V O L U M E 26, NO. 6, J U N E 1 9 5 4
1031
method of Crout (1). The application of the method to Equations l, 2, 3, and 4 results in the following series in which the pressure can be calculated directly.
P B ~= H -0.58 ~
04.05
+ 0.52 D4.e~- 3.49
Da.54
+ 14.81
D6.15
pressure. made :
The following data illustrate the kind of analysis
Measured total pressure of unknown, 100.9 mm. a t 27' C. Wave Length, 4.05 4.65 5,54 6.15
(5)
Compound
p
Absorbance 0.464 0.133 0.499 0.658 Calculated Pressure, h l m 7.8 7.0 80.8 6.4 Total 1 0 2 . 0 ~
The minimum detectable amount of diborane is about 0.2 mole
%, while for tetraborane it is about 0.6 mole %, and for dihydropentaborane, it will run about 0.7 mole yo. LITERATURE CITED
The application of these equations to unknown, impure samples of pentaborane vapor, which may contain small amounts of hexaborane not accounted for by this work, usually results in calculated pressurrs the sum of which is within 2% of the measured
(1) Crout, P. D., Trans. Am. Inst. Elec. Engrs., 60, 1235-40 (1941). CHEM.,19, 293 (2) Heigl, J. J., Bell, M. F., and White, J U., Ax.4~. (1947). (3) S o r t o n , F. J., private communication. (4) Pearson, T. G., 2. p h y s . Chem., A156, 86 (1931) R E C E I V E Dfor review November 17, 1953. Accepted hlarch 20, 1954. Work done for Army Ordnance Contract TUI-2000a.
Radiochemical Assay of Cobalt-60 ROBERT BALLENTINE and DOROTHY D. BURFORD McCollum-Pratt hstitute, Tho Johns Hopkins University, Baltimore 18,
A method of determining cobalt-60 in biological materials has been found accurate within A370 in over a thousand determinations. The sample was wet-ashed, separated as cobaltic hydroxide, and electroplated as cobalt metal from a fluoborate buffer. A special methane-flow proportional counter of cylindrical geometry is used so that a maximum precision and sensitivity of detection of cobalt-60 are obtainable. Aside from the biochemical and clinical applications of this assay, the data on the quantitative electroplating of cobalt are applicable to its standard gravimetric determination.
C
U R R E S T interest in the chemical and biological reactions of cobalt has directed attention to the use of the isotopic tracer, cobalt-60. The recent work on vitamin B12 has made this tracer of considerable biochemical and clinical importance. Cobalt-60, with a half life of 5.3 years, has many properties which should make it a very favorable isotope for experimentation. Not only is it available with high specific activity, but during decay, it emits a beta particle of 0.31 m.e.v. and two gamma rays of 1.17 and 1.33 m.e.v. energy. The major difficulty in its use has been the preparation of samples for radiochemical assay. The method employed should be both sensitive and precise. Early work on cobalt in biological systems utilized the mixture of radioactive isotopes which emitted a mixture of gamma rays, producpd with the cyclotron. Sheline, Chaikoff, and Montgomery (IO) simply dried their samples, but Comar and Davis ( 4 )first subjected their material to dry-ashing, followed by counting with a dipping counter placed in a solution of the ash. Comar, Davis, and Taylor ( 5 ) utilized electrodeposition of the cobalt; because they were only detecting gamma rays, they found no advantage in the more elaborate procedure. Others have used a combination of dry-ashing and precipitating as cobalt sulfide (6).
Md.
These techniques applied to cobalt-60 either suffer from the low sensitivity of gamma ray detection or the lack of precision when beta particles are counted in dried unprocessed samples. The serious interference of both iron and the alkaline earths complicated the plating method of Comar and Davis (6), but was avoided in biological samples by an involved and time-consuming procedure. Ballentine and Stephens ( 1j precipitated cobaltic hydroxide from wet-ashed material, detecting the beta emission with a highly efficient and accurate methane-flow beta counter ( 2 ) . They reported an error of 4 ~ 2 . 3 5 %but ~ in routine use unaccountable disagreements, as high as 25%, between duplicates are occasionally observed. An attempt to perfect a precise and dependable assay has led to the procedure described. This method has been in routine use on a thousand samples of various biological materials, covering a period of more than a year, without any of the erratic results previously experienced, and with an over-all accuracy of 4Z 97%. The necessity of controlling a number of conditions has led to the following analytical sequence : wet-ashing with perchloric and nitric acids, collection of the cobalt carrier as cobaltic hydroxide and resolution as the chloride, electrolytic plating as cobalt metal from a potassium fluoborate buffer, and counting the beta particles in a methane-flow counter of high efficiency and reproducible geometry. RESULTS AND DISCUSSION
Ashing Procedure. The original procedure ( 1 ) employed a sulfuric-nitric acid digestion. This has been abandoned in favor of a perchloric acid digestion, because it is much smoother and more rapid. However, its main advantage is in the reduction of the amount of neutral salts resulting from the neutralization in a later step. With the sulfuric-nitric acid digestion, it was necessary to employ a large dilution to prevent both the crystallization of sodium sulfate and the production of such a high solution
ANALYTICAL CHEMISTRY
1032 density that the cobaltic hydroxide precipitates could not be collected by centrifuging. These difficulties are all avoided with the perchloric acid digestion, which should present no hazard with reasonable precautions. Preliminary Separation of Cobalt. Cobalt carrier is routinely added to the samples betore ashing. While this makes subsequent procedures easier and more accurate, it is not essential. Even with the perchloric. acid digestion, the electrolyte concentration of the digest does not permit direct electroplating. Therefore, the sample is suhjected to a preliminary separation from other neutral salts.
Figure 1.
PH Rate of Plating and Current Density in Potassium Fluoborate Buffers
Voltage between electrodes, 4.0 volts Per cent of cobalt deposited i n 15 minutes from buffer solution containing 0.1 m g . cobalt per 10 ml. buffer 0 Total current den.iit). inilliamperes
Previous experience ( 1 ) showed that the nitrosonaphthol methods were inapplicable to high electrolyte solutions, because the reagent precipitated. The precipitation as cobalt metal by reduction ir-ith sodium borohydride is described by Hoekstra (9). This reduction gives excellent recoveries on numerous occasions, but is unreliable for 1,outine assay. With pure cobalt solutions, the very narrow rangr of pH and absencse of oxygen ncwssary t o achieve quantitative precipitation and to prevent resolution of the cobalt can be reproduced. However, when digests are analyzed, such control \\-as found to be impractical. The sulfide precipitation, while simple and reproducible, involves the use of a toxic and obnoxious gas. I t was found ( 1 )that cobaltic hydroxide can be quantitatively precipitated from an alkaline perborate solution. The amorphous precipitate, used before as the counting sample, is difficult to filter from a solution of high salt content, Peptization of the semicolloidal precipitate is probably the major factor contributing to the erratic behavior of the former assay. ThiF is completely avoided by centrifugation. Semicolloidal cobaltic hydroside, passing through a sintered-glass filter of medium porosit>-,is readily and quantitatively collected by moderate centrifuging. Further, the precipitate may be readily redissolved as the chloride. Several other element$-iron, silica, and the alkaline earths, which are the major contaminants in biological samples-also precipitate under t,hese conditions. The alkaline earths and silica give no trouble since they are eliminated during the electrolytic plating. The amounts of iron cncountered were insufficient t o interfere in the assay, although iron plates along with the cobalt. Therefore, no attempt was made to eliminate the iron from the samples. Should the procedure without carrier be med, it might be necessary to employ some method of eliminating this contaminant,. After resolution of the cobaltic hydroxide precipitate in 6 N hydrochloric acid, the iron may be readily removed by estraction with ether. Electrolytic Plating. Cobalt has the reputation of giving very
poor plates under the conditions, which in general were the same as those for iron, employed in the past for radiochemical assay. These plating solutions usually were alkaline ammonia solutions, used to maintain the cobalt in solution as the ammono complex. This complex is of sufficient strength to make deposition very slow, and the high electrolyte concentration leads to high current densities with etching and uneven deposition of the plate. Therefore, fluoborate was used as a complexing agent to plate from a weakly acid solution. Figure 1 shows the relationship between the pH of the potassium fluoborate buffer and both the speed of plating and current density. The pH chosen for plating is modified by two further factors. Table I shows the resolution effect of acidic fluoborate buffers on the cobalt plate. A neutral or alkaline medium is essential to abolish this effect. However, the high current densities a t a more alkaline pH lead to such a local alkaline reaction a t the cathode that a copious precipitate of cobalt hydroxide is formed with concomitant severe losses. Stirring during the plating process merely augments the resolution effect in acidic buffers, while it does not avoid the precipitation of cobalt hydroxide in the neutral range. T o balance these effects, a point of low current density was selected (pH 3.1) for the deposition of the major portion of the cobalt. After this has occurred, the pH may then be raised without danger of precipitation; a t the same time, resolution is prrvented during the removal of the electrolyte. Various
Table I.
Resolution of Cobalt Plates in Potassium Fluoborate Buffers
Buffer, p H Resolutionn 2.0 14.2 3.1 20.1 7.0 4.9 8.2b 0.06 a Per cent dissolved on 30 seconds' contact with a saturated potassium fluoborate buffer. b p H obtained when 1 rnl. of 1 N N a O H is added t o 10 ml. of plating solution of p H 3 . 1 .
;I
A-
-1.0
- 2.0
0
30
60
90
120
150
180
i 0
TIME IN MINUTES
Figure 2.
Efficiency of Plating
Plating of cobalt from potassium fluoborate buffer of pH 3.1 at 3.5 volts (current density between 2 and 4 m a . per square inch)
1033
V O L U M E 26, NO. 6, J U N E 1 9 5 4
self-absorption is also to be kept to a minimum, an advantage with beta p a r t i c l e counting, then as large an area as possible, commensurate with a minimum volume, is desirable. This is best met by a c y l i n d r i c a 1 geometry, and, therefore, the counter and plating assembly shown in Figures 4 and 6 were constructed. The equipment e m p l o y e d was the high voltage supply u s e d b y H i g i n b o t h a m (8) (Model 400 B power supply, John Fluke Engineering Co., I I Springdale, Conn.) and the 3.5 4.0 4.5 5.0 0 10 20 30 40 0 50 100 nonoverloading amplifier used KILOVOLTS DISCRIMINATOR VOLTS GAIN-RELATIVE hy Bernstein, Rrcwer, and Rubin~orl( 3 ) . Thc quantitatFigure 3. Operation Characteristics of Counter and Associated Equipment ing system w-a:' either tlir Radiation source was 0.4 m.e.v. beta radiation from cobalt-60 Rlodrl 100 counting rate metclr Brackets define operating range used by Elmore and Sands ( 7 ) A . Variation i n counting rate with voltage on center wire using gain 100 and discriminator J-5 volts or the decade version of the B . Counting rate OS. discriminator level a t gain 100 and 4.3 kv. C. Counting rate us. relative amplifier gain a t discriminator level of + 5 volts and 4.3 kv. across counter l l u l t i s c a l r r (Jfodel 1060, Atomir Instrument Co., Cambridw 3!1. lfass. ). Figwe :3 s h o w operation rui'veR for the counter. Inspertion LlLLdJ reveals that at a suitable operating point with this equipment Iin. Stainless (gain, 50 to 100; voltage, 4.1 to 4.4: tlisrriminator, +:
