Lecture 05 paper chromatography

The Power Point Presentation includes The Paper Chromatography, Its Types and Method with applications. These Slides may be helpful for master of science students. The Syllabus for the slides was prepared by following as KSV, Gandhinagar. Paper Code is CH-AC-302, Unit-01

1. Analytical Chemistry-4 By-Deepak Sharma HVHP Institute of P.G Studies and Research LECTURE – 05 PAPER CHROMATOGRAPHY For M.Sc. Analytical Chemistry Assistant Professor (Department of Chemistry) M.Sc. (Analytical Chemistry), GATE, GSET, Research Scholar at HNGU

2. Lecture Outline Deepak Sharma Paper Chromatography Introduction Principle Type Experiment Applications

3. Paper Chromatography Deepak Sharma It is a method of separating components of a mixture by differential movement through a two-phase system: the mobile phase and the secondary phase. This movement is effected by the flow of a liquid or a gas (M.P) which percolates through an adsorbent (S.P). Paper chromatography was first introduced by the German scientist, Christian Friedrich Schonbein in 1865. It is a type of a planar chromatography. It is the simplest and widely used type of chromatography procedures which runs on a specialized paper.

4. Principle Deepak Sharma Two principles of Paper Chromatography: Adsorption Chromatography- Paper impregnated with silica or alumina acts as the adsorbent (S.P) and solvent as the mobile phase. Partition Chromatography- The moisture or water present in the pores of the cellulose fibers present in the filter paper acts as the stationary phase and another solvent as the mobile phase. In General, Paper Chromatography = Paper Partition Chromatography

5. Deepak Sharma Principle Separation Cellulose layers in filter paper contains moisture which acts as stationary phase & organic solvents/buffers are used as mobile phase The principle of separation is mainly Partition rather than Adsorption

6. Deepak Sharma Ascending chromatography Descending chromatography Ascending- descending mode Radial mode/Circular Chromatography Two Dimension Chromatography Types of Chromatography

7. Deepak Sharma Ascending chromatography : As the name indicates, the chromatogram ascends. Here the development of paper occurs due the solvent movement or travel in upward direction on the paper.

8. Descending chromatography Here the development of paper occurs due to solvent travel downwards on the paper. Deepak Sharma

9. Ascending- descending mode Deepak Sharma Here solvent first travels upwards and then down wards on the paper.

10. Deepak Sharma Here the solvent travels from center(mid point) towards periphery of Circular chromatography paper. Radial mode/Circular Chromatography

11. Deepak Sharma Here the chromatogram development occurs in two directions at right angles. Two Dimension Chromatography

12. Experiment Paper Chromatography Deepak Sharma

13. Deepak Sharma Stationary Phase & Papers used Application of sample Mobile phase Development technique Detecting or Visualizing agents Requirements

14. Stationary Phase Deepak Sharma Whatman filter papers of different grades like No.1, No.2, No.3, No.4, No.20, No.40, No.42 etc. are used. In general this paper contains 98-99% of α-cellulose, 0.3–1% β–cellulose Factors that governs the choice of paper: » Nature of Sample and solvents used. » Based on Quantitative or Qualitative analysis. » Based on thickness of the paper.

15. Preparing the Paper Strips Deepak Sharma Cut the filter paper into 5 x 4 measurement. Draw a line 0.5 cm above the bottom edge of the strip with the pencil. Label each strip with its corresponding solution. Place a spot from each pen on your starting line.

16. Mobile Phase Deepak Sharma Pure solvents, buffer solutions or mixture of solvents Examples- Hydrophilic mobile phase 1. Isopropanol: ammonia:water → 9:1:2 2. Methanol : water → 4:1 3. N-butanol : glacial acetic acid : water → 4:1:5 Hydrophobic mobile phases 1. dimethyl ether: cyclohexane 2. kerosene : 70% isopropanol

17. Preparation of the solution Deepak Sharma Choice of suitable solvent for making solution is very important. Pure solutions can be applied direct on the paper but solids are always dissolved in small quantity of a suitable solvent. Biological tissues are treated with suitable solvents and their extracts obtained. Proteins can be precipitated with alcohol and salts can be removed by treatment with ion exchange resin.

18. Application of Sample Deepak Sharma The sample to be applied is dissolved in the mobile phase and applied as a small spot on the origin line, using capillary tube or micropipette. very low concentration is used to avoid larger zone The spot is dried on the filter paper and is placed in developing chamber.

19. Developing the Chromatograms Deepak Sharma Place the strips in the beakers. Make sure the solution does not come above your start line. Keep the beakers covered. Let strips develop until the ascending solution front is about 2 cm from the top of the strip. Remove the strips and let them dry.

20. Calculate the R f value and make conclusions. Deepak Sharma   R f =

21. Application Separation of mixtures of drugs Separation of carbohydrates, vitamins, antibiotics, proteins, etc. Identification of drugs Identification of impurities Analysis of metabolites of drugs in blood , urine …. Deepak Sharma

22. Deepak Sharma https://www.linkedin.com/in/deepak-sharma-a1399688 Name of Channel Deepak Sharma