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GAS CHROMATOGRAPHY

BIOCHEMISTRY

mprasadnaidu
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GAS CHROMATOGRAPHY

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

  2. Gas Liquid Chromatography • Principles • Partition of molecules between gas (mobile phase) and liquid (stationary phase).

  3. Most Common Stationary Phases • 1. Separation of mixture of polar compounds • Carbowax 20M (polyethylene glycol) • 2. Separation of mixtures of non-polar compounds • OV101 or SE-30 (polymer of methylsilicone) • Methylester of fatty acids • DEGS (diethylene glycol succinate)

  4. H RESET Air Hydrogen Gas Carrier Gas Chromatography • gas system • inlet • column • detector • data system Filters/Traps Data system Syringe/Sampler Regulators Inlets Detectors Column

  5. Schematic Diagram of Gas Chromatography

  6. Detector • Flame Ionization Detector (Nanogram - ng) • High temperature of hydrogen flame (H2 +O2 + N2) ionizes compounds eluted from column into flame. The ions collected on collector or electrode and were recorded on recorder due to electric current.

  7. Schematic Diagram of Flame Ionization Detector Exhaust Chimney Collector Electrode Igniter Column Hydrogen Effluent Inlet

  8. Thermal Conductivity Detector Measures the changes of thermal conductivity due to the sample (mg). Sample can be recovered.

  9. Thermal Conductivity Detector Principal: The thermal balance of a heated filament Electrical power is converted to heat in a filament and the temperature will climb until heat power loss form the filament equals the electrical power input. The filament may loose heat by radiation to a cooler surface by conduction to the molecules which contact with the filament.

  10. 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 Flow

  11. Thermal Conductivity Detector 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 temeprature is measured by a simple gadget and a signal is recorded

  12. Relative Thermal Conductivity

  13. Thermal Conductivity Detector

  14. Thermal Conductivity Detector • Responds to all compounds • Adequate sensitivity for many compounds • Good linear range of signal • Simple construction • Signal quite stable if carrier gas glow rate, block temperature, and filament power are effectively controlled • Nondestructive detection

  15. Electron Capture Detector Analyses for pesticide, Insecticides, vinyl chloride, and fluorocarbons in foods. Most sensitive detector (10-12 gram)

  16. Electron Capture Detector ECD detects positive ions of carrier gas by the anode electrode. 63Ni emits  particles. Ionization : N2 (Carrier gas) +  (e) = N2+ + 2e. The N2+ establish a “base line” X (F, Cl and Br) containing sample +  (e)  X- Ion recombination: X- + N2+ = X + N2, The “base line” due to the N2+ will decrease and this decrease constitutes the signal. The more the halogen containing X compounds in the sample, the less the N2+ in the detector

  17. Electron Capture Detector

  18. Electron Capture Detector

  19. Chromatogram of Compounds from Fermented Cabbage

  20. Chromatogram of Orange Juice Compounds

  21. Gas Chromatography Application

  22. Semi-Quantitative Analysis of Fatty Acids C 18 10 C 8 16 Detector Response Peak Area 6 C 4 14 2 0.5 1.0 3.0 2.0 2.5 1.5 Sample Concentration (mg/ml) Retention Time

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

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

  25. Advantages of Gas Chromatography • Very good separation • Time (analysis is short) • Small sample is needed - ml • Good detection system • Quantitatively analyzed

  26. O O R C OH CH OH H SO R C O CH 3 2 4 3 O CH O C R 2 O O CH ONa 3 R C O CH CH O C R CH OH 3 3 O CH O C R 2 Disadvantages of Gas Chromatography Material has to be volatilized at 250C without decomposition. Methylester Fatty Acids + + Reflux Volatile in Gas Chromatography 3 + Volatile in Gas Chromatography

  27. Gas Chromatogram of Methyl Esters of Fatty Acids

  28. Effects of OH groups of Carbohydrates 6 CH OH 2 O 5 4 1 OH HO OH 2 3 OH

  29. CH 3 Si 5Cl CH 3 CH 3 6 CH OH 2 O 5 4 1 OH HO OH 2 3 OH Derivation of Glucose with Trimethylchlorosilane + Glucose Trimethylchlorosilane 6 CH O-Si(CH3)3 2 O 5 + 5HCl 4 1 O-Si(CH3)3 (CH3)3-Si-O O-Si(CH3)3 2 3 O-Si(CH3)3

  30. Effects of Derivation • Time consumption • Side reaction • Loss of sample

  31. THANK YOU

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