Chromatography

By Tareq Hrahsheh

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Invention of Chromatography

Mikhail Tswett invented chromatography in 1901 during his

research on plant pigments.

He used the technique to separate various plant pigments such as

chlorophylls, xanthophyll's and carotenoids.

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How Does Chromatography Work?

In all chromatographic separations, the sample is transported in a

mobile phase. The mobile phase can be a gas or liquid.

The mobile phase is then forced through a stationary phase held in

a column or on a solid surface. The stationary phase needs to be

something that does not react with the mobile phase or the sample.

The sample then has the opportunity to interact with the

stationary phase as it moves past it. Samples that interact greatly,

then appear to move more slowly. Samples that interact weakly,

then appear to move more quickly. Because of this difference in

rates, the samples can then be separated into their components.12/20/2014 4

Chromatography is based on a physical equilibrium

that results when a solute is transferred between the mobile

and a stationary phase.

A

A

A

A

AA

A

AA

A

A

A

K = distribution

coefficient or

partition ratio

K =CS

CM

Where CS is the molar

concentration of the

solute in the stationary

phase and CM is the

molar concentration in

the mobile phase.

Cross Section of Equilibrium in a column.

“A” are adsorbed to the stationary phase.

“A” are traveling in the mobile phase. 12/20/2014 5

Common Types of Chromatography

There are now several common chromatographic methods.

These include:

*Paper Chromatography

*Thin Layer Chromatography (TLC)

*Liquid Chromatography (LC)

*High Pressure Liquid Chromatography (HPLC)

*Ion Chromatography

*Gas Chromatography (GC)12/20/2014 6

Thin Layer Chromatography

TLC is a fast, simple, and inexpensive analytical technique

used to determine or monitor:

*The numbers of components in a mixture.

*The identity of two substances.

*The effectiveness of a purification.

……………………………………..

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*TLC involves spotting a dilute solution (1%) of sample on one end of a

small sheet that has been coated with silica gel (SiO2) or alumina (Al2O3),

known as the stationary adsorbent phase.

*The sheet is placed upright inside a jar in a small pool of solvent. As the

solvent rises up the sheet by capillary action, the components travel at

different rates based on competing interactions with the mobile (solvent)

and adsorbent phases.

*SiO2 is used for separation of more polar compounds while Al2O3 is used

in the separation of non-polar compounds.

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A polar solvent will carry a polar compound farther while a

non-polar solvent will carry a non-polar compound farther.

Rf value is the ratio of the distance the spot travels from the

origin to the distance the solvent travels.

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Using thin layer chromatography to identify

compounds

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Applications and Importance of Thin Layer Chromatography

* Pharmaceuticals and Drugs

Identification, purity testing and determination of the concentration

of active ingredients, auxiliary substances and preservatives in drugs

and drug preparations, process control in synthetic manufacturing

processes.

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* Antibiotics

Penicillin's have been separated on silica gel ‘G’ by using the two

solvents, acetone-methanol (1:1) and iso-propanol-methanol (3:7). As the

detecting agent, the iodine-azide reaction was employed by spraying the

dried plates with a 0.1 iodine solution containing 3.5% of sodium azide.

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References:

1- Skoog, D.; Holler, F.; Crouch, S. Principles of Instrumental Analysis 2007.

2- Waters Corporation: History of Chromatography (accessed March 3, 2008).

Thank You

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