542 PHYSIOLOGICAL CHEMISTRY 



Procedure. Render 50 c.c. of the urine under examination faintly alkaline 

 with sodium carbonate and evaporate to one-third the original volume. Con- 

 centrate to about 10 c.c. on a water-bath, cool the residue, acidify it with a few 

 drops of concentrated 'hydrochloric acid 1 and add plaster of Paris to form a 

 thick paste. Permit the mixture to stand until it begins to "set," then break 

 it up with a stout glass rod having a blunt end and reduce the material to the 

 consistency of a fairly dry coarse meal. 2 Transfer the meal to a Soxhlet appa- 

 ratus and extract with ether for six to ten hours. At the end of this period 

 evaporate the ether-extract either spontaneously or in an air current. Dissolve 

 the residue in water, add a little boneblack, if necessary, filter until a clear 

 solution is obtained and make up the filtrate to a known volume (25 c.c. or less) 

 with water. The /3-hydroxybutyric acid should then be determined by means of 

 the polariscope. Its specific rotation is 24.12. 



This method is satisfactory where the amount of hydroxybutyric 

 acid present is not too small. Errors due to incomplete extraction of 

 the acid are partly counterbalanced by the extraction of other levo- 

 rotatory substances. 



Interpretation. Seepage 541. 



2. Method of Schaffer and Marriott. See page 539. 



Indican 



Ellinger's Method. Principle. This method for the quantitative 

 determination of indican is based upon the principle underlying JafiVs 

 qualitative test for indican. The urine after removal of interfering 

 substances with basic lead acetate is treated with Obermayers reagent 

 to oxidize the indican to indigo. The indigo is extracted with chloro- 

 form, the chloroform evaporated off and the residue titrated with 

 potassium permanganate. The method is not very accurate but is as 

 satisfactory as any. 



Procedure. To 50 c.c. of urine 3 in a small beaker or casserole add 5 c.c. 

 of basic lead acetate solution, 4 mix thoroughly, and filter. Transfer 40 c.c. of 

 the filtrate to a separatory funnel, add an equal volume of Obermayer's reagent 

 A (see page 388) and 20 c.c. of chloroform, and extract in the usual manner. This 

 extraction with chloroform should be repeated until the chloroform solution 

 remains colorless. Shake up the combined chloroform extracts two or three 

 times with distilled water in a separating funnel and complete the purification 

 by extracting with very dilute sodium hydroxide (i: 1000). Remove all traces 

 of alkali by washing with water. Now filter the combined chloroform extracts 

 through a dry filter paper into a dry Erlenmeyer flask. Distil off the chloro- 

 form, heat the residue on a boiling water-bath for five minutes in the open 



1 The residue ^ should give a distinct red color with litmus paper. 



2 Before this is accomplished it may, in some cases, be necessary to add a little more 

 plaster of Paris. 



3 If the urine under examination is neutral or alkaline in reaction it may be made ; 

 faintly acid with acetic acid before adding the basic lead acetate. 



4 For preparation of basic lead acetate solution see Appendix. 



