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GAS CHROMATOGRAPHY

BIOCHEMISTRY

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GAS CHROMATOGRAPHY

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  1. GAS CHROMATOGRAPHY M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.

  2. INTRODUCTION • 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. Principle • 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. Criteria for compounds to be analysed by GC • 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. Practical requirements • Carrier gas • Flow regulators and flow meters • Injection devices • Columns • Temparature control devices • Detectors • Recorders and integrators

  6. Carrier gas • 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. Requirements of a carrier gas • 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. Flow regulators & flow meters • 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. Injection devices • 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. Columns • 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. Types of columns • 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. B) depending on its nature:i. Packed column:

  13. ii) Open tubular columns / capillary columns / Golay columns • 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 (0.5-1µ 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. iii) SCOT columnssupport coated open tubular columns • An improved version of Golay or capillary columns • A support material is deposited (1µ) 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. Temparature control devices • 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. Types of operations • 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. Detectors • 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. Types of detectors • Katharometer / Thermal Conductivity Detector (TCD) • Flame Ionization Detector (FID) • Argon Ionization Detector (AID) • Electron Capture Detector (ECD) • Nitrogen Phosphorous Detector (NPD)

  19. Katharometer / TCD • 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. TCD • 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. FID • 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. Advantages of FID • Extremely sensitive & background noise is low • µg 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. Argon Ionization Detector (AID) • 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 α- or β- particles • α- particles can be obtained from radium-D • β- 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. AID • 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 2400C, voltages of 1000V or less are usually necessary

  25. Electron capture detector (ECD) • 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. ECD • 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. Comparision of the sensitivity of detectors

  28. Recorders & integrators • Recorders : to record the responses • They record the baseline & all peaks obtained with respect to time • Retension time for all the peaks can be found out from such recordings, but the area of individual peaks cannot be known • Integrators: improved version of recorders with some data processing capabilities • Can record the individual peaks with Rt, height & width of peaks, peak area, % of area , etc • Int provide more information on peaks than recorders

  29. Thank you

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