Dnyanasadhana College, Thane. Department of Chemistry T. Y. B. Sc Dnyanasadhana College, Thane. Department of Chemistry T.Y.B.Sc. Analytical Chemistry Paper-IV Sem-V Chromatography Dr.G.R.Bhagure

Partition Chromatography Definition No. of Phases involved Chromatography Classification of Chromatography Adsorption Chromatography Partition Chromatography

Adsorption Chromatography S+L OR S/G Partition Chromatography Classification of Chromatography Adsorption Chromatography S+L OR S/G Partition Chromatography L+L OR L+G

Adsorption Chromatography Column Chromatography S+L OR S/G Thin Layer S+L Column Chromatography Ion Exchange Gas Solid Chromatography S+G Gel Chromatography V Sem

Partition Chromatography L+L OR L+G Paper Chromatography L+L HPLC Gas Liquid L+G V Sem.

Paper Chromatography L+L Principle: In paper chromatography stationary phase is liquid as well as mobile phase is also liquid. In paper chromatography solute undergoes partition between the two liquid phases. The rate of transfer of solute and its effective separation on paper will depend on partition coefficient of the solute between the two phases. The solutes from the original mixture will have migrated along paper at different rates, forming a series of separated spots. For identification purposes spots are characterized by Rf values.

Solvent Front S O Solute front (B) L V E N t F Solute front (A) W Distance traveled by solute Rf = --------------------------------------------- Distance traveled by solvent Solvent Front S O L V E N t F W Solute front (B) 8 Rf (A) = -------=0.5 16 Solute front (A) 12 Rf(B)= ---- = 0.75 16 Original Line

Solvent Front S O L V E N t F W Solute front Solute front Original Line

EXPERIMENTAL PROCEDURE:- Preparation of the Paper. Solvent system used Preparation of the sample. Application of the sample. Development of the Chromatograms. Identifying the Spots

The paper used for chromatography Whatman no.1 is strong, medium fast, pure cellulose paper that is widely used. For the separation of polar substances special ion exchange paper (containing ion exchanging groups) For the separation of the component which is hydrophilic in nature, esters of cellulose can be used.

Preparation of Paper 15-20 Cm Sample application 3-5 Cm

Solvent system:- In paper chromatography the solvents used as stationary phase and mobile phase should have following characteristics; The solvents should not react with any component during separation. The chemical compositions of the solvents should not change with time. The Rf value for the component should be any where between 0.06 to 0.95. The distribution ratio of the component should be independent of its concentration. The solvents used may be miscible or immiscible but one of the solvent should be polar that can work as stationary phase. The paper shows affinity with polar solvent that can work as stationary phase. If water is used as stationary phase then no special impregnation is necessary. If polar solvent other than water is used then it is necessary to remove the water from the paper. Ex. Water and Ethanol

Mobile Phase Solvent system used: one main organic liquid saturated with distilled water. Polar solvent which is adsorbed on paper is used The solvent should be cheap, very pure, should not volatile by temperature Its rate of flow should not affected by temperature

Preparation of the sample. The solid sample is dissolved in organic solvent having low boiling point. The percentage of the sample in the solution should be 0.1—1%. About 10 micro liter of the sample are transferred to the paper by using capillary or micro syringe. If the sample is of biological origin, then proteins, lipids and inorganic ions present in excess are to removed for better separation.

Application of the sample. The point of the application of the sample or the origin is marked with pencil on the paper. The sample should be applied by micro pipette or capillary. 10-20 micro liter sample is to be applied. After application of sample on the marked spot solvents associated with sample solution is evaporated by using hair drier or current of hot air.

Development of the Chromatograms Ascending Descending Horizontal Radial Multiple

Ascending Paper Chromatography Mobile Phase

Solvent Front S O L V E N t F W Solute front Solute front Original Line

Descending Paper Chromatography Mobile Phase

HORIZONTAL DEVELOPMENT:- The advantage of the technique is the space required is very small. The Rf value is getting very sharp. The tank used for separation is a shallow metal, glass or container. The chromatogram is developed using paper enclosed between two aluminum or glass plates.

Radial Paper Chromatography Petri dish for mobile phase Liquid mobile phase

Separated component in terms of concentric Band Sample Solvent front Wick deep in Solvent

Two dimensional chromatography Sample A A B B X X AC is immersed in mobile phase Solvent AB is immersed in mobile phase C C Solvent front

Identifying the Spots Location of the substances: The substances separated by paper chromatography are colorless. These separated substances are detected by using visualizing agent. The paper is dried at temperature 100Oc. The spots are detected by means of physical or chemical method.

Physical Method :- In this method paper is exposed to ultraviolet light in the wave length range 240-260 nm. The compound produces florescent spots. Compounds which do not produce florescent spots are exposed to high pressure mercury vapour lamp.

Physical Method Sun rays U.V.Light Paper Chromatogram

Unsaturated Compounds Chemical Method H2S gas Iodine vapors Acid –base indicators Ninhydrin Carboxylic acids Unsaturated Compounds Metal ions Cu+2 Amino acids

Chemical Method H2S Gas Separated metal ions Paper Chromatogram

Detection of unsaturated organic compounds. Chemical Method Iodine Vapours Detection of unsaturated organic compounds. Paper Chromatogram

Detection of Carboxylic acids Chemical Method Acid-Base Indicators Detection of Carboxylic acids Paper Chromatogram

Qualitative Analysis Or Interpreting the Data:- The Rf value for each spot should be calculated. Rf stands for "ratio of fronts" and is characteristic for any given compound. Hence, known Rf values can be compared to those of unknown substances for the identifications.   Distance traveled by solute Rf = --------------------------------------------- Distance traveled by solvent Note: Rf values often depend on the temperature, solvent, and type of paper used in the experiment; the most effective way to identify a compound is to spot known substances next to unknown substances on the same chromatogram.)

Calculation of Rf Value

Application of Paper Chromatography 1 For the separation of organic, inorganic, biochemical and natural products 2 Metal ions having same chemical properties or belonging to the same group can be detected and separated 3 The technique is useful for the separation of metal ligand chelate or complexes, it’s also useful for the separation of isomer. 4. The purity of a sample can be tested .In this case pure sample can produce only one spot where as impure sample can produce two or more. 5 It is useful in the field of biochemistry. 6 It is very useful technique in the field of food, drugs and cosmetics.

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