462 CROSSLEY AND THOMAS: THE SEPARATION OF SOME [Vol. 83 The Separation of some Coal-tar Food Colours by Paper Electrophoresis BY J. CROSSLEY AND J. D. R. THOMAS* (Department of Chemistry and Biology, South-East Essex Technical College, Dagenham, Essex) The behaviour during electrophoresis on paper of some coal-tar food colours in different electrolytes is described and discussed. Results show that, in suitable electrolytes, it is possible to separate individual colours from mixtures by paper electrophoresis. LEDERER~ gives an excellent account of the basic principles and applications of paper electro- phoresis, and includes a brief survey of published work on the electrophoresis of dye-stuffs. However, the only work published on the separation of food colours by paper electrophoresis is that of Mori and Kimura2 and Mori,3 who have examined the behaviour of colours under different conditions of electrolyte, filter-paper, current and so on, and William~,~ who describes the electrophoretic isolation at pH 12 of dye-stuffs from biscuits, jams and cream confectionery.The chromatographic isolation of food colours has received greater attention, although it is only 6 years since Tilden5 remarked that, of the many applications of paper partition- chromatography, relatively few dealt with the separation and isolation of dye materials. Many papers have since appeared that describe methods for the chromatographic separation of coal-tar food colours. Some workers have used the column te~hnique,~?' but many others have dealt with separations on paper.8 to l4 Fujiil') summarises the chromatographic behaviour of no less than ninety-five artificial coal-tar dyes, and Verma and Dasll have determined the RF values, in thirteen eluting agents covering a wide pH range from acid to alkaline conditions, of forty-five dyes commonly used in foodstuffs.The identification of colouring matter in specific foodstuffs has been dealt with by some workers, and the range of foodstuffs examined includes fruit squash,8 ~aviare,~ wines, syrups and so on12 and jelly ~rysta1s.l~ * Present address : Newport and Monmouthshire College of Technology, Newport, Monmouthshire.August, 19581 COAL-TAR FOOD COLOURS BY PAPER ELECTROPHORESIS 463 Of the twenty-six food colours examined by Mori and Kimuraa and Mona by paper electrophoresis, only three are among the permitted16 coal-tar food colours, namely, amaranth, tartrazine and indigo carmine.We now describe an extension of the work to an examination of the behaviour of erythrosine BS, tartrazine, indigo carmine, ponceau MX, ponceau 4R and ponceau 3R. METHOD APPARATUS- ELECTROLYTE SOLUTIONS- An E.E.L. (Evans Electroselenium Ltd.) electrophoresis apparatus was used. Acetic acid, N. B u f e r solution, PH 4.0-Add 6.0 ml of 0.1 N sodium hydroxide to 750 ml of 0.1 M Buffer solution, pH B.O--Add 85.5 ml of 0.1 N sodium hydroxide to 750 ml of 0.1 M Buffer solution, PH 8.0-Add 702 ml of 0.1 N sodium hydroxide to 750 ml of 0.1 M Sodium tetraborate solution, 1 per cent. w l v . Ammonium hydroxide solution, 0.1 N. PREPARATION OF DYE SOLUTIONS- Prepare aqueous solutions of erythrosine BS, tartrazine, indigo carmine, ponceau MX, ponceau 4R and ponceau 3R, so that 50 ml of each solution contain 10 mg of the dye.When a mixture is to be used, prepare a solution containing 10 mg of each constituent per 50 ml. PROCEDURE- Fill the four compartments of the electrophoresis bath to the same level with the relevant electrolyte solution. Cut a Whatman No. 1 filter-paper strip (5 cm wide) to suitable length (34 cm) . Draw a faint pencil line across the filter-paper strip, 5 cm from one end. Thoroughly moisten the strip with the electrolyte solution in the bath, place it across the “bridge,” and apply evenly at the pencil line 0.02 ml of the relevant dye solution, which corresponds to 4 pg of each dye. When there is more than one strip in the bath, take care that all the samples are at the same end.Place the glass cover on the bath and connect to the power unit. Place the polarity switch in the position indicated by the position of the sample, i.e., if the pencil mark is at end A of the bath, place switch in position A also and vice versa. This makes the sample end the cathode compartment. (The dyes in each instance migrate towards the anode, although some of those examined by Mori and Kimuraz migrate towards the cathode, e g . , Bismarck brown and rhodamine 6G.) Allow electrophoresis to take place by switching on the power unit and adjusting the current to a suitable value. After a suitable time interval, end the experiment by switching off the power unit and disconnecting the electrophoresis bath.Remove the strips and dry them in an oven at 110” C for 5 minutes, the strips being suspended loopwise from a glass rod by means of “bulldog” clips at either end. potassium hydrogen phthalate, and dilute to 1.5 litres. potassium dihydrogen orthophosphate, and dilute to 1.5 litres. potassium dihydrogen orthophosphate, and dilute to 1.5 litres. The electropherogram is then ready for examination. RESULTS The dyes were subjected to paper electrophoresis singly and also as constituents of Table I shows the distances Migration distances mixtures, the observed migration distances being reproducible. migrated by the dyes, both singly and as constituents of mixtures. of less than 2 mm are shown as zero. DISCUSSIOS OF RESULTS Table I shows a number of peculiarities and trends that call for comment.Migration distances in a given time for the dyes in the nearly neutral electrolytes, i.e., buffer solutions pH 6.0 and 8.0, are relatively small when compared with the much greater distances observed under more acid or alkaline conditions. This would suggest that separa- tions can be carried out more effectively under distinctly acid or alkaline conditions. This464 CROSSLEY AND THOMAS: THE: SEPARATION OF SOME [Vol. 83 is found not to be so, as the advantage gained by greater migration distances is lost because of the considerable and often rapid tailing and fading that occurs under these extreme condi- tions. It is worthy of note, however, that erythrosine BS, tartrazine and indigo carmine have been separated in 15 minutes with N acetic acid as electrolyte, the respective migration distances being 0, 16 and 7 mm at a current density of 0.6 mA per strip, tailing and fading being only slight.TABLE I MIGRATION DISTANCES OF DYES hligration distance of- Current - c 1 density, erythro- tartra- Electrolyte mA Time, sine BS, zine, per5cm hours mm mm Acetic acid, N 0.6 2 0 130* 1.7 2 Buffer solution, Buffer solution, 1.7 2 0 23 Buffer solution, 2.0 2 0 15 Single dyes- - - pH 4.0 1.7 1.75 0 74* pH 8.0 pH 8.0 Sodium tetraborate solution, 1 per cent. 2.0 1.5 9 103t Ammonium hydroxide, 0.1 N 1.7 2 0 83* Dye mixtures (each horizontal line corresponds to a mi;vture)- Acetic acid, N 0.6 0.25 0 16 0.6 1.5 0 90* Buffer solution, pH 4.0 1.7 1.75 0 70* Buffer solution, pH 6.0 1.7 2 0 24 1.7 2 0 23 Buffer solution, pH 8.0 2.0 2 0 18 2.0 2 0 18 Sodium tetraborate solution, 1 per cent.2.0 1-5 - - 2-0 1.5 10 85 t 0.1 N 1.7 2 0 83* Ammonium hydroxide, indigo carmine, mm 52t 35s 9 8 - 38* 101 7 40 t 32t 10 8 7 7 - -: 9: ponceau 4 R mm - 37* - 26 30 100 - - - - - 27 - 30 76 80 - ponceau 3R. mm - 20* - 0 0 11 - - - - - 0 - 0 9 10 - * Tailing and fading. 7 ’Tailing. $ Fading. 11 Two bands, but the band at 5 Slight tailing and fading. mm is faint. In experiments in which electrophoresis was carried out for different time intervals, the other conditions being kept constant, an approximately linear relationship between migration distance and time was observed (com:pare Moris), e.g., for tartrazine and indigo carmine in N acetic acid for 15 and 90 minutes, the respective migration distances were 16 and 90 mm for tartrazine and 7 and 40 mm for indigo carmine.It is often observed that, when dyes are present in a mixture, they exert a “dragging” effect on one another. This is particularly marked with tartrazine and ponceau 4R in 1 per cent. sodium tetraborate solution, the migration distance of tartrazine being reduced from 103 mm for the single dye to 85 mm for the dye present in a mixture; corresponding figures for ponceau 4R are 100 and 80mm. The four red dyes, erythrosine BS, ponceau MX, ponceau 4R and ponceau 3R, were chosen for this investigation because of their similarity in colour and, in so far as the ponceaus are concerned, their similarity in structure. As .is to be expected, and as the results show, their separation by paper electrophoresis is more difficult than for the other dyes.Ponceau 4R, however, presents little difficulty, as it migrates readily in every instance. Except in 1 per cent. sodium tetraborate solution, erythrosine BS, ponceau MX and ponceau 3R didAugust, 19581 COAL-TAR FOOD COLOURS BY PAPER ELECTROPHORESIS 465 not separate, although separation would be possible in N acetic acid, except for the considerable tailing and fading that occurs in this electrolyte. When the six dyes are present in one mixture, separation into four distinct bands takes place in 1 per cent. sodium tetraborate solution, a fifth band of indigo carmine being completely faded. Two dyes that cannot be separated in this electrolyte are erythrosine BS and ponceau 3R.The difficulty in separating ponceau MX and ponceau 3R in many electrolytes can probably be attributed to the similarity in structure (ponceau 3R has one extra methyl group). The observations of Anderson and Martin,14 who examined the effect of substituents on the R, values when dye-stuffs were chromatographically extracted with isobutyl alcohol saturated with 2 N hydrochloric acid, are of interest in this connection. They report that the R, value of a mono-azo dye-stuff is not influenced by substituent groups such as methyl, but is increased by a decrease in the number of sulphonic acid groups. These trends cannot hold for paper electrophoresis, as it is possible to separate ponceau MX and ponceau 3R in both iV acetic acid and 1 per cent. sodium tetraborate solution.Further, ponceau MX, a mixture of dyes with methyl groups in different positions, has been separated into two bands in 1 per cent. sodium tetraborate solution. Also, the effect of the sulphonic acid group appears to be the reverse of that for chromatographic separation, i.e., an increase in the number of sulphonic acid groups increases the migration distance, as illustrated by ponceau 4R (three sulphonic acid groups) and ponceau MX and ponceau 3R (two sulphonic acid groups). CONCLUSIOM With a suitable choice of electrolytes it is possible to separate food-colouring materials by paper electrophoresis. Distinct separations of dyes from mixtures are possible, as long as there is a difference of 3 to 4 mm between the migration distance of each dye. This difference must of necessity be greater when tailing occurs.Two to three hours are usually sufficient to isolate the components into distinct zones, which can be cut out, the dyes eluted with distilled water and absorption spectra recorded. With suitable colour filters, which are available from the manufacturers, the E.E.L. scanner can also be used for quantitative evaluation. We thank Solmedia Ltd. for a gift of ponceau MX, ponceau 4R and ponceau 3R. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. REFERENCES Lederer, M., “Introduction to Paper Electrophoresis and Related Methods,” Elsevier Publishing Co., Amsterdam, 1955. Mori, I., and Kimura, M., J . Pharm. SOC., Jafian, 1954, 74, 179. Mori, I., Ibid., 1954, 74, 181. TVilliams. T. F.. Analvst, 1957. 82, 211. Tilden, D.“H., J. Ass: 03. Agric. Chem., 1952,35, 423. McKeown, G. G., Ibid., 1954, 37, 527. Graichen, C., Sclar, R. N., Ethelstein, N., and Freeman, K. A., Ibid., 1955, 38, 792. Mitra, S. N., and Chatterji, R. K., J . Inst. Chem., India, 1955, 27, 169. Panopoulos, G., and MBgaldoikonomos, J., Chim. Anal., 1954, 36, 68. Fujii, S., Bull. Nut. Hyg. Lab., Tokyo, 1955, 73, 335. Verma. M. R.. and Ram Ti Das. T. Sci. I n d . Res.. C. India. 1956. 15. 186. Deshusses, J.,.and DesbaGmes, g, Mitt. Lebensmitt. ‘Hyg., Bern, 1956, 47, 15. Netta, I., An%. Falsif, 1957, 50, 166. Anderson, J. R. A., and Martin, E. C., Anal. Chim. Acta, 1953, 8, 530. “The Colouring Matter in Food Regulations, 1957,” H.M. Stationery Office, London, 1957. Received March 19th, 1968