236 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS characterized by the spectrophotometric assay to be a good quality product (7). While the purity of CCC, as calculated from spectrophotometric analysis, was in a range of 78.0-94.4%, the amount of the CCC estimated by the content of copper and nitrogen was only between 42.4-46.7% and 48.2-53.1%, respectively. The poor quality of the CCC is confirmed by fractionation analysis, which reveals that the main bulk of the chlorophyll-derived constituents represents only 50% of the total product (9). No substantial analytical data on CCC have been reported in recent years. The most intense studies on the quality of CCC have been carried out by Voigtlander and Henning (6). These authors investigated commercially available CCC, samples prepared in their laboratory, and purified individual chlorophyll derivatives. The following discussion is based on these data, some of which have been modified and recalculated. It is implicit that the copper content in CCC reflects the level of chlorophyll derivatives complexed with metal. We attempted to correlate the amount of copper determined in CCC with the purity of the samples established by spectrophotometric assay (Table I). The comparison did not reveal any relationship between these two parameters. An alteration in the copper content of the samples was not accompanied by an appropriate change in their purity. While for some samples the increase in the amount of copper led to a higher quality of CCC, others were characterized by significant reduction of the purity values. The ratio between copper and nitrogen content (Cu/N index) can also be a reliable indication of the quality of CCC. Our calculations indicate that the Cu/N index for 100% coppered chlorophyll derivatives is 1.1. We estimated the Cu/N index for dif- ferent commercial samples of CCC (Table II). The data presented show that for the majority of these samples, the Cu/N index is far from the standard value of 1.1. Only four of the nine samples could be considered good quality products, since their Cu/N indices were found to be between 0.9 and 1.1. These values correspond to purities of CCC not less than 81.8%. From the lack of correlation between copper content and purity determined by spectro- photometric assay, and from the inconsistent results relative to the Cu/N index, it can be concluded that in most samples of CCC studied, copper-containing chlorophyll derivatives are accompanied by their metal-free analogs. Spectrophotometric evaluation Table I Comparative Data on the Copper Content and Spectrophotometrically Determined Purity of Commercial CCC* Sample number Cu (%) Purity (%) 1 0.80 42 2 1.27 23 3 2.90 30 4 3.05 48 5 3.10 43 6 3.33 102 7 3.68 160 8 3.86 89 9 4.70 82 * From reference 6, Table 1, section a.

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.