448 JOURNAL OF COSMETIC SCIENCE cosmetics, ink for highlighters, and fluorescent probes for biological research. To increase the maximum absorption of fluorescein dye, xanthene dyes have been halogenated to shift their spectral properties (29). Xanthene dyes have poor photostability, which means they may be fade from exposure to sunlight. For instance, the Dutch artist van Gogh enjoyed using bright xanthene dyes in his paintings, but they have faded after years of exposure to light (30). Furthermore, xanthene dyes are very sensitive to changes in their chemical environment, such as in their pH and in the solvent. Depending upon the pH of the solution, fluorescein appears in different forms: cation, neutral, monoanion, dianion (­Figure 2) (31,32). Anions, a strongly fluorescent species, are formed by the deprotonation of mainly carboxylic acid (pKa 4.31) or/and phenol (pKa 6.43) protons in sequence (31). Neutral species of fluorescein are in equilibrium with the open quinoid Table III Purity Specification Comparison of Allura Red AC Purity Specification EU United States China Korea Name CI 16035 FD&C Red No. 40 CI 16035 Red 40 Loss on drying — ≤ 14.0% (135° C) — ≤ 10.0%(135° C) Chlorides and sulfates — — ≤ 5.0% Water insoluble matter ≤ 0.2% ≤ 0.2% — ≤ 0.2% Subsidiary coloring matters ≤ 3.0% Higher sulfonated subsidiary colors — ≤ 1.0% — — Lower sulfonated subsidiary colors — ≤ 1.0% — — Disodium salt of 6-hydroxy-5- [(2-methoxy-5-methyl-4-sulfophenyl) azo] -8-(2-methoxy-5-methyl-4- sulfophenoxy)-2-naphthalenesulfonic acid — ≤ 1.0% — — Organic compounds other than coloring matters 6,6-oxybis (2-naphthalene sulfonic acid) disodium salt ≤ 1.0% ≤ 1.0% ≤ 1.0% — 6-hydroxy-2-naphthalene sulfonic acid, sodium salt ≤ 0.3% ≤ 0.3% ≤ 0.3% — 4-amino-5-methoxy-2- methylbenezene sulfonic acid (4-Amino-5-methoxy-o- toluenesulfonic acid) ≤ 0.2% ≤ 0.2% ≤ 0.2% — Unsulfonated primary aromatic amines ≤ 0.01% — ≤ 0.01% — Ether extractable matter ≤ 0.2% Heavy metals ≤ 20 ppm Arsenic ≤ 3 mg/kg ≤ 3 ppm — ≤ 2 ppm Lead ≤ 2 mg/kg ≤ 10 ppm — — Mercury ≤ 1 mg/kg — — — Cadmium ≤ 1 mg/kg — — — Pure dye content ≥ 85% ≥ 85.0% — (85.0–101.0)% Abbreviation: '—' : does not have a criteria EU: European Union. “—”: does not have a criteria.

449 COSMETIC COLORATION: A REVIEW form (major), closed lactone form, and zwitterion form. Importantly, each compound exhibits a ­different color red, colorless, and yellow, respectively. The lactone form pre- vails in an organic aprotic solvent, and the quinoid form prevails in a protic solvent such as water or alcohol (31). The equilibrium of the tautomers is affected by the solvent’s polarity. By the amount and source of halogen substituents, there are various xanthene dyes. Halogenation occurs in positions 4’ and 5’ followed by positions 2’ and 7’. Dibromofluorescein (CI 45370, D&C Orange No. 5) has two bromine substituents, tetrabromofluorescein/eosine YS (CI 45380, D&C Red No. 21/22) has four bromine substituents, and tetrachlorotetrabromofluorescein/phloxine B (CI 45410, D&C Red No. 27/28) has four bromine and four chlorine substituents. We will focus on discussing more details regarding tetrabromfluorescein (TBF CI 45380, D&C Red No. 21) which has a yellowish-red color with green fluorescence (32). TBF is prepared by condensing phthalic acid or its anhydride 4 with two moles of resorcinol 5 in the presence of zinc chloride or concentrated sulfuric acid (Figure 3) (33). The resulting fluorescein 6 is then brominated in ethanol. As a derivative of fluorescein, TBF exists in the following four possible structural forms in a medium depending on the pH of the solution: cation, neutral, monoanion, and dianion (Figure 4). Unlike the fluorescein, a phenol proton (pKa 2.85) that has preferential positions of halogen group O HO O CO2H O HO OH O O neutral open quinoid form (red) neutral closed lactone form (colorless) O HO OH CO2 neutral zwitterion form (yellow) O HO O CO2 monoanion carboxylate O O O CO2 dianion O HO OH CO2H cation +H+ -H+ +H+ -H+ +H+ -H+ O Xanthene, 3 5' 4' 7' 2' Figure 2. Structures of fluorescein by the pH of the medium. Adapted from Sjoback et al. (31) O O O H 2 O OH ZnCl 2 Br 2 + Phthalic anhydride, 4 Resorcinol, 5 Fluorescein, 6 Tetrabromfluorescein O HO O CO2H O HO O CO2H Br Br Br Br or conc. H 2 SO4 Figure 3. Synthesis of tetrabromfluorescein.