Paper chromatography

principle, requirements, method and applications of paper chromatography

1. PAPER CHROMATOGRAPHY

2. PAPER CHROMATOGRAPHY - INTRODUCTION Paper chromatography is an analytical method used to separate colored chemicals or substances. It is primarily used as a teaching tool, having been replaced by other chromatography methods, such as thin-layer chromatography . A paper chromatography variant, two-dimensional chromatography involves using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of compounds having similar polarity, for example, amino acids . The setup has three components.

3. The mobile phase is a solution that travels up the stationary phase, due to capillary action . The mobile phase is generally mixture of non-polar organic solvent, while the stationary phase is polar inorganic solvent water. Here paper is used to support the stationary phase, water. Polar water molecules are held inside the void space of cellulose network of the host paper. Difference between TLC and paper chromatography is that stationary phase in TLC is a layer of adsorbent (usually silica gel , or aluminium oxide ), and stationary phase in paper chromatography is water.

4. WHATMANN FILTER PAPER It is used as stationary phase in paper chromatography

5. TYPES OF PAPER CHROMATOGRAPHY 1. Descending 2. Ascending 3. Ascending-descending 4. Circular chromatography 5. Two-dimensional

6. DESCENDING PAPER CHROMATOGRAPHY Development of the chromatogram is done by allowing the solvent to travel down the paper. Here, mobile phase is placed in solvent holder at the top. The spot is kept at the top and solvent flows down the paper from above.

7. ASCENDING PAPER CHROMATOGRAPHY Here the solvent travels up the chromatographic paper. Both descending and ascending paper chromatography are used for the separation of organic and inorganic substances. The sample and solvent move upward.

8. Ascending-descending This is the hybrid of both of the above techniques. The upper part of ascending chromatography can be folded over a rod in order to allow the paper to become descending after crossing the rod.

9. Circular chromatography A circular filter paper is taken and the sample is deposited at the center of the paper. After drying the spot, the filter paper is tied horizontally on a Petri dish containing solvent, so that the wick of the paper is dipped in the solvent. The solvent rises through the wick and the components are separated into concentric rings.

10. RF VALUE - EXAMPLE If a compound travels 9.9 cm and the solvent front travels 12.7 cm, then R ƒ value = 9.9/12.7 = 0.779 or 0.78. R ƒ value depends on temperature and the solvent used in experiment, so several solvents offer several R ƒ values for the same mixture of compound.

11. TWO DIMENSIONAL PAPER CHROMATOGRAPHY In this technique a square or rectangular paper is used. Here the sample is applied to one of the corners and development is performed at a right angle to the direction of the first run.

12. PRINCIPLE The principle involved can be partition chromatography. Partition chromatography because the substances are partitioned or distributed between liquid phases. The two phases are water held in pores of the filter paper and the other phase is a mobile phase which passes through the paper. When the mobile phase moves, the separation of the mixture takes place. The compounds in the mixture separate themselves based on the differences in their affinity towards stationary and mobile phase solvents under the capillary action of pores in the paper.

13. PROCEDURE Selecting a suitable type of development: Usually ascending type or radial paper chromatography is used as they are easy to perform. Also, it is easy to handle, the chromatogram obtained is faster and the process is less time-consuming. Selecting a suitable filter paper : Selection of filter paper is done based on the size of the pores, and the sample quality.

14. 3. Prepare the sample: Sample preparation includes the dissolution of the sample in a suitable solvent used in making the mobile phase. 4. Spot the sample on the paper: Samples should be spotted at a proper position on the paper by using a capillary tube.

15. 5. Chromatogram development: Chromatogram development is spotted by immersing the paper in the mobile phase. Due to the capillary action of paper, the mobile phase moves over the sample on the paper. 6. Paper drying and compound detection : Once the chromatogram is developed, the paper is dried using an air drier. Also, detecting solution can be sprayed on the chromatogram developed paper and dried to identify the sample chromatogram spots.

16. Visualizing methods UV chamber/cabinet Iodine chamber Spraying agents

17. UV chamber or cabinet

18. TLC plate under UV light

19. Iodine chamber with iodine crystals

20. Spraying by sprayer

21. Specific spray reagents

22. Calculation of Rf Value

23. Calculation of Rf Value Rf = Retention or retardation factor Distance travelled by the solute Rf = ……………………………………………………. Distance travelled by solvent

24. Maximum value of Rf = 1 The retention factor (R ƒ ) may be defined as the ratio of the distance traveled by the solute to the distance traveled by the solvent. If R ƒ value of a solution is zero, the solute remains in the stationary phase and thus it is immobile. If R ƒ value = 1 then the solute has no affinity for the stationary phase and travels with the solvent front.

25. Example If distance travelled by the solute = 6 cm And distance travelled by solvent - 8 cm Then Distance travelled by the solute Rf = …………………………………………………….= 6/8 = 0.75 Distance travelled by solvent

26. APPLICATIONS To study the process of fermentation and ripening. To check the purity of pharmaceuticals. To inspect cosmetics. To detect the adulterants. To detect the contaminants in drinks and foods. To examine the reaction mixtures in biochemical laboratories. To determine dopes and drugs in humans and animals.

27. TAPAN KUMAR MAHATO [email protected]