GAS LIQUID CHROMATOGRAPHY Principles Partition of molecules between gas (mobile phase) and liquid (stationary phase)

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)

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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

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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

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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

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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

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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


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


Electron Capture Detector

For pesticide analysis (picogram).

Accept electrons of carrier gas.


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


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.


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



The TCD will respond to any substance different from the carrier gas as long as its concentration is sufficiently high enough.



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



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GAS LIQUID CHROMATOGRAPHY Principles Partition of molecules between gas (mobile phase) and liquid (stationary phase)