266 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Oxidation products are sensitive to both acids and bases used in the solvent system and produce irreversible destruction of a number of components of the product. Their use in solvent systems was avoided. Moisture.' In the resolution of oxidative dyes, a trace amount of moisture is more detri- mental for the reproducibility than other chromatographic separations. It was observed that the resolution of certain components can be eliminated completely by allowing moisture in the system through the plates or from the atmosphere. Actz'•'ation.' Developed plates, on which oxidative dyes cannot be reactivated for a second elution in a similar fashion, because they undergo irreproducible changes or cannot be controlled. Dyes to be applied on the activated plate were dissolved in DMF, since the polymeric component of the dye had a limited solubility in other less polar solvent. When preparing a sample of a solvent, that solution should never be warmed, and fresh solution should always be prepared just before applying it to the plate. Application to plate.' With oxidative dyes, it is preferable to apply the products to the plate as a sharp band, rather than as a spot, for 2 reasons. One, the left-over unreacted dye precursors, which developed during the elution process, causes heavy trailing and interferes with the chromatography of an already formed oxidized product. Second, the oxidized products undergo secondary coupling reactions on silica gel, giving grayish-brown polymeric trailing. When the dye is applied as a band, visual identifica- tion of the components present in smaller amounts can be achieved. Evaluation and documentation.' Evaluation and documentation of the thin-layer plates should be done immediately after elution to record the presence or absence of components and the relative strength of color of individual components. This is re- quired because 3 events may occur on the developed plate. One, a new color may form from untreated precursors two, color may fade from the intensely colored products and three, color may change. For example, green color changes to gray, blue changes to purple, and so on. All of these processes can be slowed down, immediately following elution, if the plates are wrapped tightly to exclude air in polyethylene bags (preferably black) or are kept in a dark place. ELUTION TECHNIQUES Single elution.' Single elution of the spotted plates should be done as soon as the spot- ting solvent has evaporated. A spotted plate cannot be dried at an elevated temperature or even at low temperature for very long. It should be developed immediately after drying. If not completely removed, DMF moved all the components until it was sufficiently diluted by the weaker eluting solvent. Aging at room temperature resulted in a dispro- portionate loss of specific components and caused the formation ofpolymeric material. Complete drying caused the destruction of some components. So it was necessary to choose between complete drying and leaving spotting solvent on the plate. These studies indicated that it was harmful to leave some spotting solvent rather then to dry completely.

THIN-LAYER CHROMATOGRAPHY 267 Two dimensional,' Two-dimensional chromatography was most useful for compositions in which individual members differ qualitatively in their response to solvents, e.g., acidic and basic amino acids. This was not the case with oxidative dyes. Any increase in solvent activity increased the mobility of all the components. No solvent compositions was found which specifically favored slow-moving components. Continuous elution.' This method was used in order to obtain single components in sufficient amount for structural determination. Continuous elution was made in an ap- paratus* (Fig. 2). Oxidation product was applied as a band at the bottom of the plates. The lids on the developing tank were positioned to form slots 4 nm wide. Plates were allowed to stand in the solvent with the upper ends projecting into the free at- mosphere. The solvent moved and evaporated at slot levels as a continuous process, so that all components, except the undesirable polymeric uneluted product, were rede- posited at lid level as a narrow line. The plate was reversed so that the streak became the origin and developed again. Two separate functions can be achieved using this method. First, broad areas of sample can be converted into hairline streaks thus, sub- sequent runs provide better separation. Second, the running length of a plate is mul- tiplied many times, which enables even the most slow-moving fractions to be separated effectively. Continuous elution was found to be more advantageous than the repeated elution tech- nique. In addition to eliminating time-wasting multiple developments, it required no continuous attention. Furthermore, in repeated elution technique, the plate had to be dried after each step. During each exposure to air and humidity, some components un- dergo polymerization, which leaves a brown residue. ISOLATION PROCEDURE From the complex mixtures, a few components were isolated and purified for structural determination by spectroscopic methods. The procedure used is shown in Fig. 3. The total dye mixture, obtained as a solid material after filtration, was applied as a band on a 2-ram preparative plate and developed a number of times. After maximum resolu- tion was obtained, single component bands were scraped from the plate and were ex- tracted with methanol at room temperature. This extract was centrifuged to remove silica gel and then filtered to remove any remaining silica. The extracts were evaporated immediately to reduce the extent of "polymerization," and then were purified by spotting on analytical plates. This process was repeated several times. Despite all precautions, single components after 3 elution and recovery cycles consistently showed the same nonmoving residue at the origin, demonstrating the chemical sensitivity of the dyes. Although column chromatography can be used for the separation of the components in large amounts, trailing after the first few components was more severe than with thin- layer plates. For this reason, column chromatography was useful as a preliminary enrichment of components for subsequent thin-layer separation. *Shundon Southern Instrument Co., Inc., 515 Broad Str, Sewickley, PA 15143. Cat. #SAB-2852.