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GAS LIQUID CHROMATOGRAPHY
• Principles
Partition of molecules between gas (mobile
phase) and liquid (stationary phase).
Gas Liquid Chromatography
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)
3. Methylester of fatty acids
DEGS (diethylene glycol succinate)
Filters/Traps
Air
Hydrogen
GasCarrier
Column
Gas Chromatography
• 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.
Thermal Conductivity Detector
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.
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
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
Relative Thermal Conductivity
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
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
Thermal Conductivity Detector
Electron Capture Detector
Analyses for pesticide, Insecticides, vinyl
chloride, and fluorocarbons in foods.
Most sensitive detector (10-12
gram)
Electron Capture Detector
ECD detects positive ions of carrier gas by the anode electrode.
63
Ni 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
Electron Capture Detector
Chromatogram of Compounds from Fermented Cabbage
Chromatogram of Orange Juice Compounds
Gas Chromatography Application
Semi-Quantitative Analysis of Fatty Acids
C
C
C
DetectorResponse
Retention Time
14
16
18
PeakArea
Sample 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 + C
100∗
14
181614
= the content % of C fatty acids14
14
Tentative Identification of Unknown
Compounds
Response
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane Decane1.6 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 - 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
+ +
Gas Chromatogram of Methyl Esters of Fatty Acids
Effects of OH groups of Carbohydrates
OH
O
OH
OH
HO
CH
2
OH
1
23
4
5
6
OH
O
OH
OH
HO
CH2 OH
1
23
4
5
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
4
5
6
5HCl+
Derivation of Glucose with Trimethylchlorosilane
Glucose Trimethylchlorosilane
Effects of Derivation
• Time consumption
• Side reaction
• Loss of sample
gas chromatography

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gas chromatography

  • 2. • Principles Partition of molecules between gas (mobile phase) and liquid (stationary phase). Gas Liquid Chromatography
  • 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) 3. Methylester of fatty acids DEGS (diethylene glycol succinate)
  • 4. Filters/Traps Air Hydrogen GasCarrier Column Gas Chromatography • gas system • inlet • column • detector • data system Data system Syringe/Sampler Inlets Detectors Regulators H RESET
  • 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.
  • 8. Measures the changes of thermal conductivity due to the sample (µg). Sample can be recovered. Thermal Conductivity Detector
  • 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. 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
  • 12. Relative Thermal Conductivity 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
  • 14. • 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
  • 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. 63 Ni 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
  • 19. Chromatogram of Compounds from Fermented Cabbage
  • 20. Chromatogram of Orange Juice Compounds
  • 22. Semi-Quantitative Analysis of Fatty Acids C C C DetectorResponse Retention Time 14 16 18 PeakArea Sample 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 + C 100∗ 14 181614 = the content % of C fatty acids14 14
  • 23. Tentative Identification of Unknown Compounds Response GC Retention Time on Carbowax-20 (min) Mixture of known compounds Hexane Octane Decane1.6 min = RT Response Unknown compound may be Hexane 1.6 min = RT Retention Time on Carbowax-20 (min)
  • 24. 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
  • 25. Advantages of Gas Chromatography • Very good separation • Time (analysis is short) • Small sample is needed - µl • Good detection system • Quantitatively analyzed
  • 26. 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 + +
  • 27. Gas Chromatogram of Methyl Esters of Fatty Acids
  • 28. Effects of OH groups of Carbohydrates OH O OH OH HO CH 2 OH 1 23 4 5 6
  • 29. OH O OH OH HO CH2 OH 1 23 4 5 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 4 5 6 5HCl+ Derivation of Glucose with Trimethylchlorosilane Glucose Trimethylchlorosilane
  • 30. Effects of Derivation • Time consumption • Side reaction • Loss of sample