Chromatography

Group MembersMasoom Shani 41 Abdul karim 12Mohammad Munawer 23Mubasher 46Mohammad Anas 47

Chromatography

Introduction

The word chromatography is derived from the two Greek words,chroma and graphein."Chroma" meaning colour and "graphein "meaning to write.So, the word chromatography means, colour writing. This technique was first discovered by Tswett in 1903.

Definition Chromatography is a physical separation technique in which the components of a mixture is separated by differences in their distribution between two phases: stationary und mobile phase. So, in simple words, we can say that chromatography is the physical separation of a mixture into its individual components.

Purpose Chromatography is used to: Analyze Identify Purify and Quantify the compounds.

Phases

There are two phases of chromatography:Stationary phase.Mobile phase.

Stationary phase A phase, which becomes adsorbed on the filter media or a surface, is called stationary phase.It may be a liquid or a solid.It may be packed in in a column.

Examples Silica gel Alumina Filter paper, etc. are some important stationary phases.

Mobile phase The solvent or mixture of solvents used for the separation of components, in chromatography, is called mobile phase. ORThe phase, which passes over stationary phase, is called mobile phase.It may be a liquid or a gas.It is also called eluent.

ExamplesAlcohol Water Ethanol Acetic acid Acetone or gas etc. are some important mobile phases

Principle This is a separation technique based upon the difference of adhesive forces (relative affinities) of solute with stationary and mobile phases. The distribution of the components of a mixture between the phases is governed by distribution co-efficient KD.It can be calculated as:KD = Conc. of components in mobile phase Conc. of components in stationary phase

Types of Chromatography

Types On the basis of stationary phase, chromatography has two types:1. Partition chromatography.2. Adsorption chromatography.

Partition Chromatography A chromatography, having liquid stationary phase, is called partition chromatography.

Example Paper chromatography is the common example of partition chromatography.

Paper ChromatographyDefinition: A chromatography, that uses paper strips or sheets as the adsorbent stationary phase through which a solution flows. OR A type of partition chromatography, that uses selective adsorption on a strip of paper.

Different ways of Paper Chromatography There are three common ways of carrying out this technique:Ascending paper chromatography.Descending paper chromatography.Radial / circular paper chromatography. But we only discuss ascending paper chromatography.

Ascending paper chromatographyMaterial : Paper, pencil, eraser, filter paper, test tube, rubber stopper, paper clip, metric ruler etc.

Procedure Take some solvent in a chromatographic tank and cover it, so that the vapours of solvent homogenize with the walls of container or tank.Take proper cut filter paper and draw a line of 2.5 cm from the bottom with the help of lead pencil. At the midpoint, put a drop of sample mixture with the help of capillary tube. Dry it for about 15-30 minutes.

After that suspend the strip in a tank, in such a way that its bottom should be dipped 1-2 cm in a solvent.

. When the solvent front rises to about 3/4 of the length of the strip or paper, then remove the strip. Mark the solvent front with the lead pencil and dry the strip. When the strip or paper is dried, the pattern on the paper is called chromatogram. Each component has a Rf value and it can be calculated as:Rf =Distance travelled by component from spot Distance travelled by solvent from original spot

Uses

Among all the chromatography methods, paper chromatography is an inexpensive and rapid method that provides graphic and clear results.It is used as a qualitative method for identifying the components in a mixture.The separated spots on the finished and dried chromatogram can be cut out and re-dissolved to obtain a pure sample of component of the sample mixtures.It is used in several scientific studies in identification of unknown organic and inorganic compounds from a mixture.

It is used as an analytical chemistry technique for identifying and separating colored mixtures like pigments.Paper chromatography can be reproduced easily as long as the conditions are controlled and maintained.

Limitations There are some disadvantages of using paper chromatography: 1. It cannot be used as a preparative technique because we can't apply a large sample quantity. 2. It can't be used in quantitative analysis. 3. It doesn't allow the separation of complex mixtures. 4.This is one of oldest method.

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Adsorption Chromatography A chromatography, having solid stationary phase,is called adsorption chromatography.

Examples TLC (thin layer chromatography) and column chromatography are the common examples of adsorption chromatography.

TLC(Thin layer chromatography)Thin layer chromatography (TLC) is a method for identifying substances and testing the purity of compounds. TLC is a chromatography technique to separate mixtures. The technique of TLC closely resembles those of column and paper chromatography

Definition A chromatographic technique in which a thin layer of solid adsorbent, supported on a glass or plastic plate is used as a stationary phase.Adsorbent used in it are silica and alumina. TLC plates are commercially available in ready form.

Procedure In thin layer chromatography, a sheet of glass of plastic plate is coated with a thin layer of adsorbent (cellulose, powder alumina, silica). This is done by mixing the adsorbent with a suitable liquid, usually water, to form a slurry. This is applied to the sheet of glass or plastic plate by spreading or dipping. After drying the plate a drop of mixture to be separated is placed just above one edge which is then placed in the air tight pool of solvent for development. After development, the developed spot can be located by holding the plate, under the UV lamp and their Rf value can be determined similar to paper chromatography.

Applications 1. Thin layer chromatography is being increasingly used for qualitative, quantitative and preparative analysis. 2.The technique of TLC is extremely suited for analysis of trace components. 3.In TLC a large number of organic and inorganic compounds have been separated and identified and whenever possible quantitatively analysed.4.The technique of TLC is very sensitive and gives sharper zones hence better resolution.5. The application of TLC include the detection of by-products in synthetic processes, determination of the presence of impurity, peptides, carbohydrates, lipids, steroids, hormones, sterols, vitamins, pigments and inorganic anions and cations, etc.

LimitationsAlthough TLC is very simple and convenient technique it can not tells the difference between enantiomers and isomers.Another disadvantage of TLC is that in order to identify specific compounds the Rf value for the compounds of interest must be known beforehand.The components of the mixture must be soluble. It is just qualitative and not quantitative. Development of the spot takes less time, so it does not provide better resolution than paper chromatography.

Column Chromatography Definition:Column chromatography is suitable for the physical separation of gram quantities of material. ORThe form of liquid chromatography in which a column is used to hold the stationery phase, whether the stationary phase is solid or liquid

Principle When a mixture of mobile phase and sample to be separated are introduced from top of the column, the individual components of mixture move with different rates. Those with lower affinity and adsorption to stationary phase move faster and eluted out first while those with greater adsorption affinity move or travel slower and get eluted out last.The solute molecules adsorb to the column in a reversible manner. The rate of the movement of the components is given as follows:R= Rate of movement of a component / Rate of movement of mobile phase. i.e; it is the ratio of distance moved by solute to the distance moved by solvent.

ApparatusSolvent acts as mobile phase. A finely divided solid surface acts as the stationary phase. The stationary phase will adsorb the components of the mixture to varying degree and adsorbent surface. This process may be described by a three-way equilibrium between the sample, the solvent and the adsorbent.

Adsorbents The most common solid adsorbents are alumina (aluminum oxide) and silica gel (silicon dioxide). Silica gel is slightly acidic while alumina may be acidic, neutral or basic.

SolventThemobile phaseoreluentis either a puresolventor a mixture of different solvents. It is chosen so that theretention factorvalue of the compound of interest is roughly around 0.2 - 0.3 in order to minimize the time and the amount of eluent to run the chromatography. For most separations, the solvent should be less polar than the compounds. The compounds must also be soluble in the solvent so they are not permanently adsorbed.

Columns The columns available in the department are simple glass tubes, varying in length and diameter.

ProcedureAdd the sample to the top of the column, either as a neat liquid, or dissolved in a minimum amount of the solvent used to pack the column. The sample should be added directly to the sand layer. Solvent is drawn from the bottom of the column until the level of the liquid is just above the level of the sandThe identity of the fractions may be determined by one of several methods. If the compounds are coloured, they can be seen to separate and visible spectroscopy can confirm the degree of separation. For colourless compounds, either TLC or GC may be used to identify the compounds present in the different fractions. The preferred method is usually TLC. Once the desired fractions are identified the solvent may be removed by rotary evaporation and the compound isolated.

Uses1. Column chromatography is best suited to separate active principle from plant materials.2. In separation of compounds after organic synthesis to obtain desired molecule.3.To separate or purify natural compound mixtures like alkaloids, glycosides.

Limitations1. Properly setting up the column requires some technical skill and manual dexterity.2. It isvery time consumingand tedious,especiallyfor large samples.3. Collectingvessels must be frequently switched andsolventlevels needto be topped up