PAPER CHROMATOGRAPHY (PC)

This presentation summarizes basic introduction, principle, procedure and applications of paper chromatography.

1. PAPER CHROMATOGRAPHY PRESENTED BY : Mr k v nanda kumar Associate professor, Department of pharmaceutical analysis, Krishna teja pharmacy college, tirupathi-517506

2. CONTENTS 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 2

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4. INTRODUCTION 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 4

5. PRINCIPLE The principle involved is partition chromatography wherein the substances are distributed or partitioned between liquid phases. One phase is the water, which is held in the pores of the filter paper used; and other is the mobile phase which moves over the paper. It is a planar method in which mobile phase comes and interacts with stationary phase against gravity due to capillary action. 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 5

6. TYPES OF CHROMATOGRAPHY PAPER The fibrous structure rather than the chemical composition characterizes the paper used in paper chromatography. Investigations have revealed that improvements in the fibrous structure can help improve the chromatographic performance. Therefore, attempts have been made to prepare paper that is utilized for chromatography by using a cellulose slurry with very short fibers or to prepare sheets from a slurry of microfilaments of glass fiber impregnated with sorbents. 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 6

7. LIST OF VARIOUS TYPES 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 7

8. UNMODIFIED CELLULOSE Most of the paper used for paper chromatography is based on unmodified cellulose. Regular filter papers are suitable for paper chromatography. The well-known examples are Whatman 1 and Schleicher and Schuell 2043b ; they have been used for a variety of separations, other examples besides the two mentioned above include Machery -Nagel 260, Munktell Chr 100, Ederdol 202, and Whatman 2. These papers are sold as rectangular or circular sheets. Easy commercial availability of these papers helps obtain reproducible results and save time. Slight changes in the width of paper at the point of entrance of solvent may affect separation. Hence, availability of standardized papers is important . 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 8

9. MODIFIED CELLULOSE PAPERS For separations of polar substances, papers with increased capacity have been introduced (e. g., Schleicher and Schuell I or II) Papers containing ion-exchange resins offer the possibilities for ion exchange, (e.g., cellulose cation- and anion-exchange papers from Ederol ) For separation of some hydrophobic substances, kieselguhr filter paper (e.g., Schleicher and Schuell 287) has been used. Other papers containing adsorbents like alumina or silica gel have also been used. 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 9

10. GLASS-FIBER PAPERS These papers have been used, in appropriate applications, because they offer the following advantages: Reagents that are too corrosive for cellulose papers can be used. Adsorption effects are minimized in some cases. Analysis time is reduced significantly . 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 10

11. MEMBRANE FILTERS Nitrocellulose membranes have been used as carrier materials in the separation of macromolecules. This technique utilizes the fact that high molecular weight materials form a homogeneous immobilized film on the surface of the microporous carrier that is capable of specific interactions with various substances. 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 11

12. TYPES OF MOBILE PHASES The useful mobile phases are listed below on the basis of their suitability for the materials that can be roughly classified as follows: Hydrophilic Hydrophobic Moderately polar 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 12

13. HYDROPHILIC SUBSTANCES The commonly used mobile phases include: n-Butanol/acetic acid/water (4/1/5) Isopropanol/ammonia/water (9/1/2) The ratio of solvents is made on a volumetric basis. The mixture with butanol yields a two-phase system. 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 13

14. HYDROPHOBIC SUBSTANCES Some of the useful mobile phases are listed here: Dimethyl formamide/cyclohexane Solution of 50% DMF in ethanol used for impregnation Paraffin oil/dimethyl formamide/methanol/water (10/10/1) Solution of 10% paraffin oil in an aromatic hydrocarbon used for impregnation 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 14

15. MODERATELY POLAR SUBSTANCES The simplest system is composed of a mixture of formamide and an aromatic hydrocarbon such as benzene or chloroform: Formamide/chloroform Formamide/toluene Varying proportions of cyclohexane may be added to aromatic hydrocarbon mixtures to improve separations. 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 15

16. WORKING PROCEDURE Selecting a suitable type of development: It is decided based on the complexity of the solvent, paper, mixture, etc. Usually ascending type or radial paper chromatography is used as they are easy to perform. Also, it is easy to handle, the chromatogram obtained is faster and the process is less time-consuming. Selecting a suitable filter paper: Selection of filter paper is done based on the size of the pores, and the sample quality. Prepare the sample : Sample preparation includes the dissolution of the sample in a suitable solvent (inert with the sample under analysis) used in making the mobile phase. Spot the sample on the paper: Samples should be spotted at a proper position on the paper by using a capillary tube. Chromatogram development: Chromatogram development is spotted by immersing the paper in the mobile phase. Due to the capillary action of paper, the mobile phase moves over the sample on the paper. Paper drying and compound detection: Once the chromatogram is developed, the paper is dried using an air drier. Also, detecting solution can be sprayed on the chromatogram developed paper and dried to identify the sample chromatogram spots. 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 16

17. DEVELOPMENT OF CHROMATOGRAMS 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 17

18. ASCENDING AND DESCENDING MODE Descending chromatography is most commonly used because chromatograms can be developed to longer distances, unhampered by gravity. It is well known that gravity affects solvent advancement by capillary action of paper in the ascending mode of paper chromatography. The longer distance development allows for better resolution because of a corresponding increase in the number of theoretical plates. Recall the concept of theoretical plates discussed earlier for distillation and column chromatography. The length of paper corresponds to the length of distillation or chromatographic column. 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 18

19. HORIZONTAL MODE The chamber design for horizontal chromatography is not difficult because all one has to assure is that solvent is fed horizontally to the paper, which is also kept in a horizontal position. 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 19

20. RADIAL MODE Radial chromatography may be carried out in a closed petri dish. In radial paper chromatography, the sample is spotted in the center of a circular paper and the solvent is applied in the center by a wicking technique so that development of the chromatogram is toward the periphery 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 20

21. DETECTION 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 21

22. QUALITATIVE ANALYSIS Paper chromatograms are developed to a predetermined distance that allows calculation of the Rf value of each component after development: In case Rf values are too low, it may be desirable to carry out overrun development, where a descending technique is frequently used and development is not stopped when the solvent front reaches the lower end of the paper. As a matter of fact, it is allowed to overrun for the desired time. 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 22

23. QUANTITATIVE ANALYSIS Quantitation requires eluting the spot of interest and then analysing the solution by colorimetric, UV, or other suitable detection techniques. Standards are similarly run and treated. The elution of substances must be done quantitatively. Densitometric techniques for visible–UV wavelengths can be used in any of the following modes: Transmittance Reflectance Fluorescence 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 23

24. ADVANTAGES 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 24

25. DISADVANTAGES 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 25

26. APPLICATIONS 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 26

27. PC VS TLC 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 27

28. QUESTIONS FOR REVIEW 1. Define paper chromatography. 2. Describe various modes of chromatography. 3. What are the advantages offered by paper chromatography? 4. Name some universal detection reagents for paper chromatography. 5. Compare TLC with PC. 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 28

29. REFERENCES 1. Macek , K. Chromatography (E. Heftman , Ed.), Van Nostrand, Reinhold, 1975. 2. Ahuja, S. Ultratrace Analysis of Pharmaceuticals and Other Compounds of Interest, Wiley, NY, 1986. 11-06-2020 B PHARM PA II - Mr K V NANDA KUMAR 29

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