M.PRASAD NAIDUMsc Medical Biochemistry,Ph.D Research scholar.

• Principles

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

1. Separation of mixture of polar compoundsCarbowax 20M (polyethylene glycol)

2. Separation of mixtures of non-polar compoundsOV101 or SE-30 (polymer of methylsilicone)

3. Methylester of fatty acidsDEGS (diethylene glycol succinate)

Filters/Traps

Air

Hyd

rog

en

Ga

s 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 (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.

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 (µg). 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.

Flo

w

Flo

w

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

Compound Relative Thermal Conductivity

Carbon Tetrachloride 0.05

Benzene 0.11

Hexane 0.12

Argon 0.12

Methanol 0.13

Nitrogen 0.17

Helium 1.00

Hydrogen 1.28

• 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 (10-12 gram)

ECD detects positive ions of carrier gas by the anode electrode.

63Ni emits β particles.

Ionization : N2 (Carrier gas) + β (e) = N2+ + 2e. The N2

+ establish a “base line”

X (F, Cl and Br) containing sample + β (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

Electron Capture Detector

Chromatogram of Compounds from Fermented Cabbage

Chromatogram of Orange Juice Compounds

Semi-Quantitative Analysis of Fatty Acids

C

C

C

Det

ecto

r R

espo

nse

Retention Time

14

16

18

Peak

Are

aSample Concentration (mg/ml)

2

4

6

8

10

0.5 1.0 1.5 2.0 2.5 3.0

The content % of C fatty acids =C

C + C + C100∗

14

181614

= the content % of C fatty acids14

14

Tentative Identification of Unknown Compounds

Res

pons

e

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

Octane Decane1.6 min = RT

Res

pons

e

Unknown compound may be Hexane

1.6 min = RT

Retention Time on Carbowax-20 (min)

Res

pons

e

GC Retention Time on SE-30

Unknown compound

RT= 4 min on SE-30

Res

pons

e

GC Retention Time on SE-30

HexaneRT= 4.0 min on SE-30

Advantages of Gas Chromatography

• Very good separation

• Time (analysis is short)

• Small sample is needed - µl

• Good detection system

• Quantitatively analyzed

Disadvantages of Gas Chromatography

Material has to be volatilized at 250C without decomposition.

R C OH CH3OH H2SO4

O

R C O CH3

O

CH2 O C R

CH O C R

CH2 O C R

O

O

O

CH3OH

O

R C O CH3

CH3ONa

Fatty Acids Methylester

Reflux

+ 3

Volatile in Gas Chromatography

Volatile in Gas Chromatography

+ +

Effects of OH groups of Carbohydrates

OH

O

OH

OHHO

CH2

OH

1

23

4

5

6

OH

O

OH

OHHO

CH2 OH

1

23

45

6

+ Si

CH3

CH3

CH35Cl

O-Si(CH3)3

O

O-Si(CH3)3

O-Si(CH3)3(CH3)3-Si-O

CH2 O-Si(CH3)3

1

23

45

6

5HCl+

Derivation of Glucose with Trimethylchlorosilane

Glucose Trimethylchlorosilane

• Time consumption

• Side reaction

• Loss of sample