M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D Research scholar.

Principles Partition of molecules between gas (mobile phase) and liquid (stationary phase).

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) 3.Methylester of fatty acids DEGS (diethylene glycol succinate)

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

Schematic Diagram of Gas Chromatography

Detector Flame Ionization Detector (Nanogram - ng) High temperature of hydrogen flame (H 2 +O 2 + N 2 ) ionizes compounds eluted from column into flame. The ions collected on collector or electrode and were recorded on recorder due to electric current.

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

Measures the changes of thermal conductivity due to the sample (  g). Sample can be recovered.

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.

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

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 Thermal Conductivity Detector

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

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 Thermal Conductivity Detector

Analyses for pesticide, Insecticides, vinyl chloride, and fluorocarbons in foods. Most sensitive detector ( gram)

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

Electron Capture Detector

Chromatogram of Compounds from Fermented Cabbage

Chromatogram of Orange Juice Compounds

Semi-Quantitative Analysis of Fatty Acids C C C Detector Response Retention Time Peak Area Sample Concentration (mg/ml)

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)

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

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

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

Gas Chromatogram of Methyl Esters of Fatty Acids

Effects of OH groups of Carbohydrates OH O HO CH 2 OH

O HO CH 2 OH Si CH Cl O -Si(CH 3 ) 3 O (CH 3 ) 3 -Si-O CH 2 O-Si(CH 3 ) HCl+ Derivation of Glucose with Trimethylchlorosilane GlucoseTrimethylchlorosilane

Time consumption Side reaction Loss of sample