, n ued ) e red (yellowet in Na2CO3), without fluorescence id (2), red violet (paler in Na:CO.0, without fluorescence Development with k5 N Hydrochloric ,'Icid put equilibration 35-38 cm. neutralization with NHa. Standards: [1) Victoria Violet 4BS [2) Sunset Yellow FCF [3) Red 2G Orange G Scarlet GN Lanafuchsin 6B Spot over (1), yellowish orange, without fluo- rescence Resorcine Yellow Spot between (2) and (3), yellowish orange, without fluorescence Spot at the level of (2), orange, without fluo- Sunset Yellow rescence FCF Orange GGN Spot at the level of (3), red, without fluores- cence Red 2G ld (4), almost at the height of both spots, red, without fluorescence Crystal Ponceau it the same height as Anthosine B, red pink (orange in NHa), without fluores- Anthosine B ce •t much lower: •elopment with odium Carbonate • N .4mmonia •ut equilibration 38 cm. length. Spot in the starting line, Spot over standard: De- velopment with $ N Per- chloric ,'Icid wi thou t equi- libration and standard. 12-15 cm. length. Neu- tralization with ammonia vapors violet red (red orange in NHa dark blue in HC104), non fl. Spot in the first segment of the chromatogram, red (yellower in NHa), with- out fluorescence Spot with a RS lower than 0.1, Benzyl Bordeaux B Fast Red E Acid Bordeaux Standard: Orange II ] Spot under standard: Consult the chromatogram developed with 1.5% Sodium Iodide violet red, nonfluorescent Spot longer than the former, with a RS always higher than 0.1 in the NaI chromatogram, red, with a brick or dull red fluo- rescence. 1 to 5 Secondary spots. elopment with odium Carbonate 3 N ,'Immonia •ut equilibration 35 cm. length. I Standard: Orange II[ Spot over standard: Consult the chromato- gram developed with Phenol Satur. with IFater Spot at the same height of Azogrenadine S, blood red, with a dull red fluorescence Spot considerably higher, ma- genta red (yellower in NH•) with a dull red fluorescence Spot under standard, red, with a dull red fluorescence Ponceau MX Ponceau 3R Azogrenadine S Red 6B Carmoisine •orescence Red FB scence Acid Yellow G at fluorescence Victoria Violet 4BS - •ver (1), crimson red, without fluorescence Ponceau 6R Between (1) and (2), yellowish red (paler in Na2COa), without fluorescence Ponceau 4R Yellow 271•75 N vetween (2) and (3), earthy yellow (orange yellow in Na,oCOa), without fluo- tenca ander (3), violet red with a dull red fluorescence •t the same height of Azogrenadine S, blood red, with a dull red fluorescence Red 10B Azogrenadine S :onsiderably lower in height, violet red, without fluorescence Amaranth

242 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Separation of mixture 3 was not possible under our conditions however, a few commercial samples of Ponceau MX can be identified by the presence of a very noticeable secondary spot, which is attributed to a characteristic impurity (see Table 3). Orange I and Orange II also show similar Rs values in the four recom- mended solvents, but the former has a characteristic pink color on fresh chromatogram 4, whereas the latter remains orange. If a complete separation of these two colors is desired, a new elution of 25 cm. with Butanol-Ethanol-Concentrated NH• (5:3:3) after equilibration of at least twelve hours will effect separation. In this solvent migration of Orange I is greatly retarded (see alkaline two-phase solvents), and Orange lI will precede it with an average difference in Rs value greater than 0.30. Secondary Spots. Many of the tested dyes showed secondary spots (Table 3), and in some instances, the same compound yielded different sub- sidiary spot spectra according to its commercial origin. The presence of these colors on the chromatogram is no serious obstacle for the interpretation of results since the intensity of their spots is generally very faint, and sometimes such spots are hardly visible. On the other hand, some of the secondary spots that are always present in tested samples, can help identify such compounds as Xylidine Orange, Ponceau MX and Ponceau 3R. From the tabulated results it may be concluded that, in most instances, the presence of secondary spots can be attributed to common impurities of intermediates or to different degrees of sulfonation during the preparation of the principal dye. In a few cases, however, it must be admitted that the secondary color is never related to the principal one (i.e., some samples of Ficloria Fiolet 4BS), and, therefo-e, this compound could have been added intentionally to maintain the standard shade of the commercial dye. Syslematic Method (Flow Sheet) This second analytical scheme is based on the successive use of different solvents until the isolation of a single dye from the other 34 has been achieved. Only ten of the tested dyes are necessary to carry out the procedme: 1. Azogrenadine S 6. Sunset Yellow FCF 2. Anthosine B 7. Red 2G 3. Victoria Violet 4BS 8. Red 6B 4. Acid Yellow G 9. Orange Ill 5. Orange G 10. Orange II These dyes are purified by wool extraction according to the previously described method this extraction, however, appears to be unnecessary if the colors are of high purity. These dyes will act as standards, and the position of all the monoazo