Separation and Determination of Millimicrogram Amounts of Cobalt RALPH E. THIERS, JOHN F. WILLIAMS*, and JOHN H. YOE Pratt Trace Analysis Laboratory, Department o f Chemistry, University o f Virginia, Charlottesville, V a .
A method is described for the separation of millimicrogram amounts of cobalt from biological materials, such as blood. The sample is dry-ashed, excess iron and alkali chlorides are removed, and the cobalt is separated on an anion exchange resin, Dowex 1. It is eluted from the resin substantially free from other elements, with recoveries of over 90%. This technique has been combined with spectrochemical analysis to determine the amount of cobalt in human blood, where its concentration is found to be below 1 part per billion. A spectrochemical method of determining cobalt is described which is sensitive to 1 my (1 X 10- gram). The direct current arc is used with the sample in the cathode. The conditions under which this sensitivity is achieved are discussed. A standard deviation of about 10% of the result ma? be expected when between 1 and 500 my of cobalt are present.
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U:iNTITXTIVE analysis, since the time of Berzelius, has progressed through the gram and milligram stages, and w i t h present instrumentation, the measurement of microgram c1u:intities is routine. The next step, measurement of millimicrograms, is accompanied by special difficulties. The quantification of millimicrogrsm aniounts of metals often requires the cmniplete separation of the desired constituent from the original matrix material. The excess of mass of extraneous elements in the sample may prevent the determination of the trace constituent by diluting it beyond the limit of concentrational sensitivity. Chemical interferences not detectable a t equimolar rat,ios arise n-hm onc constituent esceeds another by a wide margin, and the elernc~nt~ ivhich ordinarily do interfere with a given procedure ai'e certain to present significant problems when millimicrograrn amountP of an element are to be determined ( 7 ) . While a particular analytical technique may be capable of directly det'ersmall amounts of an ion when present alone in a ensitivity is generally greatly drcreased when other ions or organic matc.rials are present. The determination of cobalt in human blood is chosen as an example of such a problem. It is known that cobalt occurs in human blood since vitamin B,,, a normal blood constituent, contains this element, (10, 15). Vitamin Biz is the cyano-complex of the cobalt-containing organic compound, cobalamin, C,aHsoOlaNIIPCo, and liydroxo-, nitrito-, chloro-, bromo-, sulfato-, cyanato-, thiocyanato-, and other complexes of the cobalt of cobalamin exist and produce varying degrees of vitamin activity. The concentration of vitamin B12 in human blood has been measured by means of microbiological assays, and found t o be in the parts per billion range (Table VI). Although data have been reported on the concentration of cobalt in human blood and serum ( 1 7 , I S ) , no method of determining this concentration has been given in detail and the present communication supports the view t h a t cobalt cannot have been detected as described. T h e concentrational levels reported here are much lower than those indicated elsewhere and are below the sensitivity limits for previous methods. As vitamin Blz has been shown to play a role in protein metabolism, blood formation, etc., and is a t present the subject of a great deal of research, the determination of cobalt in blood is of real interest even though its concentrat.ion is extremely low. 1 Present address, Biophysics Research Laboratory, Harvard Medical School, Peter Bent Brigham Hospital, Boston, Mass. * Present address, Liggett and Myers Tobacco Co., Durham, N . C .
This paper describes a technique which can quantitatively separate as little as 2 m y of robalt from the other elements present in blood, and a new sensitive spectrochemical method of determining it. The cobalt can be separated so completely that spectrochemical analysis can detect no iron, nickel, or alkali metals accompanying it. The separation method has been shown t o be applicable to much larger amounts of cobalt (6) and should provide a general method of separating it as preparation for any analytical procedure %-here a pure cobalt solution is required. The spectrochemical method can determine from 1 to 500 m y of cobalt with a standard deviation about 10% (grams X of the result, and can be used whenever iron, nickel, and the alkali metals are absent. QUANTITATIVE SEPARATION OF MILLIMICROGRAM AMOUNTS O F COBALT FROM BLOOD
d sample of blood which contains a t least 2 my of cobalt is taken for analysis. It is dry-ashed, excess iron and alkali chlorides are removed, and the cobalt is separated on an anion eschangc resin, Dowex 1-X8, according to the method of I h a u s (6, 9). The effluent containing t'he cobalt is then evaporated to n small volume in hydrochloric arid, :tiid analyzed hj- spectrorhemistrv. REAGERTS
Hydrochloric Acid. hnhj-drous h j drogen chloride gas (RlatheTon Co., Inc.) i s scrubbed in a bubble tower with concentrated sulfuric acid, filtered through packed borosilicate glass wool, scrubbed again in a bubble tower by distilled water which has been saturated with the gas, and then dissolved in triply distilled water, or its equivalent, a t 0' C. t o give an acid concentration greater than 931. The acid strength is determined and 9.0Jf, 4.0Jf: and 0.01M solutions of hydrochloric acid are obtained b y proper dilution of this concentrated acid with triply distilled water. Acid thus produced is free of the traces of alkali and alkaline earth metals found in the reagent grade. Isopropyl E t h e r . Alcohol-free ether (Eastman Organic Chemical) is redistilled from an acid cleaned still. I o n E x c h a n g e R e s i n . Doiv chemical, Dowex 1-X8 resin, 50 to 100 mesh, is used in the absorption column. It is a strongly basic, quaternary amine type, styrenedivinylbenzene polymer containing approximately 7.5% of cross-linking divinylbenzene (6, 9). Cobalt-60 C h l o r i d e S o l u t i o n . This reagent was used only in checking the method. A cobaltous chloride solution having a specific activity of 4831 me. per gram and a radiochemical purity of 99.970 was Figure 1. Ion esobtained from the Oak Ridge change column Sational Laboraton. The cobalt60 was taken up into approvimatelv 60 ml. of trike distilled hydrochloric acid. The solution ~ a analyzed s colorimetrically by the nitroso-R salt procedure desciihed by Sandell ( 1 2 ) and ~ a . diluted one thousandfold, giving a final concentration of 13.3m*f of cobalt per milliliter. Tagged Vitamin BL2. This reagent 1% as used only in checking the method. One-half milliliter of a solution containing 13 y of vitamin Bl2 tagged with cobalt-60 n'as diluted to 10.0 ml. with triply distilled water. Based on the value 4.5% as the amount
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ANALYTICAL CHEMISTRY
of cobalt in vitamin BIa (b), the metal cancentrrttion was caleuIated to be 29 my per ml. This material was provided through the courtesy of Merck & Co., Ine. Sodium Heparin, Solid Salt (USP, Organon Inc., Orange, N. J.). Qualitative spectrochemical analysis of an ashed samDle showed this material to he free of cobalt. Triply Distilled Water. Laboratory distilled water was distilled twice more. The final condenser was made of fused silica, and the water was stored in polyethylene bottles. APPARATUS
Ion Exchange Column. The column shown in Figure 1 was constructed of glass. A glass wool plug was saturated with water to eliminate air and placed in the bottom of the column, after which the Dowex 1 wa8 added as a slurry in triply distilled water t o give a resin bed 11.5 cm. long and 8.0 mm. in diameter. A second plug of glass wool saturated with water was placed on top of the resin bed. Mutae Furnace. Electric furnaces operated by automatic oontrollers (Wheelco Model 241P) were used in ashing the samples. The temperature settings could he maintained within 20"
c.
half milligrams of solid sodium heparin are added to the chamber of the syringe to prevent coagulation of the sample during drawing. A volume of blood slight,ly greater than that desired for the sample is drawn into the syringe. Then the volume is adjusted t o t h a t desired by using the syringe graduations and discharging the slight excess along with any bubbles produced during drawing. Previously calibrated syringes are therefore required. The samples are then transferred directly to the silica dish for ashing, or to polyethylene bottles if the sample is to be separated into blood fractions. Samples larger than 100 ml. are drawn into soft-glass bottles regularly used for the collection of transfusion blood. The bottles (Abhott Laboratories, North Chicago, Ill.) me emptied of their anticoagulant solution, thoroughly cleaned, and after being charged with the proper amount of sodium heparin (1.5 mg. per 50 ml. of sample desired) and a few drops of triply distilled water, the bottles are re-evacuated to the vapor pressure of water. Ahhott Lahoratories blood collection sets were used in drawing the sample. They employ stainless steel needles connected h i t h polyethylene tubing. The first few milliliters of blood were discarded on the theory that it cleaned out the collection set. No actual contamination from the blood flushed through a new collection set was detected, however, when this was tried. In both methods of drawing blood i t is important t h a t the blood be shaken gently while the sample is being taken, as well as aitornards to ensure mixing of the anticoagulant or else difficulties may arise because of clotting. The samples are left in the transfusion bottles and are stored in a refrigerator until analyzed. Aliquots of the samples are measured in appropriately sized graduated cylinders and are transferred to silica dishes for ashing. Where separation of cells and serum is desired, this is done by centrifuging the fresh blood samples in a small polyethylene bottle and taking off the fractions with B syringe of the Same type as w m used for drawing small samples.
rable I. Recovery of Cobalt-60 in Blood Samples Dry Ashed at 450" C .
Figure 2.
Evapora t i o n cover
Geiger Counter. This apparatus was used only in checking the method. A Tracerlab Model SI3-2A scaler with a TGC-4 m n n i i n n +nhn n r i s n s n r l in +hn t r o n a _-_.. " ~ " " . . l v.urrr studies t o determine the gamma activity of cobalt-60. The counter wa.6 dipped into the sample solution (10 ml. in volume) contained in a l-inch-diameter test tube. A National Bureau of Standards cobalt-60 solution was used 8 s activity standard. Evaporation Cover. I n order to protect the samples from air-borne contamination, apparatus like those shown in Figure 2 were fabrioated from crystallizing dishes. Dried, filtered air was introduced through the side arm of the cover to remove vapor, thus preventing condensstion inside the apparatus, and speeding the evaporation. This method of protecting the sample during evaporation proved necessary in order to avoid high
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Sampung. It 1s necessary to draw the blood in a careful manner in order to avoid contamination. One of two sampling techniques is used, depending on the quantity of blood being taken. One technique utilizes standard transfusion equipment, the other, 50-ml. syringes.
Mitlvern. Pa.).. These needles are'size No. (9, and t h e n collars and joints are heavils plated with anid. Prior to use, they are
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gl&s jackets, which^ ha"; also'h& cleaned, and are 'sterilized by heating them in an oven at 160' C. overnight. One and one-
Compound Cos0 Added Added, my C0"CL 6.7 6.7 6.7 13.0 13.0 13.0 Vitamin BH 29.0 29.0 e High resuit attributed
Acticty Of CobQ Counts Counts Probhble added recovered Recovery, % Error, 7% 4.370 4.720 108' 4.150 4,148 - by the thiocyanate method. According to one procedure acetone is added to prevent dissociation of the cobalt thiocyanate complex, thus serving to intensify the color normally obtained in aqueous solution. The other procedure involves extraction of the colmlt thiocyanate complex n i t h isoamyl alcohol. The blue color of the cobalt thiocyanate complex has been measured spectiophotometrically a t its absorbance maximum a t about 620 mu. Thiq part of the investigation was concerned rvith studying the ultraviolet absorption spectrum of the cobalt thiocyanate complex and the effect of certain solution vaiinhles npon the
General Experimental Procedure. A definite ainount of the standard cobaltous sulfate solution was transferred to a 50-1111. volumetric flask. After the desired amount of 3376 ammonium thiocyanate solution was added, the solution \vas diluted t o 60 ml. with redistilled vater. The complexed cobalt was extracted from the water solution with an isoamyl alcohol solution ~ h i c h had been previously saturated m-ith ammonium thiocyanate. The extracts were added t o a 50-ml. volumetric flask. The extract was diluted to the graduation mark with isoamyl alcohol-thiocyanate reagent and the contents of the flask were thoroughly mixed. The absorbance measurements Tvvere made immediately using the extractant in the reference cell. The solutions were sufficiently stable to permit the necessary absorbance measurements t o be made. During the study of the effect of diverse ions, all ions were added before complexation. Effect of Cobalt Concentration. The absorption spectrum of the cohalt thiocyanate complex for various concentrations of
