Paper Chromatography
Paper Chromatography :-
Definition :- it is defined as a separation technique where
separation of a mixture of compound is mainly achieved by a flow of solvent on
a chromatographic paper.
Stationary phase has a bound or abosorbed water present with the
cellulose of the chromatographic paper and the mobile phase is an organic
solvent which is immiscible with stationary phase.
The mobile phase is rise by capillary action.
Principle :-
Paper chromatography is mainly work on principal of the adsorption & partition.
The mechanism of separation in paper chromatography is
mainly partition in Type. So, when a drop of the solute is treating with the
solvent on the paper, the more strong components back while less strong
components are move forward.
Rf value :-
The relative rate of the movement of solvent and solute is
expressed by a term Rf.
It is defined as the ratio of the distance travelled by the
compound at its maximum.
Rf= Distance travelled
by the Analyte/ Distance travelled by the solvent
Rx value :-
In many cases it has
been observed that the solvent front os run off the end of the chromatogram.
Rx value is the ratio
of the distance travelled by a substance to the distance travelled by a
reference standard.
Factor affecting Rf
value :-
It is affected by
- The temperature of environment.
- Purity of solvent used
viscosity of solvent
- The type of paper, speed
of paper, ash content.
- PH of the mobile phase.
- Chemical reaction if any during
development.
Practical
Requirements :-
- Stationary phase
- Mobile phase
- Sample application
- Selection of mobile phase
- Chromatographic development
- Drying of chromatogram
- Visualisation
1.
Stationary phase:-
Mainly paper are used
as stationary phase. Choice of filter paper depends on the 1. Thickness 2. Flow
rate 3. Purity and 4. Strength of paper.
Various types of
commercial filter paper are available. Chemically these contain α
cellulose 99%, beta cellulose 0.3-1%, pantose 0.5 to 0.8 %, ash
0.01-0.07%.
Whatmann filter paper
is widely used in paper chromatography.
2.
Preparation of solutions :-
Neat sample liquid can
be applied directly to the paper but solid are always dissolved in required
volume of suitable solvent.
Example Isopropanol:
Ammonia: water( 9:1:2), n- butanol : Acetic acid: water ( 4:1:5), water phenol.
3. Sample
application :-
Once the type of paper
and dimensions are selected, solution are applied to the paper by mean of
micropipette or disposable capillaries 2-3 cm from the bottom end of the paper.
Size of spot may range 2-5 mm depending upon number of sample to be applied to
the paper.
4.
Selection of mobile phase :-
The mobile phase which
gives Rf value range between 0.2-0.8 are selected for chromatographic work.
5.
Chromatographic development :-
There are mainly three
technique are developed
- Ascending
- Descending
- Radial
- Two dimensions
1.
Ascending technique :– In this development
the mobile phase is transferred to the chamber. The paper is suspended with the
help of suitable attachment in such a way that the lower end of the paper
containing the spot just touch the solvent layer. With the migration of the
solvent through the paper, the solute partition and equilibrium is set between
stationary phase and Mobile phase.
Before dipping the paper
into the mobile phase, pre saturation of the chamber is most essentially part.
Sometimes chamber is allowed to saturate even for 24 hour before the
development. Method give better result with low viscosity solvent.
2.
Descending technique :-
In descending
technique the mobile phase is placed at the top of the tank in a trough and the
chromatogram is so placed that the solvent flows down the paper. The
environment in the tank is saturated with the vapour of the mobile phase or
vapour of more volatile components. Since the mobile phase can be allowed to
run off the paper under influence of gravity speed of analysis is faster
compared to ascending technique.
3.
Radial or circular technique :-
Here the spot is kept
at the center of a circular paper. The solvent flow through a wick at the
center and spread in all directions uniformly. Hence the individual spot after
development look like concentric circles.
4.
Two dimensional development :-
Two dimensional
modifications can be used to increase separation. Conventional paper
Chromatography is satisfactory only for separation of mixture, coating limited
number of constituents.
If mixture contains
more number of components separation is not satisfactory with conventional
Chromatography but it can be well resolved by two dimensional technique.
The sample is placed
at one corner of the square paper and developed in the ascending direction with
solvent. This solvent is then removed by evaporation and the paper rotated 90°
following which an ascending development with solvent B is performed.
6.
Drying the chromatogram :-
The wet chromatogram after development is dried in special
cabinates under controlled temperature.