Gas chromatography by Dr. Anurag Yadav

column chromatography
mechanism of separation
gas chromatography in detail

1. Presenter Dr Anurag Yadav Morderator Dr Avinash S S C olumn C hromatography 1 Dr Anurag Yadav

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.” 2 Dr Anurag Yadav

3. Contents Gas Chromatography: Definition Types of Gas chromatography Basic principle Instrumentation Practical consideration Applications 3 Dr Anurag Yadav

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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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. 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. Basic principle GSC: adsorption chromatography GLC : partition chromatography 9 Dr Anurag Yadav

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 the column. 10 Dr Anurag Yadav

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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. 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. 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. Separation 15 Dr Anurag Yadav

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. 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. Peaks and Data 18 Dr Anurag Yadav

19. Instrumentation Carrier gas (mobile phase) supply: N2, He, H2 Flow control Injector Column Detector Computer/recorder 19 Dr Anurag Yadav

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. 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 source→GC )→uncontaminated. Molecular sieve beds → ↓moisture, hydrocarbon, oxygen content. 21 Dr Anurag Yadav

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. 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. 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. Injection techniques Split Splitless 25 Dr Anurag Yadav

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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. 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. Common problems of injection port Backflash Septum leak Adsorption of components from sample onto septum Septum heated→Decomposition 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 injection 31 Dr Anurag Yadav

32. Columns & its types Packed column Capillary (open tubular) column 1 - 4mm ID; 1 - 5 m length G lass / stainless steel coil Packed solid particles either porous/non-porous coated with thin (1 μm ) 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 μm ) film of liquid (stationary phase) 32 Dr Anurag Yadav

33. Capillary (open tubular) column 3 layers Polyamide coating – strong water proof barrier Thin fused-silica - minimize chemical reactivity, uniform surface for stationary phase Stationary phase 33 Dr Anurag Yadav

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. 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. 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 (<10 components) 60mts →complex samples 36 Dr Anurag Yadav

37. Temperature control Opertionally temp control → injector, column, detector → thermostatted chamber -Directly → heating of column, Injector & detector -Column temp maintained at constant level → isothermal operation -Varied with function of time → temperature programmed operation →mc in clinical application →solute separation in a wide range boiling point → sharp & distinct chromatographic peak in less time. 37 Dr Anurag Yadav

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39. Detection Systems The detector is the device located at the end of the column which provides a quantitative measurement of the components of the mixture as they elute in combination with the carrier gas.  39 Dr Anurag Yadav

40. Types of Gas Chromatography Detectors Non-selective Responds to all compounds present in carrier gas stream except the carrier gas itself Selective Responds to range of compounds with a common physical or chemical characteristic Specific Responds to a single specific compound only 40 Dr Anurag Yadav

41. Detectors can also be grouped into concentration or mass flow detectors Concentration Dependent The response of such Gas Chromatography detectors is proportional to the concentration of the solute in the detector such as TCD. Dilution of sample with makeup gas will lower detector response. Mass Flow Dependent Signal is dependent on the rate at which solute molecules enter the detector such as FID. Response of such detectors is not affected by makeup gas flow rate changes. 41 Dr Anurag Yadav

42. Desirable characteristics of detectors   Reproducible response to changes in eluent composition in carrier gas stream  High sensitivity   Large linear dynamic range   Low noise   Small volume to avoid peak broadening and resultant loss of resolution   Preferably non – destructive 42 Dr Anurag Yadav

43. Types of Detectors in GC To measure the separated analytes as they elute from the column. Universal unit → detect most analytes Thermal conductance detector (TCD) Mass spectrometer (MS)Selective detectors → detect specific substances Flame Ionization Detector (FID) → hydrocarbon Electron capture detector (ECD) → electronegative groups Commonly used ones are 43 Dr Anurag Yadav

44. Flame Ionization Detector (FID) Mc detector used for clinical analysis Compounds that produce ions when burned in an H 2 -air flame → organic cation → releases electron → detected by collector electrode → generation of current. Magnitude of current α mass of carbon material delivered to detector → used for detection & quantification of eluting solutes. Advantages → simple, reliable, sensitive,linearity excellent. Dis –Advantage – destroy all the sample. uses → detects hydrocarbon including fattyacids . 44 Dr Anurag Yadav

45. Thermal conductance detector (TCD) Universal detector → most of the analytes Difference in thermal conductivity between the carrier gas and sample gas causes a voltage output 45 Dr Anurag Yadav

46. Electron capture detector (ECD) Selective type of detector – electronegative groups- halogens (F, Cl , Br , I), peroxides , quinones, & nitro groups Principle – reaction b/n electronegative groups & thermal electrons (radioactive source) →Thermal electrons captured on the electrode → If electron capturing compound is present the number of thermal electrons on the electrode (standing current) is decreased. ECD Advantages Highly sensitive Easy to use reliable Selective 46 Dr Anurag Yadav

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49. GC-MS Eluted solutes introduced into a ion source of a MS, blasted with electrons, which cause them to turn into positively charged molecular ions and fragmented ions (ion source). When these charged particles passed through filter → separated according to m/e ratio → ions collected. TIC the current generated by all such ions from analytes is measured, which would be proportional to the concentration of analyte . 49 Dr Anurag Yadav

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51. Computer Regulates mobile phase composition, flow rate, column-detector temp. Electronic signals generated by detectors are recorded in the form of chromatograghic peak at varied function of time Area, height, retention time,base width of chromatograghic peak is measured to compute analyte concentration of each peak. 51 Dr Anurag Yadav

52. Resolution 52 Dr Anurag Yadav

53. Asymmetry Factor Chromatographic peak should be symmetrical about its centre If peak is not symmetrical- shows Fronting or Tailing FRONTING Due to saturation of S.P & can be avoided by using less quantity of sample TAILING Due to more active adsorption sites & can be eliminated by support pretreatment, 53 Dr Anurag Yadav

54. Practical consideration Sample extraction – ex: barbiturates Sample derivatization - - Clinically relevent compounds are nonvolitile –difficult to separate, so chemical modification or derivatization is necessary Chemical reaction – nonpolar – methylation , silylation , esterification . Derivatization enhances the specificity & sensitivity of particular separation. 54 Dr Anurag Yadav

55. Applications of GC Separation & identificaton of lipids, carbohydrates & proteins. Separation & identificaton of aminoacids in urine by GC-MS for diagnostic purpose. Measurement of drugs & other metabolites in biological fluids. Used for toxiclogical analysis of biological fluid by using ECD detectors in GC. Analysis of pesticides in soil, water, food. Forensic analysis of blood and urine alcohol levels by using PEG-SP IN GC GC can be used to identify nitro-compounds in trace quantities. 55 Dr Anurag Yadav

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57. ADVANTAGES OF G.C Very high resolution power , complex mixtures can be resolved into its components by this method. Very high sensitivity with TCD, detect down to 100 ppm It is a micro method, small sample size is required Fast analysis is possible, gas as moving phase- rapid equilibrium Relatively good precision & accuracy Qualitative & quantitative analysis is possible. 57 Dr Anurag Yadav

58. References Tietz –clinical chemistry text book Kaplan-technique text book Keith wilson -technique text book 58 Dr Anurag Yadav

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61. Split Injection Mechanisms 1.Sample syringe pierces septum which seals around needle 2.Sample rapidly introduced into heated inlet 3.Liquid sample volatilises and the gaseous ‘plasma’ is contained within a quartz glass liner 4.The sample gas is swept by the carrier gas through the liner and EITHER into the GC Column OR between the liner and inlet body and down the Split Line 5.% of sample reaching the column depends upon the relative flow rates in the column and split flow line 61 Dr Anurag Yadav

62. Split Injection Set-Up Summary 1.Used as the Default Vaporising Injector 2.Primarily used for non-trace analysis of volatile samples 3.Need to consider gas flows (particularly split flow) carefully / Don’t forget septum purge flow! 4.Increasing split flow: a.Improves peak shape b.Lowers column loading c.Lowers analytical sensitivity d.Decreases analyte inlet residence time –therefore reduces the opportunity for thermal degradation 5.Need to consider Discrimination effects 62 Dr Anurag Yadav

63. Split Injection Default / Development Conditions 63 Dr Anurag Yadav

64. Splitless Injection Mechanism 1.Same principle as Split Injection 2.DIFFERENCES INCLUDE 3.Initial injector state is SPLITLESS i.e. The split line flow is turned off 4.All sample reaches the column 5.Sample vapours trapped onto head of column (solvent and thermal effects) 6.Column temperature programmed to initiate elution 7.At some point after analyte transfer to the column the split line is turned on to empty the injector 8.Primarily used for trace and ultra-trace analysis 64 Dr Anurag Yadav

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