Analytical Techniques – Chromatography

 Chromatography is teachnique used to separate a mixture of solute by differential movement of

individual solutes through a porous medium.
 The most common chromatography technique used is paper chromatography. This technique is
used for separating mixtures of proteins or mixture of plant pigments.

How to use paper chromatography to separate chlorophyll pigments?

Paper chromatography

 The technique is halted when solvent front reaches near the top of the filter paper. 
 The distanced moved by the solvent front and each of the pigments on the chromatogram is
measured. Rf value of each component is then determined using the formula below.

 Rf value can be used to identify the components of a mixture by comparison with standard
Rf values of substances.
 To separate a mixture of proteins, a two dimensional paper chromatography can be used for
better separation.
two dimensional paper chromatography

Analytical Techniques – Electrophoresis

 Electrophoresis is a technique used to separate charged molecules such as amino acids and
proteins using an electric field.
 Charged molecules migrate in an electric field. The rate of migration depends on the size, shape
and charge of the molecule. 
 So different types of molecules will be separated in the field.
How to use a paper electrophoresis to separate a mixture of amino
acids?
Paper electrophoresis

Process of paper electrophoresis

Analytical Techniques - X-ray Diffraction

 This technique is also called an X-ray crystallography. This is used to study 3 dimensional
structures of molecules such as DNA, protein, myoglobin and other polymers.
 This technique was first used by Sir Lawrence Bragg who discovered the structure of sodium
chloride. He bombarded sodium chloride crystal with x-ray and studied the x-ray pattern that was
produced.
Technique:
X-ray diffraction technique

Process of X-ray diffraction technique

The main problem of this technique is the study and analysis of the X-ray diffraction patterns produced,
But now, with the help of computers, a more correct structure of the molecules can be deduced.