Structures of Subsidiary Colors in Food Blue No. 1 and Their Separation and Determination by High Performance Liquid Chromatography

Abstract

Food Blue No. 1 (Brilliant Blue FCF, Colour Index No. 42090, FD & C Blue No. 1) is prepared by condensation of 1mol of o-sulfobenzaldehyde (OSBA) with 2mol of N-ethyl-N-benzylaniline sulfonic acid (EBASA). This color contains many kinds of subsidiary colors. In this work, the preparation of two kinds of main lower-sulfonated subsidiary colors of Food Blue No. 1, the identification of two lower-sulfonated subsidiary colors in commercial Food Blue No. 1, and the separation and determination of the subsidiary colors by high performance liquid chromatography were investigated.One of the subsidiary colors, EA-subsidiary color was prepared by condensation of 1mol of N-ethylaniline (EA), 1mol of OSBA and 1mol of EBASA. Another one, EBA-subsidiary color was obtained by condensation of 1mol of N-ethyl-N-benzylaniline (EBA), 1mol of OSBA and 1mol of EBASA. The infrared (IR) spectra of EA- and EBA-subsidiary colors agreed with those of Bell's EA- and EBA-subsidiary colors. The absorption maxima of EA- and EBA-subsidiary colors in 0.02N ammonium acetate solution were at 629nm and 612nm, respectively (Table 1 and Fig. 3).Food Blue No. 1, EA- and EBA-subsidiary colors were able to be separated by high performance liquid chromatography (HPLC) on a reversed-phase system under the following conditions. Column, Zorbax ODS; eluant, primary 0.5% ammonium carbonate solution, secondary methanol (40-100% methanol linear gradient at 5%/min), detector, 628nm for Food Blue No. 1, 618nm for EA-subsidiary color, 633nm for EBA-subsidiary color, and 253.7nm. The retention times of Food Blue No. 1, EA- and EBA-subsidiary colors were about 7min, 9min and 11min, respectively (Fig. 7). The peak area of each color determined at the maximal absorbance wavelength was about 4 times that at 253.7nm. Linear calibration curves were obtained in the range of 10-100ppm (50-500ng) for Food Blue No. 1 standard, and EA- and EBA-subsidiary colors (Figs. 8-10). The ratio of the peak areas of EA-subsidiary color at 618nm and 628nm was 1.512 (Table 2). The minimum detectable amount was 2.5ng for Food Blue No. 1 standard, 5ng for EA-subsidiary color and 3ng for EBA-subsidiary color (Fig. 11). The recoveries of EA- and EBA-subsidiary colors added to pure Brilliant Blue FCF (free of subsidiary colors) at levels of 1-10% were 92.7-105.4% and 95.8-103.3%, respectively. (Table 3).The main lower-sulfonated subsidiary colors isolated from commercial Food Blue No. 1 were identified as EA- and EBA-subsidiary colors from their IR and absorption spectra and chromatographic behavior. Ten commercial samples of Food Blue No. 1 were analyzed by this method. EA-subsidiary color content was determined to be 2.7-10.4%, but EBA-subsidiary color was detected at only a trace level in one of the samples (Table 4 and Fig. 12). This method is considered to be suitable for the analysis of EA- and EBA-subsidiary colors in Food Blue No. 1.