1. Paper chromatography is a technique used to separate mixtures and identify unknown substances. It works on the principle of partition, where the stationary phase is the cellulose in filter paper and the mobile phase is a solvent. 2. Samples are applied to the paper and then developed using solvents like water or alcohol, which causes the compounds in the mixture to travel up the paper at different rates based on how they partition between the mobile and stationary phases. 3. The separated compounds can then be identified using detecting agents like iodine or UV light, or specific reagents that react with certain functional groups. Paper chromatography is used to analyze drugs, metabolites, proteins and more.

1. PAPER CHROMATOGRAPHY by Mr. Shaise Jacob Faculty, Nirmala College of Pharmacy Muvattupuzha Kerala, India

2. PAPER CHROMATOGRAPHY • ANALYSIS OF UNKNOWN SUSTANCES TWO TYPES 1.PAPER ADSORPTION CHROMATOGRAPHY Paper impregnated with silica or alumina 2.PAPER PARTITION CHROMATOGRAPHY Moisture / Water present in the pores of cellulose fibers present in filter acts as stationary phase & Mobile phase is used as solvent In general P.C – Paper Partition Chromatography PRINCIPLE OF SEPERATION Partition rather than adsorption Cellulose layers in filter paper contains moisture which acts as Stationary phase & Organic solvents/buffers are used as Mobile Phase

3. PRACTICAL REQUIREMENTS 1)Stationary phase 2)Application of sample 3)Mobile phase 4)Development technique 5)Detecting or Visualizing agents STATIONARY PHASE AND PAPERS USED Whatman filter papers Paper of any size can be used APPLICATION OF SAMPLE Capillary tube or Micropipette

4. MOBILE PHASE Pure solvents, buffer solutions or mixture of solvents Examples- Hydrophilic mobile phase & Hydrophobic mobile phases DEVELOPMENT TECHNIQUE 1) ASCENDING DEVELOPMENT (go up) FLOWS AGAINST GRAVITY, SPOT KEPT AT THE BOTTOM PORTION OF PAPER & KEPT IN A CHAMBER WITH MOBILE PHASE 2) DESCENDING TYPE (a downward slope) SPECIAL CHAMBER, SOLVENT HOLDER AT THE TOP- SPOT KEPT AT TOP & SOLVENT FLOWS DOWN THE PAPER ADVANTAGE IS THAT, DEVELOPMENT IS FASTER

5. 3)ASCENDING – DESCENDING DEVELOPMENT Only length of separation increased, first ascending takes place followed by descending 4)CIRCULAR / RADIAL DEVELOPMENT Spot is kept at the centre of a circular paper. The solvent flows through a wick at the centre & spreads in all directions uniformly. 5)TWO DIMENSIONAL DEVELOPMENT

8. DETECTING / VISUALISING AGENTS For detecting colorless spots, any one of the following techniques can be used Non specific methods E.g. iodine chamber method, UV chamber for fluorescent compounds – at 254 or at 365nm.

9. (b) Specific methods examples. Ferric chloride Ninhydrin in acetone Dragendroff’s reagents 3,5 dinitro benzoic acid Phenolic comp. & tannins Amino acids Alkaloids Cardiac glycosides

10. Following detecting tech. can also be categorized as 1) Destructive techniques Specific spray reagents… 2) Non-destructive techniques For radio active materials , Geiger Muller counter QUANTITATIVE ANALYSIS Direct techniques- Densitometer Indirect techniques- QUALITATIVE ANALYSIS Rf Value

11. APPLICATIONS Separation of mixtures of drugs Separation of carbohydrates,vitamins,antibiotics,proteins,…. Identification of drugs Identification of impurities Analysis of metabolites of drugs in blood , urine …. ADVANTAGES OF P.C Simple ,rapid ,inexpensive ,excellent resolving power PRECAUTIONS IN P.C Establishing the vapor solvent equilibrium Stability of solvent mixture is first ensured

12. Thank you