172 METHODS FOR THE ANALYSIS OF BLOOD 



taking care not to heat so strongly that the tube approaches 

 dryness, in which case loss of phosphoric acid may occur. The 

 sulfuric acid solution should now be clear and colorless. If it is 

 brownish in color, a drop of HNOs should be added and the heat- 

 ing continued for one minute. In the third stage the mixture is 

 allowed to cool somewhat (for about two minutes) and then one or 

 more drops of 1 per cent cane sugar solution is added. (The amount 

 added should be enough to produce a deep browning of the hot 

 solution and the color should disappear when it is boiled. If too 

 much sugar solution had been added and the brown or yellow 

 color persists after a half minute of boiling, a trace of nitric acid 

 should be added and the boiling continued.) The solution is then 

 boiled until the moisture is gone about one minute then cooled 

 and about 10 c.c. of water are added, rinsing down the sides of the 

 tube. The solution in the tube is neutralized by approximate 

 titration with 10 per cent NaOH (from sodium) using one drop of 

 0.3 per cent phenolphthalein as indicator, noting the amount of 

 alkali added, then made Just acid with a drop- or two of dilute 

 sulfuric acid (25 per cent). It is then cooled, transferred quanti- 

 tatively to a 25 c.c. glass-stoppered graduated flask, the tube 

 rinsed several tunes with water and the washings added to the 

 flask, and the volume made up to the mark with water, and the 

 whole well mixed. 



Phosphate Reagent. Sodium Molybdate. Prepared either 

 according to the direction of Kober and Egerer or, more simply, 

 in the following way. 72 gms. of molybdic acid are mixed with 

 about 300 c.c. of water and neutralized with 40 per cent sodium 

 hydroxide (free from all but traces of phosphates). Pure acid 

 requires the theoretical amount of 100 c.c., impure samples 

 require less. The molybdate, now in clear solution, is boiled for 

 about a half hour, adding water to keep the volume constant and 

 alkali if the solution becomes turbid. About 1 gm. of talcum 

 powder is added and after a further five minutes boiling the solu- 

 tion is filtered and the filter washed once with hot water, adding 

 the washings to the main filtrate. After cooling, the solution, 

 containing approximately 100 gms. of sodium molybdate, is ready 

 for use. Most of the molybdic acid now available contains ammo- 

 nia, which is volatilized during the boiling and allows some of the 

 acid to precipitate, hence the need of more alkali during the 

 boiling. 



