Chromatography

chromatography

DR SALMAN KHANSRI SAI DENTAL COLLEGE ,VKB1st Year Student

What is Chromatography?Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components.

Separate

• Analyze

• Identify

• Purify

• QuantifyComponentsMixture

CHROMATOGRAPHY

Chromatography - Definition

Uses for Chromatography

Chromatography is used by scientists to:

• Analyze – examine a mixture, its components, and their relations to one another

• Identify – determine the identity of a mixture or components based on known components

• Purify – separate components in order to isolate one of interest for further study

• Quantify – determine the amount of the a mixture and/or the components present in the sample

TYPES OF CHROMATOGRAPHY

PARTITION CHROMATOGRAPHY

Paper Chromatography Experiment

Preparing the Chromatography Strips

● Take a strips of filter paper (Whatman No. 1 or 3)

● Draw a line 2 cm above the bottom edge of the strip with the pencil

● Label each strip with its corresponding solution

● Place a spot from each pen on your starting line

Preparing the Solvent mixture

solvent mixture consisting of

•BUTANOL: ACETIC ACID :WATER

4 : 1 : 5

Developing the Chromatograms● Place the strip in the beaker● Make sure the solution does not

come above your start line● Keep the beakers covered● Let strips develop until the

ascending solution front is about 2 cm from the top of the strip

● Remove the strip and let it dry

Developing the Chromatograms

Rf = distance moved by substance distance moved by solvent front

compounds

Thin Layer Chromatography

● Thin layer of silica gel is spread on a glass plate● TLC takes 2-4hrs in whereas paper

chromatography takes 14-16 hrs● Can be performed as two dimensional

Thin Layer Chromatography

Gas Liquid Chromatography

Here the mobile phase is an unreactive gas ( eg Nitrogen) flowing through a tube.

And the stationary phase is an involatile liquid held on particles of a solid support.

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

2.Adsorption chromatography● Separation is based on

differences in adsorption at the surface of solid stationary medium

● Alumina,silica gel are used

● These are packed into columns

● Components get adsorbed at different affinity

3.Ion Exchange chromatography

● Separation of molecules on the basis of their electrical charges

● Ion exchange resins –cation exchangers and anion exchangers are used for this purpose

Ion exchange Chromatography

4.Gel filtration chromatographyAlso called molecular sieving

Hydrophilic cross linked gels –agarose ,dextran ,acrylamide are used

Technique is used for (1)seperation ,purification of proteins (2)mol.wt determination

Eg:insulin,immunoglobulins

Gel filtration chromatography

Gel Filtration Chromatography

5.Affinity chromatography

● Based on high affinity of specific protein for specific chemical group

● Eg:co-enzymes can be used to purify enzymes

6.High pressure liuid chromatography (HPLC)

● Require use of non compressible resin material and strong metal column

● Widely used for separation of all types of compounds

● This allows high flow rates

● Seperation is achieved with better resolution and high speed