Structures of Subsidiary Colors Isolated from Food Green No. 3 and Their Separation and Determination by High Performance Liquid Chromatography

Abstract

Food Green No. 3 (Fast Green FCF, Colour Index No. 42053, FD & C Green No. 3) is prepeared by condensation of 1mol of p-hydroxy-o-sulfobenzaldehyde (HSBA) with 2mol of N-ethyl-N-benzylaniline sulfonic acid (EBASA). In this work, the isolation of two major lower-sulfonated subsidiary colors in Food Green No. 3, the structures of the subsidiary colors, and the separation and identification of the subsidiary colors by high performance liquid chromatography were investigated.Two subsidiary colors were isolated from Food Green No. 3 by thin layer chromatography. One of the subsidiary colors was identified as 3-[N-ethyl-N-[4-[(4-ethylaminophenyl) (4-hydroxy-2-sulfophenyl) methylene]-2, 5-cyclohexadienylidene] ammoniomethyl] benzenesulfonate (EA-subsidiary color) from the infrared (IR), nuclear magnetic resonance and FAB mass spectra (Figs. 2, 5 and 6). The other appear to be the color (EBA-subsidiary color) obtained by condensation of 1mol of N-ethyl-N-benzylaniline (EBA), 1mol of HSBA and 1mol of EBASA as judged from the IR and absorption spectra. The absorption maxima of EA- and EBA-subsidiary colors in 30v/v% methanol were at 610nm and 624nm, respectively (Table 1 and Fig. 7).Food Green No. 3 and EA- and EBA-subsidiary colors could be simultaneously separated by reversed phase high performance liquid chromatography on a Zorbax ODS column with 0.5% ammonium carbonate solution-methanol as the eluant (J. Food Hyg. Soc. Japan 27, 30 (1986)), with detection at 624nm for Food Green No. 3 and EBA-subsidiary color and 610nm for EA-subsidiary color. The retention times of Food Green No. 3, and EA- and EBA-subsidiary colors were about 6min, 8min and 10min, respectively (Fig. 8). Linear calibration curves were obtained in the range of 10-100ppm (50-500ng) for Food Green No. 3, and EA- and EBA-subsidiary colors (Figs. 9 and 10). The minimum detectable amount was 5ng (1ppm, 5μl) for Food Green No. 3 and EBA-subsidiary color, and 25ng (5ppm, 5μl) for EA-subsidiary color. The recoveries of EA- and EBA-subsidiary colors added to pure Fast Green FCF (free of subsidiary colors) at levels of 1-10% were 98.4-101. 8% and 98.4-102.1%, respectively (Table 2).EA- and EBA-subsidiary colors in six commercial samples of Food Green No. 3 were analyzed by this method. EA-subsidiary color content was determined to be 0.4-5.7%, and EBA-subsidiary color was detected at levels of 0.2-3.5% (Table 3 and Fig. 11). This method is considered to be suitable for the analysis of EA- and EBA-subsidiary colors in Food Green No. 3.