PAPER CHROMATOGRAPHY BY Rx PHARMEDICO

PAPER CHROMATOGRAPHY

1. Paper chromatography By B.Nom Kumar Naik

2. Introduction General procedure Types of Paper chromatography Difference between partition & adsorption chromatography Different development techniques of paper chromatography Materials & methods 1.Stationary phase ( S.P ) 2. Mobile phase ( M.P ) 3.Developing chambers 4.Sample spotting & colour development 5.detective methods..i..e..Physical & chemical methods 6.Non – selective detective methods Applications of paper chromatography References Contents

3. Definition : - Paper chromatography is a chromatography technique used to separate mixture of chemical substances into its individual compounds.  It is an inexpensive method of separating dissolved chemical substances by their different migration rates across the sheets of paper. It is a powerful analytical tool that uses very small quantities of material. Paper chromatography was discovered by Synge and Martin in the year 1943 Principle : - Majorly “Partition” S.P : - Liquid ( whatmans filter paper consisting of cellulose layer which contain liquid ) M.P : - Liquid Minorly “Adsorption” S.P : - Solid ( cellulose fibres' of whatmans filter paper ) M.P : - Liquid . Note : - Partition is more predominant than adsorption , so the principle is “ Partition”. Introduction

4. Paper chromatography works in few steps : - Step 1:  A horizontal line is drawn near one end (about 1.5 cm from the bottom edge) of the paper. Step 2:   The sample needs to be separated is placed as a small drop or line on to the paper using capillary tube. Labelling the drop by a pencil with an alphabet or number help to identify the compound later.The drops are then soaked on the paper and dried. Step 3:  The paper is then placed into a sealed container with a swallow layer of  suitable solvent (M.P). The solvent level must be lower than the pencil line or drop on it. The container need to be covered to stop the solvent to evaporate. GENERAL PROCEDURE

5. Step 4:   The solvent rises up the paper chromatography taking each component of the sample with it. The components travel with the solvent depends on three things : 1.The polarity of the sample molecule. The non polar components travel faster than the polar component.

6. 2.The attraction between the sample molecule and the solvent or solvent mixture. 3.The attraction between the sample and the silica. Suppose any sample compound mixture contains three colored molecules  green, blue and red. According to their polarity, the order of these compounds is green<blue<red. Thus the most non polar green will travel first along with the mobile phase. Then blue and at last most polar compound the red one. Step 5:   When the solvent rises near the end of the paper then the paper should be taken out from sealed container and air dried. The paper with separated bands of components are then observed under UV-light.

7. Types of paper chromatography Paper partition chromatography Paper adsorption chromatography Reverse phase Paper partition chromatography Ion exchange Paper partition chromatography Based on the migration forces

8. Paper partition chromatography 1.S.P : - LIQUID (Present in the pores of the cellulose fibres in the filter paper ) 2.M.P : - LIQUID (single / mixture of the solvent ) 3. Migration of the substance id due to difference in “PARTITION Coefficient “. Difference between paper partition chromatography and paper adsorption chromatography Paper adsorption chromatography 1.S.P : - SOLID (Silica gel) 2.M.P : - LIQUID (Single / mixture of the solvent ) 3. Migration of the substance is due to “ADSORPTIVE Power “.

9. Different developing techniques of paper chromatography Ascending paper chromatography Radial paper chromatography Descending Paper chromatography Ascending – descending Paper chromatography Developing techniques Two dimension paper chromatography

10. Ascending paper chromatography Here the solvent travels up the chromatographic paper. Both descending and ascending paper chromatography are used for the separation of organic and inorganic substances. The sample and solvent move upward ( Against gravitational force ). Descending paper chromatography Development of the chromatogram is done by allowing the solvent to travel down the paper. Here, mobile phase is placed in solvent holder at the top. The spot is kept at the top and solvent flows down the paper from above.

11. Ascending-descending paper chromatography This is the hybrid of both of the above techniques. The upper part of ascending chromatography can be folded over a rod in order to allow the paper to become descending after crossing the rod. Circular chromatography paper chromatography A circular filter paper is taken and the sample is deposited at the centre of the paper. After drying the spot, the filter paper is tied horizontally on a  Petri dish  containing solvent, so that the wick of the paper is dipped in the solvent. The solvent rises through the wick and the components are separated into concentric rings.

12. Two-dimensional paper chromatography In this technique a square or rectangular paper is used. Here the sample is applied to one of the corners and development is performed at a right angle to the direction of the first run

13. Selection criteria for materials and method : - Qualitative / Quantative analysis . Analytical / preparative chromatography. Hydrophilic / Lipophilic compounds. Whatsman grade filter paper obtained from purified cotton fibre. It consist of 99% -cellulose and remain 1% -cellulose,pentose and mineral contents. Paper consist of 6% moisture. S.P ( stationary phase ) Materials & method

14. Sl.no Whatsman grade T ype of paper used Developing time of colour 1 Grade – 1 Slow / standard paper 15 – 16 min 2 Grade - 3 Preparative paper for preparative chromatography Exact 5 min 3 Grade – 4 Fast paper 9 min 4 Grade – 31T Very fast paper with in 5 min Different grades of papers

15. 1.Carbonyl paper : - oxidation Uses 2.Hydrophilic paper : - Formamide Uses Glycerol, Glycol Methanol Different types of paper Paper  Capilarity of paper  Polarity of paper separation of polar compouds Paper Hydrophilic compound separation of polar compouds

16. 3.Hydrophobic paper : - Paraffine liq. uses Note : - Here reversed phase papers are formed 4.Acetyleted paper : - Acetylation uses Esterification 5.Ion-exchange paper : - uses Paper Non-polar (S.P) Liphophilic compounds like steroids Paper  Polarity Polar Liphophilic compounds like steroids Paper for ion – exchange chromatography

17. It should be more polar than S.P. It may be single / mixture of solvents. It should not react with components and spraying reagents. There are two types of M.P 1.Hydrophilic mobile phase : - eg : - 1. n-butanol : Glacial acetic acid : water (4 : 1 : 5 ) 2. Isoproponal : Ammoia : water ( 9 : 1 : 2 ) 2.Hydrophobic mobile phase : - eg : - Cyclohexane , Diethyl ether NOTE : - If the compound is polar we can use Chloroform as solvent ( M.P ). If the compound is medium polar we can use Benzene as solvent. If the compound is low polar we can use Cyclohexane as solvent. M.P ( Mobile phase)

18. It rage between small test tubes to large jars or containers. They may be made up of material like glass , steel etc..... Developing chamber

19. 1.Spotting of the sample is done with the help of a capillary tube or automated applicator.. 2.The sample is applied as a neat spot on a horizontal line drawn with a pencil close to one edge. 3. Allow the spot to dry and then immerse the paper in the developing chamber as per the selected technique with the marked spot above the solvent level. 4.The solvent begins to move and draws the sample components differentially along with it. 5. At the end of the development take out the paper and mark the solvent front with another line. Sample spotting and development of colour

20. 6.Allow the paper to dry in drying cabinets with the provision of electrical heating before visualisation. Note : - The spotting diameter should be in range between 3 – 4 mm. For the loading of sample above edge or base line i.e.. for Ascending paper chromatography 4 – 5 cm for Descending paper chromatography 8 – 9 cm. 3. The distance between 2 spots is 3 – 4 cm.

21. Base on physiochemical nature Spraying agents or detecting agents or visualizing agents Detection Physical Chemical Non-specific Specific Auto-radioactive method Geiger-muller Radioactive method UV chamber / Fluroscence

22. A.Geiger-muller method : - Chromatogram was placed in GMC ( geiger-muller chamber ). And it is used for the identification of radioactivity. B.Autoradioactive method : - Chromatogram is pressed between glass slide and x – ray slide , from these we can identify the quantity of a compound. Physical method 1.Radioactive method

23. This method is used for the detection of “Florescent and Non – florescent compounds. Non – florescent compounds can be identified at 256 rpm . Florescent compounds produce different colours like “ Green , Blue , Violet “ with a background of Black colour. Non – florescent compounds produce “ Black colour “ with a background of different colours like Blue, Green , Violet . 2.UV chamber method : -

24. 1.Iodine chamber method 2.Uv method 3.Universal sprayer reagent ( sulphuric acid ) Note : - All the selective methods are destructive methods including universal sprayer because they destroy the original compound and gives colour when we spray reagent. All the non selective methods are non destructive except universal sprayer reagent. Non specific or Non selective method : -

25. Sl.no Name of the compounds Spraying reagent Colour obtained 1 Phenols & Tannins Ferric chloride Blue , green , Violet 2 Amino acids Ninhydrine Pink /Purple 3 sugars Aniline-hydrogenpthalate Black grey 4 Cardiac glycosides Di-nitrobenzoic acid Orange 5 Anthraquinones Ammonia Pink Chemical method Specific method : -

26. Sl.no Name of the compounds Spraying reagent Colour obtained 6 Alkaloids Dragandroff reagent Brown 7 Sulphonamides NaNO3 (0.5%) + B.M reagent (1%) Red / Purple 8 Aldehydes & Ketones 2,4-DNP Yellow / Orange 9 Barbiturates Diphenyl carbazone Pink / Purple 10 Steroids Antimony trichloride in CHCl3 Multicolours 11 Amines & Unsaturates Iodine Brown

27. All these are selective and specific reagent in chemical method of detection. Universal spraying reagent is “Sulphuric acid”( 4 ml sulphuric acid + 96 ml ethanol ). Modified papers should be taken to spray Sulphuric acid (Universal sprayer) because it is not suitable for paper chromatography and It will be suitable for modified papers like glass fibres paper After staining the reagent ( Sulphuric acid ) the paper will appear as Black colour

28. There are various applications of paper chromatography. Some of the uses of Paper Chromatography in different fields are discussed below: To study the process of fermentation and ripening. To check the purity of pharmaceuticals. To inspect cosmetics. To detect the adulterants. To detect the contaminants in drinks and foods. To examine the reaction mixtures in biochemical laboratories. To determine dopes and drugs in humans and animals Paper Chromatography Applications : -

29. A text book of pharmaceutical analysis by Dr.S.Ravisankar https://en.wikipedia.org/wiki/Paper_chromatography https://byjus.com/chemistry/paper-chromatography/ https://www.britannica.com/science/paper-chromatography Instrumental methods of chemical analysis by G.R.Chatwal . Pharmaceutical analysis Vol – 2 by A.V.Kasture . Pharmaceutical analysis by Ashutoskar . Reference