220 CALCIUM 



acid and evaporated to dryness. Small quantities of 30 per cent H2O2 

 may be added during evaporation, and the process is repeated until the 

 resultant ash is white and free of organic residues. The ash is dissolved in 

 2 A'' HCl, and an aliquot, equivalent. to less than 200 mg of ash, is trans- 

 ferred to a 50-ml centrifuge tube. One millihter of saturated (NH4)2C204 

 is added to the solution in the centrifuge tube, then 2 drops of 0.05 per 

 cent methyl red, and 4 ml acetic acid (1+4) with thorough mixing. 

 With constant stirring, NH4OH (1 +4) is added until the solution is 

 faintly alkaline, and then a few drops of acetic acid is added until the color 

 is adjusted to a faint pink (pH 5.0). The solution is allowed to stand 

 overnight for complete precipitation of the calcium oxalate. The solu- 

 tion is then centrifuged, and the supernatant can be used for phosphorus 

 determination if desired. The precipitate is dispersed in about 10 ml 

 NH4OH (1 + 49) and recentrifuged. The precipitate is then treated 

 with 2 ml H2SO4 (1 +4), heated to 80 to 90°C, and titrated with 0.02 N 

 KMn04 according to the usual practice. A standard amount of carrier 

 calcium, usually 8 mg, is added, and the solution neutralized w^ith NH4OH 

 (1 +4). Three milliliters of saturated (NH4)2C204 is added, and the 

 calcium reprecipitated as described above. A better precipitate is 

 obtained if the NH4OH is added until the calcium oxalate just begins to 

 appear and the solution is then heated at about 80°C for a few minutes; 

 the neutralization is carried on as above and the precipitate is collected 

 in the plastic-tube-and-cup assembly by centrifugation, as described in 

 Chap. 5. If chemical analysis is not required on a particular sample, 

 there is no need to reprecipitate. In this case the ash solution aliquot is 

 treated with 8 mg carrier calcium, 3 ml of saturated (NH4)2C204, and the 

 precipitation is carried out as already described, with collection of the 

 precipitated calcium oxalate for radioassay. 



BONES AND TEETH. When large amounts of material are available, a 

 representative fresh sample of at least 2 g is weighed into a tared porcelain 

 crucible and ashed in the muffle furnace; it is seldom necessary to re-ash 

 these samples. The ashed bone is ground directly in the crucible with a 

 pestle, and after redrying, exactly 500 mg is weighed out and made to 

 50 ml with dilute HCl. AHquots of 5 ml are used for the precipitation of 

 the calcium oxalate, and the precipitate is collected in the plastic-tube- 

 and-cup assembly as described. The weight of the carefully dried oxalate 

 collected in the metal cup is used to calculate the total calcium and the 

 self-absorption correction. This procedure of ashing a large amount of 

 bone and selecting an ahquot of the ground ash has been found quite help- 

 ful in permitting representative sampling of large bones and sections 

 thereof. With small samples of bone, as from rats, the total ash is made 

 to a given volume with dilute acid, and an aliquot employed which will 

 represent approximately 50 mg of bone ash. 



