INTRODUCTION:

Chromatography:

Chromatography is a non-destructive procedure for resolving a multi-component mixture of trace, minor, or major constituents into its individual fractions. Chromatography was first invented by M. Tswett; in 1906 he carried separation of chlorophyll, xanthophyll and several other colored substances by percolating vegetable extracts through a column of calcium carbonate.

The calcium carbonate column acted as an adsorbent and the different substances got adsorbed to different extent and this gives rise to colored bands at different positions. Tswett termed this system of colored bands as the chromatogram and the method as chromatography after the Greek words chroma and graphs meaning “colour” and “writing” respectively.

The column of calcium carbonate used in Tswett’s method remains stationary and is therefore termed as the stationary phase and the solution of vegetable extracts moves or flows down the column and is therefore termed as the mobile phase. Chromatography may be regarded as a method of separation in which separation of solutes occur between a stationary phase and a mobilephase¹.

Mechanism of Chromatography:

Chromatography may be defined as a method of separating a mixture of components into individual components through equilibrium distribution between two phases.

The technique of chromatography is based on the differences in the rate at which the components of a mixture move through a porous medium (called stationary phase) under the influence of some solvent or gas (called moving phase).

The chromatography method of separation involves the following steps:

1. Adsorption or retention of a substance or separation

2. Separation of the adsorbed substances by the mobilephase.

3. Recovery of the separated substances by a continuous flow of the mobile phase, the method being calledelution.

4. Qualitative and quantities analysis of the elutedsubstances².

Types of chromatography:

In chromatography, the stationary phase may be a solid or a liquid and the mobile phase may be liquid or a gas. Depending on the stationary and the mobile phase used, separation occurs because of a combination of two or more factors such as rates of migration, capillary action, extent of adsorption etc chromatographic methods can be classified on the basis of the stationary and the mobile phaseused.

Paperchromatography:

It is defined as a techniques in which the analysis of an unknown substance is carried out mainly by the flow of solvent on specially designed fitter paper one of the two solvents is immiscible or partially miscible with other solvent. The separation is effected by differential migration of the mixture of substances, which takes place due to difference in partition coefficients.

Principle:

This technique is a type of partition Chromatography in which the substance are distributed between two liquids i.e. one is stationary liquid which is held in the fibers of the paper and called stationary phase and other is the moving liquid or developing solvent and called the moving phase. The components of the mixture to be separated migrate at different rates and appear as sports at different point on the paper.

In this technique a drop of the test solution is applied as a small spot on a filter paper and the post is dried. The paper is kept in close chamber and the edge of paper is dipped into a solvent called developing solvent. As the filter paper gets the liquid through its capillary axis and when it reaches the spot of the test solution, the various substances are moved by solvent system at various appends. When reached or travelled to a suitable length the paper is dried and spot are visualized by suitable reagents called visualising reagents. The movement of substance relative to the solvent is expressed in terms of RF value³.

Types of Paper Chromatography:

1. Descending Chromatography:

When the development of the paper is done by allowing the solvent to travel down the paper it is known descending technique. The advantage of descending technique is that the development can be continued indefinitely even though the solvent runs off at the other end ofpaper.

Fig 1: Descending Chromatography

2. Ascending Chromatography:

When the development of the paper is done by allowing the solvent to travel up the paper it is known ascending technique.

Fig. 2: Ascending Chromatography

3. Ascending - DescendingChromatography:

It is a hybrid of two techniques. The upper part of ascending chromatography can be folded over a glass rod allowing the descending development to change over into the descending after crossing the glass rod.

Fig. 3: Ascending - Descending Chromatography

4. Two dimensionalchromatography:

In this a square rectangular paper is used. The sample applied on one of the corners. The second development is performed at right angle of the direction of first run. This type of chromatography can be carried out with identical solvent system in both directions or by two solvent systems.

Fig. 4: Two dimensional chromatography

5. Radial Paper Chromatography:

It is also known as circular paper chromatography. In this technique a circular filter paper is employed, various materials to be analyzed are placed in a Centre. After drying the spot the filter paper is fixed horizontally on pet dish possessing solvent, so that tongue of paper dips in solvent when solvent front has moved through a sufficient large distance the components gets separated. Forming concentric circular spots^4,
5.

Fig. 5: Radial Paper Chromatography

Experimental Details:

1. Choice of the proper chromatographictechnique:

The choice of technique is depends upon the nature of substances to be separated.

2. Choice of filterpaper:

The selection of chromatographic depends on following factors:

1. Whether the paper is being used for quantitative or qualitativeanalysis.

2. Whether it is used for analytical or preparativechromatography.

3. Whether substance used are hydrophilic or lipophilic neutral or charged species⁶.

3. Proper developingsolvent:

The choice of this depends upon the simple fact that R_F values should be different for different constituents present in mixture. A solvent or mixture of solvent, which gives a RF 0.2 – 0.8 for sample, should be selected. The solvent are listed in order of increasing polarity:⁷

Table 2: solvent listed in order to increasing polarity

Solvent	T20	T26
n-hexane	1.89	-
Cyclohexane	2.02	-
Carbon tetrachloride	2.24	-
Benzene	2.29	-
Toluene	2.44	-
Trichroroethylene	3.40	-
Diethyl ether	4.43	-
Chloroform	4.91	-
n-propanol	-	20.10
Acetone	-	20.10

4. Preparation ofsamples:

It is not possible to give any standard procedure for preparation of samples because this problem resolves around several factors of given samples. How ever sample volume of 10-220 having as many as Ng of substance is the ideal quantity to be spotted.

5. Spotting:

A horizontal line is drawn on the filter paper by a pencil. This is origin line (For ascending chromatography). The test solution are applied above the line and dried cautiously by a stream OI hot or cold air.

6. Drying the chromatograms:

The wet chromatograms after development are dried in drying cabinets, which are being heated electrically with temperature controls.⁸

7. Visualisation:

It can be done by two ways:

1. Chemical Method

2. Physical Method

1. Chemical Detection:

Chemical treatment can develop the colour of colourless solvents on paper. The reagents used for visualising spots are Chromographic reagents. The regents applied by dipping or spraying their aqueous solution.

2. Physical Detection:

The physical method involves use of UV lamp for visualization.⁹

Applications:

1. Paper chromatography is widely used for qualitative analysis of inorganic, organic and biochemical interests.

2. It is also useful is analysis of mixture of amino acid and mixture ofsugars.

3. It also offers a rapid method of separating and estimating sugars quantitatively; how ever the identification depends upon determination of their physical constants and formation of characteristic derivatives

4. Paper chromatography is also used to determine rate of ongoing reaction. Therefore, is a valuable tool in synthetic chemistry

5. Paper chromatographic technique is also used for carrying out assay of pharmaceutical compounds such as mixture of phenylephrine hydrochloride, chlorpheniramine hydrochloride and dextromethorphan hydrochloride.

6. The technique is also useful in isolation of pair of components having sample RF values using two-dimensional paper chromatography.

7. Paper chromatography also involves inorganic applications such as separation of cations like cadmium, zinc, mercury, beryllium and calcium.

8. It technique is also helpful in identification of accelerator and anti-oxidant in rubber and is useful for determining its quality.

9. Paper chromatography is widely used in detection of various plant constituents such as opium, quinine alkaloids.

10. Paper chromatography is also used to determine rate of ongoing reaction. Therefore, is a valuable tool in synthetic chemistry.¹⁰

REFERENCES:

1. Frank Settle.Handbook of Instrumental Techniques for Analytical Chemistry. Pearson Publication,2014; First Edition : 221

2. Gurudeep Chatwal, Sham Ananad, Himalaya Publiashing House, 2004; Third Edition: 520-30

3. E. Morgan and I. Wilson. An early description of paper chromatography. Chromatographia. 2004,60(1): 135-136

4. S. K. Pramod, and K. A. Navnath, A brief review on ultra-Performance Liquid Chromatography. 2017,4(2): 240-242

5. H. G. Cassidy. Investigation of paper chromatography. Analytical Chemistry, 24(1): 20-27.

6. P. W. Smit, I. Elliott, R. W. Peeling.An overview of the clinical use of filter paper in the diagnosis of tropical diseases. The American Journal of Tropical Medicine and Hygiene. 2014, 90(2): 195-210.

7. F. Kurzer. An early application of paper chromatography. Journal of Chemical Education, 1978 55(5): 321.

8. M. G. Rasul, Extraction, Isolation and Characterization of Natural Products from Medicinal Plants. International Journal of Basic Sciences and Applied Computing. 2011, 2(6): 1-6.

9. Ganesh Shinde, Godage R. K, R. S. Jadhav, Barhate Manoj, Bhagwat Aniket. A Review on Advances in UV Spectroscopy. Research J. Science and Tech. 2020; 12(1): 47-51. doi: 10.5958/ 2349-2988.2020.00005.4.

10. V. Jirgl, Paper chromatography of free amino acids in blood serum. Clinical chemistry.1957 3(1):154-155.

Received on 01.09.2020 Revised on 23.09.2020

Asian J. Pharm. Ana. 2021; 11(1):45-48.

DOI: 10.5958/2231-5675.2021.00009.0

Stationary phase	Mobile phase	Chromatography Technique
		Plane chromatography Paper chromatography (PC)
Solid	Liquid	Thin layer chromatography (TLC) Adsorption column chromatography High performance liquid chromatography (HPLC)
		Ion exchange chromatography (IEC) Gas-solid chromatography (GSC) Gel
		Permecation chromatography (Exclusion chromatography) (GPC) Gas-liquid
Solid (ion exchange resin) Solid	Liquid Gas Liquid
		chromatography (GLC)
Solid matrix	Gas Liquid	Liquid –liquid chromatography (LLC)
Liquid Liquid