Partition chromatographyPartition chromatography is carried out

on sheets of filter paper, column or thin layer of powdered cellulose, moist silica gel or kieselguhr

Silica acts as support and the water is stationary phase

It is known that filter paper consists of numerous cellulose fibre which accomodate certain percentage of moisture we can consider that fibre + water constitute cells

Partition of the substance between moisture in the cell and the solvent flowing over the cells which bring about separation

Water of cell is the stationary phase and moving solvent is the mobile phase

The extent to which the solute distribute itself between the two liquid is measured and is known as partition or distribution coefficient

K=Conc. of solute in M. Ph

Conc. of solute in St. Ph

Partition chr. IncludesA- liquid liquid extractionB- column chromatographyC- paper chromatographyD- TLCE- Gas liquid chromatography

A- Liquid liquid extractionSolute distributes itself between equal

volumes of two immiscible solvents.

Methods of extraction1- discontinuous extractionA- single contactIn separating funnel using two immiscible liquidB- multiple contactSeveral times in separating funnel.2- continuous liquid liquid extraction1- single stage by using liquid liquid extraction app.

It consists of round bottom Pyrex flaskExtraction tube connected to the flask by

another glass tubeCondenser

2- multiple stage (counter current distribution)

Several units of extraction app are specially constructed to transfer the upper phase to the subsequent tube keeping the lower stationary phase

Substance distribute itself between lower stationary phase and upper according to distribution coefficient (K)

Counter current extraction app.

K=Conc. of solute in upper phase

Conc. of solute in lower phase

The procedure:1- the app. Consists of 100 tubes

affecting 100 equilibrations2- fresh mob. Phase. Is transferred from

reservoir to tube 13- after equilibrium mob. Phase move to

fresh stationary phase, shacked and transferred to a tube containing stationary phase and so on....

It depends on1- number of shacking

2- time allowed to stand for separationNumber of equilibrium

B- column (partition chromatography)As in column (partition chr.) the

difference is the packing material which is porous material such as kieselguhr coated with layer of liquid, water, the stationary phase is the liquid layer and the solid serves as a support

More soluble substance travel more slowly down the column

Solid support as silica, diatomites, cellulose, cellulose acetate.

Preparation of the columnMixture of support + stationary phase is

stirred with some of mobile phase to be used initially

The slurry is poured in a small portion into the tube which contain small portion of the mobile phase

Application of the sample1- by use of pipette with a bent tip

which is placed against the column wall just above the adsorbent and the liquid is slowly drained from the pipette.

2- use of one or two small disks of filter paper which perforated with very small holes of a size which just to fit on the top of the column

- Solute is dissolved in volatile solvent and adsorbed on one of the filter paper

3- Mixture is dissolved in some of the Mob. Ph. And mix with some of the stationary phase , the dry and put the powder on the top of the column.

ElutionAs in adsorption columnIdentification of the separated substanceas in adsorption column

Applications of partition column1- fractionation of many alkaloids and

polyphenols2- determination of amino end group of

proteins and analysis of hormones3- separation of methylated sugars on

silica gel and free sugar on cellulose.

Paper chromatographyStationary phase is water hold by

adsorption on cellulose molecules which in turn are kept in a fixed position by the fibrous structure of the paper

Apparatus and methodTightly covered jarMethod1- choice of the paper2- purification of the sample3- application of the sample on the paper

4- development5- detection6- identification

1- Choice of the paperWhatman No 1 or 3 for analyticalWhatman No 3 or 3MM forpreparative

2- choice of solvent systemDepend on substances to be separatedAmino acid..phenol/ water Butanol / water/ acetic acidSugars...EtOAc/ pyridine/ water EtOAc/ i-prppanol/ water3- sample purificationBy column or liquid liquid extraction4- application of the sample to the paper

Clear solution is preparedTake sample by capillaryApply the spot on the paper and dry it.

4- DevelopmentA- ascending the flow upward b- descending downwardC- horizontal D- radial the flow in the radii of circular

paper

1- ascending developmentPaper is stood vertically and immersed in

the mobile phaseLevel of the spots should be above the

liquid by 1-2 cm2- descending developmentJar is provided by a trough for the mobile phase in its upper part

Advantages of descending technique A- solvent flow faster

B- long distance of development can be achieved

wire fixed to side of the tankon which trough can fest

Serrated edge to allow the solvent to flow uniformely

offthe paper

Paper suspended in trough

3- Horizontal development:Shallow tank of glass or metalThe paper is placed horizontal on glass

rods with one end of the paper is dipped in the mobile phase

support

paper

mobile phase

Direction of mobile phase

-sample is spotted on a line on the horizontal part of the paper 1-2 cm from the edge

- solvent is flows up by capillary forces4- radial developmentThe principle of this method is based on

migration of the spot from the centre of the paper toward the periphery.

.

Detection of the resolved spotsvisualization1- physical , UV, colour2- chemicalAniline phthalate...........reducing sugarsDragendorff`s..................alkaloidsFeCl3.....................phenolic compoundsNinhydrin...................amino acid, amino

sugarsAnisaldehyde H2SO4......terpene, sterols,

sugars (Not used in paper as it cause charring of the

paper)3- biological enzymatic detection of amylase

microbilogical detection of antibiotic

Uses of paper chromatography1- identification of components of

mixture2- detection of purity of components

against authentic3- major use is the separation of

flavonoids and sugars