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  • 1.Paper chromatography Presented by- Mr. Shaise JacobFaculty, Nirmala College of Pharmacy Muvattupuzha, Ernakulam, KeralaIndia, E. mail shaise@live.in1

2. PAPER CHROMATOGRAPHY Paper Chromatography (PC) was first introduced byGerman scientist Christian Friedrich Schonbein(1865). PC is considered to be the simplest and most widelyused of the chromatographic techniques because ofits applicability to isolation, identification andquantitative determination of organic and inorganiccompounds.2 3. PAPER CHROMATOGRAPHY ANALYSIS OF UNKNOWN SUSTANCESIt is carried out mainly by the flow of solventson specially designed filter paper.There are two types of paper chromatography, they are: 3 4. PAPER CHROMATOGRAPHY1.PAPER ADSORPTION CHROMATOGRAPHYPaper impregnated with silica or alumina acts asadsorbent (stationary phase) and solvent asmobile phase.2.PAPER PARTITION CHROMATOGRAPHYMoisture / Water present in the pores ofcellulose fibers present in filter paper acts asstationary phase & another mobile phase is usedas solvent In general P.C Paper Partition Chromatography 4 5. PAPER CHROMATOGRAPHYPRINCIPLE OF SEPERATIONThe principle of separation is mainly partitionrather than adsorption.Cellulose layers in filter paper contains moisturewhich acts as stationary phase & organicsolvents/buffers are used as mobile phase 5 6. PAPER CHROMATOGRAPHY PRACTICAL REQUIREMENTS1)Stationary phase & papers used 2)Application of sample 3)Mobile phase 4)Development technique 5)Detecting or Visualizing agents6 7. PAPER CHROMATOGRAPHY STATIONARY PHASE AND PAPERS USEDWhatman filter papers of different grades likeNo.1, No.2, No.3, No.4, No.20, No.40, No.42 etcare used. In general this paper contains 98-99%of -cellulose, 0.3 1% -celluloseFactors that governs the choice of paper: Nature of Sample and solvents used. Based on Quantitative or Qualitative analysis. Based on thickness of the paper.7 8. PAPER CHROMATOGRAPHY Modified Papers acid or base washed filterpaper, glass fiber type paper. Hydrophilic Papers Papers modified withmethanol, formamide, glycol, glycerol etc. Hydrophobic papers acetylation of OH groupsleads to hydrophobic nature, hence can be usedfor reverse phase chromatography. Impregnation of silica, alumna, or ion exchangeresins can also be made.8 9. PREPARATION OF PAPER Cut the paper intodesired shape and sizedepending upon workto be carried out. The starting line ismarked on the paperwith an ordinarypencil 5cm from thebottom edge. On the staring linemarks are made 2cmapart from each other.9 10. PAPER CHROMATOGRAPHY Preparation of the solution Choice of suitable solvent for making solution is veryimportant. Pure solutions can be applied direct onthe paper but solids are always dissolved in smallquantity of a suitable solvent. Biological tissues are treated with suitable solventsand their extracts obtained. Proteins can beprecipitated with alcohol and salts can be removed bytreatment with ion exchange resin.10 11. PAPER CHROMATOGRAPHYAPPLICATION OF SAMPLEThe sample to be applied is dissolved in the mobilephase 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 indeveloping chamber. 11 12. Choice of the Solvent The commonly employed solvents are the polarsolvents, but the choice depends on the nature of thesubstance to be separated. If pure solvents do not give satisfactory separation, amixture of solvents of suitable polarity may beapplied.12 13. PAPER CHROMATOGRAPHY MOBILE PHASE Pure solvents, buffer solutions or mixture of solvents Examples- Hydrophilic mobile phase Isopropanol: ammonia:water 9:1:2 Methanol : water 4:1 N-butanol : glacial acetic acid : water 4:1:5Hydrophobic mobile phasesdimethyl ether: cyclohexanekerosene: 70% isopropanol 13 14. CHROMATOGRAPHIC CHAMBERThe chromatographic chamber are made up of manymaterials like glass, plastic or stainless steel.Glass tanks are preferred most. They are available invarious dimensional size depending upon paperlength and development type.The chamber atmosphere should be saturated withsolvent vapor.14 15. PAPER CHROMATOGRAPHYDEVELOPMENT TECHNIQUE Paper is flexible when compared to glass plateused in TLC, several types of development arepossible which increases the ease of operation. The paper is dipped in solvent in such a mannerthat the spots will not dip completely into thesolvent. The solvent will rise up and it is allowed to run2/3rd of paper height for better and efficient result. 15 16. PAPER CHROMATOGRAPHY Different types of development tech. are 1) ASCENDING DEVELOPMENT (go up) Like conventional type, the solvent flows againstgravity. The spots are kept at the bottom portion ofpaper and kept in a chamber with mobile phasesolvent at the bottom. 16 17. PAPER CHROMATOGRAPHY 2) DESCENDING TYPE (a downward slope) This is carried out in a special chamber where thesolvent holder is at the top. The spot is kept at thetop and the solvent flows down the paper. In this method solvent moves from top to bottom soit is called descending chromatography. ADVANTAGE IS THAT, DEVELOPMENT IS FASTER17 18. PAPER CHROMATOGRAPHY3)ASCENDING DESCENDING DEVELOPMENTA hybrid of above two technique is calledascending-descending chromatography. Only length of separation increased, first ascendingtakes place followed by descending18 19. PAPER CHROMATOGRAPHY4)CIRCULAR / RADIAL DEVELOPMENT Spot is kept at the centre of a circular paper. Thesolvent flows through a wick at the centre & spreadsin all directions uniformly. 19 20. PAPER CHROMATOGRAPHY5)TWO DIMENSIONAL DEVELOPMENT In this method the paper is developed in onedirection and after development, the paper isdeveloped in the second direction allowing morecompounds to be separated into individual spots. in the second direction, either same solvent/different solvent system can be used for development.20 21. TWO DIMENSIONAL DEVELOPMENT21 22. PAPER CHROMATOGRAPHYDRYING OF CHROMATOGRAM After the solvent has moved a certain distance forcertain time the chromatogram is taken out from thetank & position of the solvent front is marked with apencil. They are dried by cold or hot air depending onvolatility of solvents. A simple hair dryer is aconvenient device to dry chromatograms.22 23. PAPER CHROMATOGRAPHY DETECTING / VISUALISING AGENTSIf the substance are colored they are visually detected easily.But for colorless substance, Physical and chemical methods are used to detect the spot.(d) Non specific methods ( Physical methods)E.g. iodine chamber method,UV chamber for fluorescent compounds at 254 or at 365nm. 23 24. (b) Specific methods (Chemical methods) or Spraying method - examples, Ferric chloride Phenolic comp. & tannins Ninhydrin in acetone Amino acids Dragendroffs Alkaloidsreagents 3,5 dinitro benzoic Cardiac glycosidesacid24 25. Following detecting tech. can also be categorized as 1) Destructive techniques Specific spray reagents, samples destroyed before detection e.g. ninhydrin reagent 2) Non-destructive techniques For radio active materials - Geiger Muller counter uv chamber, iodine chamberQUANTITATIVE ESTIMATIONSThe method can be divided into two main groups1. Direct techniques-2. Indirect techniques- 25 26. PAPER CHROMATOGRAPHY Direct Measurement Method (i) Comparison of visible spots A rough quantitative measurements Component in a mixture can be carried out by comparingthe intensity and size of the spot with a standardsubstance. (ii) Photo densitometry The method is used with the chromatograms of coloredcompound, instrument which measures quantitativelythe density of the spots.26 27. PAPER CHROMATOGRAPHY (iii) Fluorimetry The compound to be determined by fluorimetry mustbe fluorescent or convertible into fluorescentderivatives. (iv) Radiotracer Method The compound containing radioactive element islabeled and treated with locating reagent. UsingGeiger Muller counter. (v) Polarographic & Conductometric methods Used to measure the amount of material in the spot 27 28. PAPER CHROMATOGRAPHY Indirect Measurement Method In this technique, the spots are cut into portions andeluted with solvents. This solution can be analyzedby any techniques of analysis like spectrophotometry,electrochemical methods, etc. 28 29. Rf VALUE (Retardation Factor)In paperchromatography theresults are representedby Rf value whichrepresent the movementor migration of soluterelative to the solventfront.29 30. Factors affecting Rf VALUE i. The temperature ii. The purity of the solvents used iii. The quality of the paper, adsorbents & impuritiespresent n the adsorbents iv. Chamber saturation techniques, method of drying& development v. The distance travelled by the solute & solvent vi. Chemical reaction between the substances beingpartitioned. vii. pH of the solution 30 31. Rx VALUE In many cases it has been observed that the solventfront is run off the end of the paper. Rx value is thusused, It is the ratio of distance travelled by the sample andthe distance travelled by the standard. Rx value isalways closer to 1.31 32. Sources of Error 1. Error during application of the spots Apply minimum volume of the concentrated solutionin order to avoid diffusion through the paper whichleads to poor separation Spots should be approximately of the same diameter. 2. Development Improper adjustment of the paper in the tank leadsto this error so the paper should be held vertically. Do chamber saturation 3. Detection The spraying methods affect the final result32 33. APPLICATIONS 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 .ADVANTAGES OF P.CSimple ,rapid ,inexpensive ,excellent resolvingpower PRECAUTIONS IN P.CEstablishing the vapor solvent equilibriumStability of solvent mixture is first ensured33 34. Thank you34