374 PHYSIOLOGICAL CHEMISTRY 



or the ether-acetone filtrate from the lecithin may be used for the isolation of 

 cholesterol. In these cases it is simply necessary to evaporate the solution to 

 dryness on a water-bath.) Upon the cholesterol prepared by either of the above 

 methods make the following tests : 



(a) Microscopical Examination. Examine the crystals under the microscope 

 and compare them with those in Fig. 63, page 213. 



(b) H 2 SO 4 Test (Salkowski). Dissolve a few crystals of cholesterol in a 

 little chloroform and add an equal volume of concentrated sulphuric acid. A 

 play of colors from bluish-red to cherry-red and purple is noted in the chloro- 

 form, while the acid assumes a marked green fluorescence. 



(c) Acetic Anhydride-H 2 SO4 Test (Liebermann-Burchard). Dissolve a few 

 crystals of cholesterol in 2 c.c. of chloroform in a dry test-tube. Now add 10 

 drops of acetic anhydride and 1-3 drops of concentrated sulphuric acid. The 

 solution becomes red, then blue, and finally bluish-green in color. 



(d} Iodine-sulphuric Acid Test. Place a few crystals of cholesterol in one of 

 the depressions of a test-tablet and treat with a drop of concentrated sulphuric acid 

 and a drop of a very dilute solution of iodine. A play of colors, consisting of violet, 

 blue, green, and red, results. 



(e) Schiff's Reaction. To a little cholesterol in an evaporating dish add a few 

 drops of a reagent made by adding i volume of 10 per cent ferric chloride to 3 vol- 

 umes of concentrated sulphuric acid. Evaporate to dryness over a low flame and 

 observe the reddish-violet residue which changes to a bluish-violet. 



(/) Phosphorus. Test for phosphorus according to directions given in Chapter 

 VI, page 128. Is phosphorus present? 



3. Preparation of Cerebrin. Treat 100 grams of finely divided brain tissue, 

 in a flask, with 200 c.c. of 95 per cent alcohol and boil on a water-bath for one- 

 half hour, keeping the volume constant by adding fresh alcohol as needed or by 

 the use of a reflux condenser. Filter the solution hot and stand the cloudy 

 filtrate away for 24 hours. (If the filtrate is not cloudy concentrate it upon the 

 water-bath until it is so.) Filter off the cerebrin (cerebrin, lecithin, kephalin, 

 cholesterol) and test it as follows : 



(a) Microscopical Examination. Suspend a small portion in a drop of water 

 on a slide and examine under the microscope. 



(b) Solubility. Try the solubility of cerebrin in water, 10 per cent sodium 

 chloride and in dilute acid and alkali, and in hot and cold alcohol and hot and 

 cold ether. 



(c) Phosphorus. Test for phosphorus according to directions in Chapter 

 VI, page 128. How does the result compare with that on lecithin? 



(d) Place a little cerebrin on platinum foil and warm. Note the odor. 



(e) Hydrolysis of Cerebrin. Place the remaining cerebrin hi a small evapo- 

 rating dish, add equal volumes of water and dilute hydrochloric acid, and boil 

 for one hour. Cool, neutralize with solid potassium hydroxide, filter, and 

 test with Fehling's solution. Is there any reduction, and if so how do you 

 explain it? 



(4. Tests for Choline. (a) Rosenheim's Periodide Test. Prepare an alcoholic 

 extract of the fluid under examination, and after evaporation apply Rosenheim's 

 iodo-potassium iodide solution 1 to a little of the residue. In a short time dark 



1 Prepared by dissolving 2 grains of iodine and 6 grams of potassium iodide in 100 c.c. 

 water. 



