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 (Falwell, 1997). The methyl laurate was first compound eluted followed by methyl myristate, methyl palmitate and methyl stearate at optimum condition. According to polarity of the compounds, methyl stearate is the most retain by the column and high less nonpolar compared with others. Overall, 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. Experimental error that should be avoid is during the

preparation of the samples because dilution is important to lowering the concentration and avoid contamination of the samples.

CONCLUSION: In conclusion, The optimum condition for the separation of the methyl esters was 70 m/s gas flow rate and 210°C of column temperature. The first peak after the solvent peak was corresponds to methyl laurate followed by methyl myristate and then methyl palmitate.

REFERENCE: Driscoll, J.N. REview of Photoionization Detection in Gas Chromatography: The first Decade. Journal of CHromatographic Science , Vol 23. November 1985. 488-492. Falwell,

S.O.

(1997)

Modern

gas

chromatographic

instrumentation,

in

Analytical

Instrumentation Handbook (ed. G.W. Ewing), Marcel Dekker, New York, Chapter 23. Nor’ashikin S., Ruziyati T., Mardiana S. (2012), Analytical Separation Methods Laboratory Guide (2nd edition).