1. Gas Chromatography

2. Presented By - Mr. Shaise Jacob Faculty Nirmala College of Pharmacy Muvattupuzha, Kerala India Email – jacobshaise@gmail.com

3. What is Gas Chromatography? It is also known as…It is also known as… –Gas-Liquid Chromatography (GLC)

4. GAS CHROMATOGRAPHY  Separation of gaseous & volatile substances  Simple & efficient in regard to separation GC consists of GSC (gas solid chromatography) GLC (gas liquid chromatography Gas → M.P Solid / Liquid → S.P GSC not used because of limited no. of S.P GSC principle is ADSORPTION GLC principle is PARTITION

5. Sample to be separated is converted into vapour And mixed with gaseous M.P Component more soluble in the S.P → travels slower Component less soluble in the S.P → travels faster Components are separated according to their Partition Co-efficient Criteria for compounds to be analyzed by G.C 1.VOLATILITY: 2.THERMOSTABILITY:

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

8. The Next Generation in Gas Chromatography

9. 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.

10. Chromatographic Separation –Deals with both the stationary phase and the mobile phase. Mobile – inert gas used as carrier.Mobile – inert gas used as carrier. Stationary – liquid coated on a solid or a solid within a column.Stationary – liquid coated on a solid or a solid within a column.

11. Chromatographic Separation Chromatographic SeparationChromatographic Separation –In the mobile phase, components of the sample are uniquely drawn to the stationary phase and thus, enter this phase at different times. –The parts of the sample are separated within the column. –Compounds used at the stationary phase reach the detector at unique times and produce a series of peaks along a time sequence.

12. Chromatographic Separation (continued) –The peaks can then be read and analyzed by a forensic scientist to determine the exact components of the mixture. –Retention time is determined by each component reaching the detector at a characteristic time.

13. 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.

14. Peaks and Data

21. PRACTICAL REQUIREMENTS Carrier gas Flow regulators & Flow meters Injection devices Columns Temperature control devices Detectors Recorders & Integrators

22. 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

23. 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

24. 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

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

27. Injection Devices  Gases can be introduced into the column by valve devices  liquids can be injected through loop or septum devices

28. 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

29.  Depending on its nature 1.Packed column: columns are available in a packed manner S.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

30. 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

31. Columns Packed Capillary

34. 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

35. 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. Two types of operations  Isothermal programming:-  Linear programming:- this method is efficient for separation of complex mixtures

36. Temperature Control Isothermal Gradient Instrumentation - Oven

37. 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

38. Measures the changes of thermal conductivity due to the sample (  g). Sample can be recovered. 1.Thermal Conductivity Detector (Katharometer, Hot Wire Detector)

39. Thermal Conductivity Basics 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. The TCD is a nondestructive, concentration sensing detector. A heated filament is cooled by the flow of carrier gas. Flow

40. When a separated compound elutes from the column, the thermal conductivity of the mixture of carrier gas and compound gas is lowered. The filament in the sample column becomes hotter than the control column. The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded Thermal Conductivity Detector

41. Measures heat loss from a hot filament – filament heated to const T when only carrier gas flows heat loss to metal block is constant, filament T remains constant. when an analyte species flows past the filament generally thermal conductivity goes down, T of filament will rise. (resistance of the filament will rise).

43. Relative Thermal Conductivity Compound Relative Thermal Conductivity Carbon Tetrachloride0.05 Benzene0.11 Hexane0.12 Argon0.12 Methanol0.13 Nitrogen0.17 Helium1.00 Hydrogen1.28

44. 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

45. Flame Ionization Detector  Destructive detector  The effluent from the column is mixed with H & air, and ignited.  Organic compounds burning in the flame produce ions and electrons, which can conduct electricity through the flame.  A large electrical potential is applied at the burner tip  The ions collected on collector or electrode and were recorded on recorder due to electric current.

46. 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 DA: destroy the sample

47. FID

49. 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 → ↑ Current ADVANTAGES 1.Responds to organic compounds 2.High sensitivity DISADVANTAGES 1.Response is not absolute 2.Linearity is poor 3. Sensitivity is affected by water

50. ELECTRON CAPTURE DETECTOR 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.

51. 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. This detector is frequently used in the analysis of chlorinated compounds e.g. – pesticides, polychlorinated biphenyls

55. ADVANTAGE Highly sensitive DISADVANTAGE Used only for compounds with electron affinity

56. RECORDERS & INTEGRATORS Record the baseline and all the peaks obtained INTEGRATORS Record the individual peaks with Rt, height….

57. Derivatisation of sample  Treat sample to improve the process of separation by column or detection by detector.  They are 2 types  Precolumn derivatisation Components are converted to volatile & thermo stable derivative. Conditions - Pre column derivatisation  Component ↓ volatile  Compounds are thermo labile  ↓ tailing & improve separation

58. Post column derivatisation  Improve response shown by detector  Components ionization / affinity towards electrons is increased Pretreatment of solid support  To overcome tailing  Generally doing separation of non polar components like esters, ethers… Techniques: 1. use more polar liquid S.P 2. Increasing amt. of liquid phase 3.Pretreatment of solid support to remove active sites.

59. Parameters used in GC Retention 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 column Retention 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

60. 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 Resolution (R) The true separation of 2 consecutive peaks on a chromatogram is measured by resolution It is the measure of both column & solvent efficiencies R= 2 d W 1 +W 2

61. Retention time

63. Separation factor

64. Resolution

66. 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

67. HETP= L (length of the column) N (no of theoretical plates) HETP is given by Van Deemter equation HETP= A + B +Cu u A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column B = Molecular diffusion, depends on flow rate C = Effect of mass transfer,depends on flow rate u = Flow rate

68. Efficiency ( No. of Theoretical plates) It can be determined by using the formula n = 16 Rt 2 w 2 N = no. of theoretical plates Rt = retention time W = peak width at base  The no. of theoretical plates is high, the column is highly efficient  For G.C the value of 600/ meter

71. Asymmetry Factor  Chromatographic peak should be symmetrical about its centre  If peak is not symmetrical- shows Fronting or Tailing  FRONTING Due to saturation of S.P & can be avoided by using less quantity of sample  TAILING Due to more active adsorption sites & can be eliminated by support pretreatment,

73. Asymmetry factor (0.95-1.05) can be calculated by using the formula AF=b/a b & a calculated at 5% or 10% of the peak height

75. 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

76. Gas Chromatography vials caps

77. 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.

78. Applications of G.C G.C is capable of separating, detecting & partially characterizing the organic compounds, particularly when present in small quantities. 1, Qualitative analysis Rt & RV are used for the identification & separation 2, Checking the purity of a compound Compare the chromatogram of the std. & that of the sample

79. 3, Quantitative analysis It is necessary to measure the peak area or peak height of each component 4, used for analysis of drugs & their metabolites.

80. Semi-Quantitative Analysis of Fatty Acids C C C Detector Response Retention Time 14 16 18 Peak Area Sample Concentration (mg/ml) 2 4 6 8 10 0.51.0 1.5 2.02.5 3.0

81. Tentative Identification of Unknown Compounds Response GC Retention Time on Carbowax-20 (min) Mixture of known compounds Hexane Octane Decane 1.6 min = RT Response Unknown compound may be Hexane 1.6 min = RT Retention Time on Carbowax-20 (min)

82. Response GC Retention Time on SE-30 Unknown compound RT= 4 min on SE-30 Response GC Retention Time on SE-30 Hexane RT= 4.0 min on SE-30 Retention Times

83. Advantages of Gas Chromatography Very good separation Time (analysis is short) Small sample is needed -  l Good detection system Quantitatively analyzed

84. 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.