Chromatography

Contents Introduction, principle, Types and Uses  Paper chromatography  Thin layer chromatography  Column chromatography  Ion exchange chromatography  Affinity chromatography  Gel filtration chromatography  High performance liquid chromatography Definition: The term chromatography is derived from greek word; khromatos- colour, graph- writing. The chromatography technique is first discovered by Russian Biologists, Dr Michael Tswett in 1906 for the separation of coloured plant pigment on a column of alumina. Nowadays various types of chromatography are used to separate almost any given mixture whether coloured or colourless into its component. Chromatography may be regarded as an analytical technique employed for the purification and separation of organic and inorganic substances. It is also found useful for the fractionation of complex mixture, separation of closely related compounds, such as isomers and in the isolation of unstable substances General principle of Chromatography The basis of all forms of chromatography is the distribution or partition coefficient (Kd), which describes the way in which a compound (the analyte) distributes between two immiscible phases. For two such immiscible phases (A and B) the value for this coefficient is constant at a given temperature and is given by the expression Kd = concentration in phase A/ concentration in phase B Distribution co-efficient can be defined as the total amount of substance present in one phase divided by total amount of substance present in other phase.

Chromatography techniques separate molecules on the basis of differences of size and shape, mass, charge solubility and adsorption properties. The process invariably involves the interaction between three components. All chromatographic system consist of  mixture to be separated  stationary phase -solid/ liquid, gel, mixture of solid/liquid  mobile phase- liquid/gas During the chromatographic steps the analytes continuously pass back and forth between the two phases so that differences in their distribution coefficient results in their separation. Classification of chromatography  Gas chromatography  Liquid chromatography o Adsorption chromatography o Partition chromatography o Exclusion chromatography o Ion exchange chromatography

Based on the nature of the stationary phase and mobile phase Chromatography Adsorption chromatography Partition chromatography Gas chromatography Modes of chromatography y y Column chromatography Thin layer or planar chromatography stationary phase solid liquid solid or liquid Mobile phase liquid or gas liquid or gas mixture of gas

In column chromatography stationary phase is packed into a glass or metal column. The mixtures of analytes is then applied and the mobile phase commonly referred to as the eluent passed through the column either by gravity feed or by use of pumping system or applied gas pressure.

Paper chromatography Paper chromatography in which stationary liquid phase is supported by cellulose fibre of a paper sheet. The mobile phase passes along the paper sheet either by gravity feed or capillary action. It is an example of partition chromatography. It was first introduced by Schonbein 1961 but the technique became popular in 1963 through the work of K. Condensen, A. H Gondon, A.J. Martin and L. M. Synge. Principle This technique is a type of partition chromatography in which substances are distributed between two liquid phases i.e. one is the stationary liquid usually water which is held in the fibres eg: paper and called the stationary phase. The other moving liquid developing solvent and is known as mobile phase. Organic solvents are generally mobile phase. In paper chromatography separation of mixture of substances is mainly done by the flow of solvents on whatmann filter paper. The stationary phase is present in the cellulose of the filter paper and the mobile phase is an organic solvent. Mobile phases rises by the capillary action and by adsorption in the filter paper, separation is achieved by the differential migration of the different components which occurs due to the differences in partition coefficient. Procedure In this technique a drop of test solution is applied as a small spot on the filter paper and the spot is dried. The paper is kept in a closed chamber containing the developing solvent and the edge of filter paper is dipped into the solvent. Then the solvent is drawn up by the capillary action and carries the mixture of substances. The component of the mixtures be separated migrates at different rates and appear as spots at different points of the paper and the substances are separated according to their relative solubility in water in the organic solvent.

The component having higher solubility will move more rapidly along the strip of paper than less soluble components. RF value The movement of the substances relative to the solvent is expressed in terms of RF value. RF i.e. migration parameter - relative fraction is defined as the ratio of the distance travelled by the substance and the distance travelled by the solvent front. RF = Distance travelled by the substance (A cm)/Distance travelled by the substance(B cm) Identification of the given compound may be met on the basis of its distance moved during development relative to distance moved by the solvent front i.e. RF. For each compound, value of RF is specified and RF value is always less than 1. Thus an unknown compound is identified by comparing its RF value with the standard value. Steps involved in Paper Chromatography 1. Choice of filter paper The filter paper plays an important role in the success of paper chromatography and various types of whatmann filter paper are available. Whatmann filter paper has the chemical composition as content of 98.99% of -cellulose and rest is the minimal content. -cellulose - 0.32-1.0% pentosans - 0.4-0.8% ether soluble lactose - 0.015-0.02% Ash - 0.07% In general Paper is composed of randomly directed cellulose fibres. Cellulose itself is network of long chains of carbohydrates having molecular weight more than 10,000 daltons and possessing hydrophobic character and cross linked by a stable H-bond system. Water and other polar solvent is slightly held within the hydrophilic cellulose system. Whatmann filter paper no. 1 is mostly used for the high flow rate.

2. Choice of solvent Paper chromatography is essentially of partition chromatography and there are a wide variety of useful combinations of stationary and mobile phase.

Stationary phase: Stationary phase that can be used can be classified as  Aqueous stationary phase - water  Hydrophilic stationary phase - Ammonia, glycerol, Phenols, methanol  Hydrophobic stationary phase - Kerosene, Aromatic and Aliphatic hydrocarbons. Mobile phase: Mixture of two or more solvents is used. Generally a solvent or solvent mixture which gives the RF value 0.2 to 0.8 for the sample to be selected. Eg:  Isopropanol - ammonia - water  n- butanol - acetic acid - water  water - phenol  Formamide - chloroform  Formamide - chloroform - benzene  Formamide - benzene - cyclohexane 3. Saturation of tank (solvent tank): The inner wall of tank is rapped by filter paper before running the chromatography. 4. Preparation of sample Sample that can be used in paper chromatography can be of many types i.e. Biological material. eg. Serum, extraction from plant and animal tissues, fermented broth, cultures food and food products vitamins and antibiotics. If the sample is in solid form, it may be dissolved in volatile solvent before loading the sample. Sample should be in concentrated form. Amino acids - Isopropanol Lipids - Chloroform Plants and animal tissues - 70% ethanol 5. Sample loading or sample application: A horizontal line is drawn on filter paper by a pencil. This line is known as original line. On the origin line, cross mark are made with the pencil in such a way that each cross is at least 2 cm away from each other. The sample can be loaded by micropipette platinium loop, pasteur pipette or by microcap pipette or by micro syringe. Platinum loop can be used only for quantitative purpose and also used for most practical purposes because it can be used again and again by carefully washing

and heating strongly in a flame after each application. If a loop is 0.04 mm diameter platinium wire and cross section diameter is 1.5 mm a spot of about 10l is obtained. Microcap is used for single use. They are filled by capillary action and bulb is used to expel the sample. Sample volume of 10 l-20 l is ideal quantity to be spotted. With the help of micropipette, the sample is applied in each cross marks as spot and spots are dried continuously by stream of hot air. The spots should be 0.5 cm to 1.5 cm in diameter.

6. Development of chromatogram The loaded filter paper is dipped carefully in the solvent in such a way that it should not be dipped more than 1 cm and left undisturbed for at least 2 hours until solvent front reaches upto 15-18 cm height from the origin line. Chromatograms can be developed by different methods:  Ascending technique  Descending technique  Radial technique Ascending technique The movement of mobile phase is in upward direction. In ascending chromatogram, the mobile phase is placed in suitable container at the bottom of chamber. It consist in dipping lower end of paper contains first into the solvent so above the solvent depth then allowing to rise up to the paper by capillary action. The mobile phase moves in the upward direction against the gravitational force. Descending technique In descending technique the flow of mobile phase is in downward direction. In this technique, the solvent is kept in the trough at the top of the chamber and then allow to flow down the paper. The movement of the solvent is caused by capillary action as well as pull of gravitational force. The rate of flow of mobile phase is more rapid than ascending technique. So that chromatogram is developed in shorter time. Radial technique

This type of development is used in rare condition. In this technique a circular piece of paper is taken and a wick of about 2 cm is cut parallel to the radius from the edge to the center. The sample is deposited at the center of the paper and the upper end of the wick. The paper is then placed on the edge of circular disk with the wick dipping into the solvent at the bottom of disk. As a result, liquid ascends to wick and flowed radially through the paper. 7. Drying the chromatogram The wet chromatograms are drying on special dry cabinet which are being heated electrically with temperature control. chromatogen can be dried at 110rC for 5-10 minutes in hot air oven. 8. Visualization of spot or location of compound The colour compounds are easily located on the paper but the compounds of biological origin are usually colorless. Hence cannot be located on the chromatogen simply by visual inspections. The compound on chromatogram located either by physical method or by chemical method.

Physical method Some colorless spot when held under the UV lamp fluoresce and reveal their existence. If the compounds are invisible in ordinary light, UV lamp is to be used to locate the position of spot. Although several compound are relatively stable to UV light yet some compound like steroid, vitamins are destroyed when visualization technique is employed for identification. Radioactive method The location of substances may also be carried out by making use of radioactive substances where radioactivity can be measured by various counter devices.

Chemical methods Chemical reagents are used for the visualization of spots such reagents are chromogenic reagents. Chemical reagents used for locating spots can be gas, liquid or solid. Chemical gas H2S, methyl, ethyl or butyl alcohols are used. Potassium dichromate, Ninhydrine, aniline dyes are also used as visualizing reagents.

Spraying The chemicals are sprayed on chromatogram uniformly by using glass automizer which is held in the position normally at a distance of one feet from the chromatogram and moved slowly from top to bottom in left to right direction. Dipping A solvent is first taken in which substances are insoluble and then dipping is carried out in a trough volatile solvents ethyl and methyl alcohols are most suitable because they can be easily evaporated from chromatogram.

9. Quantitative estimation The amount of material in each spot can be measured either by direct method or elution method. Direct method Comparision of visual spot A rough quantitative measurement of a compound in a mixture can be carried out by comparing the intensity and size of spot with standard substance. For this purpose accurately measured volumes of the mixture and known amount of component being investigated are applied separately on the origin line. The spots dried, developed and treated with the reagent which will react the component to produce the colour. The size and intensity of the color spot produced by unknown component is then compared with that known standard substance by visual inspection and under UV lamp. Photosensitometry In this method, a strip of paper containing the spot is cut and placed between two glass slides then intensity of each spot is measured with a photo electric device known as photosensitometer. Elution method The spots from the developed chromatograms are eluted. The spots are cut off from the paper and then dissolve in suitable solvent in test tube. The elutes obtained from the chromatogram may be estimated quantitatively by any physical or chemical methods. The elutes obtained from the chromatogram may be estimated further by spectrophotometric method or colourimetric method.

Application of paper chromatography  Paper chromatography can be used for the quantitative analysis of inorganic, organic and biochemical interest.  This chromatography can be used for the identification of compounds in drugs, in biochemical preparation and in natural products. It can be used for checking the purity of samples or compounds.  It is ideally suited for rapid analysis of reaction mixture.  It has been widely used for the analysis of mixture of amino acids.  It can be used to detect traces of pollutants in water food or in soil  It can be used for the separation of inorganic ions.

Thin layer chromatography

The technique of thin layer chromatography was first introduced by Izmylov and Shrabier in 1938. TLC as a procedure for analytical absorption of chromatography was first introduced by Stahl in 1958 and he was mainly responsible for bringing out standard equipment for preparing thin layers. Principle A thin layer of the stationary phase is formed on a suitable flat surface such as glass or plastic plates. Since the layer is so thin, the movement of the mobile phase across the layer occurs generally by simple capillary action and is a rapid process. As the mobile phase move across the layer from one edge to the opposite, it transfer analyte movement stops either when mobile phase reaches the end of the layer and capillary action flow stops when the plate is removed from the mobile phase. The movement of the analyte is expressed by its retardation factor (Rf). Types of Thin layer chromatography There are mainly four types of TLC Partition TLC

In partition TLC, the mobile phase as well as the stationary phase are both the liquids, solid phase is liquid which is held on suitable solid such as cellulose. Adsorption TLC In case of Adsorption TLC solid phase is solid such as silica gel or alumina and the mobile phase is liquid such as organic solvents. Reverse phase partition TLC In case of this TLC plate is coated by silica which is hydrophilic and wax or paraffin making it hydrophobic. Ion exchange TLC Ion exchange TLC, a reversible exchange of ions is possible between ions in a liquid phase and a solid insoluble substances containing ionic sites i.e. Ion exchangers (Resins). Techniques of TLC 1. Adsorption or floating materials used in TLC The common absorbents used in TLC are silica gel, aluminium oxide, cellulose powder, Kieselguhr (natural adsorbent algae) etc. In general silica gel and alumina are used for partition TLC wehereas silica gel cellulose and Kieselguhr are used for Partition TLC. Silica gel: It is the most commonly used adsorbent and is slightly acidic in nature. A binding agent is usually Gypsum i.e. CaSO4 is often used added with silica gel to hold the adsorbent firmly on the plate. The silica gel is a active adsorbent and is applicable for the separation of all classes of compounds. Alumina or Aluminium oxide: It is slightly basic in nature and they can be used for the separation by bases or steroids. Kieselguhr: It is natural adsorbant. It is also known as diatomaceous earth. It is weakly adsorbant than silica gel to give a less active mixed adsorbant. It has been used for separation of sugar.

Cellulose: Cellulose is used only as supporter for the stationary liquid phase in partition thin layer chromatography. In the same way as paper of cellulose act as a support in paper chromatography, modified cellulose powder such as diethylaminoethyl triaminoethyl and carboxymethyl cellulose are useful for the separation of nucleotides, glycolipids and the pigments. Preparation of the plates or Chromatoplates Preparation of slurry Preparation of thin layer on plate