M.PRASAD NAIDU
Msc Medical Biochemistry,
Ph.D Research scholar
 2 types
 1. GSC 2. GLC
 GSC is not widely used b/cos limited no of
stationary phases available.
 Adsorption is the pr...
 Partition is the principle
 Stationary phase: liquid which is coated on to a solid
support
 Mobile phase: Gas
 Compon...
 2 important criteria are
 1. Volatility: unless a compound is volatile, it cannot be
mixed with mobile phase.
 2. Ther...
 Carrier gas
 Flow regulators and flow meters
 Injection devices
 Columns
 Temparature control devices
 Detectors
 ...
 The choice of carrier gas determines the efficiency of
chromatographic separation
 Eg: H2, He, N2, Ar
 H2: better ther...
 Inertness
 Suitable to the detector used
 High purity
 Easily available
 Cheap
 Less risk of explosion or fire haza...
 As carrier gases are stored under high
pressure, flow regulators are used to deliver
the gas with uniform pressure or fl...
 Gases can be introduced by valve devices
 Liquids can be injected through loop or
septum devices
 Most GC instruments ...
 Glass / stainless steel
 Stainless steel columns  long life & can be
easily handled without the fear of fragility
 Bu...
 A) depending on its use:
 1. analytical column: 1-1.5m of length & outer
diameter of 3-6mm
 They are packed columns & ...
Stationary phaseStationary phase NatureNature TempTemp
Polydimethyl siloxanePolydimethyl siloxane Non-polarNon-polar -60-6...
 Long capillary tubing of 30-90m
 0.025-0.075cm internal diameter
 Stainless steel & coiled
 The inner wall is coated ...
 An improved version of Golay or capillary
columns
 A support material is deposited (1µ) on the
inner wall & then coated...
 Preheaters: converts the sample into its vapour form & mix
them with mobile phase or carrier gas
 Preheaters are presen...
 Isothermal programming: same temp is maintained
throughout the process of separation
 Linear programming: in which the ...
 Heart of the apparatus
 Requirements of an ideal detector:
 Applicability to wide range of samples
 High sensitivity ...
 Katharometer / Thermal Conductivity
Detector (TCD)
 Flame Ionization Detector (FID)
 Argon Ionization Detector (AID)
...
 Principle: is based upon thermal conductivity difference b/n carrier gas &
that of component
 TCD has 2 platinum wires ...
 The thermal conductivities of some carrier gases:
 H2=32.7 ; He=33.9 ; N2=5.2; CH4=6.5; C6H12=3.0
 Advantages:
 Appli...
 Based upon the electrical conductivity of carrier gases
 At normal temp & pressure, gases act as insulators, but become...
 Extremely sensitive & background noise is
low
 µg quantities can be detected
 Stable & insensitive to small changes in...
 AID depends on the exitation of Ar atoms to a metastable
state, by using radioactive energy.
 This is achieved by irrad...
 Advantages:
 Responds to most of the org compounds
 Sensitivity is very high
 Disadvantages:
 Response is not absolu...
 ECD has 2 electrodes
 Column effluent passes b/n them
 One of the electrode is treated with a radio active isotope whi...
 The carrier gas used in this detector depends upon the e-
affinity of the compounds analysed
 For compounds with high e...
DetectorDetector KatharoKatharo
meter/meter/
TCDTCD
FIDFID AIDAID ECDECD
Min.Min.
detectabledetectable
conc %v/vconc %v/v
...
 Recorders : to record the responses
 They record the baseline & all peaks obtained with respect to
time
 Retension tim...
Gas chromatography
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Gas chromatography

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Gas chromatography

  1. 1. M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D Research scholar
  2. 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. 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. 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. 5.  Carrier gas  Flow regulators and flow meters  Injection devices  Columns  Temparature control devices  Detectors  Recorders and integrators
  6. 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. 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. 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. 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. 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. 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. 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. 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 (0.5-1µ 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. 14.  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. 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. 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. 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. 18.  Katharometer / Thermal Conductivity Detector (TCD)  Flame Ionization Detector (FID)  Argon Ionization Detector (AID)  Electron Capture Detector (ECD)  Nitrogen Phosphorous Detector (NPD)
  19. 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. 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. 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. 22.  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. 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 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. 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. 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. 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. 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. 28.  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

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