METHOD FOR IDENTIFICATION OF AZO DYES 239 A certain margin of error should be allowed for the reported Ra values. In our laboratory, the variations generally did not exceed the value of 4-0.02 in work with solvents 2, 3 and 4 for solvent 1, the errors are also low for the compounds with high or low Ra, but for any dyes of intermediate position on the chromatogram deviations as great as 4-0.05 were sometimes observed. As can be seen in the table, Rz values and shades of the 35 dyes tested vary enough to achieve the desired accuracy almost completely. With the exception of three pairs of dyes, all mixtures can be easily resolved by this method for practical purposes. The three exceptions mentioned are the following mixtures: 1. Metanil Yellow d- Orange IV 2. Sunset Yellow FCF d- Orange GGN 3. Ponceau MX d- Ponceau 3R Mixtures 1 and 2 can be separated by means of supplementary develop- ment with 50 per cent phosphoric acid and 7 N hydrochloric acid respdc-tively. In both cases, an elution of 30 cm. is recommended, and a standard solution of one of the two dyes must be spotted together with the unknown. Orange IF and Sunset Yellow FCF are always higher in position than their homo- logues, Metanil Yellow and Orange GGN, with an average difference range in Rz value near or greater than 0.10. )YES INVESTIGATED ut fluorescence Orange III over the standard, orange (violet in HC1), without fluorescence Orange IV under the standard or more frequently at its level, orange (violet in HC1), Metanil Yellow hour fluorescence Spot over (1), orange, without fluorescence Orange RN Development with 2% Sodium Carbonate in 3 N ztmmonia Without equilibration 35-38 cm. length. Standards: (1) Anthosine B ] (2) Orange III Spot between (1) and (2), orange, without fluores- cence Orange I1 Spot at the level of (2), orange, without fluorescence Monosol Orange o Spot under (2), orange, with 1-2 subsidiary spots Xylidine Orange petween (1) and (2), orange (reddish pink in NHa), with little and dull yellow •rescence under (2) or at its level, brown (orange in NHa), with orange fluorescence it the same height as Anthosine B, red pink (orange in NHa), without fiuores- ce :onsiderably higher, red (yellower in HC1), without fluorescence Orange I Neptune Brown RX Anthosine B Roccelline (Continued)

TABLE Development wi th Phenol Saturated S t with Water , po Equilibration with ½•e,tween, the same solvent for about 12 hours t ) 30-35 cm. length. Standards: (1) Anthosine B (2) Azogrena- dine S __ Spot under (2) Development with 1.5% Sodium Iodide Without equilibration: 32-35 cm. length. Standards: (1) Acid Yellow G (2) Orange III Development with 6.5 N Hydrochloric •icid •Vithout equilibration 28-30 cm. length neutralization with ammonia vapors. Standards: (1) Orange G (2) Sunset Yellow FCF (3) Red 6B Spot over (1) bright orange, without fluores Spot between (1) and (2) Spot between (2) and the starting line Development with 1% Sodium Carbonate Without equilibration 30-32 cm. length. Standards: (1) Orange G (2) Victoria Violet 4BS (3) Anthosine B (4) Orange III Development with ButanoLEthanol- Conc. NH,• (5:3:3) Equilibration with the same solvent for about 12 hours. Standard: Red 2G Spot in the starting line, red, with intense Spot over (1), yellow (orange red in HC1), wi Spot between (1) and (2), violet (pink in Spot between (2) and (3) Development with 1% Sodium Carbonate Without equilibration 30-35 cm. length. Standards: (1) Orange G (2) Victoria Violet 4BS I (3) Orange III Spot under (3) or at the height of (3): ' Consult the chromatogram developed wit Phenol Saturated with Water