paper chromatography

1. Mr. Darshan N U M Pharmacy first semester Dept. of Pharmaceutical Chemistry 1

2. INTRODUCTION Paper Chromatography (PC) was introduced by Schonbein (1865) under the name capillary analysis. PC became popular only when a team of Gorden , Martin and Syngle in England in 1944 done a outstanding work. Paper chromatography is defined as the technique in which the analysis of unknown substances is carried out mainly by the flow of solvents on specially designed filter paper. 2

3. PRINCIPLE OF SEPARATION This technique is a type of partition chromatography in which substance are distributed between two liquids , i.e., one is the stationary liquid ( usually water) which is held in the fibers of the paper & called the stationary phase; the other is the moving liquid or developing solvent and called the moving phase. The components of the mixture to be separated migrate at different rates and appear as spots at different points of paper. 3

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5. R f value R f (Retardation factor) is the fraction of an analyte in the mobile phase of a chromatographic system. R f = Distance travelled by solute / Distance travelled by solvent front The Rf value ranges from 0 to l . But the ideal values are from 0.3 to 0.8 R is a function of the partition coefficient . 5

6. R x values It is nothing but the ratio of distance travelled by the sample and the distance travelled by standard. Rx value is always closer to l. In some cases, the solvent front runs off the end of filter paper, the moment of a substance in such cases is expressed as R x . R M values R M value is used in qualitative analysis to find out whether the compounds belong to a homologous series. If they belong to a homologous series, the R M values are constant. According to Bate-smith , R M is defined as follows R M = log (1/R f -1) 6

7. PRACTICAL REQUIREMENTS Stationary phase and Papers used Application of sample Mobile Phase Development technique Detecting or visualizing agents 7

8. 1 . STATIONARY PHASE AND PAPERS USED Paper of chromatographic grade consists of  -cellulose 98 to 99%, ß-cellulose 0.3 to 1% , pentosans 0.4 to 0.8%, ether soluble matter 0.015 to 0.02%, Ash- 0.01 to 0.07% . Whatman filter papers of different grade like No. l , No.2, No.3, No.3MM, No.4. No. 17, No.20 etc are used. These papers differ in sizes, shapes, porosities and thickness. choice of filter paper depends upon thickness, flow rate, purity, technique, etc. Modified papers- Acid or base Washed filter paper, glass fibre type paper. Hydrophilic papers - Papers modified with methanol, formamide. glycol, glycerol etc. Hydrophobic papers - Acetylation of OH leads to hydrophobic nature, hence can be used for reverse phase chromatography. silicone pre treatment and organic non-polar polymer can also can be impregnated to give reverse phase chromatographic mode. 8

9. Preparation of paper Cut the paper into desired shape and size depending upon work to be carried out. The most common shape of the filter paper is rectangular, although square paper can also be used. 15 – 30 cm in length and 5 to several cm in width. The starting line is marked on the paper with an ordinary pencil 2cm from the bottom edge. On the staring line marks are made 2cm apart from each other. 9

10. 2. APPLICATION OF SAMPLE The sample to be applied is dissolved in the suitable solvent and applied using capillary tube or using micropipette. Very low concentration is used to avoid larger zone. 10

11. 3. Mobile Phase Pure solvents, buffer solutions, or mixture of solvents are used. Some of the examples of Hydrophilic mobile phases: Isopropanol : Ammonia : Water -9 : 2 : 1 n-Butanol : glacial acetic acid : water - 4:1:5 Methanol : water - 7 : 3 t-Butanol : water : Formic acid - 40:30:30 11

12. 4. Development technique Since paper is flexible when compared to glass plate used in TLC . Several types of development are possible which increases the ease and efficiency of operation . They are Ascending development: The solvent flows against gravity. The spots are kept at the bottom portion of paper and kept in a chamber with mobile phase solvent at the bottom. 12

13. Descending development: This is carried out in a special chamber where the solvent holder is at the top. The spot is kept at the top and the solvent flows down the paper. The advantage is that the flow of solvent is assisted by gravity and hence the development is faster. 13

14. Ascending-Descending development: This is a combination of ascending and descending type. Only the length of separation is increased by using a combination of techniques. In this technique, the upper part of the ascending chromatography can be folded over a glass rod allowing the ascending development to change over into the descending after crossing the glass rod 14

15. Circular/ radial development : In this technique a circular filter paper is employed. Then the various material to be analysed are placed at the centre. After drying the spot the paper is fixed horizontally on the petri-dish possessing the solvent so that the wick of the paper dips into the solvent. Cover the paper by means of petri-dish cover. The solvent rises through the wick. When solvent front has moved through a sufficient large distance, the components get separated in the form of concentric circular zones. 15

16. Two dimensional development: This technique is similar to 2 Dimensional TLC. The paper is developed in one direction and after development, the paper is developed in the second direction allowing more compounds or complex mixtures to be separated into individual spots . In the second direction, either the same solvent system or different solvent system can be used for development 16

17. Drying the chromatogram After the solvent has moved a certain distance for a certain time the chromatogram is taken out from the tank . They are dried by cold or hot air depending on volatility of solvents. A simple hair dryer is a convenient device to dry chromatograms. 17

18. 5 . DETECTING OR VISUALISING AGENTS After the development of chromatogram the spots should be visualised. Detecting coloured spots can be done visually. But for detecting colourless spots, any one of the following techniques can be used. Non specific methods: Where the number of spots can be detected, but not the exact nature or type of compound. Examples Iodine chamber method: where brown or amber spots are observed when the plates are kept in a tank with few iodine crystals at the bottom. UV chamber for flourescent compounds: When compounds are viewed under UV chamber, at 254nm (short  ) or at 365nm (long  ), flourescent compounds can be detected. Bright spots are seen against a dark background. 18

19. B. Specific methods: Specific spray reagents or detecting agents or visualising agents are used to find out the nature of compounds or for identification purposes. Example: Ferric chloride - for Phenolic compounds and tannins Ninhydrin in acetone - for amino acids. Dragendroff”s reagent - for alkaloids 3,5 - Dinitro benzoic acid - for cardiac glycoside 2,4 - Dinitrophenyl hydrazine - for aldehydes and ketones 19

20. For radioactive materials, detection is by using autoradiography or Geiger muller counter. For antibiotics, the chromatogram is layed on nutrient agar inoculated with appropriate strain and the zone of inhibition is compared. 20

21. Factors affecting Paper Chromatography Quality of paper used:  Some papers are better absorbed and retained than the other , so the different types and brands of paper s are used. Length of the paper:  Usually, separation will be better on long paper.  Type of Solvent used:  The solubility of each compound have different, so for different effects of separation occur in the different solvent.  The thickness of the paper:  Paper thickness is difficult to travel the spot upwards.  The concentration of the sample or spot:  the concentrated spot cannot separate properly and if the very diluted so it cannot be seen properly.  Effect of Temperature:  Temperature can affect the separation of analytes.  21

22. Precautions in Paper Chromatography Minimum volume of the concentrated solution of the sample should be applied on the chromatographic paper so as to avoid diffusion through the paper. Complexing agents should not be unnecessarily used as they form a complex with the solute having different properties resulting in multiple spots. Solvent for elution should be properly selected. Vapor – solvent equilibrium should be properly established. Natural salts should be avoided because they disturb the cellulose water which results in separation of water on to the paper. 22

23. Advantages over other methods The equipment is very simple and is easily available. It has high efficiency of separation. Separation can be effected on macro, micro and semi-micro scale. Closely related homologues, isotopes, isomers and very labile and reactive substances can be separated readily and satisfactorily. 23

24. APPLICATION OF PAPER CHROMATOGRAPHY 24

25. Paper chromatography has been applied to the separation of many organic and biochemical products. For example, it has been utilized in the determination of indoles in urine and in the study of barbiturates, antibiotics, carbamyl phosphates, hormones and amino acids, It also been used in the study of inorganic metal salts and complex ions 25

26. Separating mixture amino acids Paper chromatography is especially useful in characterizing amino acids. The different amino acids move at differing rates on the paper because of differences in their R groups. The rate of movement of a biomolecule during paper chromatography is reported as its relative mobility . alanine 0.38 Methionine 0.55 arginine 0.20 leucine 0.73 26

27. FOODS Both natural and synthetic food colours are added to foods to improve their acceptability and to make them more popular. Paper chromatography has been primarily used for analysis of food colors in ice creams, sweets, drinks and beverages, jams and jellies. To ensure that no non-permitted coloring agents are added to the foods, only edible colors are permitted for use. 27

28. Pathology And Forensic Science For investigation of crimes, paper chromatography is useful in the field of forensic science, as this process can be successfully carried out with even very small quantities of material. Using this technique, samples from crime scenes are collected to be analyzed and identified. Used in DNA and RNA fingerprinting. Moreover, to detect the presence of alcohol or chemicals in blood, pathological laboratories use paper chromatography. 28

29. Separation of Biological components Several compounds of biological origin, such as carbohydrates, phosphorylated sugars, lipids, steroids, bile acids, hormones, nucleosides and nucleotides, alkaloids etc., have been studied by PC. Pesticides, halogenated insecticides and organo-phosphorous insecticides can be identified by PC. 29

30. Nowadays, microfluidic paper-based devices (μPAD) technology has become widely used in almost every field of science due to several advantages of paper such as low-cost, mechanical flexibility and strength, easy to store. Doing paper chromatography on μPADs has been reported by Murphy et al. for separation of ascorbic acid and dopamine . 30

31. Separation and Purification of Dolichol and Dolichyl Phosphate by Anion-Exchange Paper Chromatography Dolichyl phosphate 31

32. REFERENCES Text book of pharmaceutical analysis by Dr .S. Ravishankar Instrumental Method of Chemical Analysis by Gurdeep R. Chatwal Journal by Fereshte Mohamadi Gharaghani, Morteza Akhond, Bahram, Hemmateenejad , A. Kupferberg , M. L. Tomassoni , and M. Mersel * 32

33. Important Question Add a note on paper Chromatography. 5M ( 2018-2019) 33

34. THANK YOU 34