Gas chromatography and Gas- Liquid chromatography

Contains information about the Principle, Components and Parts, Working, Applications and Pictures of Gas chromatography.

1. GAS CHROMATOGRAPHY AND GAS-LIQUID CHROMATOGRAPHY By, Tharrun Daniel Paul , Aakash Reddy, Roshan Kumar Sah . B.tech -Biotechnology, II yr , Dr. M. G. R Educational and Research Institute University, Chennai.

2. History : Mikhail Semyonovich Tswett (a Russian botanist) regarded as ‘ FATHER OF CHROMATOGRAPHY ’ carried out the first chromatographic separation in 1903 with chlorophyll pigments in a column of chalk powder. Martin and Synge pioneered GC as an analytical technique in 1941.

3. INTRODUCTION: Chromatography: C h r o m a -color; graphos - writing (by Mikhail Tswett ) An analytical technique used for the separation of compounds using a mobile phase (moving) and a stationery phase (immovable). Sample to be separated is mixed with mobile phase and is made to flow along the stationary phase. Due to characteristic affinity of molecules towards the stationary phase molecules in sample travel at different velocities and distances, thus getting separated. e.g. paper chromatography- paper is stationary phase and a solvent is the mobile phase.

4. Gas chromatography: It is an analytical, separation technique which is used to separate volatile components. A gas as a mobile phase . Hence the name gas chromatography. Sample is made volatile and it is mixed with a carrier gas (mobile phase) and passed through a stationary phase. Principle: It follows the principle of adsorption-sample components due to different affinity towards the stationary phase, travel at different velocities. And it is understood that components travelling slowly, have more affinity and those which travel quick, have less affinity towards the stationary phase.

5. Driving forces: Boiling point/ volatility: low boiling liquids get vaporized easily and pass though the stationary phase quickly and high boiling liquids vaporize slowly and travel slow (samples can be separated according to their boiling points). Components coming out first can be collected and the following components can be collected accordingly. Polarity: polar and polar molecules have a great attraction. A . If stationary phase is polar and sample is also polar, then there will be good attraction so it will travel slow. B . If the sample has a little non-polarity then there will be lesser attraction and the sample will travel a little quick. C . If the stationary phase is polar and the sample is non polar there will be no attraction and the sample will flow freely without any lag in time.

6. Chromatograph: Chromatogram:

7. Elution : process in which sample comes out as separated components. Separated components are called elutes. Retention time: difference in time between the injection and elution of the sample or the particular component. Derivitization: making non-volatile components into volatile components.

8. Components of a GC machine: A typical GC machine consists of the following components: 1. Mobile/ carrier gas cylinders 2. Purification system 3. Injector 4. Column 5. Oven 6. Detector 7. Recorder

9. MOBILE GAS CYLINDERS: Mobile gases (carrier gases) are the gases which carry the volatile mixture to be separated. Common gases used are N 2, H 2 , He 2 , Ar which are kept way from the apparatus in cylinders, as H 2 is explosive. Most used carrier gas is N 2 . He 2 is effective in separation but it is expensive. H 2 is the best separation carrier gas but it is explosive in high temperature conditions. The amount of gases coming out of the cylinders can be regulated by regulators provided in the cylinders.

10. PURIFICATION SYSTEM: The carrier gases should have 99.9995% of purity so three stages of filters are used to purify them. 1. Oxy trap 2. Hydrocarbon trap 3. Activated carbon trap

11. OVEN: Oven is the place where separation takes place. Oven consists of the following parts; 1. Injector 2. Column 3. Detector As volatile samples are used, the oven and its components should be kept at a particular temperature so that the volatile liquid gets vaporized before entering the separation process. The oven helps in maintaining a constant temperature, so that the volatile components are maintained in their vapor state. The specifications about the temperature, split ratio and other required specifications are setup in the oven.

12. INJECTOR: The sample is injected through the injector which is maintained at the vaporization temperature of the volatile sample. Sample is injected using a micro syringe, as only a small amount of sample is needed(0.1- 1.0 micro litres ). Injector consists of a septum (rubber), to prevent the back flow of the vaporized sample. A carrier gas inlet, vaporization chamber, outlet to column, split outlet is provided with the injector.

13. Micro syringe Rubber septum

14. COLUMN: Column is a coiled tube like structure which can range in length from 1- 120 m and contains the stationary phase embedded in it. A column may be either packed or capillary. Packed column: Small particles of stationary phase (silica gel on a rigid support) are packed inside throughout the inside of column. If damaged the packing alone can be refilled. Capillary column: hollow tube inside which the stationary phase ( polysiloxane /PEG) is embedded either as a thick or thin film. a) thick film: high capacity, increased retention, but more bleed. b) thin film: quick analysis, better separation, low bleed, but low capacity.

15. DETECTOR: The common detector used is the flame ionization detector (FID). It consists of a) A collector: collects the gas coming out from column. b) ignitor: produces flame. c) H 2 and air/o 2 : for ignition of flame. d) column exit e) outlet port: for signal processing. f) two electrodes: for detection of ions. An FID is used to detect the ions produced by the flame inside it. When sample reaches the detector H 2 , air/O 2 is mixed and the sample is ignited. The ions produced due to ignition are detected and the signal is generated which is amplified by the amplifier placed outside the GC machine. This signal is displayed as an output in a monitor. The concentration of the organic component in the sample is proportional to the ions generated.

16. RECORDER: A recorder is nothing but a software that can transform the amplified signal from the detector into a readable graph (chromatogram). The graph is produced in the form of peaks. A sharp peak denotes a good separation. Sharp peaks are always preferable as they give appropriate values and make the calculation easier and accurate. Blunt peaks denote that there is error in the injection of the sample which leads to poor separation.

17. WORKING: The carrier gas cylinders are opened. Temperature of the injector, column, oven, detector are set along with the split ratio in the chromatograph (The column temperature- above the dew point of the sample; injection temperature- 10º - 30º C greater than the boiling point of the samples). [ split ratio: parts of gaseous sample thrown out/ parts of gaseous sample going into the column] Now the sample is quickly injected into the injector using a micro syringe (delay in injection may cause sample in syringe needle to get vaporized). A portion of the sample passes through the split outlet remaining enters the columns as it avoid overloading is column.

18. Samples move according to their boiling point inside the column (low boiling samples move quickly and the remaining move increasing sequence) and come out (Also, samples having less affinity to the stationery phase travel quickly). After reaching detector the signals are recorded. And the separated gases are isolated accordingly as they come out (Time taken by the between the sample injection and elution (coming out) is called as the retention time). Each compound in the mixture has a characteristic retention time. By knowing the retention time , the specific sample can be identified (from graph) The height and area of the peaks is proportional to the concentration of the component in the mixture.

19. APPLICATIONS: Used for the analysis of components in a sample. Used In the separation of volatile compounds in a sample. Quantification of pollutants in drinking and waste water. Quantification of the drugs and their metabolites in blood and urine for both pharmacological and forensic applications. Identification of unknown organic compounds in hazardous waste dumps. Analysis of industrial products for quality.

20. GAS-LIQUID CHROMATOGRAPHY (GLC): GLC is a similar technique to GC, in that in GLC a liquid is used as a mobile phase. Retention time is shorter in GLC than in GC. At least one among two is a liquid phase in GLC. High concentration of samples can be used. Capillary columns are used.

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