Paper chromatography

Paper chromatography is partition chromatography in which solutes distribute between two liquid phases(water in paper and eluting solvent)

1. Techniques of separation Module : Chromatography Topic : Paper Chromatography By Dr. M. T. Bachute Dept. of Chemistry K.B.P. Mahavidyalaya , Pandharpur

2. Paper Chromatography Paper chromatography is a type of Planar chromatography in which paper is the support for stationary phase. It is a partition chromatography. Stationary Phase : Liquid, deposited in network of cellulose fibers. Mobile Phase : Liquid, solvent(single or mixture) It is a liquid – liquid chromatography

3. Principle It is based on the principle of partition of a compound between two liquid phases. The water hold in cellulose fibers of the paper acts as the stationary phase and acts as one of the solvents (liquids). When a non–aqueous mobile phase moves along the paper, the components of the loaded mixture distribute themselves between two phases in the ratio characteristic of their distribution coefficients. The component which is more soluble in stationary phase moves slowly while the component which is more soluble in mobile phase moves fast. As a result of this differential movement, components get separated. Distribution coefficient = (solute in stationary phase) ÷ (solute in mobile phase)

4. Important Aspects of Chromatography Filter Paper Solvent System Spraying reagents

5. Filter Paper Usually Whatman paper No. 1 is used. Characters : Uniform flow rate Grain size and texture Some times the papers are acid washed to remove traces of impurities which may affect the analysis. Strip of whatman No. 1 paper

6. Solvent system Solvent is prepared by saturating organic solvent with water. But several organic liquids incorporate only small amount of water. In such binary solvent system polar components of the mixture fail to separate. In such cases a third liquid like acid( HCl , AcOH , HCOOH) or base(NH 3 or pyridine) or a complexing agent is often added to retain more water in organic solvents. Hence, generally three component solvent systems are used

7. Some important solvent systems Compounds Solvent system Spraying reagent 1. Amino acids Butanol : Pyridine:Water (33 :33 :33) Ninhydrin Methanol:Pyridine:Water (25 : 12 :63) Butanol : AcOH : Water (40 : 19 50) Phenol : Water (80 : 20) 2. Carbhydrates (mono , di ) Butanol : AcOH : Water Butanol : Pyridine:Water:AcOH (8:8:4:1) Ammoniacal AgNO 3 Alkaline KMnO4 3. Chlorophils and carotenoids Propanol : Pet. Ether Chloroform : Pet. ether Self 4. Metal ions Ethanol: 5M HCl (90 : 10) Rubeanic acid, NH 3

8. Spraying reagents Coloured components do not require spraying reagent, they are visible. Colourless components are located by spraying with specific reagents. These reagents chemically react with the components and develop colour in the region where they are present.

9. Methodology Sample loading Preparation of sample solution : 1-2% solution is prepared in suitable solvent. A reference line at 2cm . Distance from the edge of the paper is drawn and points of application are marked. The solution of mixture under test is spotted either as a fine line or fine spot. Spotting is repeated twice or thrice. Reference Line .

10. Methodology 2. Developing Saturation of the paper strip: The sample loaded strip is hanged for some time in the chromatographic jar containing the solvent but without dipping in the solvent. The strip is developed by running the solvent through the paper. Depending on the direction of flow, there are two types paper chromatography. Ascending paper chromatography Descending paper chromatography

11. Ascending P. C. Solvent moves upward(ascends) from the bottom to top by capillary action. The paper strip is placed vertically in the solvent keeping the reference line above the solvent level. Pencil Filter Paper Ink Mark Tape – Label with marker

12. Descending P. C. Solvent moves downward(descends) from the top to bottom. The paper strip is hanged vertically downward from the trough containing solvent, keeping the spotted end at the top. The flow of solvent is faster than that in ascending P.C. But A.P.C. is preferred than D.P.C. due to simplicity of set up.

13. Radial or Circular chromatography The plane of paper is kept horizontal and solvent moves laterally(toward the periphery) from the center.

14. Methodology 3. Spraying : After movement of the solvent to expected distance(15 to 18 cm), paper is removed, dried and sprayed with suitable reagent.

15. Descending paper chromatography

16. Rate of Flow : R f value R f value : It is the ratio of distance travelled by the solute to the distance travelled by the solvent front. i.e. Distance moved by solute R f = Distance moved by the solvent front Rf value ……. characterises the distance moved by the solute in the direction of solvent flow under standard conditions it closely reflects the distribution coefficient and it is characteristic of that compound

17. Rf value Factors affecting R f value Quality and dimension of the paper. Solvent system, its flow rate and direction of flow. Temperature of the environment Size of the vessel in which chromatogram is developed. If these factors are kept constant, R f values of known and unknown compounds can be compared.

18. Applications of Paper Chromatography Separation of amino acids, peptides, alkaloids, sugars, lipids etc in biological samples. To limited extent as a preparative method. To study the structure and amino acid composition of proteins Analysis of blood, hemoglobin, urine etc which is of great diagnostic value.

19. Advantages Method is simple, inexpensive and sensitive. Separations can be carried out under ordinary lab. conditions.

20. Disadvantages Time required is large. It can not be used as a preparative method. Heating or corrosive reagents can not be used for detection.

21. Advantages of TLC over PC Versatility in the use of stationary phase. It offers choice between wide range of adsorbents to suite the separation of variety of components. 2. Adsorbent layer of variable thickness( upto 5mm)can be obtained. Higher sample load is possible.(higher load on paper not possible) 3. Method is very rapid & most of the separations can be completed within an hour. (PC requires 18 to 24 hrs.) 4. Separated spots are very compact, hence detection at lower concentration is possible. (In PC spots are diffused) 5. Higher temp. and corrosive sprays can be used to detect unknown solutes.( higher temp and corrosive sprays can not be used with paper.) 6. Since it is simple, rapid and inexpensive, it can be used to check purity of synthesised or isolated chemicals.

22. Disadvantages of TLC R f values are not reproducible. Rf value is affected by thickness of the layer below 0.2mm and also by uneven thickness of the layer.