CHROMATOGRAPHY

1. According to IUPAC, chromatography is a physical method of separation where the components to be

separated are distributed in two phases – one remains stationary and the other moves in a definite
direction.
2. It is one of the most widely used separation techniques in chemistry. Purity of a sample can also be
analysed using chromatography. The word comes from chroma – a Greek word meaning colour.
Originally it was used for separation of plant components.
3. A mixture of substances are applied onto a stationary phase, and the movable components are allowed
to move on a definite direction. The moving phase is called mobile phase. Based on the principle
involved it is classified into –
 Adsorption chromatography – The component to be separated
moves on a stationary phase that is an adsorbent. Various
components in mobile phase move in defined direction based
on their adsorbing degrees. It is again classified into –
i. Column chromatography –
 It involves chromatographic action on a column of adsorbent
packed in a glass tube with a stopcock at the lower end.
 After the process an eluant is allowed to flow down the
column slowly. Based on their degree of adsorption,
complete separation occurs.
 Degree of adsorption α compounds on top
ii. Thin layer chromatography –
 It involves chromatographic action on a thin layer of
adsorbent coated on a glass plate. The glass plate is
known as thin layer chromatography plate or
chromaplate.
 The substance to be separated is applied as a dot
over the TLC plate (2 cm above) in a closed jar
containing eluant.
 As the eluant rises, various substances are adsorbed
and separation takes place. The extent of separation
is expressed as retardation factor (Rf).
 The colourless compounds are analysed by exposing
the plate to UV light.
 They can also be detected by placing it in a jar
containing iodine. The substances which adsorb
iodine show up as brown spots.
 Amino acids to be detected are sprayed by ninhydrin
solution. The substances move by capillary action.

 Partition chromatography –
  It involves continuous portioning of different components between the mobile and stationary
phase. Common one is paper chromatography which uses a chromatography paper.
 The chromatography paper has water trapped inside that act as the stationary phase. The
mixture to be separated is applied as spot on the paper and is suspended in the eluant.
 The eluant rises by capillary action and the spot gets drenched in it. This separates the mixture
into various components.
 The colourless components are analysed using a reagent or spray or exposure to UV.
4.

In chromatography, substance equilibrates between a mobile and a stationary phase. The more the
interaction of substance with the stationary phase, slower is its movement.
5. In paper chromatography the eluant can be hexane, toluene, acetone or methanol water mixture. Based
on solubility of various components and mobile phase carries them along with it.
6. The extent to which the components of a mixture get separated at a temperature and eluant is
expressed in terms of retardation factor.

7. Retardation factor has no units and is always less than one because the solvent moves faster than the
component to be separated. Colourless compounds are determined by reagent (also called developer).
8. The important characteristics of the developer are that it should impart a colour without reacting with
the components and should be volatile.
9. Precautions
 Use good quality pencil for drawing the reference line so that the mark does not dissolve in the
solvent in which TLC is run.
 Dip the paper strip in the solvent in such a way that the spot of the mixture is above the solvent
level and the movement of the solvent front is not zig-zag.
 While spotting the test solution on the paper, do not allow the spots to spread. Use finely drawn
capillary to put the spot on the paper.
 Ensure that the filter paper strip hangs freely in the jar.
 Once the experiment is set, do not disturb the jar as long as the chromatogram is being
developed
 Keep the jar covered with the lid when the chromatogram is being developed.
 Make the paper strip perfectly dry before developing the spots.
 Handle the organic solvent/solvents, with care.