34 MILK ANALYSIS 



and allowing it to remain overnight. Annatto will cause a red- 

 dish-yellow stain on the paper. 



/Leys gives the following method for detecting annatto: 

 50 c.c. of the sample are shaken with 40 c.c. of 95 per cent, 

 alcohol, 50 c.c. of ether, 3 c.c. of water, and 1.5 c.c. of am- 

 monium hydroxid solution (sp. gr. 0.900), and allowed to stand 

 for 20 minutes. The lower layer, which in presence of annatto 

 will have a greenish-yellow tint, is tapped off and gradually 

 treated with half its measure of 10 per cent, solution of sodium 

 sulfate, the separator being inverted without shaking, after 

 each addition. By this treatment the casein separates in flakes 

 which conglomerate and rise to the surface, when the adjacent 

 liquid is tapped off, strained through wire gauze, and placed in 

 four test-tubes. To each of these amyl alcohol is added, and 

 the tubes shaken and immersed in cold water, which is gradually 

 raised to 80. This causes the emulsion to break up, and the 

 alcohol, holding the annatto in solution, to come to the surface. 

 The alcoholic layer is separated from the lower stratum, evapo- 

 rated to dryness, and the residue dissolved in warm water con- 

 taining a little alcohol and ammonium hydroxid. A bundle of 

 white cotton fibers is introduced and the liquid evaporated 

 nearly to dryness on the water-bath. The fiber, which is colored 

 a pale yellow, even with pure milk, is washed and immersed 

 in a solution of citric acid, when it will be immediately colored 

 rose-red if the milk contained annatto. Saffron, turmeric, and 

 the coloring-matter of marigolds do not give a similar reaction. 

 Coal-tar colors may often be detected by the wool-test (see 

 under " Butter"), but Lythgoe has devised the following method, 

 which is satisfactory: 15 c.c. of the sample are mixed in a 

 porcelain basin with an equal volume of hydrochloric acid (sp. 

 gr. i. 20), and the mass shaken gently so as to break the curd 

 into coarse lumps. If the milk contains an azo-color, the curd 

 will be pink; with normal milk the curd will be white or yellow- 

 ish. (See next page.) 



