2 94 PHYSIOLOGICAL CHEMISTRY 



alizarin indicator solution (0.2 per cent). Titrate back until faintly acid with 

 approximately o.5/N hydrochloric acid. McCrudden's method is then followed. 

 Add from a burette 2.5 c.c. of the hydrochloric acido.5/N mentioned above and then 

 the same amount of 2.5 per cent oxalic acid. To the boiling solution add dropwise 

 in two portions 2.5 c.c. of 3 per cent ammonium oxalate. Digest at near boiling 

 for 15 minutes. 



Cool in ice water to room temperature or lower. Add another drop of alizarin 

 and also dropwise from a burette 2.5 c.c. of 20 per cent sodium acetate solution 

 while rotating the flask, or if found necessary add until the alizarin just begins 

 to change color. Allow to stand over night (or at least 4 hours after 10 minutes' 

 shaking). 



Transfer completely to a 50 c.c. round bottom centrifuge tube with the aid 

 of a little water and whirl for about 3 minutes at 1500 revolutions per minute. 

 With an automatic siphon draw off the supernatant liquid at first rapidly and then 

 gently to within a drop or two. Wash with cold distilled water (i5-2oC.) first 

 adding about 20 c.c., washing down the sides and rotating the tube. Then add 

 more water from the wash bottle to within about 1.75 cm. of the top of the tube 

 (approximately 50- c.c.). Placing the metacarpal portion of the palm of the hand 

 at the thumb over the mouth of the tube shake vigorously for 5 to 10 seconds. 

 Centrifuge again. Siphon off to within one or two drops and titrate. 



C. Titration. To the precipitates in 50 c.c. centrifuge tubes add, with shaking, 

 4 c.c. of 5 per cent sulphuric acid (very faintly tinged with potassium permanganate). 

 Place in a water-bath at 65C. until the tubes approach the temperature of the bath. 

 Remove and titrate rapidly with an approximately o.oi33/N potassium per- 

 manganate solution, 1 with moderate shaking using a white background. A 

 burette of 10 c.c. capacity with which readings can be readily estimated to o.oi 

 c.c. is desirable. 



The end point is attained when a faint but definite pink color persists for a 

 minute or longer on gentle shaking and standing. If the precipitate has not been 

 contaminated the end point will be sharp to o.oi c.c. The sulphuric acid must be 

 brought in contact with all parts of the tube as far up as the original solution ex- 

 tended. The burette reading should be corrected for the small amount of per- 

 manganate required to titrate 4 c.c. of the sulphuric acid to the same end point. 



D. Calculation. i c.c. of b.oi33/N potassium permanganate is equivalent to 

 0.267 m g- of calcium. The exact factor for a given solution must be determined 

 by standardization. Multiply the number of c.c. used by this factor to obtain 

 the amount of calcium in the 25 c.c. of filtrate. If blank on reagents is not negligible 

 deduct. Multiply by 28 to get mg. of Ca per 100 c.c. of serum or plasma. 



Nephelometric Methods 



The Nephelometer. The nephelometer is an instrument for 

 measuring the density of precipitates and thus determining the amount 

 of any substance which can be obtained in the form of a suitable 

 suspension. It is somewhat similar in form and principle to a colori- 

 meter. It differs from the latter in that the light which reaches the 

 eye is not transmitted light, which, on the contrary, is excluded, but 



1 For the method of preparing this dilute standard permanganate solution see Halverson 

 and Bergeim: Jour. Ind. Eng. Chem., 10, 119, 1918. 



