ANALYTICAL CHEMISTRY
828
DeMerre, L. J., and Brown, W: S., Arch. Biochem., 5, 181
of 25 grams of crude alloxantin dihydrate, thus prepared, from 400 ml. of (boiled) boiling water gave 22 grams of alloxantin dihydrate. ] For the fluorometric determinations, solutions of the various samples of commercial alloxan monohydrate (1.0 microgram ml. in solution B) were freshly prepared. A series of red test tug:; waa then made up to contain 1.0 ml. of the respective alloxan monohydrate solution Ius 2.0 ml. of fresh R.HC1 solution, a t least three such tubes geing prepared for each alloxan monohydrate sample. After preservation at 70" F. in the dark for 3 days, the fluorescence was determined aa previously described. Two volumes of diluent solution were necessary in every case. The results are given in Table I.
(1944).
Euler, H., v., Karrer, P., Malmberg, M., SchBpp., K., Bene, F., Becker, B., and Frei, P., Helv. Chim. Acta, 18, 522 (1935). Hanson, 9.W. F., and Weiss, A. F., Analyst, 70, 48 (1945). Kamlet, J. (to Miles Laboratories, Inc.), U. 9. Patent 2,406,774
(June 28, 1943). Karrer, P., and Fritzsche, H., Helv. Chim. Acta, 18, 911 (1935). Karrer, P., SchBpp, K., and Benz, F., Ibid., 18, 426 (1935). Kuhn, R., Angeu. Chem., 49, 6 (1936). Kuhn, R., Chem-Ztg.. 59, 604 (1935). Kuhn, R., and Maruzzi, G., Ber., 67,888 (1934). Kuhn, R., Reinemund, K., Kaltechmitt, H., Strobele, R., and Trischmann, H., N a t u r w ~ s e n s c ~ f23, m , 260 (1935). Kuhn, R., Reinemund, K., Weygand, F.,and Strobele, R., Ber.,
ACKNOWLEDG.MENT
68, 1765 (1935).
Kuhn, R., and Weygand, F.. Ibid., 68, 1282 (1935). Kuhn, R., Weygand, F.. and Cook, A. H. (to Winthrop Chemical Co.), U. S. Patent 2,238,874 (April 15, 1941). Loofbourow, J., and Harris, R. S., Cereal. Chcm., 19, 151 (1942). Prout, W., Phil. Trans. Roy. Soc. London, 108, 420 (1818). Ruben, J. A., and Tipson, R. S., Science, 101, 536 (1945). Scott, M. L., Hill, F. W., Norris, L. C., and Heuser, G. F., J.
The authors are grateful to J. A. Aeschlimann, Hoffmann-La Roche, Inc., Nutley 10, N. J., for his kindness in presenting the samples of ~-l-ribitylsmino-2-amino-4,5-dimethyIbenzene and its hydrochloride used in the present study.
Bid. Chem., 165, 65 (1946).
LITERATURE CITED
(1) Abderhalden, R., 2.physiol. Chem., 252, 81 (1938). (2) Banerjee, S., Dittmer, K., and du Vigneaud, V., Science, 101, 647 (1945). (3) Cohn, E,J., Heyroth, F. F., and Menkin, M. F.. J . Am. Chem. SOC.,50, 696 (1928).
Tipaon, R. S., J. Am. Phann. Assoc., Sn'. Ed., 34, 190 (1945). Tipuon, R. S., and Ruben, J. A., Arch. Biochem., 8, 1 (1945). Tipson, R. S., and Ruben, J. A., Science, 103, 634 (1946). Trager, W., Ibid., 107, 175 (1948). Wahler, F., and Liebig, J., Ann., 26, 265 (1838). RECEIVED December 14, 1949.
Determination of Traces of Beryllium in Biological Material F. W. KLEMPERER
AND
A. P. MARTIN
Trudeau Foundation for the Clinical and Experimental Study of Pulmonary Disease, Trudeau, N . Y .
A procedure is described for the isolation and determination of traces of beryllium in biological material. After deatruction of organic material, the excess of calcium is precipitated as sulfate and beryllium is separated from the mlution by precipitation with ammonia together with iron phosphate which L added as a collector. Iron and other interfering cationr are removed by electrolysis with a mercury cathode, and beryllium phosphate L then separated from the solution by ammonia precipitation using aluminum as a collector. Beryllium is estimated quantitatively by the fluorometric method with morln. The smallest amount of beryllium that can be detected reliably by this method is 0.05 microgram. Because beryllium can be isolated from large samples, it can be determined quantitatively in concentrations of less than 1 part in IOLo parts of urine.
T
OXICOLOGICAL studies of beryllium have necessitated the development of analytical methods for its determination in biological material (1-4, 9). Of these procedures, only the spectrographic pries (8, 3, 9 ) possess the sensitivity necessary to detect beryllium in the small concentrations present in the urine of exposed persons. Because the expensive equipment required for spectrographic determination is not available in most Iaboratories, the development of a sensitive chemical procedure appeared dasirable. Such a method, in order to be useful for estimating the degree of exposure of workers to beryllium compounds, should, according to Cholak and Hubbard (S), permit the determination of beryllium in concentrations of 0.2 microgram per liter of urine. Of all reactions devised for the estimation of beryllium, only the fluorometric one with morin is sufficiently sensitive to allow quantitative estimation of such small amounts of beryllium. This method, first described by Zermatten (11), waa carefully investigated by Sandell and adapted to the determination of beryllium in ores (6-8). Its use for biological material, however, was
discouraged by subsequent workers (1, 4 ) because of ita lack of specificity. In order to apply the morin reaction to urine and other biological materials, complete separation of beryllium from interfering substances proved necessary. I n the following report a procedure is described which permits concentration of traces of beryllium from large quantities of biological material, its separation from interfering compounds, and its fluorometric determination by the morin method. REAGENTS
All reagents were of C.P. grade: nitric acid, concentrated; hydrochloric acid, 3 N ; sulfuric acid, concentrated, 3 N and 1 N ; hydrofluoric acid; ammonia, distilled, Concentrated, and 3 N ; sodium hydroxide, 1 N ; a saturated solution of ammonium sulfate; potassium sulfate, 3%; bromocresol green, 0.04% sium ferricyanide, 1%; potassium ferrocyanide, 5%; ethyP%z hol, 95 and 60%. Iron Phosphate Reagent (1% iron). ( a ) A 2 molar solution of phosphoric acid. ( b ) 43.2 grams of FeNH,(SO,)z.l2HzO and 50
829
V O L U M E 2 2 , NO. 6, J U N E 1 9 5 0 ml. of 10 N sulfuric acid, diluted with water to 250 ml. Equal parts of ( a ) and (a) are mixed. Only a week's supply of this
reagent should be mixed, because iron phosphate precipitates after this period. Acetate Buffer, 230 grams of ammonium acetate and 270 ml. of glacial acetic acid, diluted with water to lo00 ml. Aluminum Sulfate (1% aluminum)! 12.3 grams of AL(SO& 18H20and 10 ml. of 1 N sulfuric acld, diluted to 100 ml. and filtered. Aluminum Sulfate (0.2% aluminum), made by diluting the 1% aluminum solution with water. Aluminate Solution.. To a 50-ml. centrifuge tube are added 8 ml. of 1% aluminum solution, 5 ml. of 2 M hosphoric acid, and water to about 30 ml. After addition of 5 8 0 p s of bromocresol green, the solution is neutralized approximately with ammonia and 5 ml. of acetate buffer are added. It is heated in boiling water bath for 10 minutes, and centrifuged, and the precipitate is washed twice with 3% potassium sulfate. The preci itate is dissolved in 40 ml. of 1 N sodium hydroxide, transferre: to a 1Wml.volumetric flask, diluted to volume with water, filtered, and kept in a glass-stoppered bottle. Stannite Solution. A,l% solution of stannous chloride dihydrate in 0.5 N sodium hydroxlde is filtered and kept in a glass-stoppered bottle. Morin Solution. A stock solution is made by shaking 500 mg. of finel ground morin (Eastman, technical) m t h 100 ml. of 95% alcozol overnight, and filtering the mturated solution. This solution keeps a t least 3 months in the refrigerator. The working reagent is made by dilutin 5 ml. of the stock solution with water to 100 ml. It is kept in gkss-stop ered bottles. concentrated standard conBeryllium Standard Solution. taining 10 micrograms of beryllium per ml. is made by dissolving 196.6 mg. of beryllium sulfate tetrahydrate in water, adding 10 ml. of 3 N hydrochloric acid, and diluting to 1 liter. The dilute working standard containing 0.2 micro ram per ml. is made by diluting 2 ml. of this solution to 1M) mf. with water. If kept in borosilicate glass bottles, these solutions will not change their titer.
tities of 60% alcohol. The filtrate and washings are evaporated in a beaker on a water bath either until most of the alcohol has If been eliminated or, more conveniently, overnight to d dry, the salts are dissolved in water and acidified w i r E m a l l amount of hydrochloric acid. To this solution, from which most of the calcium has been removed, 2 ml. of iron phosphate reagent are added, followed by enough concentrated ammonia to cause fading of the yellow color of ferric chloride. Before precipitation of iron phosphate begins, 10 drops of bromocresol green are added and the solution is neutralized under constant stirrin by slowly. adding dilute (3 N ) ammonia until the indicator c%anges to light green. About 1 volume of acetate buffer is then added for every 5 volumes of solution, and the mixture is heated to near boiling and placed on the steam bath for 10 minutes. The solution should not be boiled or heated on the steam bath for a prolonged period, as volatilization of acetic acid ma cause appreciable change in the h dro en ion concentration. $he precipihte which contains the geryl!!ium is then collected in a conical qml. centrifuge tube by repeated centrifugation and washed tmce with 40 ml. of 3% potassium sulfate solution. It is important to d@ erse the precipitate comEletely in the washing solution, whicg is best done with the elp of a thin-footed glass rod. Between washings the centrifuge tube is inverted and drained.on a pad of filter paper. After the last washing the precipitate 1s dissolved in 1 ml. of 3 N sulfuric acid.
PROCEDURE FOR DETERMINATION OF BERYLLIUM IN URINE
For determination of minute amounts of beryllium such as may occur in the urine of exposed persons, it is best to ash a specimen collected during a 24-hour period, regardless of its volume. Ordinary dry-ashing procedures cannot be used, as shown by Cholak and Hubbard ( 2 ) , presumably because of the volatility of beryllium chloride. Losses are avoided, however, if hydrochloric acid is first expelled by evaporating with nitric acid. The urine is transferred from its original coqtainer into one or more large Erlenmeyer flasks and the container is rinsed with a total volume of 100 ml. of concentrated nitric acid. The combined urine and nitric acid rinsings are concentrated on a hot plate. If, on boiling, the mixture does not turn a light yellow and boiling becomes uneven, more nitric acid must be added. When the volume has been reduced sufficiently, the solution is transferred to a 350ml. Vycor evaporating dish and taken to dryness on a steam bath. The contents of the dish are then moistened with 10 ml. of concentrated nitric acid, and the dish is placed into a radiator and heated over a low flame. (Iron containers 10 cm. in diameter and 4 cm. in height, as used for packing 1Wfoot rolls of 35mm. hotographic film, are ideal for this purpose.) When the mixture gecomes dry, the flame is gradually increased until charring occurs and no vapors are given off. The dish is then heated in a muffle furnace to 500" C. until the ash is pure white. The ash is dissolved in a minimal amount of 3 N hydrochloric acid, usually less than 100 ml., and heated until the small amount of silica coagulates. It is then filtered through a small ashless pa er and washed once with water and twice with dilute (1 N ) sufIuric acid. The paper is ignited in a platinum crucible and to the residue are added a few drops of concentrated sulfuric acid and about 1 ml. of hydrofluoric acid. This is heated until dense fumes of sulfuric acid appear, water is added, and the clear solution is combined with the main filtrate. The beaker containing this filtrate is then covered with a watch glass, heated, and kept boiling for 20 minutes in order to convert any meta- and pyrophosphates to orthophosphates. A few glass beads may be added to prevent bumping. If, on cooling, crystals are formed, they are brought back in solution by the addition of water, Then 10 ml. of saturated ammonium sulfate solution are added for every 50 ml. of the solution and calcium sulfate is precipitated by adding 1.3 volumes of 95% alcohol. The solution is allowed to stand for about 30 minutes and filtered through a Buchner funnel, and the precipitate is washed two or three times with small quan-
Cell for Electrolysis w i t h Mercury Cathode
Figure 1.
The electrolysis vessel shown in Figure 1 has proved particularly convenient for electrolysis of small volumes of solution. This cell, a modification of the one described by Melaven ( 5 ) , permits separation of the electrolyte from the mercury without the use of a leveling bulb. [Recently an electrolysis cell has been described in which the mercury is separated from the solution by a somewhat similar stopcock arrangement (IO).] In addition to the greater ease of operation, this has the advantage that contaminated mercury is never returned to the reservoir. (Used mercury may be cleaned sufficiently by aeration.) For electrolysis enough mercury is run into the cell from the reservoir to cover the lower paddle of the motor-driven stirrer and the flow of mercury is stopped by closing the lower stopcock while the upper remains open. The sulfuric acid solution of the phosphates is then transferred to the electrolysis vessel with the help of several small washin with water. The volume a t this point should be about 9 ml. #e platinum anode is adjusted to the top level of the solution and electrolysis is carried out with stirring, using a current of 0.2 to 0.3 ampere (a direct current source of about 20 volts is required). When most of the iron has been re
ANALYTICAL CHEMISTRY
830 Table I. Recovery of Beryllium Added to 24-Hour Urine Collections and to Tissues of Individuals Not Exposed to Beryllium Sample
Amount
M1. Urine
1300 I7ml
820 910 1500 1500 1650 1300 2000 1 Rnn 1900 1900 2200 2600
____
Weight of Ash Grama 14.9
Beryllium Added
Beryllium Found
7
7
Recovery
%
... ...
0
0
0.05 0.05 0.2 0.2 0.4 0.4 0.6
6.05 0.05 0.16 0.17 0.34 0.33 0.47
i .O
49
72
12.0 14.5 13.2 20.0
1 .o 1.2 1.2
0.6. 0.67 1.06 0 96
SO 67 88 80
0.4 0.1 6.6 14.0
0.4 0.4 0.5 0.5
0.34 0.33 0.41 0.40
16 5
11.5 12.8 16.5 13.9 10.9 16.4 17.2 19. . 1 .
n
n
n 6
1000 100' 80 85 85 83 78
low intensities of fluorescence are measured, i t is necessary to calibrate the cells and to apply the usual corrections to the readings. If the reading is higher than 100, the determination is r e peated, using a smaller aliquot of the unknown solution. To this aliquot (n ml.) are added an amount of aluminate solution equal to4-n 7 ml. and enough water to bring the volume to 4 ml.
R
The amount of beryllium in the original sample is - X 0.2 X 100 10 1 - X - micrograms, where R is the corrected reading and n the n 0.8 1 volume of aliquot in milliliters. The factor of - isintroduced to 0.8 correct for the loss of beryllium, because the average recovery during the procedure is 80% (see Table I). DETERMINATION OF
Grama
Liver
85 83 82 l3 80 25 Av. 80.5 Because of relatively large error in determination of very small intensities of fluorescence, these values are not included in average. Bone
53 20
Table 11. Recovery of Beryllium Added at Various Stages during Analysis of Beryllium-Free Urine Samples
Ashing Cas04 precipitation Preci itation of beryllium a n i i r o n phosphate Precipitation of beryllium and aluminum phosphate
0.42 0.43 0.43
84 86 86
0.46
92
moved (about 10 minutes) 2 ml. of 1 N sodium hydroxide are added to the electrolysis vessel, and any spray is rinsed from the sides of the vessel and from the stirring rod. Electrolysis is then continued until spot tests with ferrocyanide as well as with ferricyanide are negative. This usually takes another 10 minutes. At this point the spray is again washed down with a small portion of water and electrolysis is continued for another 15 minutes. The mercury is then lowered to the level of the upper stopcock by draining i t through the lower stopcock. During this process the anode is lowered in order not to interrupt the flow of current. The solution is then transferred to a conical 30-ml. centrifuge tube through the open end of the up er stopcock, and the electrolysis vessel is washed with repeatecf small portions of water. The volume of solution and washings should be 15 ml. To the iron-free solution are added 1 ml. of 0.2% aluminum solution and 2 drops of bromocresol green. Neutralization with dilute (3 N ) ammonia is then carried out slowly and with vigorous stirring until the first permanent precipitate of aluminum phoshate forms. The indicator a t this point should still be ellow. b i l e stirring i? continued 3 ml. of acetate buffer are added: The pH of the solution will then be exactly 4.4. The centnfuge tube IS laced in a boiling water bath for 5 minutes and centrifuged. &he supernatant liquid is decanted and the precipitate is suspended in 15 ml. of 3% otassium sulfate solution with the help of a footed stirring rod. $he tube is then placed in a boiling water bath until the precipitate coagulates and is centrifuged while hot. The washing procedure is repeated a second time. The precipitate of aluminum phosphate and beryllium is dissolved in 1 ml. of 1 N sodium hydroxide and heated briefly in a boilin water bath. It is then diluted to 10 ml. m t h water and centrifu ed If the procedure has been carried out correctly, there wifl be a t this point only a minimal amount of undissolved residue which consists of silica. Four milliliters of the clear solution are pi etted into an E-mm. test tube, followed by 1 ml. of stannite sorution. At the same time the blank is prepared by addin 1 ml. of aluminate solution and 1ml. of stannite solution to 3 mf of water. The standard contains 1 ml. of aluminate, 1 ml. of stannite 1 ml. of standard beryllium solution (0.2 microgram), and 2 mi. of water. These sohtions are allowed to stand at mom temperature for about 15 minutes, after which 1ml. of morin solution is added to all tubes simultaneously. The authors have measured the fluorescence with a Beckman photofluorometer, setting the blank at 0 and the standard to 100 on the transmittance scale with the selector switch in the 0.1 position. Because
BERYLLIUM M MATERIAL
OTHER
BIOLOGICAL
The procedure outlined above is easily adapted to the determination of beryllium in all kinds of biological material, including bone. The tissue to be analyzed is weighed and dissolved in 50% nitric acid, taken to dryness and ashed as described for urine. If heating in the muffle to 500 for 2 hours does not provide a clean whiteash, the ashis moistened with a small amountof nitric acid or 30% hydrogen peroxide and again heated .to.500 . The amount of tissue which can be subjected to analysis is essentially limited by the inconvenience of ashing large quantities. Up to 50 grams of soft tissues or bone have been ashed, however, without undue difficulty. The ash is treated exactly as in 'the procedure described for urine, except that in the case of most tissues other than bone the preliminary separation of calcium sulfate may be omitted, b e cause small concentrations of calcium are not carried down by the iron hosphate precipitate. d e n small amounts of tissues (
