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Understanding Paper Chromatography: Theory, Types, and Applications

Paper chromatography is a separation technique discovered in 1943 by Martin and Synge. It involves the distribution of substances between a stationary liquid (held in paper fibers) and a moving liquid (developing solvent). The process relies on factors like partition coefficient and retention factor. This method offers advantages like simplicity and low cost but has limitations in effectively separating complex mixtures and volatile components. Different types of paper chromatography include descending, ascending, ascending-descending, radial, and two-dimensional chromatography. Filter paper selection, sample preparation, and visualization techniques play crucial roles in successful chromatographic analysis.

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Understanding Paper Chromatography: Theory, Types, and Applications

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  1. Paper Chromatography: Introduction • Paper chromatography is a separation technique in which paper or paper covered with a thin film of solvent is used as a stationary phase. • Martin and Synge discovered paper chromatography in 1943 as a means to survey constituents of plants and separate and identify them. Martin and Synge

  2. Paper Chromatography: Theory • The resolution of mixtures of solutes on filter paper may depend upon: 1. surface adsorption 2. Ion exchange 3. Partition between two solvents • The predominant factor of separation, however, is partition. • The substances are distributed between two liquids, i.e., one is stationary liquid, held in the fibers of paper (Stationary Phase) and other is moving liquid or developing solvent (Mobile Phase).

  3. Retention Factor • Partition coefficient (Kd) describes the way in which a compound distributes itself between two phases. • ‘R’ is a function of partition coefficient and is related to the migration of solute front relative to the solvent. • RFdefines the movement of the substances relative to the solvent front in a given chromatographic system.

  4. Retention Factor • RF is constant for a given substance, provided the conditions of chromatographic system are kept constant (temperature, type of paper, duration and direction of the development etc.) • RFvalue of a substance depends upon: a. Solvent employed b. Medium used for separation c. Nature of the mixture d. Temperature e. Size of the vessel in which chromatography is carried out.

  5. Advantages & Disadvantages Advantages • Simpler and Cheaper, Easy to set up and handle • Can be used for both organic and inorganic substances Disadvantages • Separation of complex mixtures is difficult • Not compatible with large amount of samples • Not suitable for separation of volatile components. • Chromatograms can’t be saved for a longer period of time. • Not as accurate as other chromatographic techniques

  6. Paper Chromatography: Types • Types of Paper Chromatography: a. Descending Chromatography b. Ascending Chromatography c. Ascending-Descending Chromatography d. Radial Paper Chromatography e. Two Dimensional Chromatography

  7. Descending Chromatography • Development of paper is done by allowing the solvent to travel down the paper. • First reported successful use by Consden, Gordon and Martin in 1944. • Advantages: Development can be continued indefinitely though solvent runs off at other end. even

  8. Ascending Chromatography • Development of paper is done by allowing the solvent to travel up the paper. • First used Goppelsroeder et al. • Advantages: Simplicity of apparatus, consistency of results and easy • Preferred if RFvalues are similar by Schoenbein,

  9. Ascending-Descending Chromatography • Hybrid of ascending and descending chromatography. • In this technique upper part of ascending chromatography can be folded over a glass rod allowing ascending development to change into descending. • First used by Block (1960)

  10. Radial Paper Chromatography • Also paper chromatography. • Substances to be analyzed are resolved into circular zones. • Various materials analyzed are placed at the centre and paper is fixed horizontally on petri-dish possessing solvent. • Tongue or wick of the paper dips in the solvent. known as circular to be

  11. Two Dimensional Chromatography • In this technique, square or rectangular paper is used. • The sample is applied to one of the corners development is performed at right angle to the first run. • It can be carried out with same solvent system in both direction or by two solvents. and second

  12. Experimental Details 1. Choice of proper chromatographic technique. 2. Choice of Filter paper 3. Proper Developing Solvent 4. Preparation of Samples 5. Spotting 6. Drying the chromatogram 7. Visualization 8. Calculation of RFvalues

  13. Filter Paper • Chromatography papers are prepared from cotton linters selected to be especially low in organic and inorganic impurities and uniform in physical characteristics. • Choice of paper depends on problem under investigation: a. Quantitative or Qualitative analysis b. Analytical or Preparative c. Substances used are hydrophilic or lipophilic, neutral or charged species

  14. Whatmann Filter Papers • These are the most commonly used paper for paper chromatography. • It usually contains α cellulose (98-99%), β cellulose (0.3-1%), pentosans (0.4- 0.8%), ether soluble matter (0.015-0.03%), ammonia (0.001-0.06%), organic nitrogen (<0.1%) and inorganic materials (0.008-0.06%) with slight variation according to grade. • Capillarity of paper can be increased by partial hydrolysis (soaking paper in 7% HCl for 24 hours and then washing with water and ethanol).

  15. Modified Papers Types Typical Uses Carboxyl Papers Cationic separation of protonated amines and amino acids. Acetylated Papers Reversed phase chromatography of lipophilic substances; Metal cations Kieselguhr Papers, Alumina Papers, Zirconia Papers, Silica Papers Separation of low polarity substances such as amines, fatty acids, steroids, triglycerides, vitamins and pesticides Ion exchange papers Ion exchange paper chromatography

  16. Proper Developing Solvent • The following general criteria is used to choose a proper solvent system: a. RFvalue of sample should lie between 0.05 and 0.85 in the system. b. Minimum difference between RFvalue of two components must be 0.05 in the system. c. The solvents should not undergo chemical reaction with any of the components of the sample mixture. d. The solvent should not interfere with the detection of spots on developed chromatogram. e. The composition of solvent system should not alter with time.

  17. Preparation of Sample & Spotting • No standard procedure as it depends on several factors of the given samples. • Sample volume : 10 – 20µ • A horizontal line is drawn on filter paper by lead pencil. (Origin line) • Cross marks (x) are made with pencil in such a way that each cross (x) is atleast 2 cm away from each other. • Test solutions are applied on cross (x) marks using graduated micro pipette and spots are dried.

  18. Drying of Chromatogram • Chromatograms after development are dried in special cabinets which are being heated electrically with temperature controls.

  19. Visualization • Visualization can be done in two ways: a. Chemical Means: Chemical reagents are applied, either by pressure-spraying or dipping the chromatogram to visualize it and the spots are then dried. b. Physical Methods: Some colourless spots, when subjected to UV light, fluoresce and reveal existence.

  20. Calculation of RFValues • The distance of chromatographed species is measured from its centre to line of origin. • RFvalue is calculated using the formula for retention factor.

  21. Quantitative Analysis After Extraction from paper a. Isolation of separated components from paper chromatography: Cutting out the appropriate part of filter paper having solvent and soak it in suitable solvent b. Determination: Microanalysis of resultant eluate by Gravimetric estimation, UV Spectrophotometry, Colorimetry, Polarography, Coulometry, Flame photometry etc. In situ Methods • Visual assessment, measurement of area of spots or zones, by Densitometer and Potentiometry.

  22. Forensic Significance • For separation of components of Ink • For determination of indoles in whole urine and in study of barbiturates, carbamyl phosphates and amino acids. • The identification of chloride, chlorite, chlorate and perchlorate in explosive residues • To detect adulteration in food and drinks

  23. Explosive Residues Paper Eluent Visualizing Reagent RFand Colour Chloride 0.2N aq. AgNO3 ~0.25 Purple Whatman No. 1 filter paper 5 x 43cm, spotted 4 cm from end Propanol: water: Pyridine: Concentrated ammonium hydroxide: :15: 2: 2: 2. Chlorite 3M aq. hydrochloric acid : acetone :: 1 :1 and 5% ethanolic diphenylamine ~0.36 Blue Chlorate ~0.54 Green Perchlorate Aq. Sodium Acetate and aq. Methylene blue ~0.71 Violet spot on a blue background

  24. References • Block, R.J., LeStrange, R., Zweig, G. (1952). Paper chromatography: A laboratory manual. Academic Press. New York. • Chatwal, G.R., Anand, S.K. (2019). Instrumental methods of chemical analysis. Himalaya Publishing House. • Clatworthy, A.J., DeBree, P.K., Edwards, R.W.H., Ersser, R.S., Goodwin, H.J., Jackson, J.V., Lake, B.D. (1976). Chromatographic and electrophoretic techniques. A William Heinemann Medical Books Publication. Chicago. • Zweig, G., Whitaker, J.R. (1971). Paper chromatographic and electrophoresis. Academic Press. New York. NOTE: These slides are only for academic purposes

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