Chromatography- method discovered by Tswett in 1903, used
for the separation of plant leaf pigments such as chlorophyll,
xanthophylls through the use of solid
adsorbents. it can be used for the separation of polar as well as non polar
compounds. (chroma = color, graphein =to write)
It includes two phases
i)mobile phase-containing the mixture of substances to be
ii)stationary phase-solid porous matrix
through which mixture travels ( chromatogram).
Types of chromatography
1)paper chromatography-used for the
separation of polar molecules.mixtures of components is applied on a filter
paper 2cm above the one end.that end is dipped in a solvent containing aqueous
and organic components(water/butanol/acetic acid) in a jar which is also equillibriated with the vapors of solvent.different
components in a mixture travels differently wrt to their relative solubility
in polar stationary and non polar mobile phase.
Rate of migration of
substance is expressed as the ratio of distance travelled by substance to the
distance travelled by solvent front.non polar moving faster than polar one.
2) ion exchange chromatography-used for
the separation of charged particles.
i)cation exchanger(acidic ion exchanger) -bears negative charge and separates cation.
ii)anion exchanger(basic ion exchanger)-bears
positive charge and separates anion.
3) gel filtration chromatography-molecules
are separated on the basis of size n shape.
known as size exclusion /molecular sieve chromatography.large molecules
the column more rapidly as compared to small molecules.
4)adsorption chromatography-separates non
polar compounds rather than proteins. molecules are adsorbed on the surface and
then eluted by pure solvent such as chloroform,hexane. separation is based on
partition of various substance between polar column and non polar solvent.
5)thin layer chromatography- solid
material (silica) is spread on a glass plate.
6)reverse phase chromatography-stationary
phase is non polar while mobile phase is polar, reverse of paper chromatography.initially
used for separation of lipids but later on used for proteins as well as
7)metal chelation affinity chromatography-divalent
metal ion is covalently attached to chromatographic matrix such that proteins
bearing cys or his tags are retained on the matrix.these tags are created by
using recombinational techniques.by
changing the ph, proteins can be removed from the matrix.
8) hydrophobic interaction chromatography-
used for separation of native proteins.
9) high performance liquid chromatography-separation
is greatly improved in this. narrow and long columns are used filled with non
compressible silica beads.solvent is forced through the column under the force
of upto 15000 psi, reducing the analysis time.
Advantage-high resolution, less time,high sensitivity.