The Microdetermination of Calcium G. H. ELLIS, Laboratory of Animal Nutrition, Cornell University, Ithaca. N. Y.
D
URIiYG the course of a biological investigation, the
need arose to determine calcium in certain tissues of the rat. I n many instances, the amount of tissue available necessitated a method requiring not more than 5 or 10 gamma of calcium per determination. The technic of Miller and Kirk (5),based on the oxalatepermanganate volumetric method, was tried, but was discarded because of the difficulty in determining the end point in the titration. Lindner and Kirk (2) later surmounted the difficulties encountered in the earlier method by replacing potassium permanganate with ceric sulfate, which is added in excess to the calcium oxalate precipitate dissolved in dilute sulfuric acid. The ceric sulfate not reduced by the oxalate is then determined by titration with standard ferrous ammonium sulfate, using phenanthroline-ferrous sulfate as an indicator.
TABLEI. Yolume of Sample A
COMPARISON OB
Calcium Present
r
h l . 4 ~ ~ 0A-S D
Volume of 0.01 'V Cerate Solution
x
Mean
&lICROMETHODS
Calciuin Found
Deviation froiii Amount Present
Y
%
7.36
This method proved very satisfactory. A somewhat simpler procedure is afforded by the use of hexanitrato ammonium cerate (obtainable from the G. Frederick Smith Chemical Company, Columbus, Ohio), which was suggested by G. Frederick Smith, of the University of Illinois. The cerate solution is relatively stable. -4 0.01 N solution in 1 A' perchloric acid stored in clear glass a t room temperature lost 3 per cent in strength over a period of 4 months. In the titra-
tion of oxalate ion the end point is sharp, and the titration can be carried out a t room temperature. The technic of the calcium determination is essentially that of Lindner and Kirk, with the exceptionof the titration of the calcium oxalate, which is dissolved in about 1 ml. of a 3.5 er cent solusolution of tion of perchloric acid and titrated with a 0.01 hexanitrato ammonium cerate which is 1 N n-ith perchloric acid. Orthophenanthroline ferrous complex (0.0005 Izr) or setopaline C ( 1 per cent aqueous solution) is a satisfactory indicator, particularly the latter, since it uses up practically no cerate solution. When calcium is precipitated in the presence of proteins the calcium oxalate crystals are very small and, in spite of long digestion losses of the precipitate through t'he filter stick wed by lliller and Kirk, are practically unavoidable. To overcome this difficulty, the precipitate was separated by centrifuging. The precipitation was carried out in pointed tubes of 1- or 2-ml. capacity. After standing for an hour or more the tubes were centrifuged, the supernatant liquid was drawn off by means of a long-tipped medicine dropper, and the precipitate was washed once or twice with ammonium hydroxide solut'ion (1 to 9) saturated with calcium oxalate. The washed precipitate wa5 then dissolved as above, transferred to a 1- or 2-ml. beaker, and titrated. When the material is ashed as recommended by Lindner and Kirk, either filtration or centrifugation may be used for the isolation of the precipitate of calcium oxalate.
%
Table I gives a comparison of the micromethod with the macromethod of Ilramer and Howland (1). The calcium present in a solution of bone salts was determined by the macromethod, and after suitable dilution samples were taken for microanalysis.
Literature Cited (1) Kratner, B., and Howland, J., J . B i d . Chem., 68, 711 (1926). (2) Lindner, R., and Kirk. P. A , Mikrochemie, 22, 291 (1937). (3) Miller, R. P., and Kirk, P. L., Ibid., 14, 15 (1933). KECEIYEDSeptember 25, 1937. Btudy supported by funds from the Rookefeller Foundation Grant for Research in Longevity.
Fume Tube for Micro-Kjeldahl Digestions J. S. BLAIR, Research Department, .inierican Can Company-, JIagwood, 111.
For use in such cases, fume tubes (Figure 1,upper) are on the market. These are supposed to carry the acid fumes to an
