CHLOROPHYLLIN COPPER COMPLEX 237 Table II Copper/Nitrogen Index and Content of Metal-Free Chlorophyll Derivatives in Commercial CCC* Metal-free derivatives Sample number Cu/N as % of the total 1 0.3 72.7 2-4 O.6 45.5 5 O.7 36.4 6-7 O.9 18.2 8 1.0 9.1 9 1.1 0 * Based on data from reference 6, Table 1, section a. of purified trisodium copper chlorine 6 (one of the main components of CCC) and its metal-free analog, following the procedure currently used for CCC (5), revealed the purity as 106% for the former compound and 380% for the latter (6). By comparison of these data, it may be estimated that 100% copper-free trisodium chlorine 6 exhibits a value of specific absorbance 3.6 times greater than that of its coppered counterpart. Investigations on the spectral properties of various metal-containing chlorophyll deriv- atives corroborate the conclusion of higher absorptivity if the metal is eliminated from these compounds (10). Hence, specific extinction coefficients determined at the absorp- tion maxima between 400-450 nm for pheophytin a and its coppered analog were 126 and 67.8, respectively. Similarly, values of 182.2 and 93.9 were obtained for pheo- phorbide a and its coppered derivative. Therefore, the presence of even small quantities of metal-free chlorophyll-derived compounds in CCC can result in increased values of apparent purity, determined by spectrophotometric assay. Valuable conclusions can be made from the study of chlorophyll-derived compounds intended to mimic commercially available CCC (Table III). Although the Cu/N indices of such samples are close to 1.1 (as required for completely coppered chlorophyll derivatives), the purity determined spectrophotometrically and on the basis of copper content is different. The values of purity exceeding 100% may be due to the presence of carotenoids, which have an intense absorptivity at 350-500 nm (11). On the other hand, those values below this level may come about because of samples containing colorless impurities. These two groups of compounds have been already found in CCC (7,9). Table III Percent Purity of Laboratory Preparations of CCC* Determined by Sample number Copper content Spectrophotometry 1 52 62 2 62 89 3 78 107 4 88 115 5 92 147 6 96 163 * Based on data from reference 6, Table 1, section b.

238 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS The present data clearly indicate that the currently used spectrophotometric assay er- roneously estimates the purity of CCC as a result of metal-free chlorophyll derivatives having even higher extinction coefficients than their coppered counterparts and/or car- otenoids. In order to control the quality of CCC, the Cu/N index should be employed in conjunction with the absolute percentage of copper and nitrogen content. These values have to be in range of those calculated for a 100% pure CCC. Interference by carotenoids can be avoided by obtaining the total absorptivity of the red peak for the preparation, rather than spectrophotometric measurements in the blue region of the spectrum, as presently employed. REFERENCES (1) A.M. Humphrey, Chlorophyll, Food Chem., 5, 57 (1980). (2) S. A. Chernomorsky and A. B. Segelman, Biological activities of chlorophyll derivatives, NJ Med., 85, 669 (1988). (3) M. E. Wall, Preparation of chlorophyl derivatives for industrial and pharmaceutical use, US Agri- cultural and Chemistry Bureau, 299, 1 (1951). (4) J. C. Kephart, Chlorophyll derivatives: Their chemistry, commercial preparation and uses, Economic Botany, 9, 3 (1955). (5). New and Nonofficial Remedies (J. B. Lippincott Co., Philadelphia, 1953), pp. 57-58. (6) H. W. Voigtlander and H. M. Henning, Das Desodorans "Chlorophyllin," Arzneim. Forsch., 3, 182 (1953). (7) F. D. Pickel, J. J. Scalan, and R. Heggie, Water-soluble copper derivatives of chlorophyll, Drug Stand., 22, 173 (1954). (8) M. Strell, A. Kalojanoff, and F. Zuther, Zur Analytik von Chlorophyll--Praparaten des Handels, Arzneim. Forsch., 6, 8 (1956). (9) M. Sato, I. Fujimoto, T. Sakai, T. Aimoto, R. Kimura, and T. Murata, Effect of sodium copper chlorophyllin on lipid peroxidation. IX. On the antioxidative components in commercial preparations of sodium copper chlorophyllin, Chem. Pharm. Bull., 34, 2428 (1986). (10) S. H. Schanderl, G. L. Marsh, and C. O. Chichester, Color reversion in processed vegetables. II. Model system studies, J. Food Sci., 30, 317 (1965). (11) R. F. Boyer, Modern Experimental Biochemistry (Benjamin/Cummings Publishing Company, Redwood City, California, 1993), p. 383.