Determination of Calcium in Biological Materials 1
Wethiod f o r Semimicrodet,erminati,on of
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Cad0
and Radioassay of C a M
WILLIAM P. NORRIS AND BLANCHE J. LAWRENCE Division of Biological and Medical Research, Argonne National Laboratory, Lemont, I1 This study was initiated to investigate methods for radioassay of oaloium-45 and determination of calcium-40 in biological materials. It was found that calcium precipitated as the oxalate ma) be mounted quantitatively by filtration in a manner well adapted to radioaetiv,e counting as well as to further steps in analysis of total calcium. Quantities of calcium-40 varied from 20 y to 120 mg., with identical recoveries in all oases. 1mean recoveries and associated standard errors for calcium-40 and caloium-45 were 99.45 0.13 and 98.54 0.28%, respectively. This combination of te,:hniques results in reduction of effort required for both analyses and at the sa1me time affords increased sensitivity and accuracy in tracer studies with calcium-45. I
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inr. use of radioactive tracers to study dynamic systems, the specific activity-that is, the ratio of tracer isotope to total element-is generally a moat informative quantity. Thus methods which permit the determination of both values necessary to this ratio in a single over-all procedure are most advantageous. Because most methods for calcium determination require isolation a8 the oxalate, i t should he possible to utilize the precipitate for radioassay of calcium-45 prior to the final step in determining calcium-40. Also in counting radioactivity the use of samples having constant chemical composition is desirable, as it eliminates variations in the scatter contribution which occur as a function of the atomic number of the sample. The main problem encountered was that of mounting the calcium oxalate precipitate suitably for counting radioactivity and without introducing interferences in the analysis of calcium-40. The low energy of the beta rays from calcium-45 [Emli.= 0.254 m.e.v. (9)lrequires that the sample be mounted uniformly and reproducibly, in order that appropriate corrections may be made for scattering and absorption of beta rays as a function of sample thickness. The early portion of the self-absorption curve is complicated by self-scattering, a point which has not been adequately demonstrated in many earlier investigations. Good counting conditions have been obtained by mounting calcium oxalate on fritted glass by filtration. The sample may be recovered for total calcium determination by dissolution in acid. The use of hesauitratoammonium cerate as described by Ellis (5)was found preferable to permanganate, as the titration may be done a t room temperature and the end point is sharp. The method may be applied with facility over a wide range of IY
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20 y to 120 mg., 2 to 4 mg. being most convenient. Theamount of calcium45 needed will depend upon the type and characteristics of the counter. I n this laboratory a counting rate of 50 t o 75 counts per second (approximately 0.08 pc.) was considered optimum, as Geiger tube resolution time does not contribute appreciable error below 75 counts per second. To the aliquot add sufficirnt bra in ethyl aI~.iihol,to ncr as an intlic; xeric arid iirr ml. of Iiolurioo. H saturated immonium oxalate soliution su&cient t o Drecioitate : heating for 10 to 15 minutes Nxalate, and then raise the plI d ammonium hydroxide 8 0 1 ~ tiou io ;final value of 4.5 to 5.0. This is indicated bv a vellowgreen color. Allow the precipitate to stand for a t least" 1 hhur before filtration. I n precipitating 20-r quantities of calcium, the final volume, after addition of reagents, should not exceed 1 ml. With 500-7, 1-mp., 10-mr.. and 100-mr. auautities of calcium. convenient
ANALYTICAL PROCEDURE
Figure 1. Filtration Apparatus Used to Collect Precipitated Calcium Oxalate on Fritted Disk of Crucible
Ashing of Samples. Samples of bone or fecal material eontained in porcelain or borosilicate glass are ashed in en electric muffle furneoe a t %temperatureof 550' C. Twenty-four hours is usually sufficient, although the time for complete ashine is dependent upon the size of the sample.
with f d n g Gtric acia and evaporated to dryness. 'The addition of a small quantity of Superasol (30% hydrogen peroxide) during evaporation accelerates ashiug. The process is repeated until the resulting ash is white and free from organic residues. Precipitation of Calcium Oxalate. The ash is dissolved and made np to volume in 1N hydrochloric acid. Because calcium-40 and calcium-45 are both determined in the same aliquot, the aliquot must be estimated so as to bring both values within the range of the method. Quantities of calcium-40 may range from
Filtration and Mounting of Sample for Counting. The apparatus (Figure 1)used in filtration of the sample is a modified version of that used by Anthony (1) and by Sacks (12).
The stopcock fitted with ball joints forms au adapter useful in controlling suction and in compensating for variations in height of the filter flasks. The filter is made from a 30-ml., high-form, fine-porosity, Gooch crucible (Corning Catalog No, 32940) hy cutting off tbe top 1 em. above the disk. The bottom edge of the crucible is ground flat, so that it will produce a tight seal when placed on the filter apparatus. The effective diameters of the disks are very uniform, the average being 28.2 mm., which .corresponds to an area of 6.25 sq. cm. The precipitated calcium oxalate is collected on the fritted disk 956
V O L U M E 25, NO, 6, J U N E 1 9 5 3
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sides and to remove residual water. Dry the sample a t 100" C. The solid has the composition CaCz04.H20. Counting of Radioactivity. The mounted sample is held in position below a shielded, thin mica window, Geiger tube (ff) using the holder shown in Figure 2. The filter disk of the crucible rests on the Lucite plug, which also functions as a centering device. The distance from the surface of the fritted disk to the mica window of the Geiger tube is 1.7 om. The Geiger tubes (Tracerlab Type TGC-2) have windows 2.8 cm. in diameter with thicknesses of 1.6 to 2.9 mg. per sq. em. Alternatively, the samples may he counted using a proportional counter as shown in Figure 3. Such a counter has the advantages of higher sensitivity and higher resolution, which allow measurement over a wider range of calcium-45 content. The end of the counting volume is closed with a thin (37 y per sq. cm.) nylon film painted on the inside with colloidal graphite (Aquadag, Acheson Colloids Co.). The chamber is flushed slowly during operation With s miu+.,-n A OnW. o - ~ n na n r l I n 4 rnethana
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by filtration using suction. Inasmuch as the precipitate must be uniformly distributed in order to achieve proper conditions for counting, the folloning procedures are advisable. Check to be sure the surface of the filter disk is level (it is convenient to mount the filter assembly on a plate equipped with I
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placedon the filter disk without suction)
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811 washings through the filter.
becessary h deposit it in Ijyer6 allowing eseh'iaver to &ck raiher dry before conhuing). Wash down the i d e s of the crucible gently with 95% ethyl sleohol just before the last of the ammonium hydroxlde wash has filtered. Continue the alcohol wash, being careful not to disturb the precipitate, to remove any material adhering to the
Figure 4.
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R h W E Of MEASUREMENTS GEIGER-MUELLER COUNTER PROPORTlONIL COUNTER
MASS/CM?(M~J
Self-Absorption Curve for Calcium-45
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