1. 1. Presenter Dr Anurag Yadav Morderator Dr Avinash S S Column Chromatography 1 Dr Anurag Yadav
2. 2. Capsule: Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components. Supporting medium planar column MECHANISM ION EXCHANGE PARTITION ADSORPTION AFFINITY SIZE EXCLUSION 2 Dr Anurag Yadav
3. 3. Contents Gas Chromatography: Definition Types of Gas chromatography Basic principle Instrumentation Practical consideration Applications 3 Dr Anurag Yadav
4. 4. The father of modern gas chromatography is Nobel Prize winner John Porter Martin, who also developed the first liquid- gas chromatograph. (1950) First separated compound was fatty acid. History 4 Dr Anurag Yadav
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6. 6. Column chromatography GC LC 6 Dr Anurag Yadav
7. 7. Gas chromatography Gas chromatography is a type of column chromatographic technique that can be used to separate volatile organic compounds. Mobile phase: inert gas: nitrogen, helium, hydrogen carrier gas. Stationary phase: liquid/solid. 7 Dr Anurag Yadav
8. 8. Types of Gas chromatography GSC : Packed columns filled with solid sorbent (stationary phase) support particles. GLC : Support particles coated with thin liquid layer 8 Dr Anurag Yadav
9. 9. Basic principle GSC: adsorption chromatography GLC : partition chromatography 9 Dr Anurag Yadav
10. 10. How a Gas Chromatography Machine Works First, a vaporized sample is injected onto the chromatographic column. Second, the sample moves through the column through the flow of inert gas. Third, the components are recorded as a sequence of peaks as they leave the10 Dr Anurag Yadav
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12. 12. Chromatographic Separation Deals with both the stationary phase and the mobile phase. Mobile inert gas used as carrier. Stationary liquid coated on a solid or a solid within a column. 12 Dr Anurag Yadav
13. 13. Sample to be separated is converted into vapour And mixed with gaseous M.P Component more soluble in the S.P travels slower Component less soluble in the S.P travels faster Components are separated according to their Partition Co-efficient Criteria for compounds to be analyzed by G.C 1.VOLATILITY: 2.THERMOSTABILITY: 13 Dr Anurag Yadav
14. 14. Chromatographic Separation Chromatographic Separation In the mobile phase, components of the sample are uniquely drawn to the stationary phase and thus, enter this phase at different times. The parts of the sample are separated within the column. Compounds used at the stationary phase reach the detector at unique times and produce a series of peaks along a time sequence. 14 Dr Anurag Yadav
15. 15. Separation 15 Dr Anurag Yadav
16. 16. Chromatographic Separation (continued) The peaks can then be read and analyzed to determine the exact components of the mixture. Retention time is determined by each component reaching the detector at a characteristic time. 16 Dr Anurag Yadav
17. 17. Chromatographic Analysis The number of components in a sample is determined by the number of peaks. The amount of a given component in a sample is determined by the area under the peaks. The identity of components can be determined by the given retention times. 17 Dr Anurag Yadav
18. 18. Peaks and Data 18 Dr Anurag Yadav
19. 19. Instrumentation Carrier gas (mobile phase) supply: N2, He, H2 Flow control Injector Column Detector Computer/recorder 19 Dr Anurag Yadav
20. 20. Carrier gas supply Function: to provide carrier gas to chromatographic column Carrier gas carries sample to column. Tank, needle valve, flow meter, pressure gauge Type of carrier gases depends on column & detector Capillary columns: H2, He. Packed columns: N2 TCD, ECD: N2 FID: He 20 Dr Anurag Yadav
21. 21. Carrier gas supply Ideal carrier gases: pure & dry Impure & moisture: harm the column, performance of detectors, adversely affect quantification of trace analysis. Measures: Tubing (gas sourceGC)uncontaminated. Molecular sieve beds moisture, hydrocarbon, oxygen content. 21 Dr Anurag Yadav
22. 22. Requirements of a carrier gas Inertness Suitable for the detector High purity Easily available Cheap Should not cause the risk of fire Should give best column performance 22 Dr Anurag Yadav
23. 23. Flow control Regulates the carrier gas flow in GC Constant flow of carrier gas column efficiency & reproducible elution time. Magnitude of carrier gas flow rate depends type of column Packed column 10-60ml/min Capillary column 1-2ml/min 23 Dr Anurag Yadav
24. 24. Injection port The injection port consists of a septum through which a syringe needle is inserted to inject the sample. The sample is injected into a stream of inert gas usually at an elevated temperature by a microsyringe. The vapourized sample is carried into a column packed with the stationary phase. To ensure rapid & complete solute volatilization temp of injector 30-50 degree celsius>column temp 24 Dr Anurag Yadav
25. 25. Injection techniques Split Splitless 25 Dr Anurag Yadav
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27. 27. Split Injection Advantages & Disadvantages Advantages 1.Simple to Use 2.Rugged Design 3.Narrow analyte band on column 4.Protects column from involatile sample components 5.Easy to Automate Disadvantages 1.Not suitable for ultra- trace analysis 2.Suffers from Discrimination 3.Liner geometry dictates injector settings 4.Analytes susceptible to thermal degradation 27 Dr Anurag Yadav
28. 28. Splitless Injection Advantages & Disadvantages Advantages 1.Simple to Use 2.Rugged Design 3.Excellent for trace analysis 4.Less Risk of Analyte Discrimination than Split Mode 5.Easy to Automate Disadvantages 1.Need to carefully optimise conditions 2.Risk of backflash 3.Analytes susceptible to thermal degradation 28 Dr Anurag Yadav
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31. 31. Common problems of injection port Backflash Septum leak Adsorption of components from sample onto septum Septum heatedDecomposition products leak into column ghost peak (chromatogram) Measures Back flash: Septum purge, small injector volume, larger vol injector liners. Teflon coated low leak septum is used Inner surface is purged continuously with carrier gas Septum should be replaced 100 injection31 Dr Anurag Yadav
32. 32. Columns & its types Packed column Capillary (open tubular) column 1 - 4mm ID; 1 - 5 m length Glass/stainless steel coil Packed solid particles either porous/non-porous coated with thin (1 m) film of liquid 0.1 - 0.5 mm I.D. (ID); 10 - 150 m length Thin fused-silica. Inner wall coated with thin (0.1-5 m) film of liquid (stationary phase) 32 Dr Anurag Yadav
33. 33. Capillary (open tubular) column 3 layers 1. Polyamide coating strong water proof barrier 2. Thin fused-silica- minimize chemical reactivity, uniform surface for stationary phase 3. Stationary phase 33 Dr Anurag Yadav
34. 34. Stationary phase Polymer inner surface of fused silica layer Thickness, uniformity, chemical nature influences the separation of components in sample. Mc stationary phase silicon polymer used polysiloxane. 34 Dr Anurag Yadav
35. 35. GC Columns : Composition of stationary phase 100% dimethyl polysiloxane: non polar; for drugs and amino acid derivatives Polyethylene glycol : Polar ; for acids, ketones and alcohols. Disadvantages: high susceptibilty of structural damage by oxygen at high temperatures. GSC: PLOT: Polystyrene, aluminium oxide, molecular sieve Separation: partition/adsorption 35 Dr Anurag Yadav
36. 36. Selection criteria for capillary column Stationary phase close to polarity of solutes. Column diameter : small diamter(0.25mm) when sample overloading is not a problem. Film thickness: thin high boiling point solutes (TG, steriods) thick low boiling point solutes Column length: 30mts most application 15mts simple samples (