Itlethods for Testing Stains 299 



Sudan III, C. I. No. 248 



Identification: Sudan III is beiizene-azo-beiizene-azo-2-naphthol, C22H16N4O. It 

 is identified by the following method: Dissolve 50 mg. in 250 ml. of benzene. Dilute 

 2 ml. of this solution to 50 ml. with benzene. Read in Beckman spectrophotometer. 

 Absorption maximum 508-511 m/x; ratio P-15/P+15 is from 0.97 to 1.01. 



Method of Analysis: The low solubility of this dye interferes with its determina- 

 tion, and it is therefore sulfonated before titration. Weigh about 1 g. of dye into 

 a 125 ml. beaker-flask, add 25 ml. of 23% fuming sulfuric acid, and heat on the steam 

 bath for about one hour. Allow the mixture to cool and pour into 500 ml, of dis- 

 tilled water in a one-liter volumetric flask. After cooling to room temperature, the 

 solution is brought up to the mark.* Pipette a 100 ml. aliquot of the sulfonated so- 

 lution into the titration flask, add 100 ml. of distilled water and 30 g. of sodium acid 

 tartrate, heat to boiling, and titrate with 0.05A'^ TiClj. The color change near the 

 end point is very gradual, going from orange to a lemon-yellow. The disappear- 

 ance, however, of the last trace of orange is easily detected. The following data 

 are used in calculating the percentage of anhydrous dye in the original sample: 



Molecular weight 352.380 



Hydrogen equivalents per mol of dye 8. 



ml. of N/10 TiCla per gram of dye 227.026 



Samples of this stain should contain not less than 75% anhydrous dye. 



Biological Tests: Sudan III is tested as a fat stain. The method employed is the 

 same as that given under the following dye. Sudan III, however, is not expected to 

 give as intense a coloration as Sudan IV. 



Sudan IV, C. I. No. 258 



Identification: Sudan IV is o-toluene-azo-o-toluene-azo-2-naphthol, C24H20N4O. 

 It is identified as follows: Dilute 1 ml. of sulfonated solution of Sudan IV to 200 ml. 

 with distilled water. Sulfonating procedure is outlined under Sudan III. Read in 

 Beckman spectrophotometer. Absorption maximum 522-529 myu; ratio P— 15/P-|-15 

 is from 0.9i to 0.99. 



Method of Analysis: The low solubility of this dye interferes with its determina- 

 tion, and it is therefore sulfonated before titration, by the same procedure as that 

 outlined for Sudan III. The percentage of dye in the original sample is calculated 

 from the following data: 



Molecular weight 380.432 



Hydrogen equivalents per mol of dye 8. 



ml. of N/10 TiCla per gram of dye 210.286 



For certification, samples must contain not less than 80% anhydrous dye. 



Biological Tests: Sudan IV is tested as a fat stain. Frozen sections of formalin- 

 fixed tissue containing fat, or portions of the mesentery of some animal, serve for 

 material. Sections are dipped for an instant in 70% alcohol, stained 2-5 minutes in 



*The red solution obtained on the dilution of the blue sulfonation product should 

 be clear. If the sulfonation has been incomplete, the solution is cloudy. 



