PPD AND RESORCINOL IN HAIR DYES 855 Separation of Dye Components Thin-layer Chromatography Analytical (250-m/•) thin-layer plates with silica gel* coating and with or without fluorescent indicator were used. The vacuum dried precipitate was dissolved in N,N-dimethylformamide (DMF) and spotted in a band form on the preconditioned (100øC, 1 hour) plates. The air- dried plates were eluted in an ascending mode with one of the following solvent systems. Chloroform: ethyl acetate: methanol, 6: 2: 2 Chloroform: DMF, 3: 1 Chloroform: methanol, 2: 1 The thin-layer elution was satisfactory for qualitative recognition of spe- cific components in the product mixture according to their color and mobility (Table I). Table I Values of the Oxidation Products ofp-Phenylenediamine and Resorcinol (1:1)• Band Color R/ , 100 Brown (origin) 0 Blue 9 Brown 17 Pink 22 Green 29 Yellow 33 Yellow 45 Yellow 50 '• Chloroform: ethyl acetate: methanol (6: 2: 2) solvent system. Preparative Layer Chromatography Separation of components in quantities sufficient for analysis was car- ried out by preparative thin-layer chromatography (2-mm thick silica gel). The band-spotted, dried plates were repeatedly eluted with sol- vents of increasing activity (increasing alcohol or DMF content) until sufficient resolution of the specific components was achieved. The silica gel zones containing the eluted bands were separately scraped off the glass plate and extracted with cold methanol. The solution was filtered or centrifuged, and concentrated under vacuum at room temperature. * Merck & Co., Inc., Rahway, N.J. 07065.

856 .1OURNAL OF THE SOCIETY OF COSMETIC CHEMISTS These solutions were tBrther purified by repeated elutions on analytical tic plates. In spite of all the precautions, some nonmoving residue was always present or formed on the silica gel, demonstrating the inherent instability of the dyes. Column Chromatography Coluinn chromatography (acidic silica gel) was used only for the pre- liminary enrichment of the components in large amounts for the subse- quent thin-layer separation. In column chromatography, component trailing occurred to a greater extent than on the preparative plates. Structure Elucidatzon The major component which was resolved sharply during chromato- graphic separation was green and showed medium mobility (R• 0.3-0.4) on silica gel with the solvents mentioned above. The isolated sample was a very dark green crystalline material. The analytical work in- volved for identification of this component is described below. Ultraviolet and Visible Spectra The ultraviolet spectrum of the aqueous solution of this dye showed a bathochromic shift of the 190-mt• and 242-rest bands to 203 and 276 m/•, respectively (Fig. 3), at alkaline pH. This shift is commonly asso- ciated with the presence of phenolic hydroxyls (24). Figure 3. ,.0[• __ ,.: •.0 [• eepH: 2.0 200 250 300 350 Wavelength (millimicrons) Effect of pH on uv spectrum of green component from p-phenylenediamine and resorcinol oxidation The presence of amino groups was indicated by the hypsochromic shifts of the 420-mt• and 600-mt• bands to 380 and 560 mt•, respectively (Fig. 4), when the pH of the solution was lowered to 2.