V O L U M E 2 8 , NO. 1 2 , D E C E M B E R 1 9 5 6 however. For example, in Figure 5 there are clean maxima both at 190.3 mp and a t 187.0 mp, with apparent e values of 3715 liters per mole-cm. (Table V) and 2370 liters per mole-cm., respectively. The DK-2 instrument recorded the maximum a t 190.3 mp with an apparent e value of 1106 liters per mole-cm. but failed to record the maximum a t 187.0 mp. At 204.3 mp both instruments are capable of detecting about 5 y (7 p.p.m.) of ammonia per liter of air or nitrogen under optimum instrument conditions with IO-cm. cells Mulliken (6) states that, in regard to the electronic configuration of ammonia, the structure, [sall2[~el4 [zal] (3saJ, 'AI, indicates that the XHa + core per ee should be stable because the electron removal is essentially nonbonding, but that the possibility of predissociation of excited NH, exists. Thus, the excited electron may interact with the core as follows:
KH,+
+e
-+ Ir;Ha
+H
but predissociation should in general tend to diminish with increase in the principal quantum number of the excited electron. To test the effect of prolonged radiation a t 204.3 mp upon ammonia gas, two 1-cm. cells carefully matched a t this wave length Rere filled a t i24 mm. with a mixture of ammonia gas (3 mg. per liter) and dry nitrogen. One cell was exposed continriously to the ultraviolet energy, whereas the companion cell v a s exposed only for the few seconds necessary to make readings. Typical results against dry nitrogen are shown in Table VI. At the end of this period the two cells were quickly scanned from 200 to 206 mp, with essentially parallel absorption curves resulting. These data and curves indicate that prolonged irradiation of ammonia gas in this region does decrease the effective concentration of the absorbing species of interest, unless this predissociation is reversible in that concentration varies with elapsed time b e k e e n exposure and concentration measurement.
1989 Table VI.
Photodecomposition of Ammonia Gas at 204.3 M p and 26" C. Elapsed Transmittance Time, Minutes Unexposed Exposed 0 31.2.31.1 31.1.31.3 20 31.3;31.4 38.2i38.2 80 31.4,31.5 40 .O,40.1 110 31.8.31.9 42.6.42.5 130 31.9;31.9 43.8;44.0
ACKNOWLEDGMENT
The authors wish gratefully to acknowledge technical and other assistance extended by many members of Beckman Instruments, Inc., especially Lee Cahn, R. Pat Connor, George Kincaid, J. G. Myers, and Henry Noebels. LITERATURE
(1) Bahner, F.,Chem.-Ing.-Tech. 25, 89 (1953). (2) Carman, G. E., Gunther, F. A., Blinn, R. C., Garmus, R. D., J . Econ. Entomol. 45, 771 (1952). (3) Huguenard, M. E.,Compt. rend. 213,21 (1941). (4) Kruse, J. SI.,Rlellon, AI. G., ANAL.CHEM.25, 1188 (1953). (5) hlagill, P.L.,Am. I n d . Hyg. Assoc. Quart. 11, 55 (1950). (6) Mulliken, R. S ,J. Chem. Phys. 3, 506 (1935). (7) Scheurer, P.G., Smith, F., ANAL.CHEM.27, 1616 (1955). (8) Schwarz, N., A p p l . Sca. Research Al, 47 (1947). (9) Tannenbaum, E.,Coffin, E. AI., Harrison, a.J., J . Chem. Phys. 21, 311 (1953). (10) Thompson, R.J., Duncan, -1.B. F., Ibid., 14,573 (1946). (11) Ususovskaya, L. G.,Frank-Kamenetskii, D. A , Zuvodskaya Lab. 14, 12 (1948). R E C E I V E for D review M a y 14, 1956. Accepted September 6, 1956. Paper No. 918, University of California Citrus Experimental Station, Riverside,. Calif.
Determination of Thorium in Urine R. W.
PERKINS and D. R. K A L K W A R F
General Electric Co., Richland, Wash.
A bioassay procedure can be used to determine thorium in submicrogram amounts in urine. Thorium is separated from interfering materials by coprecipitation with lanthanum fluoride, followed by an extraction using 2thenoyltrifluoroacetone in benzene. The amount of thorium is determined colorimetrically as the thoriumniorin complex.
I
N ORDER to determine the relationship between urinary excretion of thorium and the amount of thorium present in the body, a procedure must be available to detect the small amount of thorium that can be expected in the urine. A maximum permissible limit of 89 mg. (0.01 pc.) of thorium in the body has been recommended by the International Commission on Radiological Protection (9). The available information on the rate of excretion of thorium has been recently summarized (8); however, it is not sufficient to define the sensitivity required of a bioassay procedure for determining a given body burden of thorium. ;In estimate based on the biochemical similarity of thorium and plutonium (7) indicated that a sensitivity of 0.27 of thorium would be sufficient to detect any significant thorium content in an individual and thus be suitable for future studies of the relationship between urinary excretion and body burden of thorium.
Several possible analytical techniques for estimating the thorium content of urine samples were considered. An alphaparticle counting procedure sensitive to 0.05 disintegration per minute (0.2 y of thorium-232) can be realized; however, complicating factors in the case of thorium make this approach undesirable. One of the thorium-232 decay products is thorium228, which is also an alpha emitter. The relative amounts of the two isotopes present are a function of the time since the decay chain between them had been broken. The alpha emissions of thorium might be counted immediately on separation and then again 1 to 4 weeks later, when the amount of thorium-228 could be determined by the build-up of alpha-particle-emitting daughters of thorium-228. However, this would be difficult to evaluate a t present because only nuclear track counting has the required sensitivity and 1 week or more of exposure would be required for each count, further confusing the analysis. The direct determination of as little as 0.3 y of thorium in animal tissue samples with an emission spectrograph has been reported (6). Because urine contains the same inorganic salts as tissue but in a higher concentration (by a factor of about loa), a spectrographic analysis might be performed after a preliminary separation of the thorium from the bulk of the urine salts. A recently reported mass spectrometric determination of thorium indicated (19) that 0.1 y of thorium could be detected; hon-ever, a preliminary separation would again be required.
ANALYTICAL CHEMISTRY
1990 Both of these methods require elaborate instrumentation which, together with the technical difficulties associated with sample preparation, make their use undesirable. Of the colorimeteric reagents for thorium reported in the literature, morin, 2’,3,4’,5,i-pentahydroxyflavone,is the most sensitive. The sensitivity of this reagent has been reported (8) to be such that 0.1 to 0.2 y of Tho2 in 50 ml. of solution can be determined. -4s this sensitivity compares favorably with those of the other methods, a colorimetric procedure utilizing morin appeared to be most suitable for routine bioassay application. OUTLINE OF METHOD
The urine sample is first freed from organic matter by a series of wet ashings with concentrated nitric acid. Two lanthanum fluoride precipitations remove the thorium from the bulk of urine salts. Thorium in 0 . 2 S nitric acid is separated from any remaining urine salts and the lanthanum carrier by extraction with 0.45.V 2-thenoyltrifluoroacetone (TTA) in benzene. Thorium is stripped from the 2-thenoyltrifluoroacetone-benzene solution with 2N nitric acid and evaporated to dryness with perchloric acid to remove the last traces of organic material. Then the thorium is taken up in p H 2.0 perchloric acid and determined colorimetrically as the thorium-morin complex. A direct precipitation method was developed to replace the wet-ashing procedure, which is time-consuming as well as a possible source of an occasionally low result, possibly due to the formation of a difficultly soluble, refractory thorium oxide during the heating. The acidified urine sample is boiled do-irn to a small volume and the lanthanum fluoride is precipitated directly from this solution. This procedure gave a higher yield and precision than the a et-ashing procedure. REAGENTS AND APPARATUS
Standard Thorium Perchlorate. -4solution of thorium perchlorate %-as prepared and purified by the method of Willard and Gordon ( 1 1 ) . The solution was standardized gravimetrically by the oxalate procedure ( 1 7 ) and dilutions of this stock solution were prepared using doubly distilled water. Lanthanum Nitrate Solution. .4 solution containing 25 grams of lanthanum nitrate tetrahydrate (Lindsay Light and Chemical Co.) in 50 ml. of water was washed for 10 minutes with each of three 10-ml. portions of 0.45M 2-thenoyltrifluoroacetone in benzene. The purified lanthanum nitrate solution was then diluted with doubly distilled water to give a concentration of 10 mg. of lanthanum per ml. 2-Thenovltrifluoroacetone (TTA) Solution. A solution of 2thenoyltrifhoroacetone (Midcontinent Chemicals Corp.) was prepared by dissolving 10 grams in redistilled benzene to give a total volume of 100 ml. Portions of this solution required for the extractions were washed 5 minutes with each of three 20-ml. portions of 2N nitric acid in the separatory funnel just prior to use. Morin Solution. Morin (2’,3,4’,5,i-pentahydroxyflavone) was purified from technical grade morin (Eastman T44i5) in the following manner. A sample of the technical grade material was stirred with ten times its weight of absolute ethyl alcohol a t room temperature and the suspension was allowed to settle overnight. The alcoholic extract was filtered and evaporated under a heat lamp t o one tenth its volume. An equal quantity of water was added and the precipitated morin was filtered. The precipitate was dissolved in a minimum amount of ethyl alcohol and again precipitated with an equal volume of water. The precipitate was then filtered, washed with water, and dried at 110’ C. for 1 hour. A 2.5% yield w-as obtained; the product was light yellow Kith a melting point of 289-92” C. The solution for the colorimetric measurements was prepared by dissolving 25 mg. of purified morin in 100 ml. of absolute ethyl alcohol. Acid Solutions. Nitric acid and perchloric acid Tyere both redistilled and all dilutions were made with doubly distilled water. Hydrofluoric acid (ACS Code 1100, General Chemical Division, Allied Chemical and Dye Corp.) was used without further purification. Glassware. Erlenmeyer flasks were cleaned with boiling nitric acid and rinsed with distilled water. Separatory funnels and Vycor (No. 7900) evaporating dishes were boiled in chromic acid cleaning solution for 10 minutes, followed by boiling in concentrated nitric acid for 5 minutes and rinsing with doubly distilled
water. Silicone grease (Dow Corning) was used on the separatory funnel stopcocks. Other Equipment. A Beckman Model DU spectrophotometer and 1-em. Corex cells were used to obtain the absorbance messurements. A Beckman Model H-2 pH meter was used for the pH determinations. A Burrell Model D D shaker was used in the extraction operations. An International centrifuge Model V was used in the centrifugation operations. DETAILED PROCEDURE
Ashing Procedure. To a 500-ml. urine sample in a 1000-nil. Erlenmeyer flask add 1 ml. of lanthanum nitrate solution (10 mg of lanthanum) and 50 ml. of concentrated nitric acid and evap3rate to dryness. Add 10 ml. of concentrated nitric acid and evaporate. Repeat this step until only white salts remain. Dissolvc the salts in 60 ml. of 2LVnitric acid, add 1ml. of lanthanum nitratc solution, and stir thoroughly. Transfer flask contents to a 100ml. Lusteroid centrifuge tube. Wash the flask with three IO-ml. portions of 2147 nitric acid, adding washings to the centifuge tuhv. Direct Precipitation Modification. To a 500-ml. urine sample in a 1000-ml. Erlenmeyer flask add 2 ml. of lanthanum solution and 100 ml. of concentrated nitric acid and evaporate to 25 to 35 ml. Allow the sample to cool 5 minutes. Transfer the samplr, to a 100-ml. Lusteroid centrifuge tube containing 10 to 20 ml. of distilled water. Wash the flask with three 10 to 15 ml. portions of 212: nitric acid, adding washings to the centrifuge tube. The first lanthanum fluoride precipitation can be made directly on this solution. Thorium Separation and Determination. Add 5 ml. of concentrated hydrofluoric acid, stir thoroughly, and allow it to stand 1 hour. Centrifuge a t 1600 r.p.m. for 2 minutes. Decant the solution, taking care to leave the precipitate undisturbed, and dissolve the precipitate in 10 to 20 ml. of 2N nitric acid directed in a fine stream from a wash bottle: dilute to 80 to 90 ml. with 2N nitric acid. Add 5 ml. of concentrated hydrofluoric acid, stir, and allow to stand 5 minutes. Centrifuge a t 1600 r.p.m. for 2 minutes. Decant the solution and transfer the precipitate to a 45-ml. Vycor evaporating dish with the aid of 10 to 15 ml. of 2 S nitric acid. Add 3 ml. of concentrated perchloric acid and evaporate to dryness on a hot plate. Dissolve the residue in 3 ml. of 1 S nitric acid and transfer the solution to a separatory funnel containing 10 ml. of 0.45M 2-thenoyltrifluoroacetone in benzene. Wash the T-ycor dish with three 1-ml. portions of water, adding the washes to the separatory funnel. Shake for 5 minutes a t maximum agitation on a mechanical shaker, then add 10 ml. of distilled water and shake for 15 minutes. Discard the aqueous layer. Kash the inside of the separatory funnel with 25 to 35 ml. of distilled water by spraying from a wash bottle, then discard the rrash xater. Wash the 2-thenoyltrifluoroacetone-benzene layer by shaking it with three 5-ml. portions of 0.2‘47 nitric acid 5 minutes each, discarding the aqueous layer each time. Wash the inside of the separatory funnel with 25 to 35 ml. of distilled water by spraying from a wash bottle and discard the m*ash water. Add 10 ml. of 2N nitric acid and shake 15 minutes. Let stand for 30 minutes to allow complete separation of the phases. (Incomplete separation here may lead to a mild explosion and loss of the sample during the next steps.) Withdraw the aqueous phase into a 45-nil. Vycor dish COIItaining 3 ml. of concentrated peichloric acid. Place the samples
H h O )
h O R M A L i T Y
Figure 1. Thorium distribution between nitric acid and 0.45M 2-thenoyltrifluoroacetone as a function of nitric acid concentration
1991
V O L U M E 2 8 , NO. 1 2 , D E C E M B E R 1 9 5 6 on an asbestos-covered hot plate and cover them with an inverted rectangular borosilicate glass dish raised slightly at the back to allow the fumes to escape while preventing entrance of dust from the hood ventilating air. Evaporate to dryness. Remove the dishes from the hot plate and allow them to cool. Add 3.3 ml. of perchloric acid a t pH 2.0, swirl 0.5 minute, allow to stand 5 minutes, and swirl again, Transfer 3.0 ml. of the sample t o the spectrophotometer cell and add 0.50 ml. of morin solution. Prepare a blank solution containing 3.0 ml. of perchloric acid a t pH 2 and 0.50 ml. of morin solution. Place the cap on the cells and invert several times to mix. Allow to stand 1 hour and measure the absorbance a t a wave length of 412 mp and a slit width of 0.03 mm. on a Beckman Model DU spectrophotometer. DISCUSSIOh OF METHOD
A preliminary study of possible methods of separating thorium from urine samples indicated that a lanthanum fluoride carrier precipitation of thorium followed by a solvent extraction separation of the thorium from the carrier should be suitable. Several organic solvents and chelating agents were considered for the extraction, among which 2-thenoyltrifluoroacetone-benzene (4, 6, 16) mesityl oxide (1, 8, 4, 11, 1 4 ) and methyl isobutyl ketone ( 1 8 ) seemed most promising. A brief study of these systems indicated that 2-thenoyltrifluoroacetone-benzene required no salting agent, which could introduce contamination, and greater qelectivity for thorium could be obtained. The distribution of thorium between 0.25 1112-thenoyltrifluoi-oacetone-benzene and nitric acid solutions as a function of pH has been determined (6). For the present work a 0.4531 2thenoyltrifluoroacetone-benzene solution (10 grams per 100 ml.) was chosen to permit the use of a more acid solution for the initial extraction step. To determine suitable acid concentrations for thorium extraction into and out of 0.45M 2-thenoyltrifluoroacetonebenzene, thorium distribution as a function of nitric acid concentration was studied. Thorium-228 was used as a tracer to permit the use of low thorium concentrations, similar to the thorium-232 concentrations to be studied in urine, and a t the same time have a sufficient quantity of radioactivity for accurate measurements. Thorium distribution was studied R ith thorium-228 added both as Th(N0,)r and as Th(TTA)s in benzene. Equal volumes of the acid and the organic solutions were equilibrated 20 minutes by vigorous shaking; then the phases were separated and their alpha activities measured. The results of this study are summarized in Figure 1.
To determine the approximate loss of thorium in each of the separation steps (exclusive of wet ashing) of the analytical procedure, an analysis was performed using thorium-228 tracer on a '75-ml. aliquot of 2N nitric acid. The thorium present in the various solutions normally discarded in the analysis was measured and is tabulated in Table I. The data in Table I indicate the size of the small thorium losses that occur in the procedure, and that an over-all yield of about 85% could be expected. The major loss of thorium is due to its equilibrium distribution between the orgaiiic and aqueous phases in the extraction and rrashing steps. These washing steps, while reducing the yield slightly, were required to give a product of sufficient purity for colorimetric analysis. Using thorium-228 as a tracer the yields were determined on eight 500-ml. urine samples for both the v-et-ashing and direct precipitation procedures. The average yield for the wet-ash procedure was about '70% and for the direct precipitation procedure above 80%. In the 2-thenoyltrifluoroacetone-benzene extraction step the extraction proceeds initially from 0.5-V nitric acid and finally (Tq-ithout separation) from 0.2N nitric acid. Shaking with the more concentrated acid a t first prevents an occasional formation of a solid material a t the interference, which is redissolved only with difficulty. This solid material has not been identified but may be a lanthanum-2-thenoyltrifluoroacetone complex. In dilute perchloric acid solutions, thorium forms a complex n i t h morin which has an absorption maximum a t 412 mp, as shown in Figure 2. Because thorium precipitates or is absorbed on the container walls in solutions with p H greater than 3.0 ( I S ) , a pH of 2.0 was chosen for the medium on which to make the absorbance measurements. This value is a compromise to ensure keeping the thorium in solution while maintaining a high absorptivity.
0
I
Im. 10 13 14 18 18
mum), mum), Mm. hlm. 70 37 70 39 72 39 75 39 75 39
F, Tolerance,
311. 10.010 10.015 f0.015 10,020 10,020
To be marked “T.C. fcanaoitv) 20° C.” 1-ml. size to weigh leis &an l”9 grams empty (stopper included). Shape of bases may be either round or hexagonal. Dimensions given in column E are maximum permitted for distance between parallel sides of hexagonal bases and are maximum diameters of round bases.
(-) 3 8 STOPPER
