Module 3 - Strategic case studies in practice

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2. Identification of water solubles dyes A thin layer chromatography technique is used. The dyes present are confirmed by the method of «Co-chromatography" after an initial chromatogram has been run which indicates the dyes probably present. In co-chromatography, the standard dyes are run under the same. conditions as are the extracted dyes; thus, any changes in Rf values due to co-extracted materials are overcome. This is achieved by overspotting the extract with solutions of standard dyes. Solvent Trisodium citrate "Analar"- 2 grams Ammonia solution- 5 ml (wt. per ml- 0.88) Distilled water to - 100 ml Apparatus Method a) The extract obtained from method A or 8 is spotted onto a 5 x 10 em cellulose plate. b) A load of 0.2-5.0 micro litre is applied with a micropipette 2.5 em from the base of the plate. The load depends on the concentration of the extract. c) The plate is run in the solvent for a distance of about 5 em. d) The plate is examined and compared with chromatograms of standard dyes, and the dyes probably present are chosen. e) The extract is spotted on two places on a 5 x 20 em cellulose plate. A load of 0.2-0.5 microlitre is used and it is applied 2.5 em from the base of the plate. f) Onto the plate is spotted 0.2-0.5 micro litre of 0.1 % w/w aqueous solutions of standard dyes as chosen in "d". g) All the dyes thought to be present are overspotted onto the spot of extract on the plate. h) The plate is run in the solvent for a distance of about 15 em. i) The dyes are identified by a comparison of the Rf values and the combined spot should show only the same number of spots as the known dyes added. Thin-layer chromatography plates, cellulose layers. Pipettes, 1 microlitre; e.g. «Microcaps, Drummond.

3. Identification of pigments A. Titanium dioxide identification Reagents Sulphuric acid Hydrogen p_eroxide solution (3 %) EP Procedure

a) To the residue of ashes obtained from the «ash" content test. add 4 ml of sulphuric acid. Boil for 5 minutes: cool. Add 2 ml of hydrogen peroxide and mix. A yellow-orange colour will develop. b) To a 500 mg sample, add 5 ml of sulphuric acid. Heat gently until fumes of sulphur trioxide appear. Cool the suspension and cautiously dilute with water to 100 mi. Filter; and to 5 ml of the clear filtrate, add a few drops of hydrogen peroxide solution. An orange-red colour develops immediately.