Simple experiments to illustrate the
techniques of paper and thin layer chromatography (preparation of plates not
required) leading to an understanding of Rf values; two way
chromatography.
Chromatography
This
is an analytical technique for the sepctration of a mixture of solutes.
Separation is achieved by the differential
movement of individual solutes through a porous medium, under the influence of
a moving solvent. The degree of
separation depends on four forces which operate independently of each other.
·
Rate
of flow of solvent.
·
Solubility
of the substance in the solvent.
·
Partition
effects.
·
Adsorption
effects.
The
first two of theses are responsible for pushing the substance along the support
material while the last two are responsible for retarding the movement of the
substance.
Chromatography
may be achieved by either a partition or adsorption mechanism.
Partition
chromatography
This
involves the separation of a mixture of substances by the partition between the
moving solvent (mobile phase) and a stationary liquid (stationary phase) held
on a suitable solid support.
Paper
chromatography
Paper
has associated with it moisture (10%) bound to the cellulose fibres which act
as the stationary phase. The solvent is
the mobile phase. The more soluble the
component in the solvent the faster it will be washed along.
0 0 0
·
Mark
a pencil line about 2cm from the end of the chromatography paper.
·
Place
spots of coloured dyes about 2cm apart along the line, avoiding the edges.
·
Record
the original colour of each spot, in pencil, below the spot itself.
·
Wrap
the chromatography paper into a cylindrical shape and clip the ends together
with a paper clip or staple ( alternatively the flat sheet con be placed into a
chromatography developing tank).
·
Place
a small amount of a suitable solvent in a large beaker (or chromatography tank).
·
Carefully
place the cylinder of chromatography paper into the beaker ensuring that the
coloured spots are above the level of the solvent and that there is no contact
between the cylinder and sides of the beaker.
·
Leave
undisturbed until the solvent has travelled nearly to the top of the paper.
beaker
Coloured spots
Solvent level
·
Remove
the paper and allow to dry before opening out flat.
Solvent line
0 0
0 0 0
The
separated components can be obtained by cutting the paper into strips and dissolving out each compound.
Gas-Liquid
Chromatography
Here
the mobile phase is an inert gas (usually helium or nitrogen) and the
stationary phase is a non-volatile liquid held on a finely divided solid
support. The stationary phase is chosen
according to the nature of the sample to be separated. The material under test (either gas or
volatile liquid) is injected into the apparatus and is carried by the stream of
carrier gas into a long heated column packed with a porous solid impregnated with a non-volatile liquid or
oil. Gas-liquid partitioning occurs, the retention time being characteristic of
a particular substance.
Detection
is usually achieved by measuring the changes in thermal conductivity of the
effluent gases.
Adsorption
Chromatography
Separation
is achieved by differences in the adsorption behaviour of substances between a
moving liquid solvent and a station" solid phase.
The
substances experience varying interactions with the surface of the solid
adsorbent due to weak electrostatic forces.
Thus for two solute molecules A and B, if A is more strongly adsorbed it
will move in the solvent more slowly than B and separation can be achieved.
Thin
Layer Chromatography
A
thin coating of an adsorbent material (silica gel, alumina A1203, cellulose,
chalk) is spread evenly on a glass plate.
A solution of the mixture to be separated is spotted onto the adsorbent
surface at one
end.
The plate is then placed in a closed vessel containing an appropriate
solvent so that the spotted end comes in contact with the solvent, which then
moves across the adsorbent material.
The mixture separates into its components depending on the relative
affinities for the adsorbent material compared with the migrating solvent.
Rf
values
The
movement of any substance relative to the solvent front in a given system is
constant and characteristic of that substance.
The
Rf value is used to express the relative distance that a substance
has travelled (Rf<l).
distance solvent front has
travelled from origin
solvent front
no Rf (A)=
X
X
Rf (B)= b
X
AO b
base
line
Fluorescence
- many organic compounds fluoresce in u.v. light.
Radioactivity
- detected using Geiger counter.
Chemical
- colourless compounds are converted to a coloured product by treatment with a
locating agent such as H2S or ninhydrin.
Two
way chromatography
This
is an adoption of chromatography in order to achieve better separation of the
components of a mixture. Chromatography
is carried out twice with the chromatography paper being turned through 90o
between the two developments.
1 st
separation 0
vent 1
Turn 90o
v
nd
2 -1
separation
solvent 2
From
the Rf values for solvents 1 and 2 components can be identified.
Chromatography
is used for testing the purity of a sample (and for analysis) as well as for
separation.
Partition
chromatography can be used for the analysis of dyes, the identification of
sugars and the separation of cations e.g. Cu2+, Hg2+, Bi3+.