The quantitative thin layer chromatographic method of a greenish fluorescent substance, 2, 3, 4-trichloro-5, 7-dibromo-6-hydroxyxanthone-1-carboxylic acid found in commercial phloxine (Food Red No. 104) was studied.The maximum wave length of this substance was at 251mμ and 368mμ in 0.1N sodium hydroxide solution. The calibration curves were linear in the range of 1-10μg/ml at 251mμ and 1-20μg/ml at 368mμ (Fig. 1). The maximum of the spectrum shifted from 368mμ to 383mμ by addition of ethyl alcohol. Also the determination of this substance was readily performed with a fluorometric method. The calibration curves were linear in the range of 1×10-7M-4×10-6M at 494mμ (emission maximum) (Fig. 3). The fluorescence intensity was increased by addition of ethyl alcohol, and the maximum of intensity was obtained the ratio 1: 1 between 0.2N sodium hydroxide solution and ethyl alcohol (Fig. 2).The quantitative thin layer chromatographic method was applied for the determination of 2, 3, 4-trichloro-5, 7-dibromo-6-hydroxyxanthone-1-carboxylic acid in commercial phloxine. The amount of this substance was measured with an ultraviolet spectrophotometric and fluorometric methods after it is separated on chromatoplates using a solvent system of methyl ethyl ketone, n-propyl alcohol and distilled water (20: 2: 3) and eluted with distilled water. The recovery in these methods was satisfactory to both an ultraviolet spectro-photometric and fluorometric methods, and the results obtained are shown in Table 3. In case of the ultraviolet spectrophotometric method the absorbance at 251mμ and 368mμ of the material eluted from the silica gel layer has influence on the determination of this substance. Therefore, the absorbance at these wave lengths was investigated using various silica gel adsorbent (Tables 1 and 2). On the silica gel layer the minimum detectable amount was found to be 0.01μg of 2, 3, 4-trichloro-5, 7-dibromo-6-hydroxyxanthone-1-carboxylic acid (Table 4). Commercial phloxine was found to contain 1-3.02 per cent of this substance (Table 5).