Gas Chromatography (GC) By Faizan Akram

Gas Chromatography

1. 1 Faizan Akram

2. Gas Chromatography 2 REAL PHARMACIST GROUP (RPG) Faizan Akram

3. Table Of Contents History & Introduction Advantages Of Gas Chromatography Components Of Gas Chromatography Detectors Gas Chromatography-Mass Spectrometry 3 Faizan Akram

4. What Is Gas Chromatography? 4 Faizan Akram

5. History 5 The father of modern gas chromatography is Nobel Prize winner John Porter Martin , who also developed the first liquid-gas chromatograph. (1950) Faizan Akram

6. Introduction 6 Gas chromatography – “It is a process of separating component(s) from the given crude drug by using a gaseous mobile phase”’ It involves a sample being vaporized and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid. Two major types • Gas-solid chromatography (stationary phase: solid) • Gas-liquid chromatography (stationary phase: immobilized liquid) Faizan Akram

7. Gas - Solid Chromatography (GSC) 7 The stationary phase, in this case, is a solid like silica or alumina. It is the affinity of solutes towards adsorption onto the stationary phase which determines, in part, the retention time. The mobile phase is, of course, a suitable carrier gas. Most useful for the separation and analysis of gases like CH 4 , CO 2 , CO, ... etc. The use of GSC in practice is considered marginal when compared to gas liquid chromatography. Faizan Akram

8. Gas - Liquid Chromatography (GLC) 8 The stationary phase is a liquid with very low volatility while the mobile phase is a suitable carrier gas. GLC is the most widely used technique for separation of volatile species. The presence of a wide variety of stationary phases with contrasting selectivities and easy column preparation add to the assets of GLC or simply GC. Faizan Akram

9. Advantages of Gas Chromatography 9 The technique has strong separation power and even complex mixture can be resolved into constituents The sensitivity of the method is quite high It gives good precision and accuracy The analysis is completed in a short time The cost of instrument is relatively low and its life is generally long The technique is relatively suitable for routine analysis Faizan Akram

10. Types of Column 10 Open tubular (capillary) column When the stationary phase (liquid or solid) is uniformly distributed on the interior surface of column it is called an open tubular column. When the stationary phase is uniform film of few μm thick liquid that is uniformly coat the inner capillary tubing. The type of column is capillary column. They are further classified into three types i. Wall-coated open tubular column (WCOT) ii . Support-coated open tubular column (SCOT) iii. Porous-layer open tubular column (PLOT) Faizan Akram

11. b).Packed Columns These columns are fabricated from glass, stainless steel, copper, or other suitable tubes. Stainless steel is the most common tubing used with internal diameters from 1-4 mm. The column is packed with finely divided particles (<100-300 mm diameter), which is coated with stationary phase. However, glass tubes are also used for large-scale separations . 11 Faizan Akram

12. Components Of Gas Chromatography 12 Carrier gas Flow regulators & Flow meters Injection devices Columns Temperature control devices Detectors Faizan Akram

13. schematic diagram of a gas chromatograph Syringe Injector Detector Carrier Gas Cylinder Column To Waste or Flow Meter Flow Controller Two-Stage Regulator 13 Faizan Akram

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15. Carrier Gas 15 » Hydrogen ( H 2 ) better thermal conductivity Disadvantage: it reacts with unsaturated compounds & inflammable » Helium ( He ) excellent thermal conductivity it is expensive » Nitrogen ( N 2 ) reduced sensitivity it is inexpensive Faizan Akram

16. Requirements Of A Carrier Gas 16 Inertness Suitable for the detector High purity Easily available Cheap Should not cause the risk of fire Should give best column performance Faizan Akram

17. Impurities in the carrier gas degrade the stationary phase. High-quality gases should be used , and even they should be passed through purifiers to remove O 2 , H2O , and traces of organic compounds prior to entering the column. Steel or copper tubing, rather than plastic or rubber tubing, should be used for gas lines because metals are less permeable to air and do not release volatile contaminants into the gas stream. Impurities in the carrier gas 17 Faizan Akram

18. Flow regulators & Flow meters 18 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 Faizan Akram

19. Soap Bubble Meter Similar to Rota meter & instead of a float, soap bubble formed indicates the flow rate 19 Faizan Akram

20. Sample injection- Direct Injection 20 1)Direct injection : into heated port (>T oven) using micro syringe (i) 1-20 uL packed column ( ii) 10 -3 uL capillary column Faizan Akram

21. Sample Injection- Rotary Sample Valve With Sample Loop 21 Split injection: routine method 0.1-1 % sample to column remainder to waste The injector temperature is kept high (for example,350 ) to promote fast evaporation. Quantitative analysis with split injection can be inaccurate because the split ratio is not reproducible from run to run. Faizan Akram

22. Split Ratio:- The proportion of sample that does not reach the column is called the split ratio and typically ranges from 50:1 to 600:1. 22 Faizan Akram

23. Split less injection: all sample to column best for quantitative analysis only for trace analysis, low [sample] Injector temperature for splitless injection is lower (220 ) than that for split injection, because the sample spends more time in the port and we do not want it to decompose . 23 Faizan Akram

24. On-Column Injection On-column injection is used for samples that decompose above their boiling points and is preferred for quantitative analysis . Solution is injected directly into the column, without going through a hot injector 24 Faizan Akram

25. Gas Chromatography - Columns 25 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 Faizan Akram

26. Depending on its nature a)-Packed columns contain a finely divided, inert, solid support material (  diatomaceous earth ) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm . 26 Faizan Akram

27. b).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 27 Faizan Akram

28. They are further classified into three types Wall-coated Open Tubular Column (WCOT) Support-coated Open Tubular Column (SCOT) Porous-layer Open Tubular Column (PLOT) b).Open tubular or Capillary column or Golay column 28 Faizan Akram

29. A:- Wall-coated Open Tubular Column (WCOT) Inner coated with the liquid stationary phase The most common type of wall coated open tubular column used is fused-silica, because it is stronger, inert, reliable, easy to use, and flexible. 29 Faizan Akram

30. b :- Support-coated Open Tubular Column (SCOT) Inner layer is of Solid support coated with liquid stationary phase Open tubular column has a greater amount of stationary phase than the wall coated column, so it can handle a larger quantity of sample. 30 Faizan Akram

31. C);- Porous-layer OpenTubular Column (PLOT) Inner layer is coated with stationary solid phase particles. The only difference b/w SCOT & PLOT is that a PLOT does not have a liquid stationary phase. 31 Faizan Akram

32. Capillary columns advantages compared to packed columns higher resolution shorter analysis times greater sensitivity Capillary columns disadvantage compared to packed columns smaller sample capacity Need better experience 32 Faizan Akram

33. Temperature Control Devices 33 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. Faizan Akram

34. Detectors 34 Definition:- “ D etector , sensor, sensing element any device that receives a signal or stimulus (as heat or pressure or light or motion etc.) and responds to it in a distinctive manner” Detectors can be grouped into:- a)-concentration dependant detectors   &   b)-mass flow dependant detectors Faizan Akram

35. The signal from a concentration dependant detector is related to the concentration of solute in the detector, and does not usually destroy the sample Dilution of with make-up gas will lower the detectors response. Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. The response of a mass flow dependant detector is unaffected by make-up gas 35 Faizan Akram

36. G C – IDEAL DETECTORS 36 Sensitive (10 -8 -10 -15 g solute/s ) Operate at high Tempreture (0-400 °C ) Stable and reproducible Linear response Wide dynamic range Fast response Simple (reliable ) Nondestructive Uniform response to all analytes Faizan Akram

37. Types Of Detectors 37 Thermal conductivity (TCD) Flame Ionization Detector (FID) Electron capture Detector (ECD) Nitrogen-phosphorus Detector (NPD) Flame photometric Detector (FPD ) Photo-ionization Detector (PID ) 7. sulfur chemiluminescence detector Faizan Akram

38. 1-Thermal conductivity (TCD) 38 ( Katharometer , Hot Wire Detector) In the past, thermal conductivity detectors were most common in gas chromatography because they are simple and universal. Principle:- “TCD is based upon changes in the thermal conductivity of the gas stream brought about by the presence of analyte molecules” The sensing element of TCD is an electrically heated element whose temperature at constant electrical power depends upon the thermal conductivity of the surrounding gas. Faizan Akram

39. Helium is the carrier gas commonly used with a thermal conductivity detector. Helium has the second highest thermal conductivity (after H2), so any analyte mixed with helium lowers the conductivity of the gas stream . The heated element may be a fine platinum, gold, or tungsten wire or a semiconducting thermistor. 39 Faizan Akram

40. Thermal Conductivity Basics 40 The TCD is a nondestructive, concentration sensing detector. A heated filament is cooled by the flow of carrier gas. Flow When the carrier gas is contaminated by sample , the cooling effect of the gas changes. The difference in cooling is used to generate the detector signal Flow Faizan Akram

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42. Advantages of Katharometer Linearity is good Applicable to most compounds Non destructive Simple & inexpensive Disadvantages Low sensitivity Affected by fluctuations in temperature and flow rate Biological samples cannot be analyzed 42 Faizan Akram

43. 2-Flame Ionization Detector 43 It operates by the principle that by change in conductivity of the flame as the compound is burnt. The change in conductivity of the flame does not arise by simple ionization of the compound , it is partial or complete stripping of the compound to give charged hydrogen-deficient polymers or aggregates of carbon of low ionization potential Carbon atoms (except carbonyl and carboxyl carbons) produce CH radicals, which are thought to produce CHO ions and electrons in the flame . CH + O CHO + e Faizan Akram

44. Nitrogen gives best detection limit • signal proportional to number of susceptible carbon atoms • 100-fold better detection than thermal conductivity • 107 linear response range Flame Ionization Detector The flame ionization detector is sensitive enough for narrow-bore columns. It responds to most hydrocarbons and is insensitive to nonhydrocarbons such as H2, He, N2, O2, CO , CO2, H2O, NH3, NO, H2S, and SiF4. 44 Faizan Akram

45. FIDs are mass sensitive rather than conc. Sensitive ADVANTAGES : µg quantities of the solute can be detected Stable Responds to most of the organic compounds Linearity is excellent DISADVANTAGE: destroy the sample 45 Faizan Akram

46. FID 46 Faizan Akram

47. Remember that FID characteristics include: • Rugged • Sensitive (10-13 g/s ) • Wide dynamic range ( 107) • Signal depends on number of carbon atoms in organic analytes which is referred to as mass sensitive rather than concentration sensitive • Weakly sensitive to carbonyl, amine, alcohol, amine groups • Not sensitive to non-combustibles – H2O, CO2, SO2, NOx • Destructive 47 Faizan Akram

48. 3-Electron-Capture Detectors(ECD) 48 Principle:- “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’’ Mechanism:- The detector consists of a cavity that contains two electrodes and a radiation source that emits  - radiation (e.g. 63 Ni, 3 H ) The collision between electrons and the carrier gas ( methane plus an inert gas) produces a plasma containing electrons and positive ions. Faizan Akram

49. If a compound is present that contains electronegative atoms, those electrons are captured and negative ions are formed, and rate of electron collection decreases The detector selective for compounds with atoms of high electron affinity. APPLICATION:- This detector is frequently used in the analysis of chlorinated compounds e.g . – pesticides, polychlorinated biphenyls 49 Faizan Akram

50. ECD 50 Faizan Akram

51. Remember the following facts about ECD : Electrons from a b-source ionize the carrier gas (nitrogen ) 2. Organic molecules containing electronegative atoms capture electrons and decrease current 3. Simple and reliable 4. Sensitive (10 -15 g/s) to electronegative groups (halogens ) 5. Largely non-destructive 6. Insensitive to amines, alcohols and hydrocarbons 7. Limited dynamic range (10 2 ) 8. Mass sensitive detector 51 Faizan Akram

52. 4-Nitrogen-phosphorus Detector 52 (alkali flame detector) The nitrogen-phosphorus detector , also called an alkali flame detector , is a modified flame ionization detector that is especially sensitive to compounds containing N and P. Its response to N and P is times greater than the response to carbon. “It is particularly important for drug, pesticide, and herbicide analyses” . Ions such as NO2, CN and PO2 , produced by these elements when they contact a Rb2SO4 containing glass bead at the burner tip, create the current that is measured. N2 from air is inert to this detector and does not interfere. The bead must be replaced periodically because RbSO4 is consumed shows a chromatogram from a nitrogen-phosphorus detector . Faizan Akram

53. Nitrogen-phosphorus Detector 53 Faizan Akram

54. 5-flame photometricdetector (FPD) 54 A flame photometric detector measures optical emission from phosphorus, sulfur, lead, tin , or other selected elements . When eluate passes through a H2-air flame, as in the flame ionization detector, excited atoms emit characteristic light. Phosphorus emission at 536 nm or sulfur emission at 394 nm can be isolated by a narrow-band interference filter and detected with a photomultiplier tube. Faizan Akram

55. FPD 55 Faizan Akram

56. 6-Photo-ionization Detector (PID) 56 A photoionization detector uses a vacuum ultraviolet source to ionize aromatic and unsaturated compounds, with little response to saturated hydrocarbons or halocarbons . Electrons produced by the ionization are collected and measured. Faizan Akram

57. 7-sulfur chemiluminescence detector 57 A sulfur chemiluminescence detector takes exhaust from a flame ionization detector, in which sulfur has been oxidized to SO, and mixes it with ozone to form an excited state of that emits blue light and ultraviolet radiation . A nitrogen chemiluminescence detector is analogous. Combustion of eluate at 1800°C converts nitrogen into NO, which reacts with Ozone to create chemiluminescent product. The response to N is 107 times greater than the response of Cabon (C) Faizan Akram

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59. Gas Chromatography-Mass Spectrometry (GC-MS) 59 Faizan Akram

60. GC-MS Contents 60 GC-Mass Spectrometry Introduction Mass Spectrometer & Their Components Application Of GC-MS Sample Preparation Method Development in Gas Chromatography Faizan Akram

61. A:-Mass Spectrometry (MS) 61 Mass spectrometry (MS) is an  analytical chemistry  technique that helps identify the amount and type of chemicals present in a sample by measuring the  mass-to-charge ratio  and abundance of gas- phase ions Faizan Akram

62. Principle of GC-MS 62 The sample solution is injected into the GC inlet where it is vaporized and swept onto a chromatographic column by the carrier gas (usually helium). The sample flows through the column and the compounds comprising the mixture of interest are separated by virtue of their relative interaction with the coating of the column (stationary phase) and the carrier gas (mobile phase ). The latter part of the column passes through a heated transfer line and ends at the entrance to ion source where compounds eluting from the column are converted to ions and detected according to their mass to charge m/z ratio Faizan Akram

63. B:-Mass Spectrometer & Their Components 63 “ Mass spectrometry is a technique used for measuring the molecular weight and determining the molecular formula of an organic compound ” In general a mass spectrometer consists of an ion source, High-vacuum system a mass-selective analyzer, and an ion collector Faizan Akram

64. C:-Application Of GC-MS 64 Petrochemical and hydrocarbons analysis Geochemical research Forensic (arson, explosives, drugs, unknowns ) Environmental analysis Pesticide analysis, food safety and quality Pharmaceutical and drug analysis Faizan Akram

65. GC-MS can analyze the particles from a human body in order to help link a criminal to a crime. accelerant is significant evidence in a fire investigation because it suggests that the fire was set intentionally. Criminal forensics 65 Faizan Akram

66. Law enforcement 66 GC-MS is increasingly used for detection of illegal narcotics marijuana, cocaine, opioids Clinicians oxycodone and oxymorphone Piperazines are not detectable by typical immunoassay testing, but they may be detectable via GC-MS Sports anti-doping analysis Faizan Akram

67. Astrochemistry 67 Several GC-MS have left earth. Two were brought to Mars by the Viking program. Venera 11 and 12 and Pioneer Venus analysed the atmosphere of Venus with GC-MS. Faizan Akram

68. Sample Preparation 68 Sample preparation is the process of transforming a sample into a form that is suitable for analysis. This process might entail extracting analyte from a complex matrix, preconcentrating very dilute analytes to get a concentration high enough to measure, removing or masking interfering species, or chemically transforming ( derivatizing ) analyte into a more convenient or more easily detected form . Solid-phase microextraction Purge and trap Thermal desorption Faizan Akram

69. Solid-phase microextraction 69 Solid-phase microextraction is a method to extract compounds from liquids, air, or even sludge without using any solvent.16 The key component is a fused-silica fiber coated with a film of stationary phase similar to those used in gas chromatography . Faizan Akram

70. Purge and trap 70 Purge and trap is a method for removing volatile analytes from liquids or solids ( such as groundwater or soil), concentrating the analytes , and introducing them into a gas chromatograph . In contrast with solid-phase microextraction , which removes only a portion of analyte from the sample, the goal in purge and trap is to remove 100% of analyte from the sample . Quantitative removal of polar analytes from polar matrices can be difficult. Faizan Akram

71. Purge and trap 71 Faizan Akram

72. Thermal desorption 72 Thermal desorption is a method to release volatile compounds from solid samples. Aweighed sample is placed in a steel or glass tube and held in place with glass wool. The sample is purged with carrier gas to remove O2 , which is vented to the air, not into the chromatography column. The desorption tube is then connected to the chromatography column and heated to release volatile substances that are collected by cold trapping at the beginning of the column. The column is then heated rapidly to initiate chromatography. Faizan Akram

73. Thermal desorption 73 Faizan Akram

74. Method Development in Gas Chromatography 74 Order Of Decision:- Goal Of Analysis Sample Preparation Choosing the Detector Selecting the Column Choosing the Injection Method Faizan Akram

75. Goal Of Analysis 75 What is required from the analysis? Is it qualitative identification of components in a mixture ? Will you require high-resolution separation of everything or do you just need good resolution in a portion of the chromatogram? Can you sacrifice resolution to shorten the analysis time? Do you need quantitative analysis of one or many components? Do you need high precision? Will analytes be present in adequate concentration or do you need preconcentration or a very sensitive detector for ultratrace analysis? How much can the Analysis cost? Faizan Akram

76. Sample Preparation 76 The key to successful chromatography of a complex sample is to clean it up before it ever sees the column . solid-phase microextraction , purge and trap & thermal desorption to isolate volatile components from complex matrices. Other methods include:- liquid extraction, supercritical fluid extraction, and solid-phase extraction, Faizan Akram

77. Choosing the Detector 77 The next step is to choose a detector. Do you need information about everything in the sample or do you want a detector that is specific for a particular element or a particular class of compounds? The most general purpose detector for open tubular chromatography is a mass spectrometer. Thermal conductivity (TCD ) Flame Ionization Detector (FID ) Electron capture Detector (ECD ) Nitrogen-phosphorus Detector (NPD ) Flame photometric Detector (FPD ) Photo-ionization Detector (PID) Faizan Akram

78. Selecting the Column 78 The basic choices are the stationary phase, column diameter and length, and the thickness of stationary phase. A nonpolar stationary phase is most useful. An intermediate polarity stationary phase will handle most separations that the nonpolar column cannot. For highly polar compounds, a strongly polar column might be necessary. Optical isomers and closely related geometric isomers require special stationary phases for separation. Faizan Akram

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80. Choosing the Injection Method 80 The last major decision is how to inject the sample. Split injection: • concentrated sample • high resolution • dirty samples (use packed liner) • could cause thermal decomposition Splitless injection: • dilute sample • high resolution • requires solvent trapping or cold trapping On-column injection: • best for quantitative analysis • thermally sensitive compounds • low resolution Faizan Akram

81. Application Of Gas Chromatography 81 1)- Qualitative Analysis – by comparing the retention time or volume of the sample to the standard / by collecting the individual components as they emerge from the chromatograph and subsequently identifying these compounds by other method 2)- Quantitative Analysis- area under a single component elution peak is proportional to the quantity of the detected component/response factor of the detectors . 3)- Volatile Oils, official monograph gives chromatography profile for some drugs. E.g. to aid distinction between anise oil from star anise and that from Pimpinelle anisum 4)- Separation of fatty acids derived from fixed oils Faizan Akram

82. 5 )- Miscellaneous-analysis of foods like carbohydrates, proteins, lipids, vitamins, steroids, drug and pesticides residues, trace elements 6 )- Pollutants like formaldehyde, carbon monoxide, benzene, DDT etc 7 )- Dairy product analysis- rancidity 8 )- Separation and identification of volatile materials, plastics, natural and synthetic polymers, paints, and microbiological samples 9 )- Inorganic compound analysis 82 Application Of Gas Chromatography Faizan Akram

83. Forensic Pathology 83 gas chromatography is useful in identifying the individual elements and molecules present in a compound, it has been applied in forensic pathology to determine which fluids and compounds are present inside a human body after death Faizan Akram

84. Crime Scene Testing 84 Gas chromatography can also be used to test samples found at a crime scene, whether these be blood samples or fibre samples from clothing or other materials. This allows scientists to pinpoint exactly what (and perhaps who) was present at the scene of the crime Faizan Akram

85. Barbiturates & Benzodiazepines 85 Faizan Akram

86. blood and serum 86 It can also be used for the volatile samples such as human breathe, blood, saliva and other secretions which contains organic volatiles and can be easily analyzed with Gas Chromatography. Faizan Akram

87. Cosmetics 87 Manufacturers of Cosmetics also use gas chromatography to effectively measure how much of each chemical is used for their products. Faizan Akram

88. Radioactive Isotopes 88 Faizan Akram

89. pesticides and fungicides 89 Faizan Akram

90. vitamins 90 Faizan Akram

91. Volatile Oils 91 Faizan Akram

92. References 92 1)- Quantitative Chemical Analysis 7 th Edition Daniel C. Harris Page # 528 ( Accessed at December 22,2014) 2)- Chromatographic methods 5 th Edition By A. Braithwaite & F.J.Smith Page # 166 (Accessed at December 23,2014) 3)- Chemical Analysis Modern Instrumentation Methods and Techniques 2 nd Edition Francis Rouessac and Annick Rouessac Page # 31 (Accessed at December 24,2014) 4)- UNDERGRADUATE INSTRUMENTAL ANALYSIS 6th Edition By James W. Robinson Page # 750 (December 26,2014) Faizan Akram

93. References 93 5)- VOGES Textbook Of Quantitaive Chemical Analysis By MENDHAM. Page # 317,745 (December 27,2014) 6)- Organic Spectroscopy & Chromatography By M.YOUNAS 7)- Mass Spectrometry Principles & Applications By ED Hoffmann & Stroobant Page # 217 (December 27,2014) 8)- Mass Spectrometry By H-GROSS Page # 482 9)- Principles Of Instrumental Analysis By Holler & Skoog Page # 788 (Accessed at December 28,2014) 10)- Instrumental Techniques For Analytical Chemistry By FRAUT SELLER Page # 125 (Accessed at December 29,2014) Faizan Akram

94. 94 References 11)- Dr.S . Ravi Sankar.Text book of pharmaceutical analysis:Rx Publication , Tiruneveli : Edition 1997,1999,2001 . 12)- A Textbook Of Analytical Chemistry Mahinder Singh Page # 110 (Accessed at December 29,2014) 13)- Analytical Chemistry- A Modern Approach To Analytical Science 2 nd Edition By M. Mermet , M.Otto Page # 536,553 14)- Analytical Chemistry By Gary D. Christian 6 th Edition Page # 584,591,593 (December 30,2014) 15)- Chromatography CONCEPTS & CONTRASTS By James M. Miller Page # 141,165,278,285 (December 30,2014) Faizan Akram

95. References 95 16)- Forensic Applications Of Gas Chromatography By Michelle Groves Carlin. 17)- Amirav , A.Gordin , A. Poliak , M. Alon , T. and Fialkov , A. B. (2008), "Gas Chromatography Mass Spectrometry with Supersonic Molecular Beams ". Journal of Mass Spectrometry 43: 141–163 . 18)- A. Hites and K. Biemann , Analytical Chemistry, 855 19 )- H.F.Waltor,J.Reyes: Modern Chemical analysis and instrumentation. Faizan Akram

96. References 96 20)- Structural Identification Of Organic Compounds With Spectroscopic Techniques By Richard R.Ernst Page# 252 21)- Introduction To Pharmaceutical Chemical Analysis By Steen Hansen Page # 191-200 22)- G Chatwal,S anand.Ihstrumental Methods of Chemical analysis: Himalaya publishing house,Delhi:Edition June 1995 . 23)- www.GasChromatography/Slideshare.com 24)- www.Gas Chromatography/Authorstream.com 25)- www.GasChromatography/Orgchem.com Faizan Akram

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99. 99 Presented to: Respected Mam Saharish & 3rd Prof (Eve) Presentation Given by Members of: ‘‘ Real Pharmacist Group (RPG )’’ Made By: Faizan Akram ( Faizy ) Faizan Akram

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