738 PHYSIOLOGICAL CHEMISTRY. 



color is seen at the point of union between the two fluids, the colors 

 changing from green to blue, violet-red, and yellow or yellowish- 

 green. While the appearance of the green at the beginning is indis- 

 pensable to prove the presence of bile, the presence of all the other 

 colors is not essential. (Plate VIII., 7.) 



The above test may be made in a somewhat modified form by mix- 

 ing the urine with a concentrated solution of sodium nitrate, and 

 pouring down the sides of the test-tube concentrated sulphuric acid in 

 such a manner as to form two distinct layers ; the colors are seen at 

 the point of contact as above. 



If the urine be very dark in color, it should be diluted with water 

 before applying the above tests. 



2. Add a few drops of sodium carbonate solution to the urine until 

 it has a distinct alkaline reaction, then add calcium chloride and 

 shake well. The precipitated calcium carbonate carries down the 

 pigments and leaves the urine nearly colorless or of its normal color. 

 Collect the precipitate on a filter, wash, and transfer it with alcohol 

 to a test-tube. Dissolve by the addition of hydrochloric acid and 

 boil the clear solution, when it turns green. Allow to cool and add 

 nitric acid, when the green solution turns blue, violet, and red. 

 (This test may show the presence of biliary coloring-matters when 

 Gmelin's test fails to do so, and is recommended when the urine con- 

 tains a large amount of indican.) 



While bile acids are always present with bile-pigment in urine, 

 their demonstration is usually difficult. 



Pettenkofer's test for biliary acids is made by dissolving a few 

 grains of cane-sugar in urine contained in a test-tube, and allowing 

 concentrated sulphuric acid to trickle down the side of the inclined 

 test-tube ; a purple band is seen at the upper margin of the acid, and 

 on slightly shaking the liquid becomes at first turbid, then clear, and 

 almost simultaneously it turns yellow, then pale cherry-red, dark 

 carmine-red, and finally a beautiful purple violet. The temperature 

 must not be allowed to rise much above 38 C. (100 F.). 



As many substances (other than biliary acids) show a similar 

 reaction, it is often necessary to separate the bile acids by the process 

 described in connection with the consideration of bile itself. 



In case the quantity of biliary constituents is so small that they 

 cannot be noticed by the tests mentioned, the urine should be shaken 

 with about one-fourth of its volume of chloroform, which dissolves 

 the biliary matters. Some of this solution is dropped upon blotting 

 paper, and after evaporation a drop of red fuming nitric acid is 



