View
122
Download
0
Tags:
Embed Size (px)
DESCRIPTION
good
Citation preview
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