PAPER CHROMATOGRAPHY

INTRODUCTION TO PAPER CHROMATOGRAPHY
Cellulose filter paper is often used as the statioary phase in the paper chromatography. Since it is hydrophillic, it is usually covered with thin film of water. The procedure is often regarded as liquid-liquid cromatography
Other liquids can be encorporated in place of water, thus provides different type of stationary phase. Eg. Paper treated with silicone or paraffin oil permits reverse phase-paper chromatography, in which mobile phase is a polar solvent.
There are some commercially available papers that contain an adsorbent or an ion-exchange resin, thus permmits adsorption and ion-exhange paper chromatography.
PRINCIPLE OF CHROMATOGRAPHY
This is type of partition chromatography in which the substance are distributed between two liquids that is one is the stationary liquid (usually water) which is held in the fibres of paper and called the stationary phase; the other is the moving liquid or developing solvent and called the mobile phase. The components of mixture to be separated migrates at different rates as its solubility between two phases and appear as spot at different points on the paper.
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 close chamber and the edge of filter is dipped into a solvent called as developing solvent. As soon as filter paper gets liquids via capillary action and reaches to the spot of the test solution then various substances are moved by solvent with various speeds. When solvent move up to suitable height (15-18) the paper is dried and various spot are visualised by suitable reagent called visualising reagent.
MIGRATION PARAMETERS
1) RF VALUE(RETENSION FACTOR) :- It is ratio of the solute’s distance travelled to solvent’s distance travelled.

It is constant for a given substance, provided the conditions of chromatographic system are kept constant with respect to tempreture, type of paper, duration and direction of development, nature and the shape and the size of the wick used (i.e., radial chromatography), the amount of liquid in the reservoir, humidity etc.

The Rf of of a substance depends upon a number of factors which are:
The solvent employed
The medium used for separation i.e., the quality of paper chromatography
The nature of mixture
The tempreture
Size of vessel in which operation has been carried out

It is possible to compare the Rf Values of different substances keeping above factor constant

types of paper chromatogtaphy
ascending, descending, ascending-descending, radial , two diamentional chromatography

1. PAPER CHROMATOGRAPHY REPRESENTED BY :- PARMESHWAR M. PATHADE

2. TABLE OF CONTENTS 05 04 02 01 03 INTRODUCTION TO PAPER CHR0MATOGRAPHY PRINCIPLE OF PAPER CHROMATOGRAPHY MIGRATION PARAMETERS TYPES OF PAPER CHROMATOGRAPHY METHODOLOGY AND APPLICATION

3. INTRODUCTION TO PAPER CHROMATOGRAPHY Cellulose filter paper is often used as the statioary phase in the paper chromatography. Since it is hydrophillic , it is usually covered with thin film of water. The procedure is often regarded as liquid-liquid cromatography Other liquids can be encorporated in place of water, thus provides different type of stationary phase. Eg . Paper treated with silicone or paraffin oil permits reverse phase-paper chromatography, in which mobile phase is a polar solvent. There are some commercially available papers that contain an adsorbent or an ion-exchange resin, thus permmits adsorption and ion- exhange paper chromatography.

4. PRINCIPLE OF CHROMATOGRAPHY This is type of partition chromatography in which the substance are distributed between two liquids that is one is the stationary liquid (usually water) which is held in the fibres of paper and called the stationary phase; the other is the moving liquid or developing solvent and called the mobile phase. The components of mixture to be separated migrates at different rates as its solubility between two phases and appear as spot at different points on the paper. 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 close chamber and the edge of filter is dipped into a solvent called as developing solvent. As soon as filter paper gets liquids via capillary action and reaches to the spot of the test solution then various substances are moved by solvent with various speeds. When solvent move up to suitable height (15-18) the paper is dried and various spot are visualised by suitable reagent called visualising reagent.

5. MIGRATION PARAMETERS 1) R F VALUE(RETENSION FACTOR) :- It is ratio of the solute’s distance travelled to solvent’s distance travelled. It is constant for a given substance, provided the conditions of chromatographic system are kept constant with respect to tempreture , type of paper, duration and direction of development, nature and the shape and the size of the wick used (i.e., radial chromatography), the amount of liquid in the reservoir, humidity etc. The R f of of a substance depends upon a number of factors which are: The solvent employed The medium used for separation i.e., the quality of paper chromatography The nature of mixture The tempreture Size of vessel in which operation has been carried out It is possible to compare the R f Values of different substances keeping above factor constant

6. Distance travelled by the solute from the origine line R f = Distance travelled by the solvent from the origine line

7. 2) R x VALUE :- In some cases, the solvent front runs off the end of filter paper, the movement of substance in such cases is expressed as R x Distance travelled by the substance from the origine line R x VALUE= Distance travelled by the standard substance x from the origine line

8. 3) R M VALUE :- The term R M value is additive and it is composed of the partial R M value of the individual functional groups or other grouping atoms in the molecules. R M VALUE = log (1/ R f -1)

9. TYPES OF PAPER CHROMATOGRAPY 1) Descending Chromatography :- When the development of the paper is done by allowing the solvent to travel down the paper, it is known as descending technique. The advantage of descending chromatography is that the development can be continued indefinitely even though the solvents runs off at the other end of the paper

10. 2) ASCENDING HROMATOGRAPHY :- When the development of the paper is done by allowing the solvent to travel up the, it is known as ascending chromatography. In ascending chromatography, the mobile phase is placed in a suitable container at the bottom of the chamber or in the bottom itelf . The sample are applied a few centimeters from the bottom edge of the paper suspended from a hook

11. ASCENDING CHROMATOGRAPHY 3) Ascending-descending chromatography:- It is hybrid of the above two techniques. In this technique, the upper part of the ascending chromatography can be folded over a glass rod allowing the ascending development to change over into the descending after crossing the glass rod

12. Ascending-Descending chromatography 4) RADIAL PAPER CHROMATOGRAPHY:- This is also known as circular paper chromatography. This makes use of radial development. In this technique a circular filter paper is employed. Then the various material to be analysed are placed at the centre. After drying the spot the paper is fixed horizontally on the petri -dish processing the solvent so that wick of paper dips into the solvent. Cover the paper by means of petri -dish cover. The solvent rises through the wick. When solvent front has moved through a sufficient large distance, the components get separated in the form of concentric circular zones

13. 5) TWO DIAMENTIONAL CHROMATOGRAPHY :- In this, a square or rectangular paper is used. The sample is applied to one of the corners. The second development is performed at right angle to the direction of first run. This type of chromatography can be carried out with identical solvent system in both direction or by two solvent system.

14. This technique can be understood by spotting the sample at the lower corner of a rectangular sheet of filter paper so that the spot is situated above the surface of the developing solvent in the trough. The dried paper is then kept with its edge in the solvent and developed by either ascending or descending technique. When the solvent reaches the opposite edge of the paper, it is removed from the tank and dried. The solvent system is now changed to a second liquid. The filter paper is now rotated through 90° so that the edge having the series of spots is now at the bottom, just above the solvent trough and the chromatogram is run as before. The chromatogram is now having spot of the solute scattered all over the paper.

15. METHODOLOGY 1) CHOICE OF THE PROPER CHROMATOGRAPHIC TECHNIQUE :- The first job is to select the mode of paper chromatographic technique, i.e., ascending, descending, ascending-descending, radial or two-dimensional technique. The choice of technique depends upon the nature of the substances to be separated. 2) CHOICE OF THE FILTER PAPER :- The filter paper plays an important role in the success of paper chromatography. The choice of paper is dependent on the type of problem under investigation. The prime factors, that govern the choice, are as follows: ( i ) Whether the paper is being used for quantitative or qualitative analysis; (ii) Whether it is used for analytical or preparative chromatography; or

16. (iii) Whether the substances used are hydrophilic or lipophilic neutral or charged species. Various types of Whatmann chromatography papers are available Coarser/ faster Whatmann 31 ET Used when R f values are wide apart Slow Whatmann 20 schleicher , Schull 3045 a, Edrol 208, Machery Nagel 261. Used when R f values are close Heavy Schleicher, Schull 2071. Used for preaparative purpose

17. Rate of flow fast Medium slow No.4 No.7 No.2 Thin papers No.54 No.1 No. 20 No. 540 Thick papers No.31 No.3 No.17 No. 3MM Characteristics of whatmann chromatography paper are summarised in below table

18. 3) PROPER DEVELOPING SOLVENT :- The best possible developing solvent is generally selected for the separation of substances under examination. The choice of this depends upon the simple fact that R values should be different for different constituents present in a mixture. With Many combination of solvent systems can be used provided they are not completely miscible one another. The following are usual general criteria for a good solvent system: The R f values of the sample should lie between 0.05 and 0.85 in the system. The difference between the R f values of any two components must be 0.05-the minimum value necessary in order to separate any two components. The distribution ratios of the components in the solvent system should be independent ofconcentration so as to get circular spots The solvents should not undergo chemical reaction with any of the components of the sample mixture. The solvent should not interfere with the detection of the spots on the developed chromatogram. The composition of the solvent system should not alter with time.

19. Solvents in their increasing polarity ; N- hexane→Cyclohexane→carbon tetrachloride→Benzene→Tolune→Trichloroethylene→Diethyl ether→Chloroform→Ethylacetate→n-butanol → n- propanol→Acetone→Ethanol→Methanol→Water . 4) PREPARATION OF SAMPLES :- It is not possible to give any standard procedure for preparation of samples because this problem revolves around several factors of the given samples, especially the presence of other systems like fats, salts, protein etc. However, the sample volume of 10-20 μ having as many μg of the substance is that ideal quantity to be spotted.

20. 5) SPOTTING :- For ascending technique, a strip of Whatmann filter paper of suitable size (25 cm x 7 cm) is generally used. A horizontal line is drawn on the filter paper by a lead pencil. This line is known as origin line. On the origin line, cross marks (x) are made with a pencil in such way that each cross (x) is at least 2 cm away from each other as shown in Fig. 32.10. By the help of a graduated micro pipette. the test solutions are applied on cross (x) marks and the spots are dried cautiously by a stream of hot or cold air.

21. 6) DRYING THE CHROMATOGRAM :- THE WET CHROMATOGRAM AFTER DEVELOPMENT ARE DRIED IN SPECIAL DRYING CABINETS WHICH ARE BEING HEATED ELECTRICALLY WITH TEMPERATURE CONTROLS. 7) VISUALISATION :- visualisation of the spots can be done in two ways: either by chemical means or by using physical methods CHEMICAL DETECTION .:- chemical treatment can develop the colour of colourless spots on the paper the reagents used for visualising the spots are known as chromatogenic reagents or visualisingthe reagents are applied in one of two ways, either by the pressure spraying or by dipping the chromatogram. reagents in organic solvents are more suitable for spraying than aqueous solutions then, the spots are dried. PHYSICAL METHODS.:- Some colourless spots, when held under a UV lamp, fluoresce and rev their existence. When the substance is coloured, the spots can be observed either by reflected or transmitted ligh The former is more selective but the latter more sensitive.(h) Calculation of R, Values. The distance of chromatographed species is noted from its centre to the origin line. The distance of solvent front from the origin line gives the R f values.

22. 8) CALCULATION OF R F VALUES :- The distance of chromatographed species is noted from its centre to the origin line. The distance of solvent front from the origin line gives the R f values.

23. APPLICATIONS Paper chromatography has been applied to the separation of many organic and biochemical products. For example, it has been utilized in the determination of indoles in whole urine and in the study of barbiturates, antibiotics, carbonyl phosphates, hormones, and amino acids, among others. It has also been used in the study of inorganic metal salts and complex ions. The locations of the migrated spots are identified readily if colored . Otherwise, it may be necessary to detect fluorescence which is initiated with an ultraviolet lamp or "develop" colored compounds by spraying the separated spots with a color -forming reagent. For example, ninhydrin is used for producing color with the amino acids. At times it may be desirable to form colored derivatives which are then separated and identified. chromatography has also been employed for the analysis of mixture of sugars.

24. REFERENCES https://microbenotes.com/paper-chromatography/ Chatwal , G.R. and Anand , S.K.J. (2011) Instrumental Methods of Chemical Analysis. Himalaya Publishing House, Mumbai, 2.587-2.598. https://www.differencebetween.com/difference-between-ascending-and-descending-paper-chromatography/ https://pharmainformationzone.blogspot.com/2020/04/descending-chromatography.html https://www.google.com/search?q=radial+chromatography&source=lnms&tbm=isch&sa=X&ved=2ahUKEwjegpH85-_7AhUxsFYBHdc0Aw4Q_AUoAXoECAEQAw&cshid=1670701495402465&biw=1458&bih=606&dpr=0.94#imgrc=8Sncnbi522JPbM https://www.researchgate.net/figure/1113-Ascending-Descending-Paper-Chromatography-d-Radial-or-Circular-Paper_fig18_339662342 https://www.google.com/search?q=two+dimensional+paper+chromatography&source=lnms&tbm=isch&sa=X&ved=2ahUKEwjvzqP16O_7AhXYrVYBHVDXAegQ_AUoAXoECAEQAw&biw=1458&bih=606&dpr=0.94#imgrc=XLpIbNm9wKEMyM