1 / 29

GAS CHROTOGRAPHY NEW

BIOCHEMISTRY

Download Presentation

GAS CHROTOGRAPHY NEW

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


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

  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

More Related