Paper Chromatography

A chromatogrpahic technique widely employed for identification of certain organic compounds. Applied in laboratories of colleges as a teaching tool for easy understanding the thechnique of chromatography.

1. PAPER CHROMATOGRAPHY Mrs.Divya Narla Asso.professor Aditya College of Pharmacy

2. INTRODUCTION In 1961 paper chromatography was first discovered by SEHON BEN . in  analytical   chemistry , technique for separating dissolved chemical substances by taking advantage of their different rates of migration across sheets of paper. Paper chromatography is an chromatography technique used to separate mixture of chemical substances into its individual compounds.

3. PRINCIPLE The principle involved is Partition Chromatography   wherein the substances are distributed or partitioned between liquid phases. One phase is the water, which is held in the pores of the filter paper used; and other is the mobile phase which moves over the paper .

4.   In paper chromatography, substances are distributed between a stationary phase and a mobile phase. The stationary phase is the water trapped between the cellulose fibers of the paper. The mobile phase is a developing solution that travels up the stationary phase, carrying the samples with it. Components of the sample will separate readily according to how strongly they adsorb onto the stationary phase versus how readily they dissolve in the mobile phase .

5. TYPES/MODES OF PAPER CHROMATOGRAPHY Based on the development of chromatogram on paper ,PC has 5 types. Ascending paper chromatography Descending paper chromatography Ascending Descending paper chromatography Radial / Circular paper chromatography Two-Dimensional paper chromatography

6. ASCENDING TECHNIQUE As the name indicates, the chromatogram ascends. Here, the development of paper occurs due to the solvent movement or upward travel on the paper . The solvent reservoir is at the bottom of the beaker. The paper tip with sample spots just dips into the solvent at the bottom so that spots remain well above the solvent.

7. DESCENDING CHROMATOGRAPHY Here, the development of paper occurs due to solvent travel downwards on the paper . The solvent reservoir is at the top. The movement of the solvent is assisted by gravity besides the capillary action.

8. ASCENDING-DESCENDING MODE Here solvent first travels upwards and then downwards on the paper .

9. RADIAL/CIRCULAR MODE the solvent moves from the center (mid-point) towards the periphery of circular chromatography paper. The entire system is kept in a covered Petri dish for the development of the chromatogram . The wick at the center of paper dips into the mobile phase in a petri dish, by which the solvent drains on to the paper and moves the sample radially to form the sample spots of different compounds as concentric rings.

10. TWO DIMENSIONAL CHROMATOGRAPHY Here the chromatogram development occurs in two directions at right angles . In this mode, the samples are spotted to one corner of rectangular paper and allowed for first development. Then the paper is again immersed in the mobile phase at a right angle to the previous development for the second chromatogram.

11. STEPS INVOLVED Stationary phase/choice of filter paper Mobile phase Sample preparation Sample application Development chamber Development of Chromatogram Detection & Visualization

12. Choice of Filter paper Whatmann filter papers are used. Papers are made up of 98-99% of α – cellulose. Various grades and types of paper are available for sample separation. Factors governing choice of paper Nature of sample & solvent Based on thickness of paper Based on quantitative or qualitative analysis.

13. Preparation of paper The paper is cut into desired shape and size depending upon work to be carried out. A base line is drawn on the paper with an ordinary pencil at about 1cm from the bottom edge. On the base line cross marks are made with certain distance.

14. Mobile Phase/Solvent system Several organic liquids can be used as Mobile phase. The solvent system can be a single solvent or mixture of solvents. The solvent are selected based on nature of sample components to be separated. Eg ., Ethyl alcohol Benzene N-hexane Toulene Water Chloroform

15. Sample Preparation & Application The sample should be dissolved in a small amount of mobile phase or any other suitable solvent to obtain solution. The concentration of sample in the solution should be adjusted as per requirement.

16. Sample Application The sample solution is applied with micropipette or micro capillary tubes as a small spot on the origin line. The sample can be applied for 2-3 times. The spot is air dried before placing in development chamber.

17. Development chamber The chromatographic chamber is made up of many materials such as Glass, Plastic or Stainless steel . Glass tanks are mostly preferred. They are available in various dimensions, depending on paper length and development type. SATURATION of development chamber: The development chamber should be saturated with solvent vapour.

18. Development of Chromatogram The paper spotted with sample is placed inside the saturated chamber. The paper dips inti the solvent. Make sure that the baseline is above the level of solvent. The solvent travels in upward direction by capillary action & is allowed to run 3/4 th of paper height. After development the paper is taken out of chamber carefully.

19. Drying of chromatogram The chromatogram is dried after its development. They are dried by cold or hot air depending on volatility of solvent. A simple hair dryer is a convenient device to dry chromatograms.

20. Detection of Spots If the substance is coloured , they are visually detected easily . But if the substance is colourless, physical or chemical methods are used to detect spot. PHYSICAL METHODS: The paper is observed under UV light if the sample fluorescent compound. Radioisotope measurements can be done.

21. CHEMICAL METHODS: Chemical reagents are used to develop colour. Amino acids - Ninhydrin reagent Alkaloids - Dragendorffs reagent Phenols - ferric chloride Aldehydes - 2,4-Dinitro phenyl hydrazine.

22. Rf value: It represents the movement or migration of solute relative to the solvent front. Used to indicate the position of the separated components. Rf value ranges between 0 – 1 Ideal Rf value ranges from 0.3 – 0.8.  

23. APPLICATIONS It is specially used for separation of mixture having polar and non polar compounds. For separation of Amino acids, Peptides, Alkaloids, Sugars, Lipids etc. To determine organic compounds in biological samples such as urine. For evaluation of inorganic compounds like salts and complexes. Used for both Qualitative & Quantitative analysis.

24. ADVANTAGES Simple method Inexpensive Sensitive Separations can be carried out under ordinary lab conditions.

25. DISADVANTAGES Time consuming Cannot be used as Preparative method. Corrosive reagents cannot be used for detection .

26. THANK YOU