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.
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
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
- 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.
1. Which type of m.p. are used in paper chromatography ?
A. The mobile phase which gives Rf value range between 0.1-0.3
B. The mobile phase which gives Rf value range between 0.2-0.8
C. The mobile phase which gives Rf value range between 0.8-1
D.The mobile phase which gives Rf value range between 0.6.
2. When there are to complex mixture are separated which development technique of paper Chromatography is used ?
D. Two dimensional
3. How much time is required to saturate of chamber ?
A. 12 hour
B. 24 hour
C. 6 hour
D. 10 hour
4. Which force is responsible for the separation of the components in descending paper chromatography?
D. All of the above
5. Which is not development technique of paper Chromatography ?
A. Two dimensional
6. What is the principle of the paper chromatography ?
C. A and B
D. None of the above
7. Rf value is
A. Distance travelled by the compound at it’s point of maximum.
B. Distance travelled by the standard.
C. Solvent travelled
D. None of the above
8. Which type of filter paper are mostly used in paper chromatography?
A. Butter paper
B. Sample paper
C. Whatmann filter paper
D. Filter paper
9. Which factor is not affect in stationary phases in paper chromatography ?
B. Flow rate
D. Freezing point
10. Size of spot in paper chromatography is
A. 2-5 mm
B. 1-2 mm
C. 7-8 mm
D. 6-8 mm
11. What is the ratio of Isopropanol: Ammonia: water in mobile phase?
12. How many percentage of the Beta cellulose in stationary phase?
Reference:text book of p.analysis of third edition Dr.s.ravi sankar(15.1TO15.8)