164 METHODS FOR THE ANALYSIS OF BLOOD 



again evaporated to dryness and ashed as before. A crystalline 

 material is obtained which is readily soluble in not more than 1 

 c.c. of 0.1 N H 2 SO 4 . (About 0.5 c.c. of 0.1 N sulfuric acid is 

 added and solution of the ash facilitated by warming over the 

 steam bath. The solution is then transferred with a small pipette 

 to the Pyrex tube and the process repeated by using two addi- 

 tional portions of 0.25 c.c. of 0.01 N sulfuric acid.) The solution 

 is transferred to a Pyrex tube 100 mm. in length and 10 mm. in 

 diameter calibrated for 1 and 2 c.c. The tube is heated for a few 

 minutes in the water bath and a drop of 0.01 N potassium perman- 

 ganate is added to prove the absence of any oxidizable material. 

 The pink color should persist for at least one minute. One drop 

 of 0.01 per cent phenolsulfonephthalein is added followed by one 

 drop of concentrated ammonia. The tube is then heated on the 

 water bath to drive off the excess of ammonia. At this point a 

 fluffy precipitate forms. To the tube while hot is added exactly 

 0.3 c.c. of 0.1 N oxalic acid in 0.05 N sulfuric acid. This is added 

 in three portions with shaking after each 0.1 c.c. The fluffy 

 precipitate dissolves and is replaced by a copious fine crystalline 

 precipitate. The reaction is usually acid at this point. If it is 

 not, sufficient 0.1 N sulfuric acid is added until the color is lemon- 

 yellow which corresponds to a pH of 6.4 to 6.6. Heating is then 

 continued for a few minutes. The tube is then cooled and 0.1 

 c.c. of a saturated solution of sodium acetate is added and the 

 tube well shaken. The volume is made up to 2.0 c.c. and 

 the tube allowed to stand several hours or over night. The 

 material is then filtered through hardened filter paper. 1 c.c. of 

 the filtrate is measured into a small beaker. To this, 1 c.c. of 20 

 per cent sulfuric acid (20 c.c. of concentrated acid diluted to 100) 

 is added. (Hydrochloric acid should be avoided as it is oxidized 

 to free chlorine by permanganate.) The beaker is heated on the 

 water bath for a few minutes and then titrated in good daylight to ' 

 a definite pink that persists for at least thirty seconds. For very 

 accurate work the end-point is determined by comparison with 

 an equal volume of water in a beaker of the same size. The amount 

 of permanganate necessary to give the same intensity of color 

 with an equal volume of water is also determined and this volume 

 is subtracted from the titration. A blank determination should 

 also be made on the reagents. For most purposes the subtraction 

 of 0.04 c.c. of 0.01 N permanganate is all that is necessary. 



