Gas chromatography

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Transcript of Gas chromatography

  • 1. M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D Research scholar

2. 2 types 1. GSC 2. GLC GSC is not widely used b/cos limited no of stationary phases available. Adsorption is the principle GSC is used only in case where there is less solubility of solutes in stationary phase, which are rare. GLC only 3. Partition is the principle Stationary phase: liquid which is coated on to a solid support Mobile phase: Gas Components are separated according to their partition coefficients Partition coefficient is the ratio of solubility of a substance distributed between two immiscible liquids at a constant temp. 4. 2 important criteria are 1. Volatility: unless a compound is volatile, it cannot be mixed with mobile phase. 2. Thermostability: All the compounds will not be in the form of vapour.(solids & liquids) Hence to convert them to a vapour form, they have to be heated. At that temp the compounds have to be thermostable If they are not thermostable, the compounds cannot be analysed by GC, since they will be decomposed. 5. Carrier gas Flow regulators and flow meters Injection devices Columns Temparature control devices Detectors Recorders and integrators 6. The choice of carrier gas determines the efficiency of chromatographic separation Eg: H2, He, N2, Ar H2: better thermal conductivity, low density Used in thermal conductivity detector / FID Demerits: 1. it reacts with unsaturated compounds 2. inflammable He: excellent thermal conductivity, but expensive Used in thermal conductivity detector. N2: inexpensive but has reduced sensitivity 7. Inertness Suitable to the detector used High purity Easily available Cheap Less risk of explosion or fire hazards Should give best performance Consistent with the required speed of analysis Compressible, gases are stored under high pressure in cylinders N2, He are the most commonly used 8. As carrier gases are stored under high pressure, flow regulators are used to deliver the gas with uniform pressure or flow rate Flow meters to measure the flow rate of carrier gas 1. Rotameter: 2. Soad bubble meter: 9. Gases can be introduced by valve devices Liquids can be injected through loop or septum devices Most GC instruments have a high quality rubber septum Solid samples are dissolved in a suitable solvent and injected through a septum 10. Glass / stainless steel Stainless steel columns long life & can be easily handled without the fear of fragility But some samples react with them Hence in such cases, glass columns are used Eg: steroids Glass columns are inert but highly fragile and are difficult to handle 11. A) depending on its use: 1. analytical column: 1-1.5m of length & outer diameter of 3-6mm They are packed columns & are made up of glass or stainless steel Demerit: Only small quantity of sample can be loaded 2. Preparative columns: larger & large amount of sample can be loaded 3-6m of lengthy, outer diameter 6-9mm 12. Stationary phaseStationary phase NatureNature TempTemp Polydimethyl siloxanePolydimethyl siloxane Non-polarNon-polar -60-6000 -320-32000 CC Poly(diphenyl) dimethyl siloxanePoly(diphenyl) dimethyl siloxane Non-polar bondedNon-polar bonded phasephase -60-6000 -320-32000 CC Polycyano propyl phenyl dimethyl siloxanePolycyano propyl phenyl dimethyl siloxane IntermediateIntermediate polaritypolarity Upto 280CUpto 280C Polyalkylene glycolPolyalkylene glycol PolarPolar 30-22030-220 Polyethylene glycolPolyethylene glycol PolarPolar 50-28050-280 PEG modified with Nitroterephthalic acidPEG modified with Nitroterephthalic acid Polar bonded phasePolar bonded phase 60-20060-200 Poly bis cyano propyl siloxanePoly bis cyano propyl siloxane Very polar non-Very polar non- bonded phasebonded phase Upto 250Upto 250 13. Long capillary tubing of 30-90m 0.025-0.075cm internal diameter Stainless steel & coiled The inner wall is coated with the st. phase liquid (0.5-1 thin film) these columns offer least resistance to flow of carrier gas More efficient than packed columns ( offers more resistance) Demerit: more sample cannot be loaded 14. An improved version of Golay or capillary columns A support material is deposited (1) on the inner wall & then coated with a thin film of liquid phase Have a low resistance to flow of carrier gas Advantage: more sample load 15. Preheaters: converts the sample into its vapour form & mix them with mobile phase or carrier gas Preheaters are present along with injecting devices Thermostatically controlled oven: In GC partition is the principle Since partition coefficient as well as solubility of a solute depends upon temp, temp maintenance in a column is highly essential for efficient separation Hence column & injecting devices should be maintained at a particular temp. 16. Isothermal programming: same temp is maintained throughout the process of separation Linear programming: in which the oven is heated linearly over a period of time This is required when a sample has a mixture of low bp & high bp compounds Separation of complex mixtures 17. Heart of the apparatus Requirements of an ideal detector: Applicability to wide range of samples High sensitivity to even small conc Rapidity of response Linearity: i.e., less response to low conc & vice versa Response should be unaffected by temp, flow rate or characteristics of carrier gas Non destructive to the sample in case of preparative work Simple & easy to maintain inexpensive 18. Katharometer / Thermal Conductivity Detector (TCD) Flame Ionization Detector (FID) Argon Ionization Detector (AID) Electron Capture Detector (ECD) Nitrogen Phosphorous Detector (NPD) 19. Principle: is based upon thermal conductivity difference b/n carrier gas & that of component TCD has 2 platinum wires of uniform size which form part of Wheatstone bridge Through one of them, pure carrier gas always flows & through the other the effluents of the column passes 2 Pt wires are heated electrically When pure carrier gas passes through both or them, there is no diff in temp or resistance & hence baseline is recorded When a component emerges from the column, it alters the thermal conductivity & resistance of the wire Hence this produces a diff in resistance So conductivity b/n wires, which is amplified & recorded as a signal. 20. The thermal conductivities of some carrier gases: H2=32.7 ; He=33.9 ; N2=5.2; CH4=6.5; C6H12=3.0 Advantages: Applicable to most compounds Linearity is good Sample is not destroyed & used in preparative scale Simple, easy to maintain & inexpensive Disadvantages: Low sensitivity Affected by fluctuations in temp & flow rate Response is only relative & not absolute Biological samples cannot be analysed 21. Based upon the electrical conductivity of carrier gases At normal temp & pressure, gases act as insulators, but become conductive if ions are present H2 is the carrier gas used in FID If the carrier gas is either N2/Ar, it can be mixed with H2 Anode: Ag gauze placed over the burner tip Cathode: burner tip made up of Pt capillary When pure carrier gas alone passes, there is no ionization & no current flows When a component emerges, no. of ions are produced b/cos of ionization by the thermal energy of the flame This causes a potential diff & causes a flow of current which is amplified & recorded as a signal 22. Extremely sensitive & background noise is low g quantities can be detected Stable & insensitive to small changes in the flow rate of carrier gas & water vapour Responds to most of the org compounds Linearity is excellent 23. AID depends on the exitation of Ar atoms to a metastable state, by using radioactive energy. This is achieved by irradiating the carrier gas with either - or - particles - particles can be obtained from radium-D - particles can be obtained from Sr90 / H3 These high E particles ionize the Ar atoms & hence they are exited to metastable state These molecules collide with the effluent molecules and ionizes them These ions when reach the detector will cause an increase in current Thus the components are detected 24. Advantages: Responds to most of the org compounds Sensitivity is very high Disadvantages: Response is not absolute & it is relative Linearity is poor Sensitivity is affected by water & is much reduced for halogenated compounds The response varies with the temp of the detector High temp like 2400 C, voltages of 1000V or less are usually necessary 25. ECD has 2 electrodes Column effluent passes b/n them One of the electrode is treated with a radio active isotope which emits electrons as it decays. These emitted electrons produce 2o electrons which are collected by the anode, when a PD of 20V is applied b/n them When carrier gas alone flows through, all the 2o electrons are collected by the +vely polarised electrode Hence a steady baseline is recorded Effluent molecules which have affinity for electrons, capture these e- when they pass through the electrodes Hence the amount of steady state current is reduced This diff is amplified & recorded as output signal 26. The carrier gas used in this detector depends upon the e- affinity of the compounds analysed For compounds with high e- affinity, Ar is used For low e- affinity , N2, H2, He or CO2 can be used Advantages: highly sensitive (10-9 ) Disadvantage: ECD can be used only for compounds with e- affinity Halogenated compounds, pesticides etc can be detected by ECD 27. DetectorDetector KatharoKatharo meter/meter/ TCDTCD FIDFID AIDAID ECDECD Min.Min. detectabledetectable conc %v/vconc %v/v 1010-6-6 1010-11-11 1010-11-11 1010-12-12 28. Recorders : to record the responses They record the baseline & all peaks obtained wit