156 METHODS FOR THE ANALYSIS OF BLOOD 



minutes, after which they are transferred to the cups of the color- 

 imeter (Duboscq) and compared as usual, setting the standard 

 at 15 mm. The cement of the colorimeter cups must, of course, 

 not be soluble in chloroform; plaster of Paris has been found 

 satisfactory, or even ordinary glue if the cups are not used for any 

 other purpose. The error of the above method when carried out 

 with ordinary care is 4 to 5 per cent. If greater accuracy is desired 

 it may be obtained, at the expense of more material and tune, by 

 using 50 c.c. of the alcohol-ether extract, evaporating as above, 

 extracting with larger quantities of chloroform, making the extracts 

 to 25 c.c., and taking an aliquot of 5 c.c. for the determination. 



CHOLESTEROL 



Lichtenthaeler Modification of the Method of Autenrieth and Funk 1 



Principle. The blood or serum is boiled with strong alkali to 

 saponify the fats. The alkaline solution is extracted with chloro- 

 form to separate the cholesterol. After being purified, dried and 

 clarified, the chloroform extract is treated with sulphuric acid 

 and acetic anhydride, and the characteristic blue-green color of 

 the Liebermann-Burchard test for cholesterol is thus obtained. 

 The color is then compared, in a colorimeter, with that produced 

 by a known amount of cholesterol. 



Procedure. With an accurate pipette transfer 2 c.c. of whole 

 blood, serum or corpuscles to a 100 c.c. Erlenmeyer flask provided 

 with a straight tube reflux condenser, and into which has pre- 

 viously been introduced 20 c.c. of a 25 per cent potassium hydroxide 

 solution. Heat on the water-bath, ^baking frequently, continuing 

 the digestion until the liquid is colorless or greenish and 'contains 

 suspended solids derived from certain decomposed substances. 

 This usually requires from three to six hours. Transfer the 

 undiluted mixture to a separatory funnel and add 25 to 30 c.c. 

 of chloroform. Shake vigorously for fifteen minutes and separate. 

 Extract with three additional 25 c.c. portions of chloroform, 

 shaking from five to ten minutes each time. Return the combined 

 chloroform extracts (which are turbid and usually colorless but 

 may be greenish or brown) to the separatory funnel. Wash once 

 or twice by shaking with 15 c.c. portions of distilled water (shaking 

 1 Autenrieth and Funk: Munch. Med. Woch., 1913, 60, 1243. 



