Methods of Purification 2

Question 1

what is the basic principle behind chromatography?

Answer 1

Chromatography is an important technique
extensively used to separate mixtures into
their components, purify compounds and also
to test the purity of compounds.

In this technique,
the mixture of substances is applied onto a
stationary phase, which may be a solid or a
liquid. A pure solvent, a mixture of solvents,
or a gas is allowed to move slowly over the
stationary phase. The components of the mixture get gradually separated from one
another. The moving phase is called the mobile
phase.
Based on the principle involved,
chromatography is classified into different
categories. Two of these are:
(a) Adsorption chromatography, and
(b) Partition chromatography.

Question 2

what is adsorption chromatography

Answer 2

Adsorption chromatography is based on the fact
that different compounds are adsorbed on an
adsorbent to different degrees. Commonly
used adsorbents are silica gel and alumina.
When a mobile phase is allowed to move
over a stationary phase (adsorbent),
the components of the mixture move by
varying distances over the stationary
phase. Following are two main types of
chromatographic techniques based on the
principle of differential adsorption.

(a) Column chromatography, and
(b) Thin layer chromatography.

Question 3

explain column chromatography

Answer 3

Column
chromatography involves separation of a
mixture over a column of adsorbent
(stationary phase) packed in a glass tube. The
column is fitted with a stopcock at its lower The mixture adsorbed on adsorbent is placed on the top of the adsorbent
column packed in a glass tube. An appropriate
eluant which is a liquid or a mixture of liquids
is allowed to flow down the column slowly.
Depending upon the degree to which the
compounds are adsorbed, complete separation
takes place. The most readily adsorbed
substances are retained near the top and others
come down to various distances in the column

Question 4

explain thin layer chromatography: what is a chromoplate

Answer 4

Thin layer
chromatography (TLC) is another type of
adsorption chromatography, which involves
separation of substances of a mixture over a
thin layer of an adsorbent coated on glass
plate. A thin layer (about 0.2mm thick) of an
adsorbent (silica gel or alumina) is spread over
a glass plate of suitable size. The plate is
known as thin layer chromatography plate or
chromaplate.

Question 5

explain thin layer chromatography:

Answer 5

The solution of the mixture to
be separated is applied as a small spot about
2 cm above one end of the TLC plate. The
glass plate is then placed in a closed jar
containing the eluant eluant rises up the plate, the components of
the mixture move up along with the eluant to
different distances depending on their degree
of adsorption and separation takes place.

Question 6

what is retardation factor

Answer 6

The
relative adsorption of each component of the
mixture is expressed in terms of its
retardation factor i.e. Rf
value

Rf= distance moved by the substance from base line/ distance moved by the solvent from ase line

Question 7

how do we detect the compounds on the chromaplate

Answer 7

The spots of coloured compounds are visible
on TLC plate due to their original colour. The
spots of colourless compounds, which are
invisible to the eye but fluoresce in ultraviolet
light, can be detected by putting the plate under
ultraviolet light. Another detection technique is
to place the plate in a covered jar containing a
few crystals of iodine. Spots of compounds,
which adsorb iodine, will show up as brown
spots. Sometimes an appropriate reagent may
also be sprayed on the plate. For example, amino
acids may be detected by spraying the plate with
ninhydrin solution

ninhydrin gives purple blue with all amino acids except proline with which it gives yellow=orange

Question 8

describe parititon chromatography

Answer 8

Partition
chromatography is based on continuous
differential partitioning of components of a
mixture between stationary and mobile
phases. Paper chromatography is a type of
partition chromatography. In paper
chromatography, a special quality paper
known as chromatography paper is used.
Chromatography paper contains water
trapped in it, which acts as the stationary
phase