CHROMATOGRAPHY

Reference
Boyer, D. 2000. Modern Experimental
Biochemistry. 3rd Ed. Pearson Education Inc.

Online Link on Google Books

GAS CHROMATOGRAPHY
 Partition chromatography
 For volatile compounds
 Mobile phase is gaseous
 Stationary phase is a liquid coated on inert solid particles
 Stationary phase is present in stainless steel or glass tube called
column
 Principle: sample is injected into gaseous phase and swept
through stationary phase. Components of sample mixture that
have affinity for stationary phase are retarded in their
movement and vice versa
COMPONENTS
 Carrier gas (helium, nitrogen or argon)
 Column
 Injection port
 Detectors
 Data processor/recorder
 Column, injection port and detectors are in individual
oven maintained at elevated temperature so that sample
components remains vaporized through their residence
time in the system
 CARRIER GAS

Helium, nitrogen, and argon

Gas supply are pressure regulators, gauges, and flow meters
Molecular sieve to remove water or other impurities.
 COLUMNS
 Two types of columns
 Packed columns and open tubular or capillary columns
 Packed columns
 Filled with inert, solid particles-coated with liquid stationary
phase,
 diameter is 0.5cm
 length varies from 1m to 20 m
 Solid supports are diatomaceous earth, teflon powder and glass
beads
 COLUMNS
 Capillary columns:
 Coating the inner wall of column with stationary liquid phase
 Inside diameter of tube is 0.25 mm
 Length ranges from 10 to 100 meters
 Direct relationship between length of columns resolution
 Made of fused silica glass
DETECTORS
 Several types but two most common
 Thermal conductivity cell
 Flame ionization detector
THERMAL CONDUCTIVITY DETECTOR

 Measures temperature dependent electrical resistance of wire

 Made of platinum or platinum alloy wire
 Sensitivity (5 ug)
 Most widely used due to its response to all organic compounds
 Do not destroy the sample
 Sample can be collected back
FLAME IONIZATION DETECTORS
(FID)
 Organic vapors are burnt in flame supported by hydrogen gas
and air
 Electron and ionic fragments
 Production of electrical current , then fed to printer
 Sensitive- about 10-5 ug
 Sensitive to only oxidize able compounds
 Destructive
RECORDER
 Electrical signal from detector amplified and fed to recorder.
 Peak formation
 Peak represents a components in mixture
 Peak is identified by retention time
 Retention time: time lapse b/w injection of sample and
maximum signal from recorder
 Constant for particular compound
ANALYSIS OF GC DATA
 Qualitative and quantitative analyses
Retention time and peak enhancement
By comparing with peaks of know standards
SELECTION OF
OPERATING CONDITIONS
 Proper column
 Temp for column oven
 Adjustment of gas flow rate
 Selection of stationary phase
 Detector and injector temp is about 10 centigrade above
column temp
 A suitable flow rate for a 0.5cm packed column is b/w 50
and 120 ml/min.
ADVANTAGES & DISADVANTAGES

Excellent separation, simple, versatile, rapid and

comparatively less inexpensive

Only volatile compounds or compounds which can be

made volatile with in suitable temp range