paper chromatography

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Submitted by PRAKASHAIAH.B.G.

What is Chromatography?:

What is Chromatography? Chromatography is a laboratory technique for separating components within mixtures . Mixture: a material composed of two or more elements or parts. Component: a constituent, element, or part

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Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components. Separate Analyze Identify Purify Quantify Components Mixture Illustration of Separating a Mixture Analyze – examine a mixture, its components, and their relations to one another Identify – determine the identity of a mixture or components based on known components Purify – separate components in order to isolate one of interest for further study Quantify – determine the amount of the a mixture and/or the components present in the sample

Uses for Chromatography:

Uses for Chromatography Real-life examples of uses for chromatography: Pharmaceutical Company – determine amount of each chemical found in new product Hospital – detect blood or alcohol levels in a patient’s blood stream Law Enforcement – to compare a sample found at a crime scene to samples from suspects Environmental Agency – determine the level of pollutants in the water supply Manufacturing Plant – to purify a chemical needed to make a product

Definition of Chromatography:

Definition of Chromatography Detailed Definition: Chromatography is a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass. Terminology : Differential – showing a difference, distinctive Affinity – natural attraction or force between things Mobile Medium – gas or liquid that carries the components ( mobile phase ) Stationary Medium – the part of the apparatus that does not move with the sample ( stationary phase )

Definition of Chromatography:

Simplified Definition : Chromatography separates the components of a mixture by their distinctive attraction to the mobile phase and the stationary phase. Explanation : Compound is placed on stationary phase Mobile phase passes through the stationary phase Mobile phase solubilizes the components Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases Definition of Chromatography

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Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase) Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads ( stationary phase ) Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel ( stationary phase) Types of Chromatography

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PAPER CHROMATOGRAPHY Paper chromatography was first introduced by schonbein (1865) under the name capillary analysis But it become popular only after the outstanding work by Gorden , Martin and Synge in 1944. PC is considered to be the simplest and the most widely used chromatography technique PRINCIPLE: The principle involved in separation by paper chromatography is largely by partition coefficient phenomenon. Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and stationary phase.

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Types of paper chromatography PAPER PARTITION CHROMATOGRAPHY Filter paper –act as an inert support Water –stationary phase Organic solvent –mobile phase Mechanism –partition between the two phases Adsorption paper chromatography Impregnated filter paper with alumin or silica stationary phase Alumina or silica –stationary phase Organic solvent –mobile phase Mechanism - absorrption

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Theory Two types of forces operate when a drop of solution is applied on the filter paper and treated with a solvent (a) 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


In paper chromatography the results are represented by Rf value which represent the movement or migration of solute relative to the solvent front. Rf VALUE: The Rf value is calculated as : Distance travelled by the solute Distance travelled by the solvent front Retarding force

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Factors affecting R f 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 P of the solution The concentration of the separated substance

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Techniques of paper chromatography The various operations involved in PC are Choice of filter paper The choice of filter paper depends on the Thickness Flow rate Purity Net strength of paper. Generally Whatman filter papers are used Chemical composition of Whatman paper α - cellulose 99% β-cellulose 0.3-1% Pentosans 0.5-0.8% Ash 0.01-0.07% Ether soluble substance 0.01-0.1%

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PREPARATION OF PAPER: Cut the paper into desired shape and size depending upon work to be carried out . The most common shape of the filter paper is rectangular. Although square paper can also be used on the paper with an ordinary pencil 5cm from the bottom edge.

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Preparation of the solution Pure solution can be applied directly on the filter paper but solids are always dissolved in small quantity of a suitable solvent Concentrated solutions are usually applied on the filter paper to avoid diffusion.

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Application of sample The sample mixture to be separated is applied as a small spot on the origin line. The spot is dried on the Filter paper and is placed in developing chamber. Micropipette or glass capillary is used for sample application.

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A number of solvents can be used in the paper chromatography. The solvent selection depends upon nature of substance to be separated. Some Eg of solvents, Ethyl alcohol n- Butanol N-Hexane water Benzene methanol Toluene chloroform These solvents are used in different ratio with different mixture….


CHROMATOGRAPHIC CHAMBER : The chromatographic chamber are made up of many materials like glass, plastic or stainless steel. Glass tanks are preferred most. They are available in various dimensional size depending upon paper length and development type. The chamber atmosphere should be saturated with solvent vapour .


DEVELOPMENT OF CHROMATOGRAM: 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 by capillary action. It is allowed to run 2/3 rd of paper height for better and efficient result. After development is complete paper is taken out of the chamber carefully .

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DRYING OF CHROMATOGRAM: The chromatogram is dried after its development. They are dried by cold or hot air depending on volatility of solvents. A simple hair dryer is a convenient device to dry chromatograms.

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LOCATION OF SPOT: If the substance are coloured they are visually detected easily. But for colourless substance. Physical and chemical methods are used to detect the spot. Physical method: In this method observation are done under UV light, detection of fluorescence and radioisotope measurements. Chemical method: colourless compound can be detected by converting them into coloured compound by reaction with some reagents. >Amino Acids- Ninhydrin Reagent >Alkaloids- Dragendroff’s Reagent


DEVELOPMENT TECHNIQUE: There are various methods of development of paper chromatography… DESCENDING CROMATOGRAPHY: In this method solvent moves from top to bottom so it is called descending chromatography. ASCENDING CROMATOGRAPHY: In this case the solvent migrates upward by capillary action .

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ASCENDING-DESCENDING CHROMATOGRAPHY: A hybrid of above two technique is called ascending-descending chromatography. Initial ascending chromatography is performed, often crossing the glass rod changes to descending. RADIAL CHROMATOGRAPHY: This is rarely used method, in this case a circular piece of paper is taken which has a wick cut parallel to the radius from the edge to centre . The sample is applied at the centre of the paper. The paper is then laid on the edge of circular disk with wick dipped into the solvent at the bottom of the dish.

Two dimensional paper chromatography :

Two dimensional paper chromatography one dimensional chromatography is quite satisfactory for separation for separation of many mixtures But for complex mixtures, two dimensional developments (i.e. the development of the chromatography in the other direction at right angle to the first, using another solvent) can be carried out. First solvent flow D B C A D C B A ABCD Second solvent front


advantages Simplicity and availability of materials and equipment High efficiency of separation The characterization of substances in mixtures without prior separation of individual components Good separation of homologues Its applicability to structural analysis The study the reaction kinetics

Quantitative estimation:

Quantitative estimation There are several methods for quantitative determination of a particular compound in a mixture. The choice of method depends upon properties, composition of the compound and degree of accuracy required. The methods can be divided into two types Direct measurement methods Comparison of Visible Spots Photodensitometry Fluorimetry Radiotracer method Polarographic and conductometric methods

2. Elution method:

2. E lution method S M S S M S S M S X X X X X X X X X Standards Origin line Spot containing known volume of mixture Paper before development Area to be eluted Paper After development

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Experiment1-to separate cations (Pd 2+, Ag + , Hg 2+ ) Solvent: Distilled water or ethyl alcohol Locating agent:0.25 M potassium chromate solution Colours : Pd-Yellow,0.08, Ag-Orange red,0.16 Hg-Orange,0.85 Experiment2- To separate mixture of amino acids,arginine glutamic acid, Lycine and aspartic acid Solvent: Ethanol water ammonia in the ratio of 20:2.5:2.5 Locating agent:300mg ninhydrin dissolved in 100ml acetone Colour: Blue spots compare the R f value with the literature value Experiment 3- To separate the cations (Hg 2+ .CU 2+ .Cd 2+ .Bi 3+ ) Solvent: n- butanol alcohol and HCl Locating agent: Conc solution of dithizone in chloroform Colour: Cu-brown. Hg-pink. Cd-purple. Bi-brown

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