ayes Materials and Apparatus required : Alumina (for column chromatography) 5. Stand and Clamp Methylene blue 6. Ethanol Fluorescein 7. Cotton Glass chromatographic column 8. Dropper (Lem ~ 1.5 em diameter and 30cm long) 9. Conical flasks (100 ml) 3. Prepare the solution of the dye mixture as described in the experiment No. &, mal of this solution for column chromatographic separation, Mount the glass chromatography column (a 50 ml burette may be used) to a stand using a clamp. Place a small wisp of cotton or glass wool at the bottom of the column using a long piece of glass rod. :—€) Add alumina on the top of the column to fill up about 15 em of this column. Keep the ‘stopper open. Now add the solution of the dye-mixture (~2 ml) on the top of the column evenly with the help of a dropper and allow the solution to run down the column completely. ) ~— Elute with 5 ml of ethanol and allow the eluant to run down the column, When the ethanol level is ~ 2-3 mm above the top of the column, add more ethyl alcohol to develop the coloured bands. 1) Blue band of methylene blue begins to separate and moves down the column while fluorescein remains at the top. Continue addition of ethanol till the blue band reached the bottom of the column. Collect this fraction in a 100 ml conical flask until the lower end becomes colourless. © Elutethe column with water, when the yellow band of fluorescein immediately moves down. Continue elution with water till the yellow band reaches near the bottoms of the column. Collect this fraction in another 100 ml conical flask until the effluents appear colourless. ) The dyes may be obtained in the solid state on careful evaporation of the extracts. Note ; If salica gel (60-120 mesh) is used as the stationary phase instead of alumina, the Sequence of coloured bands is just reversed, i.c., fluorescein begins to come out of the column first with ethanol as eluant while methylene blue remains at the top. ‘Methylene blue moves down the column when the column is eluted with water-acitic acid (70 : 30) as the eluant. 5L awk wp sa | fa) Experiments based on paper chromatography Experiment No.4 : Separation and identification of amino acids in a mixture (DL-alanine, L-lysine and L-teucine) by paper chromatography : Materials and Apparatus required : Amino acids (DL-alanine, L-lysine, Leucine) Acetic acid Measuring cylinders (25 ml, 10 ml) ‘Test tubes Electrical air oven (100°-110°C) Solvent chamber (development jar) (20cm x 4 em) 7. 8. 9. 10. IL. 1-Butanol Sprayer Distilled water Fine capillary tubes Whatman No. | chromatography paper strip (20 cm x 4m) 12. Spraying reagent : 0.3% ninhydrin solution in 95% ethanolProcedure : (a) Preparation of the sample solutions : Prepare the solutions by dissolving 10-15 mg of each of pure samples of the. » acids and of the given unknown mixture in ~ | mi of distilled water in separate test tubes. ‘and label them accordingly. (b) Application of the sample : Draw with a pencil a base line (~3 cm) above the lower end of the chromatography Paper strip. Put four pencil dots on the base line at equal distances apart. Using separate fine capillary tubes put spots of the solutions of three known amino acids and that of the unknown: mixture on the pencil dots (diameters of the spots should be ~2-3 mm) and record their Tespective positions. Allow the spots to dry in air for S minutes. (©) _ Preparation of the developing solvents : Prepare the required quantity of the developing solvent by mixing 1-butanol, acetic acid and distilled water Tespectively in 12: 3 : 5 (v/v/v) proportion, Pour the solvent into the ‘developing jar, cover the same with the lid, swirl the solvent inside and allow to stand fora few minutes, (d) Development of the chromatogram : ‘Open the jar and suspend the Paper strip into the developing solvent toa depth of ~ Lom keeping the solvent level well below the base line (Fig. 1(a)}. Care must be taken so fe) Location of the spots : Spray the ninhydrin reagent on both side of the dried Paper chromatogram and then dey the paper at 100°-110° by suspending ‘vertically in an electrical air oven (or dry theR, (Di-alanine) | Unknown mixture | R,(A) me - From comparison of R, values of the components A and B (in the unknown mixture) with those of the standards (1,2 and 3) the amino acids A and B may identified to be L-lysine and L-leucine respectively. Approximate R, values of the amins acids : L-leucine (0.62) L-lysine (0.17) DL-alanine (0.35)Experiment No.§: Separation and identification of sugars (glucose fructose and sucrose) by paper chromatography. Materials and Apparatus required : 1, Sugars (glucose, fructose and sucrose) 6. Test tubes 2 Whatman No. | chromatography paper 7. ‘Distilled water strip (20 cm x 4 cm) 8, |-Butanol 3. Fine capillary tunes 9. Acetic acid 4. Measuring cylinders (25 ml, 10 ml) 10. Solvent chamber 5. Spraying reagent : 11. Sprayer Aniline oxalate : Dissolve 0.093 g (~ 9-10 drops of aniline) in 50 ml of 95% ‘ethanol and mix with 50 ml of 0.2 (M) aq. oxalic acid,Procedure : ‘Set up the apparatus as discussed in the experiment No. 4. Prepare the solvent by mixing I-butanol, acetic acid and water in the ratio 4 rl: 5 (wlviy) i Prepare the solutions of glucose, fructose and Sucrose by dissolving ~ 30 mg of each sugar in ~0.25 ml of water in separate test tubes. Using fine capillary tubes, put spots of the three Sugar solutions and of the unknown solution Separately on the paper strip and dry the same inair, Develop the chromatogram using the developing solvent mentioned above (as described. in experiment No. 1}. Dry the chromatographic Paper and spray the same with the aniline ‘Oxalate reagent, when yellow Spots will be visible, Determine the R,, values of the pure