Paper chromatography

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1. Paper chromatography

2. Definition: Paper chromatography is defined as the technique in which the analysis of unknown substances is carried out mainly by the flow of solvents on a specially designed filter paper. There are two types: Paper adsorption chromatography: Paper impregnated with silica or alumina, it act as adsorbent (stationary phase) and solvent as mobile phase. Paper partition chromatography: in which moisture water present in the pores of the cellulose paper and another mobile phase is used as solvent. In general PC refers to P partition C only since most separation is based on partition type only.

3. Principle: The principle of separation is mainly partition rather than adsorption. The whatmann filter paper is used as stationary phase. It is made of 99% of cellulose. Cellulose is very good adsorbent and it adsorb atmospheric moisture. This moisture develop as thin film of liquid on the surface of the filter paper and this behaves as stationary liquid. Instead of water as stationary phase other organic solvents can be used by suitable modification.

4. Mobile phase – organic solvent or mixture of organic solvents (mixed in a specific ratio) and buffers are used. It is also called moving liquid or development solvent. The distribution of analyte takes place between stationary liquid (aqueous phase) and mobile liquid (organic phase) . Hence called partition paper chromatography.

5. In this technique a drop of the test solution is applied as a small spot on a filter paper and the spot is dried. The paper is kept in a closed chamber and the edge of the filter paper is dipped into a solvent called development solvent. Now the development solvent moves against the gravitational force (upwards) by capillary action of filter paper. The minute pores present in the stationary phase will provide the capillary action. When the solvent reaches the spot (mixture of 2or more substances), the various substances are moved by solvent system at various speeds and the solvent is allowed to move upto 3/4 th of the paper, then stopped.

6. This process of movement of m/p and separation of analyte mixture is called development process. Now the paper is dried and various spots are visualized by suitable detection processes.

7. Steps: Selection of stationary phase. Selection of mobile phase. Chamber saturation (preparing the developing chamber). Preparation and application of sample. Development. Drying. Detection.

8. Selection of stationary phase : Paper of chromatographic grade consists of α - cellulose – 98.99%, β -cellulose – 0.3-1%, pentosans – 0.4-0.8%, ether soluble matter – 0.015-0.02%, ash – 0.01-0.07%. Whatman filter papers of different grade like no.1, no.2, no.3, no.3MM, no.4, no.17, no.20 etc are used. These papers differ in size, shapes, porosities and thickness. Choice of filter paper depends upon thickness, flow rate, purity, technique, etc. Modified papers: acid or base washed, glass fiber type paper.

9. Hydrophilic papers: papers modified with methanol, formamide, glycol, glycerol etc. Hydrophobic papers: acetylation of OH group leads to hydrophobic nature, hence can be used for reverse phase chromatography. the filter paper of suitable size that can be kept in the chamber is taken and a line is drawn leaving 2cm from bottom and also another line at 3/4 th length of the paper is drawn with a pencil.

10. Selection of mobile phase: Organic solvents, buffers, or mixture of solvents are used. Generally in PC mixture of organic solvents prepared in some ratios are used based the polarity of analyte mixture. Hydrophilic mobile phases: Methanol : water – 3:1 or 4:1 n-butanol : glacial acetic acid : water – 4:1:5 Hydrophobic mobile phases: Kerosene : 70% isopropanolol Dimethyl ether : cyclohexane

11. Chamber saturation: A development chamber is taken and mobile phase is added to it and closed. This setup is kept aside for 20 to 25 minutes . During which between the components of the developing solvent and their vapor, an equilibrium will be established eventually. This equilibrium is called chamber saturation. It the process of replacing of atmospheric air present with vapors of mobile phase. Use of chamber saturation: Edge effect doesn’t occur Due to the presence of air the separation favors to edges of the paper and is not proper. It is prevented by chamber saturation.

12. Preparation and application of sample: Analyte mixture is dissolved in a suitable solvent to make about a one percent solution (0.01 g sample/1g solvent). Less than one milliliter of solution will be needed for the experiment. Spotting: Touch the tip of a clean drawn out capillary tube to the sample and let about 3mm of sample rise into the tube. To spot the sample, touch the capillary to the line. Make sure the spot does not exceed 1cm in diameter. Allow the sample to evaporate. Spots should be 2 to 2.5cm away from the edges of the paper and from each other.

13. Development: After preparing the chamber and spotting of the samples, the sample is developed. The paper is dipped into the mobile phase and the mobile phase moves upward due the capillary action due to pores present in the paper. As the solvent reaches the 3/4 th length of the paper the development is stopped. Development technique: Ascending: conventional type Development occurs against the gravity.

14. Descending: Flow of solvent is assisted by gravity and hence development is faster. Solvent holder is on top. Ascending – descending: Combination of ascending and descending type. First ascending takes place then descending. Length of separation is increased. Circular or radial development: Filter paper is circular. The spot is kept at the center. The solvent flows through a wick at the centre and spreads in all directions Spots appear as concentric circles.

15. Two dimensional development: First the plates are developed in one axis and the plates after drying are developed in other axis (i.e., the plates are rotated to 90 degree). When large number of compounds or complex mixtures are need to be separated this method can be followed. Either same solvent or different solvent system can be used.

16. Drying: Gently remove the paper out of the chamber by handling it in the corners And dry it in a well ventilated area.

17. Detecting or visualizing agents: Colored spots can be visually detected. But for detecting colorless spots, the following techniques are used: Non specific methods: no. of spots can be detected but not exact nature or type of compound. Iodine chamber method : where brown or amber colored spots are observed when the paper are kept in a tank with few iodine crystals at the bottom. UV chamber for fluorescent compounds: when viewed under UV chamber at 254 or at 365 nm, fluorescent compounds can be detected.

18. Specific methods: specific spray or detecting or visualizing agents are used to find out the nature of compound or for identification purposes. Eg. Fecl3 for phenolic and tannin compounds ninhydrin for amino acids. The detecting techniques can also be categorized as: Destructive technique: when specific spray agents are used the samples are destroyed before detection. Eg. Ninhydrin reagent Non destructive technique: methods like UV chamber, iodine chamber, densitometry method doesn’t destroy the sample even after detection.

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

20. Quantitative analysis: Direct technique: by densitometer measure density of spots This method is also called as in-situ method. Indirect technique: The papers are cut into portions and eluted with solvents. The solution is analyzed by spectrophotometry, electrochemical techniques, etc.

21. Qualitative analysis: Rf value: Rf= dist travelled by solute/dist travelled by solvent front value ranges from 0-1 Ideal values are 0.3-0.8. It is characteristic to each compound in a particular combination of sp and mp. The unknown compound can be identified by comparing its rf values with standard’s. Rx value: Distance travelled by sample/dist travelled by standard. It is always closer to 1.

22. Rm values: To find whether compounds belong to a homologous series. It is a combined value. Delta Rm = log (1/Rf – 1).

23. Applications: Useful for analysis of amino acids, sugars, natural products, etc. Separation of mixture of drugs of chemical or biological origin, plant extracts, etc. Separation of carbohydrates , vitamins, antibiotics, proteins, alkaloids, glycosides, amino acids, etc. Study of inorganic metal salts and complex ions. Identification of decomposition products. Analysis of metabolites of drugs in blood, urine etc. Identification of impurities. Identification of foreign substances in drugs.