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GAS LIQUID CHROMATOGRAPHY
Principles
Partition of molecules between gas (mobile phase) and liquid (stationary phase).
Gas Chromatography –
Gas Chromatography
Uses
Separation and analysis of organic compounds
Testing purity of compounds
Determine relative amounts of components in mixture
Compound identification
Isolation of pure compounds (microscale work)
Similar to column chromatography, but differs in 3 ways:
Partitioning process carried out between Moving Gas Phase and Stationary Liquid Phase
Temperature of gas can be controlled
Concentration of compound in gas phase is a function of the vapor pressure only.
GC also known as Vapor-Phase Chromatography (VPC) and Gas-Liquid Partition Chromatography (GLPC)
04/19/232
Gas Chromatography –
Gas Chromatograph Microliter Syringe Heated injection port with rubber septum for
inserting sample Heating chamber with carrier gas injection port Oven containing copper, stainless steel, or glass
column. Column packed with the Stationary Liquid Phase
a non-volatile liquid, wax, or low melting solid-high boiling hydrocarbons, silicone oils, waxes or polymeric esters, ethers, and amides
Liquid phase is coated onto a support material, generally crushed firebrick
04/19/233
Gas Chromatography –
Principals of Separation Column is selected, packed with Liquid Phase, and installed.
Sample injected with microliter syringe into the injection port
where it is vaporized and mixed into the Carrier Gas stream
(helium, nitrogen, argon).
Sample vapor becomes partitioned between Moving Gas
Phase and Stationary Liquid Phase.
The time the different compounds in the sample spend in the
Vapor Phase is a function of their Vapor Pressure.
The more volatile (Low Boiling Point / Higher Vapor Pressure)
compounds arrive at the end of the column first and pass into
the detector
04/19/234
Gas Chromatography –
Principals of Detection
Two Detector Types
Thermal Conductivity Detector (TCD)
Flame Ionization
TCD is electrically heated “Hot Wire” placed in carrier gas stream
Thermal conductivity of carrier gas (helium in our case) is higher than most organic
substances.
Presence of sample compounds in gas stream reduces thermal conductivity of
stream
Wire heats up and resistance decreases.
Two detectors used: one exposed to sample gas and the other exposed to reference
flow of carrier gas.
Detectors form arms of Wheatstone Bridge, which becomes unbalanced by sample
gas.
Unbalanced bridge generates electrical signal, which is amplified and sent to
recorder
04/19/235
Gas Chromatography –
Factors Affecting Separation
Boiling Points of Components in Sample
Low boiling point compounds have higher vapor pressures.
High boiling point compounds have lower vapor pressures
requiring more energy to reach equilibrium vapor pressure, i.e.,
atmospheric pressure.
Boiling point increases as molecular weight increases.
Flow Rate of Carrier Gas
Choice of Liquid Phase
Molecular weights, functional groups, and polarities of component
molecules are factors in selecting liquid phase.
Length of Column
Similar compounds require longer columns than dissimilar
compounds. Isomeric mixtures often require quite long columns
04/19/236
Most Common Stationary Phases
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
Gas C
arrier
Column
Gas ChromatographyGas Chromatography
gas system
inlet column detecto
r data
system
Data system
Syringe/Sampler
Inlets
Detectors
Regulators
H
RESET
Schematic Diagram of Gas Chromatography
Schematic Diagram of Gas Chromatography
DETECTORS
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
Polarizing Electrode
Collector Electrode
Schematic Diagram of Flame Ionization DetectorSchematic Diagram of Flame Ionization Detector
Schematic Diagram of Flame Ionization Detector
Collector
Jet
Flame
Detector electronics
- 220 volts
Column
Chassis ground
Signal output
Thermal Conductivity Detector
Measures the changes of thermal conductivity due to the sample (g). Sample can be recovered.
Thermal Conductivity DetectorPrincipal: The thermal balance of a heated filament
Electrical power is converted to heat in a resistant 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 and by conduction to the molecules coming into contact with it.
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 .
Flo
w
Flo
w
When a compound elutes, the thermal conductivity of the gaseous mixture of carrier gas and compound gas is lowered, and the filament in the sample column becomes hotter than the other control column.
Its resistance increased, and this imbalance between control and sample filament resistances is measured by a simple gadget and a signal is recorded
Thermal Conductivity DetectorThermal Conductivity Detector
Thermal Conductivity Detector
Relative Thermal Conductivity
CompoundRelative 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 provided carrier gas glow rate, block temperature, and filament power are controlled
• Nondestructive detection
Thermal Conductivity DetectorThermal Conductivity Detector
Electron Capture Detector
For pesticide analysis (picogram).
Accept electrons of carrier gas.
Electron Capture Detector
ECD detects ions in the exiting from the gas chromatographic
column by the anode electrode.
3H or 63Ni which emits particles.
Ionization : N2 (Nitrogen carrier gas) + (e) = N2+ + 2e
These N2+ establish a “base line”
X (F, Cl and Br) containing sample + (e) X-
Ion recombination : X- + N2+ = X + N2
The “base line” will decrease and this decrease constitutes the signal.
Insecticides, pesticides, vinyl chloride, and fluorocarbons
Electron Capture DetectorElectron Capture Detector
Electron Capture Detector
Gas Chromatography Application
SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS
C
C
CDetector Response
Retention Time
14
16
18 Peak Area (cm )
Sample Concentration (mg/ml)
2
4
6
8
10
0.5 1.0 1.5 2.0 2.5 3.0
2
The content % of C fatty acids =C
C + C + C
= 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
OctaneDecane
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
GLC ADVANTAGES
1. Very good separation
2. Time (analysis is short)
3. Small sample is needed - l
4. Good detection system
5. 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
The Effects of OH groups of Carbohydrates
OH
O
OH
OHHO
CH2OH
1
23
45
6
OH
O
OH
OHHO
CH2OH
1
23
45
6
OH
O
OH
OHHO
CH2OH
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
CH2O-Si(CH3)3
1
23
45
6
5HCl+
Derivation of Glucose with Trimethylchlorosilane
Glucose Trimethylchlorosilane
Effects of Derivation
1. Time consumption
2. Side reaction
3. Loss of sample
THIN LAYER CHROMATOGRAPHY
Stationary Phase ---------> Silica Gel
Mobile Phase -------------> Solvent (developing)
SOLVENT
SPOT
DEVELOPINGCHAMBER
Origin
SolventFront
1.1 cm
5.5 cm
Rf =Distance from starting origin to center of zone
Distance from starting origin to solvent front
=5.5
11= 0.5
THIN LAYER CHROMATOGRAPHY
The detector contains two filaments: one exposed only to carrier gas, while the other is exposed to the carrier gas for sample analysis.
When the gas for the sample analysis is only carrier gas , the two filaments can be balanced.
Instead of a direct measurement of filament temperature, the filament resistant, which is a function of temperature, is measured.
Thermal Conductivity DetectorThermal Conductivity Detector
The ability of a colliding molecule to carry off heat depending on its thermal conductivity. Hydrogen and helium have high thermal conductivity and therefore will be more efficient at “cooling” a heated filament than other gases will
Thermal Conductivity DetectorThermal Conductivity Detector
Thermal Conductivity Detector
The TCD will respond to any substance different from the carrier gas as long as its concentration is sufficiently high enough.
Thermal Conductivity Detector
Thermal Conductivity Detector
Electron capture compound, X (highly electonegative element), tends to capture free electrons and increase the amount to ion recombination
X (F, Cl and Br) + e X-
Ion recombination : X- + N2+ = X + N2
The current will decrease and this decrease constitutes the signal.
Halogens, lead, phosphorous, nitro groups, silicone and polynuclear aromatics.
Insecticides, pesticides, vinyl chloride, and fluorocarbons
Electron Capture DetectorElectron Capture Detector
Electron Capture DetectorElectron Capture Detector