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BACKGROUND Paper chromatography was first introduced in 1865 by Schonbein under the name capillary analysis . It become popular after the outstanding work by Gorden, Martin and Syng e in 1944. Paper chromatography is considered to be the simplest and most widely used of the chromatographic techniques because of its applicability to isolation, identification and quantitative determination of organic and inorganic compounds.


DEFINITION It is one type of partition chromatography in which substances are distributed between two liquids, i.e., the stationary liquid (usually water) which is held in the fibres of the paper and called stationary phase; the other is the moving liquid or developing solvent and called the mobile phase.


PRINCIPLE OF SEPARATION PARTITION The principle involved is partition chromatography where in the substances are distributed or partitioned between to liquid phases. One phase is the water which is held in pores of filter paper used and other phase is that of mobile phase which moves over the paper. Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and stationary phase.


PRINCIPLE OF SEPARATION ADSORPTION The principle can also be adsorption chromatography between solid and liquid phases, where in the stationary phase is the solid surface of paper and the liquid phase is of mobile phase. Paper impregnated with silica or alumina acts as adsorbent (stationary phase) and solvent as mobile phase. Most of the applications of paper chromatography work on the principle of partition chromatography.


THEORY Two types of forces operate when a drop of solution is applied on the filter paper and treated with a solvent ; Propelling force Retarding force

Propelling force:

Propelling force It tries to drag the substances in the direction of the flow of solvent. This depends upon; The rate of the solvent flow The solubility of the substance in the solvent The component with higher solubility will move rapidly along the filter paper than the less soluble component

Retarding force:

Retarding force In paper chromatography the results are represented by Rf value which represents the movement or migration of solute relative to the solvent front. Rf VALUE:

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FACTORS AFFECTING Rf VALUES: The temperature The solvent used Quality of the paper Techniques employed The distance travelled by the solvent and the solute Chemical reactions between the substance to be separated and the solution The concentration of the separated substance


TYPES Based on the way, the development of chromatogram on the paper is done in the procedures, there are 5 types of chromatography.

Descending chromatography:

Descending chromatography When the development of paper is done by allowing the solvent to travel down the paper, it is known as descending chromatography. APPARATUS: Well-sealed glass tank of suitable size and shape Trough for mobile phase in upper portion Paper with upper end in the trough Glass jar equilibrated with mobile phase prior to elution

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ADVANTAGES: Separation is quick and easy due to gravity and capillary action. Descending technique is preferred if Rf values of various constituents are almost same. Development can be continued indefinitely even through the solvent runs off at the other end of paper. DISADVANTAGES: The descending technique is a complex set up .

Ascending chromatography:

Ascending chromatography When the development of paper is done by allowing the solvent to travel up the paper, it is known as ascending chromatography. APPARATUS: Well-sealed glass jar containing mobile phase at the bottom of chamber Hook to suspend paper in jar. Paper may also be rolled in jar, held together by staples, stings or plastic clips. Paper

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ADVANTAGES: Apparatus is simple and easy to construct DISADVANTAGES: Slow development compared to descending technique.

Ascending-Descending chromatography:

Ascending-Descending chromatography It is hybrid technique. Upper part of ascending chromatography is folded over a glass rod allowing the ascending development to change over into descending after crossing the glass rod. Solvent first travels upwards and then down wards on the paper.

Radial paper chromatography:

Radial paper chromatography This is also called circular paper chromatography. This makes use of radial development. APPARATUS: Circular filter paper Wick 2 Petri dishes

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PROCEDURE: Spot is applied on filter paper using capillary. After the spot is dried, paper is fixed horizontally on the petridish containing mobile phase. The wick at the center of paper dips into mobile phase in a petridish and solvent rises through this wick. The solvent travels from center towards periphery of circular chromatography paper. The entire system is kept in a covered petridish for development of chromatogram. Components get separated in the form of concentric circular zones.

Two dimensional chromatography:

Two dimensional chromatography In this technique, chromatogram development occurs in two directions at right angles. APPARATUS: Rectangular/square filter paper Well-sealed glass Jar 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 mobile phase at right angle to previous development for second chromatogram.

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ADVANTAGES: This type of chromatography can be carried out with identical solvent systems in both directions or by two solvent systems.


PAPER FOR CHROMATOGRAPHY A wide variety of papers are available commercially in different sizes, shapes, porosities, thickness and chemical treatments. COMPOSITION: Paper is composed of randomly directed cellulose fibers. Cellulose itself is a network of polymeric carbohydrate chains possessing hydrophilic character and cross linked by stable hydrogen bonded system.

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TYPES: Glass fiber type papers Modified cellulose papers Whatman papers GLASS FIBER TYPE PAPERS If very corrosive eluting conditions are required, a glass fiber type paper can be used. By special treatments, adsorption effects due to glass can be minimized.

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MODIFIED CELLULOSE PAPERS Cellulose paper can be modified in several ways to alter its behavior. Several types of modified cellulose paper and their characteristics are as followed; Types Characteristics Uses Carboxyl papers Exchange capacity of paper is increased by increasing carboxyl content by partial oxidation For efficient separation of polar substances. Cationic separation of potential amines and amino acids Acetylene papers Such paper has hydrophobic property Reverse phase chromatography of lipophilic substances like steroids, insecticides, pigments and metal cations

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WHATMAN PAPERS Whatman papers commonly used for chromatography purpose have a content of 99% α-cellulose. There are various types of Whatman papers available. The choice of Whatman chromatography paper depends on the type of separation . Types Characteristics Uses Kieselguhr papers, Alumina papers, Zirconia papers, Silica papers Adsorbent action Separation of low polarity substances like amines, fatty acids, steroids, triglycerides, vitamins and pesticides Ion exchange papers - Ion exchange paper chromatography

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Slow papers are rarely used and they facilitate better resolution of substances with close Rf values. Some examples are; Whatman 20 Schleicher and Schull 2045 a Machrey Nagel 261 Edrol 208 Faster papers are used for substances which have wide-apart Rf values. Example is Whatman 31 ET. Whatman 3MM is generally used for preparative purposes . Type Rate of flow Fast Medium Slow Thin paper No. 4 No. 7 No. 2 No. 54 No. 1 No. 20 No. 540 Thick paper No. 31 No. 3 No. 17 No. 3MM



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Phase Solvent Uses Aqueous stationary phase Water Separation of moderately polar to very polar (ionic) mixture Hydrophilic stationary phase Methanol, Formamide, Glycols, Cellosolves and Glycerol Hydrophobic stationary phase Dimethylformamide, Kerosene, Aromatic and aliphatic hydrocarbons, oxygenated solvents


MOBILE PHASE Numerous combinations are possible for the mobile phase. Mixtures of two, three or more solvents, solutions of salts and solutions of buffers can be used. SOLVENT USED IN CHROMATOGRAPHY LISTED IN ORDER OF INCREASING POLARITY n-hexane < cyclohexane < carbon tetrachloride < Benzene < Toluene < Trichloroethylene < Diethyl ether < Chloroform < Ethyl acetate < n-butanol < n-propanol < Acetone < Ethanol < Methanol < Water

General criteria for good solvent system:

General criteria for good solvent system Rf values of sample should lie between 0.05 and 0.85 in the system . The difference between the Rf 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 of concentration so as to get circular spots. The solvents should not undergo chemical reaction with any of the components of the sample mixture .

General criteria for good solvent system:

General criteria for good solvent system The solvent should not interfere with detection of spots on the developed chromatogram. The composition of solvent system should not alter with time.

Suitable solvents for paper chromatography:

Suitable solvents for paper chromatography If a pure solvent is not satisfactory, solubility of suitable polarity may be obtained by trying out mixtures in various proportions of solvents . Stationary phase Mobile phase Water Isopropanol-Ammonia-Water (9:2:1) Water n-butanol-Acetic acid-Water (4:1:5) Water Phenol saturated with water Formamide Chloroform Formamide Benzene Formamide Benzene-Cyclohexane Dimethyl Formamide Cyclohexane Phenoxy ethanol Heptane Kerosene 70% isopropanol Liquid paraffin Dimethyl Formamide-Methanol-Water (10:10:1)


EXPERIMENTAL DETAILS FOR QUALITATIVE ANALYSIS Two important things are to be considered; Choice of paper chromatographic technique Choice of filter paper

Choice of paper chromatographic technique:

Choice of paper chromatographic technique Choice of technique depends upon the nature of the substances to be separated. Techniques may be; Ascending chromatography Descending chromatography Ascending-descending chromatography Radial paper chromatography Two dimensional chromatography

Choice of filter paper:

Choice of filter paper Choice of paper depends on type of problem under investigation. Prime factors, that govern the choice, are; Whether the paper is being used for qualitative or quantitative purpose Whether it is used for analytical or preparative chromatography Whether the substances used are hydrophilic or lipophilic, neutral or charged species


PRACTICAL REQUIREMENTS Preparation of paper Preparation of sample solution Application of sample preparation of solvent Preparation of environment of glass jar Development Drying Detection Physical methods Chemical methods

Preparation of paper:

Preparation of paper Cut the paper into desired shape and size depending upon work to be carried out . The starting line is marked on the paper with an ordinary pencil 5cm from the bottom edge. On the starting line marks are made 2cm apart from each other.

Preparation of sample solution:

Preparation of sample solution Choice of suitable solvent for making solution is very important. Pure solutions can be applied directly on the paper but solids are always dissolved in small quantity of a suitable solvent. Biological tissues are treated with suitable solvents and their extracts obtained. Proteins can be precipitated with alcohol and salts can be removed by treatment with ion exchange resin.

Application of sample:

Application of sample The sample to be applied is dissolved in the mobile phase and applied as a small spot on the origin line, using capillary tube or micropipette. Very low concentration is used to avoid larger zone. The spot is dried on the filter paper and is placed in developing chamber.

Preparation of solvent:

Preparation of solvent The commonly employed solvents are the polar solvents, but the choice depends on the nature of the substance to be separated . If pure solvents do not give satisfactory separation, a mixture of solvents of suitable polarity may be applied.

Preparation of environment of glass jar:

Preparation of environment of glass jar The chromatographic jars may be made up of many materials like glass, plastic or stainless steel. Glass tanks are preferred most. They are available in various dimensional sizes depending upon paper length and development type. Chamber is filled with solvent and lid is closed. The chamber atmosphere is allowed to saturate with solvent vapor.

Development :

Development Several types of development are possible which increases the ease of operation. The paper is dipped in solvent in such a manner that the spots will not dip completely into the solvent. The solvent will rise up and it is allowed to run 2/3 rd. of paper height for better and efficient result. Various development techniques are ; Ascending chromatography Descending chromatography Ascending-descending chromatography Radial paper chromatography Two dimensional chromatography

Drying of chromatogram:

Drying of chromatogram After the solvent has moved a certain distance for certain time, the chromatogram is taken out from the glass jar. Position of the solvent front is marked with a pencil. Paper is dried by cold or hot air depending on volatility of solvents. A simple hair dryer is a convenient device to dry chromatograms.

Detection :

Detection If the substances are colored they are visually detected easily. But for colorless substance, physical and chemical methods are used to detect the spot. NONSPECIFIC METHODS (PHYSICAL METHODS) Iodine chamber method UV chamber for fluorescent compounds – at 254 or at 365nm

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SPECIFIC METHODS (CHEMICAL METHODS) SPRAYING METHODS Spraying agents used are; Ferric chloride Ninhydrin in acetone Dragendroff’s reagents 3,5 dinitro benzoic acid These methods are used for identification of; Phenolic compounds and tannins Amino acids Alkaloids Cardiac glycosides



Direct measurement methods:

Direct measurement methods COMPARISON OF VISIBLE SPOTS A rough quantitative measurement Component in a mixture can be carried out by comparing the intensity and size of the spot with a standard substance. PHOTODENSITOMETRY This method is used for chromatograms of colored compound. This instrument measures quantitatively the density of the spots.

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FLUORIMETRY The compound to be determined by fluorimetry must be fluorescent or convertible into fluorescent derivatives. RADIOTRACER METHOD The compound containing radioactive element is labeled and treated with locating reagent i.e. Geiger Muller counter. POLAROGRAPHIC & CONDUCTOMETRIC METHODS These are used to measure the amount of material in the spot .

Indirect measurement methods:

Indirect measurement methods In this technique, the spots are cut into portions and eluted with solvents. This solution can be analyzed by any techniques of analysis like spectrophotometry, electrochemical methods etc. Rf Rx

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Rf It refers to the movement or migration of solute relative to the solvent front. Rf value is always closer to 1.

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Rx In many cases it has been observed that the solvent front runs off the end of the paper. Rx value is thus used. Rx value is always closer to 1.


SOURCES OF ERROR ERROR DURING APPLICATION OF THE SPOTS Apply minimum volume of the concentrated solution in order to avoid diffusion through the paper which leads to poor separation. Spots should be approximately of the same diameter. DEVELOPMENT Improper adjustment of the paper in the tank leads to this error so the paper should be held vertically. Do chamber saturation


SOURCES OF ERROR DETECTION The spraying methods may affect the final result


APPLICATIONS Separation of mixtures of drugs Separation of carbohydrates, vitamins, antibiotics, proteins, etc. Identification of drugs Identification of impurities Analysis of metabolites of drugs in blood, urine etc.


ADVANTAGES Simple Rapid Inexpensive Excellent resolving power


LIMITATIONS Establishing the vapor solvent equilibrium Stability of solvent mixture is first ensured

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