Chromatographic __——+—____Techniquesoe ey SOE s jc techniques were coloured components 1 de the name “chromatography: onc from colour. ays it column instead of glass column and by irradiati gUV li the bands with a Os phic separation is based ales jeter) phases of which one era : n distribution of crude product hed eaten) and another is moving (mobile), ‘The stationary phase may be either columan or plate Of is 8*'rpe mobile phase (liquid or gas) moves through the stationary phase when different components are PRE ed due to differential migration. Differential migration is again dependent on their affinity—le~ fttn or solubility 10 the stationary and mobile phase, the Somporcat which is atracied less BY iphase will move faster in the mobile phase, and vice versa, ome cromatouaphic methods can broadly be classified into five categories depending upon the separatio que employed : ow son chromatography ii) Partition chromatography Gi fon-exchange chromatography z eon nding upon the nature of stationary phase, three chromatographic techniques have been developed ey in the laboratory for everyday use. They are : ee A. Column chromatography or Liquid-solid adsorption chromatography (LSC), Paper chromatography (PC), and C. Thin-layer chromatography (TLC). ‘The discussions about other chromatographic techniques are beyond the scope of this book. 4. Column Chromatography : ‘A mixture of different compounds can be separated through the following steps : (a) Adsorbent selection (b) Solvent selection (© Column preparation (@ Sample placement (©) Column (chromatogram) development Adsorbent selection : The following criteria give more or less exact selection of adsorbent : (i) Adsorbent must be insoluble in solvents to be used. (ii) Adsorbent must be non-reactive towards compounds to be separated. Prac, Chem. — 24 adepends on nature and number of polar idea can be given as : carboxylic acids moot Pesan fides > unsaturated hyérocarbong eta Meiy s Be a Polarities of solvents. It is purely trial and error method, 4olled Boece pai and poor separation is obtained, either the adsorbent is made ‘ce comer adsorbent fo used A net hands if the elution is very slow then solvent polarity seg OME = used. As @ whole, non-polar solvents are used for weakly adsorbed con wunds anauiler vents are used for strongly adsorbed compounds a ‘The order of polarity of common solvents is : Petroleum ether < carbon Mixture sample spot Matching of spots of unknown mixture with standard sample Conclusion : By comparing the Ry values of the constituents of unknown mixture with standard ‘samples A and B it is found that the constituent X is ...B... and Z is ...A... and Y is rest of the three, N.B.: (i) When two different plates are used for standard samples and unknown mixture in Table 1 = dy, d = dp, Rp = dy/dy and in Table-2, dy = d3, d = da, Rr = dy/dy because for different plate a’ will be different (ii) Another way of detection of amino acids in a mixture is to compare observed Ry values with the standard tables provided, solvent used is same in both the cases and Ry values are sufficiently different. For this purpose table of Ry values for amino acids in TLC with two sets of solvent are given in the next page :Valine QUESTIONS FOR VIVA VOCE |, What are the factors those must be considered for the choice of an adsorbent in chromatographic separation? Hint. See Page No. 371-372. 2, How the chromatographic separation is influenced by : (a) Nonuniform packing of column? (b) Not maintaining the eluent above the adsorbent in the column? (©) Too rapid flow rate of the solvent?” @ (¢)_Adsorbent having too strong adsorption power? (Application of too large volume of the sample solution? (@) Use of solvent having high polarity? Hint. See Page No. 371-373, 3. Which one adsorbent either high or low activity would you prefer to separate a mixture of highly polar compound by TLC? Too large a fraction collected at a time? Hint. High activity. 44. What are the two phenomena on which the basis of chromatography depends? Hint. See Page No. 371 5, Why thin layer chromatography is Superior to paper chromatography? Hint. This can be used for preparative separation in small scale, a ._...._..__; : Quntiative Reagents : Solution A is prepared ’ ; is disso! line copper sulphate vsHGh lat woriniter Solgice Bl pemseat Oe = Sodium citrate and 50 of anhydrous sodium carbonate in 400 ma of water Now solu st : on x solution B, the volume is made up to 500 ml and is mixed well. Aw . Bial's Reagent : 1 g of orcinol is dissol concentrated hydrochloric dope of 10% fer chloride solution i added wer Comremumted 96d and ay 4. Brady's Reagent : | g of powdered 2,4-dinitrophen) ine is r , . wylhydrazine is added sulphuric acid, 20 ml of methanol is added cautiously, stirred estar ial eee er eed After adding 5 ml of water, the mixture is stirred thoroughly and filtered, if necessary ee _- Bromine Water : 5 ml of bromine ii added to 100 ml aki bromine is allowed to romain in solution (Gone, abSUSW)- Toros rere re ae 6. Chlorine Water : Prepared by passi i i i Se eee passing chlorine gas in 100 ml of cold water until satuateg 7. Cerric Ammonium Nitrate Reagent : Prepared by dissolvi ic ammoni in 100 ml of warm dilute (2N) nitric acid coe ae 8. Chromic Anhydride Reagent : Prepared by dissolving 25 g of chromic anhydride i - ry . “ concentrated sulpluiric acid! Poured cautsousiy inbo'7s tar OPWaler*Witt tring Ved the bokeh ae 9. Denige's Reagent : Prepared by dissolving 5 g of powdered yellow mercuric oxide in 20 ce. concentrated HsSO, by stirring. Then this solution is poured gradually with stirring into 100 c.c. of water 10. Diphenylamine (2%) : Prepared by dissolving 2 g solid in 100 c.c. of strong H3SO4. 11, Erdmann's Reagent : Prepared. by mixing 5 ml of concentrated nitric acid with 100 ml of concentrated sulphuric acid. 12, Febling's Solution : Solution A (blue) prepared by dissolving 34.6 g of pure CuSO4'SH0 in distilled water and diluted to 500 c.c, Prepared by dissolving 170 g of Rochelle Salt (sodium potassium Solution B (Colourless) : tartarate) and 71 g of NaOH in water and diluted to 500 c.c. 13, Iodine Potassium Iodide Solution : Prepared by grinding 1 g of iodine and 5 g of Potassium iodide with 15 c.c. of water ina mortar, until all the solids have dissolved Prepared by dissolving 68 g of HgClz in a litre of water. Its 14. Mercuric Chloride Solution : is (N Bream (7). 15. Molisch's Reagent : Prepared by dissolving 10 g of a-naphthol in 100 ml ethyl alcohol. 16. B-Naphthot : Prepared by dissolving 10 g of substance in one litre of caustic soda solution (10%). — |lamine + Prepared by dissolving 3 g of reagent in cod ie yylander's Reagent : Prepared by ‘ Tartaratein 100 ml of 10% NaOH sauton © > & of PSS bismath gchiff's Reagent : Prepared by dissolvin rosanil ) 05 i ae water. After cooling, soln. is saturated vith apn ere cs ‘becomes yw) and diluted to 500 ml with water, ae ; Seliwanoff's Reagent : Pre issolvi a. aa ‘pared by dissolving 0.05 g of resorcinol in 100 ml of dilute Sodium Nitroprusside Solution : ae Bc. of water. ion : Prepared by dissolving 0.1 g of sodium nitroprusside ery 23, Tollen's Reagent : Prepared by dissolving 6 g of silver nitrate i ‘olution is mixed with 2 ml of 10% sodium hydroxide before use and tntoniui bye So Jaded to it drop by drop until the ppt. formed just dissolves. TE a ———— INDICATORS |. Bromophenol Blue : Prepared by dissolving 0.2 g of bromophenol blue in 100 ml of rectified spirit. 2. Diphenylamine : Prepared by dissolving 10 g of diphenylamine in 100 ml of concentrated sulphuric acid, 3. Methylene Blue : Prepared by dissolving 0.2 g of methylene blue in 100 ml of water. 4. Methyl Orange : Prepared by dissolving 0.1 g of methyl orange in 100 ml of water. Methyl Red : Prepared by dissolving 0.1 g of methyl red in 100 ml of rectified spirit. 6. Methyl Violet : Prepared by dissolving | g of methyl violet in 25 ml of 95% alcohol and the Volume is made up to 100 ml with water 7. Phenolphthalein : Prepared by dissolving 1 g of phenolphthalein in 100 mi of rectified spirit. 8. Starch Solution + Prepared by suspending 2 g of soluble starch in 100 ml of water, 90 + is added 10 it, Then the solution is boiled 1 Bet clear solution with constant stirring, boiling wate