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.
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?

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.
 Light Microscope
Illuminating source is the Light.
Specimen preparation takes usually few
minutes to hours.
Live or Dead specimen may be seen.
Condenser, Objective and eye piece lenses are
made up of glasses.
It has low resolving power (0.25µm to 0.3µm).
It has a magnification of of 500X to 1500X.
The object is 5µm or thicker.
Image is Colored.
Vacuum is not required.
Electron Microscope
Illuminating source is the beam of electrons.
Specimen preparation takes usually takes few
days.
Only Dead or Dried specimens are seen.
All lenses are electromagnetic.
It has high resolving power (0.001µm), about
250 times higher than light microscope.
It has a magnification of 100,000X to 300,000X.
The object is 0.1µm or thinner.
Image is Black and White.
Vacuum is essential for its operation.