CALCIUM IN BLOOD 159 



and the unknown into the 25 c.c., and pour back twice. Stopper 

 and let stand ten minutes. Fill both nephelometer tubes with 

 the standard, set the left side at 32 mm., and take a careful read- 

 ing to be sure that the two sides of the instrument are balanced. 

 Replace the standard on the left with the unknown and read. 

 Care should be taken before reading to remove with a glass rod 

 any bubbles adhering to the walls of the nephelometer tubes. 



Calculation. // the unknown is set at 32 mm. and the standard 

 is read, the reading divided by 4 will equal the number of mg. of 

 calcium in 100 c.c. of blood. Note that calcium is calculated as 

 calcium and not as the calcium oxide. 



Reagents, etc. Trichloracetic Acid Solution, 6.5 Per Cent. 

 This reagent, as well as all other materials, should be tested 

 for calcium before use. 



Filter Paper. As the filter paper used for common laboratory 

 purposes contains considerable calcium, a paper must be selected 

 for filtering the coagulated blood or milk which has been washed 

 in acid. For this purpose Baker and Adamson's paper " A " 

 washed in hydrochloric and hydrofluoric acids was employed. 

 For filtering the reagents absorbent cotton washed first with 

 hydrochloric acid, 10 per cent, then with water, until the wash 

 water is no longer acid to litmus, and finally dried, may be used. 



Indicator, Methyl Orange, 0.1 Per Cent. Dissolve 0.1 gm. 

 of methyl orange in 10 c.c. of alcohol and make up to 100 c.c. 

 with water. 



Nitric Acid, 0.1 N and 0.05 N. The concentrations need not 

 be exact, provided the same strength of acid is used in all steps 

 of the process, including the making up of the standard. For 

 convenience a stock solution of 2 N may be prepared and the lower 

 concentrations made up from this as needed. 



Ammonium Hydrate, 2 N. This need not be accurate. 13.5 

 c.c. of ammonium hydrate, sp. gr. 0.9, made up to 100 c.c, with 

 water will serve the purpose. 



Oxalic Acid, 4 Per Cent. As 1 c.c. of this solution is used to 

 precipitate the calcium in only 2.5 c.c. of blood, it will easily be 

 seen that the excess over the theory is very large. Unless such an 

 excess is present, however, precipitation does not begin promptly, 

 owing to the low concentration of calcium oxalate. 



Sodium Acetate, 20 Per Cent. 20 gm. of crystallized sodium 

 acetate dissolved in 100 c.c. of water. 



