Gc mpharm sud


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Gc mpharm sud

  1. 1. Gas Chromatography Presented By - Sudheer kumar kamarapu Assistant professor Sri Shivani college of Pharmacy sudheerkumar kamarapu 1
  2. 2. What is Gas Chromatography?• The father of modern gas chromatography is Nobel Prize winner John Porter Martin, who also developed the first liquid-gas chromatograph. (1950) sudheerkumar kamarapu 2
  3. 3. GAS CHROMATOGRAPHY Separation of gaseous & volatile substances Simple & efficient in regard to separation GC consists of GSC (gas solid chromatography) GLC (gas liquid chromatographyGas → M.PSolid / Liquid → S.PGSC not used because of limited no. of S.PGSC principle is ADSORPTIONGLC principle is PARTITION sudheerkumar kamarapu 3
  4. 4. Sample to be separated is converted into vapourAnd mixed with gaseous M.PComponent more soluble in the S.P → travels slowerComponent less soluble in the S.P → travels fasterComponents are separated according to their Partition Co-efficientCriteria for compounds to be analyzed by G.C1.VOLATILITY:2.THERMOSTABILITY: sudheerkumar kamarapu 4
  5. 5. 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. sudheerkumar kamarapu 5
  6. 6. 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. sudheerkumar kamarapu 6
  7. 7. Peaks and Datasudheerkumar kamarapu 7
  8. 8. PARTS OF INSTRUMENT1. Carrier gas2. Flow regulators & Flow Meters3. Injection devices4. Columns5. Temperature control devices6. Detectors7. Recorders sudheerkumar kamarapu 8
  9. 9. Schematic diagram of gas chromatography sudheerkumar kamarapu 9
  10. 10. sudheerkumar kamarapu 10
  11. 11. CARRIER GAS» Hydrogen better thermal conductivity disadvantage: it reacts with unsaturated compounds & inflammable» Helium excellent thermal conductivity it is expensive» Nitrogen reduced sensitivity it is inexpensive sudheerkumar kamarapu 11
  12. 12. 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 sudheerkumar kamarapu 12
  13. 13. Flow regulators & Flow meters deliver the gas with uniform pressure/flow rate flow meters:- Rota meter & Soap bubble flow meter Rota meterplaced before column inlet it has a glass tube with a float held on to a spring. the level of the float is determined by the flow rate of carrier gas sudheerkumar kamarapu 13
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  15. 15. Soap Bubble Meter◊ Similar to Rota meter & instead of a float, soap bubble formed indicates the flow rate sudheerkumar kamarapu 15
  16. 16. Injection Devices Gases can be introduced into the column by valve devices liquids can be injected through loop or septum devices sudheerkumar kamarapu 16
  17. 17. COLUMNS• Important part of GC• Made up of glass or stainless steel• Glass column- inert , highly fragile COLUMNS can be classified Depending on its use 1. Analytical column 1-1.5 meters length & 3-6 mm d.m 2. Preparative column 3-6 meters length, 6-9mm d.m sudheerkumar kamarapu 17
  18. 18. Depending on its nature1.Packed column: columns are available in a packed mannerS.P for GLC: polyethylene glycol, esters, amides, hydrocarbons, polysiloxanes…2.Open tubular or Capillary column or Golay column Long capillary tubing 30-90 M in length Uniform & narrow d.m of 0.025 - 0.075 cm Made up of stainless steel & form of a coil Disadvantage: more sample cannot loaded sudheerkumar kamarapu 18
  19. 19. 3.SCOT columns (Support coated open tubular column Improved version of Golay / Capillary columns, have small sample capacity Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column Then coated with a thin film of liquid phase sudheerkumar kamarapu 19
  20. 20. Columns• Packed• Capillary sudheerkumar kamarapu 20
  21. 21. Equilibration of the column Before introduction of the sample Column is attached to instrument & desired flow rate by flow regulators Set desired temp. Conditioning is achieved by passing carrier gas for 24 hours sudheerkumar kamarapu 21
  22. 22. Column temperature and temperature program The column(s) in a GC are contained in an oven, the temperature of which is precisely controlled electronically. The rate at which a sample passes through the column is directly proportional to the temperature of the column. The higher the column temperature, the faster the sample moves through the column. A method which holds the column at the same temperature for the entire analysis is called "isothermal Programming" Most methods, however, increase the column temperature during the analysis,. " Gradient Temperature programming - Start at low temperature and gradually ramp to higher temperature sudheerkumar kamarapu 22
  23. 23. Temperature Control DevicesPreheaters: convert sample into its vapour form, present along with injecting devicesThermostatically controlled oven: temperature maintenance in a column is highly essential for efficient separation. sudheerkumar kamarapu 23
  24. 24. DETECTORSHeart of the apparatus The requirements of an ideal detector are- Applicability to wide range of samples Rapidity High sensitivity Linearity Response should be unaffected by temperature, flow rate… Non destructive Simple & inexpensive sudheerkumar kamarapu 24
  25. 25. Types of Detectors1. Thermal Conductivity Detectors (TCD)2. Flame Ionization Detectors (FID)3. Photo Ionization Detectors (PID)4. Argon Ionization Detectors (AID)5. Electron Capture Detectors (ECD). sudheerkumar kamarapu 25
  26. 26. 1.Thermal Conductivity Detector (Katharometer, Hot Wire Detector)• A TCD detector consists of an electrically-heated wire or thermistor.• The temperature of the sensing element depends on the thermal conductivity of the gas flowing around it.• Changes in thermal conductivity, such as when organic molecules displace some of the carrier gas, cause a temperature rise in the element which is sensed as a change in resistance.• The TCD is not as sensitive as other detectors but it is non-specific and non- destructive. sudheerkumar kamarapu 26
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  28. 28. Thermal Conductivity Basics When the carrier gas is contaminatedThe TCD is a nondestructive, by sample , the cooling effect ofconcentration sensing detector. A the gas changes. The difference inheated filament is cooled by the flow cooling is used to generate theof carrier gas. detector signal. Flow Flow sudheerkumar kamarapu 28
  29. 29. Relative Thermal Conductivity Relative Thermal Compound ConductivityCarbon Tetrachloride 0.05Benzene 0.11Hexane 0.12Argon 0.12Methanol 0.13Nitrogen 0.17Helium 1.00Hydrogen 1.28 sudheerkumar kamarapu 29
  30. 30. Advantages of KatharometerLinearity is goodApplicable to most compoundsNon destructiveSimple & inexpensiveDisadvantages Low sensitivity Affected by fluctuations in temperature and flow rate Biological samples cannot be analyzed sudheerkumar kamarapu 30
  31. 31. Flame Ionization Detectors (FID)  It also called as Destructive detector (destructive of sample) The principle involved in the detectors are based upon the electrical conductivity of carrier gases. At normal temperature and pressure gases act as insulators, but become conductive of ions if electrons are present. sudheerkumar kamarapu 31
  32. 32. Flame Ionization Detectorsudheerkumar kamarapu 32
  33. 33.  The effluent from the column is mixed with hydrogen and air and then ignited electrically. Most organic compounds, when pyrolyzed at the temperature of a hydrogen-air flame, produce ions and electrons that can conduct electricity through the flame A potential of a few hundred volts is applied. The resulting current (~10-12 A) is then measured. The flame ionization detector exhibits a high sensitivity (~10-13 g/s), large linear response range (~107), and low noise. A disadvantage of the flame ionization detector is that it is destructive of sample. sudheerkumar kamarapu 33
  34. 34. ADVANTAGES:• µg quantities of the solute can be detected• Stable• Responds to most of the organic compounds• Linearity is excellent• DA: destroy the sample sudheerkumar kamarapu 34
  35. 35. 35 P HOTO IONIZATION DETECTOR Principle  A PID is an ion detector which uses high-energy photons, typically in the UV range, to produce ions.  As components elute from the GCs column they are bombarded by high-energy photons and are ionized.  The ions produce an electric current, which is the signal output of the detector.  The greater the concentration of the component, the more ions are produced, and the greater the current. SUDHEERKUMAR KAMARAPU
  37. 37. Argon ionization detector Depends on the excitation of argon atoms to a metastable state, by using radioactive energy.Argon→ irradiation Argon + e- →collision Metastable Argon→ collision of sub. → Ionization →↑CurrentADVANTAGES1.Responds to organic compounds2.High sensitivityDISADVANTAGES1.Response is not absolute2.Linearity is poor3. Sensitivity is affected by water sudheerkumar kamarapu 37
  38. 38. Electron-Capture Detectors(ECD) The electron capture detector is composed of a radioactive source which emits electrons, a cathode which repels the electrons, an anode and wire which collects the electrons.  The ECD has two electrodes with the column effluent passing between them. One of the electrode is treated with a radioactive isotope which emits electrons as it decays. Electron-Capture Detector sudheerkumar kamarapu 38
  39. 39.  These emitted electrons produce secondaryelectrons which are collected by the anode, when apotential of 20V is applied between them. When carriergas alone flows through, all the secondary electronsare collected by the positively polarised electrode. An important application of the electron-capturedetector has been for the detection and determinationof chlorinated insecticides. It is insensitive to functional groups such asamines, alcohols, and hydrocarbons. sudheerkumar kamarapu 39
  40. 40.  The electron-capture detector is selective in its response being highly sensitive to molecules containing electronegative functional groups such as halogens, peroxides, quinones, and nitro groups.ADVANTAGE Highly sensitiveDISADVANTAGE Used only for compounds with electron affinity sudheerkumar kamarapu 40
  41. 41. RECORDERS & INTEGRATORSRecord the baseline and all the peaks obtained INTEGRATORSRecord the individual peaks with Rt, height…. sudheerkumar kamarapu 41
  42. 42. Derivatisation of sampleTreat sample to improve the process of separation by column or detection by detector.They are 2 types Precolumn derivatisationComponents are converted to volatile & thermo stable derivative.Conditions - Pre column derivatisationComponent ↓ volatileCompounds are thermo labile↓ tailing & improve separation sudheerkumar kamarapu 42
  43. 43. Post column derivatisation Improve response shown by detector Components ionization / affinity towards electrons is increasedPretreatment of solid supportTo overcome tailingGenerally doing separation of non polar components like esters, ethers…Techniques: 1. use more polar liquid S.P2. Increasing amt. of liquid phase3.Pretreatment of solid support to remove active sites. sudheerkumar kamarapu 43
  44. 44. Parameters used in GCRetention time (Rt) It is the difference in time b/w the point of injection & appearance of peak maxima. Rt measured in minutes or seconds(or) It is the time required for 50% of a component to be eluted from a columnRetention volume (Vr) It is the volume of carrier gas which is required to elute 50% of the component from the column. Retention volume = Retention time ˣ Flow rate sudheerkumar kamarapu 44
  45. 45. Separation factor (S)Ratio of partition co-efficient of the two components to be separated.If more difference in partition co-efficient b/w two compounds, the peaks are far apart & S is more.If partition co-efficient of two compounds are similar, then peaks are closer & S is less.Resolution (R)The true separation of 2 consecutive peaks on a chromatogram is measured by resolutionIt is the measure of both column & solvent efficiencies R= 2d W1+W2 sudheerkumar kamarapu 45
  46. 46. Retention timesudheerkumar kamarapu 46
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  48. 48. Separation factorsudheerkumar kamarapu 48
  49. 49. Resolutionsudheerkumar kamarapu 49
  50. 50. Resolutionsudheerkumar kamarapu 50
  51. 51. THEORETICAL PLATE An imaginary unit of the column where equilibrium has been established between S.P & M.P It can also be called as a functional unit of the column HETP – Height Equivalent to a Theoretical Plate Efficiency of a column is expressed by the number of theoretical plates in the column or HETP If HETP is less, the column is ↑ efficient. If HETP is more, the column is ↓ efficient sudheerkumar kamarapu 51
  52. 52. HETP (length of the column) (No of theoritical plates)HETP is given by Van Deemter equation HETP= A + B +Cu uA = Eddy diffusion term or multiple path diffusion which arises due to packing of the columnB = Molecular diffusion, depends on flow rateC = Effect of mass transfer,depends on flow rateu = Flow rate sudheerkumar kamarapu 52
  53. 53. Efficiency ( No. of Theoretical plates)It can be determined by using the formula n = 16 Rt2 w 2N = no. of theoretical platesRt = retention timeW = peak width at baseThe no. of theoretical plates is high, the column is highly efficientFor G.C the value of 600/ meter sudheerkumar kamarapu 53
  54. 54. Asymmetry Factor Chromatographic peak should be symmetrical about its centre If peak is not symmetrical- shows Fronting or Tailing FRONTINGDue to saturation of S.P & can be avoided by using less quantity of sample TAILINGDue to more active adsorption sites & can be eliminated by support pretreatment, sudheerkumar kamarapu 54
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  56. 56. Asymmetry factor (0.95-1.05) can be calculated by using the formula AF=b/ab & a calculated at 5% or 10% of the peak height sudheerkumar kamarapu 56
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  58. 58. 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 sudheerkumar kamarapu 58
  59. 59. Applications of G.C• G.C is capable of separating, detecting & partially characterizing the organic compounds , particularly when present in small quantities.1, Qualitative analysisRt & RV are used for the identification & separation2, Checking the purity of a compoundCompare the chromatogram of the std. & that of the sample sudheerkumar kamarapu 59
  60. 60. 3, Quantitative analysisIt is necessary to measure the peak area or peak height of each component4, used for analysis of drugs & their metabolites. sudheerkumar kamarapu 60
  61. 61. 2/5/2013 sudheerkumar kamarapu 61