(EXPERIMENT 1) GAS CHROMATOGRAPHY (GC), OPTIMIZATION OF FLOW RATE AND COLUMN TEMPERATURE NAME:
MOHAMAD NOR AMIRUL AZHAR BIN KAMIS
STUDENT ID:
2014647344
PARTNERS’ NAMES: 1.
MOHAMAD HAMIZAN BIN MOHD ISA
2.
MOHAMAD SHAFIQ BIN PARMAN
3.
MOHAMAD AZMIZAM BIN MOHAMAD NOOR
DATE OF EXPERIMENT:
16/10/2014
DATE OF SUBMISSION:
8/12/2014
INTRODUCTION: Gas chromatography is a term used to describe the separation techniques used to analyse volatile substances in the gas phase. In gas chromatography, the components of a sample are dissolved in a solvent and vaporized in order to separate the analytes by distributing the sample between two phases: a stationary phase and a mobile phase such as helium or hydrogen. The mobile phase is a chemically inert gas that serves to carry the molecules of the analyte through the heated column. The mobile phase goes through a glass column packed with silica that is coated with a liquid. Materials that are less soluble in the liquid will elute faster than the material with greater solubility. The rate of which compound travels in the column depends on the volatility of compound, column temperature, flow rate of the mobile phase and the length of column. Low boiling point compound (volatile) will elutes faster than the high boiling point compound. High column temperature and high mobile phase flow rate will reduce the time of solute in the column. Longer column length will take longer time to elute all compounds but will give better separation due to high number of theoretical plates and smaller plate height. The liquid stationary phase is adsorbed onto a solid inert packing or immobilized on the capillary tubing walls in a thin layer. In a capillary column, the tubing walls are coated or bonded with the stationary phase liquid. A typical gas chromatograph consists of an injection port, a column, carrier gas flow control equipment, ovens and heaters for maintaining temperatures of the injection port and the column, an integrator chart recorder and a detector. The technique propose in this experiment has been employed in isothermal gas chromatography with split injection since, the concentration of the analyte is high. Isothermal gas chromatography is an analysis where the column temperature is remained constant throughout the analysis. Resolution (Rs) is used to describe how well the species in the mixture are separated. Ideal resolution is 1.5 that indicates adequate separation between species. Lower than 1.5 resolutions 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.
Where
t : retention time of analytes W: peak base width
EXPERIMENTAL: a. Isothermal elution was set up with condition of: Injection port: split (40:1) Injection port temperature: 250°C Column temperature: varies (170°C, 190°C, 210°C) Carrier gas flow rate: varies (30m/s, 50m/s, 70m/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 0.4μL at 210°C isothermally and 30m/s of gas flow rate. 2. For the second injection with 0.4μL volume of standard mixture, the gas flow rate was increased to 50m/s with same temperature of 210°C. 3. The same procedure was repeated with 70m/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.
RESULT AND DISCUSSION: *Calculation of resolution is based on peak 2 and 3 as references. A.
Effects of the variation of the gas flow rate on the resolution:
Condition
Injection
1 30m/s, 210°C
2 1
50m/s, 210°C
2 1
70m/s, 210°C
2
Retention time of peak 2 and 3 (min) 2.812, 4.015 2.807, 4.003 1.684, 2.412 1.680, 2.406 1.190, 1.706 1.199, 1.714
Peak width of peak 2 and 3 (min)
Resolution
0.1070, 0.2052 0.1084, 0.2056 0.0755, 0.1480 0.0746, 0.1447 0.0578, 0.1137 0.0509, 0.1042
7.71
Average resolution
7.67 7.62 6.51 6.57 6.62 6.02 6.33 6.64
The optimized separation time of methyl esters mixture is at 70m/s of gas flow rate. B.
Effects of the variation of column temperature at optimized column temperature on the resolution:
Condition
Injection
Retention Peak width Resolution Average time of of peak 2 resolution peak 2 and and 3 (min) 3 (min) 1 2.311, 0.1588, 8.99 70m/s, 4.655 0.3626 9.05 170°C 2 2.302, 0.1513, 9.10 4.656 0.3661 1 0.505, 0.0868, 7.65 7.80 70m/s, 2.519 0.1783 190°C 2 10548, 0.0842, 7.95 2.596 0.1793 1 1.190, 0.0578, 6.02 70m/s, 1.706 0.1137 6.33 210°C 2 1.199, 0.0509, 6.64 1.714 0.1042 The optimized column temperature at 70m/s of gas flow rate is 210°C column temperature because it produced the resolution nearest to the ideal resolution value that is 1.5 and also shorter analysis time.
C.
Retention time of standard compounds of the methyl esters: Standard compound Methyl laurate Methyl myristate Methyl palmitate Methyl linoleate Methyl stearate
D.
Retention time (min) 1.199 1.721 2.815 4.667 5.070
Sample calculation:
*condition of 70m/s gas flow rate at 210˚C: Rs (2,3) =
= 6.02 (first trial) Rs (2,3) = = 6.64 (second trial) Average resolution (2,3) = = 6.33 The variation of the mobile phase flow rate will affect the retention time of the compounds in which slow mobile phase flow rate will give better separation but very long analysis time. In contrast, high flow rate will shorten the analysis time but will cause broadening due to the mass transfer (C-term) in Van Deemter plot, because the solute does not completely interact with the stationary phase. To reduce the analysis time and to produce better separation, the optimum gas flow rate must be used. In this experiment, the optimum mobile phase flow rate is 70m/s that give good resolution of 6.33 compared to the others that are far from the ideal resolution value that 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 order to separate each compounds adequately. In this case, 210°C of column temperature is the best temperature to separate each of the compounds. Based on this experiment, the best condition to separate the methyl ester mixture is by using 70m/s gas flow rate at 210°C column temperature that will give adequate separation between compounds and also shorter analysis time.
Optimum flow rate and optimum column temperature will produce better separation, high efficiency, good resolution and short analysis time for the separation. Since the separation of gas chromatography is based on the boiling point of the compounds, it can be concluded that methyl laurate has lowest boiling point followed by methyl myristate and highest boiling point is the methyl palmitate.
CONCLUSION: The optimized condition for the separation of the methyl esters is 70m/s of 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 methyl palmitate.
REFERENCES: 1. Nor’ashikin S., Ruziyati T., Mardiana S. (2012), Analytical Separation Methods Laboratory Guide (2nd edition), 3/10/2014. 2. Y. Guillaume and C. Guinchard (January 1997), France, Prediction of Retention Times, Column Efficiency, and Resolution in Isothermal and Temperature-Programmed Gas Chromatography: Application for Separation of Four Psoralens, 3/10/2014, http://chromsci.oxfordjournals.org/content/35/1/14.full.pdf. 3. Gas Chromatography, 4/10/2014, http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chro matography/Gas_Chromatography. 4. Mardiana Saaid, Gas Chromatography Lecture Notes, 5/10/2014.
