Chromatography

1. Dnyanasadhana College, Thane.Department of ChemistryT.Y.B.Sc. Analytical ChemistryPaper-IV Sem-V ChromatographyDr.G.R.Bhagure

2. Chromatography

4. V Sem

5. V Sem.

6. Paper ChromatographyL+L • Principle: In paper chromatography stationary phase is liquid as well as mobile phase is also liquid. In paper chromatography solute undergoes partition between the two liquid phases. The rate of transfer of solute and its effective separation on paper will depend on partition coefficient of the solute between the two phases. The solutes from the original mixture will have migrated along paper at different rates, forming a series of separated spots. For identification purposes spots are characterized by Rf values.

7. Distance traveled by solute Rf = --------------------------------------------- Distance traveled by solvent Solvent Front S O L V E N t F L O W Solute front (B) 8 Rf (A) = -------=0.5 16 Solute front (A) 12 Rf(B)= ----= 0.75 16 Original Line

8. Solvent Front S O L V E N t F L O W Solute front Solute front Original Line

9. EXPERIMENTAL PROCEDURE:- • Preparation of the Paper. • Solvent system used • Preparation of the sample. • Application of the sample. • Development of the Chromatograms. • Identifying the Spots

10. The paper used for chromatography • Whatman no.1 is strong, medium fast, pure cellulose paper that is widely used. • For the separation of polar substances special ion exchange paper (containing ion exchanging groups) • For the separation of the component which is hydrophilic in nature, esters of cellulose can be used.

11. Preparation of Paper 15-20 Cm Sample application 3-5 Cm

12. Solvent system:- • In paper chromatography the solvents used as stationary phase and mobile phase should have following characteristics; • The solvents should not react with any component during separation. • The chemical compositions of the solvents should not change with time. • The Rf value for the component should be any where between 0.06 to 0.95. • The distribution ratio of the component should be independent of its concentration. • The solvents used may be miscible or immiscible but one of the solvent should be polar that can work as stationary phase. • The paper shows affinity with polar solvent that can work as stationary phase. • If water is used as stationary phase then no special impregnation is necessary. If polar solvent other than water is used then it is necessary to remove the water from the paper. • Ex. Water and Ethanol

13. Mobile Phase • Solvent system used: one main organic liquid saturated with distilled water. • Polar solvent which is adsorbed on paper is used • The solvent should be cheap, • very pure, • should not volatile by temperature • Its rate of flow should not affected by temperature

14. Preparation of the sample. • The solid sample is dissolved in organic solvent having low boiling point. The percentage of the sample in the solution should be 0.1—1%. About 10 micro liter of the sample are transferred to the paper by using capillary or micro syringe. If the sample is of biological origin, then proteins, lipids and inorganic ions present in excess are to removed for better separation.

15. Application of the sample. • The point of the application of the sample or the origin is marked with pencil on the paper. The sample should be applied by micro pipette or capillary. 10-20 micro liter sample is to be applied. After application of sample on the marked spot solvents associated with sample solution is evaporated by using hair drier or current of hot air.

17. Ascending Paper Chromatography Mobile Phase

18. Solvent Front S O L V E N t F L O W Solute front Solute front Original Line

19. Descending Paper Chromatography Mobile Phase

20. HORIZONTAL DEVELOPMENT:- The advantage of the technique is the space required is very small. The Rf value is getting very sharp. The tank used for separation is a shallow metal, glass or container. The chromatogram is developed using paper enclosed between two aluminum or glass plates.

21. Radial Paper Chromatography Petri dish for mobile phase Liquid mobile phase

22. Separated component in terms of concentric Band Sample Solvent front Wick deep in Solvent

23. Two dimensional chromatography Sample A A B B X X AC is immersed in mobile phase Solvent AB is immersed in mobile phase C C Solvent front

24. Identifying the Spots • Location of the substances: • The substances separated by paper chromatography are colorless. • These separated substances are detected by using visualizing agent. • The paper is dried at temperature 100Oc. • The spots are detected by means of physical or chemical method.

25. Physical Method :- • In this method paper is exposed to ultraviolet light in the wave length range 240-260 nm. • The compound produces florescent spots. Compounds which do not produce florescent spots are exposed to high pressure mercury vapour lamp.

26. Physical Method Sun rays U.V.Light Paper Chromatogram

27. Carboxylic acids Unsaturated Compounds Metal ions Cu+2 Amino acids

28. Chemical Method H2S Gas Separated metal ions Paper Chromatogram

29. Chemical Method Iodine Vapours Detection of unsaturated organic compounds. Paper Chromatogram

30. Chemical Method Acid-Base Indicators Detection of Carboxylic acids Paper Chromatogram

31. Qualitative Analysis Or Interpreting the Data:- • The Rf value for each spot should be calculated. Rf stands for "ratio of fronts" and is characteristic for any given compound. Hence, known Rf values can be compared to those of unknown substances for the identifications. • Distance traveled by solute • Rf = --------------------------------------------- • Distance traveled by solvent • Note: Rf values often depend on the temperature, solvent, and type of paper used in the experiment; the most effective way to identify a compound is to spot known substances next to unknown substances on the same chromatogram.)

32. Calculation of Rf Value

33. Application of Paper Chromatography

34. Thank you