Chromatography

Chapter 4-4

1Dr Gihan Gawish

Principles

Partition of molecules between liquid (stationary phase) and gas (mobile phase)

Gas-liquid chromatography (GLC)

Dr Gihan Gawish

The mobile phase is a carrier gas, usually an inert gas such as helium or an

uncreative gas such as nitrogen

The stationary phase is a microscopic layer of liquid or polymer on an inert solid

support, inside glass or metal tubing, called a column.

The instrument is called a gas chromatograph

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In the animation below the red molecules are more soluble in the liquid (or less volatile) than the green

molecules .

Gas-liquid chromatography (GLC)

Most Common Stationary Phases of GC

Dr Gihan Gawish

1. Separation of mixture of polar compounds (polyethylene glycol)

2. Separation of mixtures of non-polar compounds (polymer of methyl silicone)

3. Methyl ester of fatty acidsDEGS (diethyl glycol succinct)

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Gas Chromatography

gas system

inlet column detector data

system

Dr Gihan Gawish

Filters/Traps

Air

Hyd

rog

en

Gas C

arrier

Column

Data system

Syringe/Sampler

Inlets

Detectors

Regulators

H

RESET

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A gas chromatograph is a chemical analysis instrument for separating chemicals in a complex sample.

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Schematic Diagram of Gas Chromatography

GC analysis

Dr Gihan Gawish

A gas chromatograph uses a flow-through narrow tube

known as the column, through which different chemical

constituents of a sample pass in a gas stream (carrier

gas, mobile phase) at different rates depending on:

1. Their various chemical and physical properties

2. Their interaction with a specific column filling, called

the stationary phase.

1.

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Dr Gihan Gawish

As the chemicals exit the end of the column, they are detected and identified electronically.

The function of the stationary phase in the column is to separate different components at

a different time (retention time).

Other parameters that can be used to alter the order or time of retention are the carrier

gas flow rate, and the temperature and strength of adsorption.

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GC analysis

Physical Components

Dr Gihan Gawish

1. Auto samplers The auto sampler provides the means to introduce automatically a sample into the

inlets.

Manual insertion of the sample is possible but is no longer common.

Automatic insertion provides better reproducibility and time-optimization.

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Dr Gihan Gawish

2- The column inlet (or injector) provides the means to introduce a sample into a continuous flow of carrier gas.

The inlet is a piece of hardware attached to the column head.

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Physical Components

Dr Gihan Gawish

3. ColumnsTwo types of columns are used in GC:

3-1- Packed columns They are 1.5 - 10 m in length and have an internal diameter

of 2 - 4 mm.

The tubing is usually made of stainless steel or glass and contains a packing of solid support material that is coated with a liquid or solid stationary phase.

The nature of the coating material determines what type of materials will be most strongly adsorbed.

Thus numerous columns are available that are designed to separate specific types of compounds.

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Animation

Dr Gihan Gawish

3-1- Capillary Columns They have a very small internal

diameter, on the order of a few tenths of millimeters, and lengths between

25-60 meters are common.

The inner column walls are coated with the active materials.

Most capillary columns are made of fused-silica with a polyimide outer

coating. These columns are flexible, so a very long column can be wound into

a small coil.

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Physical Components

4.Detector

Dr Gihan Gawish

4- Detector is used to monitor the outlet stream from the column; thus,

the time at which each component reaches the outlet

the amount of that component can be determined.

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Physical Components

Dr Gihan Gawish

Types of Detector in GC

4.1.Flame Ionization Detector (sensitivity 10 µg)

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.

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Dr Gihan Gawish

4.2.Thermal Conductivity Detector (sensitivity 10 µg)

Measures the changes of thermal conductivity due to the sample (g).

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Types of Detector in GC

4.3.Electron Capture Detector (sensitivity 10 µg)

Dr Gihan Gawish

• which uses a radioactive (electron) source to measure the degree of electron capture.

• Sensitive to electronegative groups

• Insensitive to amines, alcohols

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Types of Detector in GC

4.4. GC-MS Detector

Dr Gihan Gawish

Some gas chromatographs are connected to a mass spectrometer

which acts as the detector.

The combination is known as GC-MS.

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Types of Detector in GC

Dr Gihan Gawish

SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS

C

C

CDetector Response

Retention Time

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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

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GC-Application

Dr Gihan Gawish

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)19

GC-Application

Dr Gihan Gawish 20

GC-Advantages

1. Very good separation

2. Time (analysis is short)

3. Small sample is needed - l

4. Good detection system

5. Quantitatively analyzed

Dr Gihan Gawish

DISADVANTAGES OF GAS CHROMATOGRAPHY

Material has to be volatilized by oxidation, acylation and alkylation. 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

+ +

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Dr Gihan Gawish

Gas Chromatogram of Methyl Esters of Fatty Acids

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