,2-5,
2.0 i3.0
Figure 2.
G
\
Microliter pipet for 5- to 500-PI. sizes
Dpliverv Stem .. I.D. Approx. Mas. hlin. Wall I.D. a t end, Min. Cap., length, O.D., length, at end, Tubing, Size! 1.11. Over-all Mm. Arm. Safety Bulb mm. mm. mm. Mm. Length, (Calibd. t o Contain) Mm. B D E F G C Ul. 65 4 55 0.5-0.7 0.15-0.25 50 0.18-0.25 5 140 zk 5 Fio 65 4 55 0.5-0.7 0.15-0.25 0.18-0.25 6 140 f 5 65 4 55 0 5-0 7 0 15-0 25 50 0 18-0 25 140 i.5 50 0 18-0 25 65 4 55 0 5 0 7 0 15-0 25 140 i.5 50 65 4 55 0 5-0 7 0 15-0 25 0 18-0 25 9 140 i.5 50 65 4 55 0 5-0 7 0 15-0 25 10 140 i.5 0 20-0 35 55 0.5-0.7 0.15-0.25 50 65 4 15 140 i.5 0,25-0,40 50 0,35-0,50 65 4 55 0.5-0.7 0.25-0.50 20 140 i.5 0.35-0.50 65 4 55 0.5-0.7 0.25-0.60 50 25 140 i.5 50 0.35-0.50 65 4 55 0.5-0.7 0.25-0.50 35 140 i.5 50 65 4 55 0.5-0.z 0.25-0.50 50 140 1 5 0,35-0.50 0.30-0.50 50 65 4 55 0.5-0.r 60 140 f 5 0.40-0.55 75 0,40-0,60 6.5 4 55 0,5--0.7 0.30-0.50 75 140 f 5 100 140 i.5 0.50-0.75 65 4 55 0.5-0.7 0.30-0.50 IS 150 140 i.5 0.75-1.00 65 4 55 0.5-0.7 0.40-0.60 100 0.75-1.00 65 4 55 0.6-0.8 0.40-0.60 100 200 145 i.10 0.75-1.00 65 4 55 0.6-0.8 0.40-0.60 100 250 145 i.10 300 145 i.10 5-4 0.75-100 65 4 55 0.6-0.8 0.40-0. T O 200 400 150 zt IO 6-7 1.00-1.25 70 6 GO 0.6-0.8 0.40-0.70 200 590 100 1 10 6-7 1.23-1.50 70 6 60 0.6-0.8 0.40-0.70 200 a Closer volumetric standardization must be carried out by user with substances riiider actual conditions of use. ~
r
;
O.D. Tubing, Mm. A 5-6 5-6 5-6 5-6 5-6 5-6 5-6 5-6 5-6 5-6 5-6 5-6 5-6 5-6 5-6 5-6 5-6
%
Xko 5
f0 5 i.0 5
1 0 5 1 0 5 3x0 5 10 5 10 5 10 5 =IC0 3 i.0 3 i.0 3 10 3 1 0 3 iro 3 1 0 2 10 2 i.0 2
r -
understood that the vendor marks the pipets on the basis of a similar method employing a n adjusted volume of mercury. zkdditional reports be made other microvolumetric apparatus.
Vol. ToLa,
On
LITERATURE CITED
(1) Chem. News26, 883 (1948). (2) Committee on hlicrochemical Apparatus. Division of Analvtical Chemistrv. American Chemical Society, ANA:: CHEM.21, 1283,1555 (1949);22, 1228 (1950);23, 523, 1689 (1951);26, 1186 (1954);28, 112,1993 (1956).
+o
2 10 2
(3) L’pson, U. L., Hanford Works, Genera1 Electric Co., Richland, Wash., private communication. RECEIVEDfor review May 26, 1958. Accepted May 26, 1958. Division of Analytical Chemistry, 133rd Meeting, ACS, San Francisco, Calif., April 1958.
Semimicrodetermination of Bromides Application to Physiological Fluids DAVID KAPLAN and ISAAC SCHNERB Research laboratory, Dead Sea Works, Itd., Jerusalem, Israel
.A
semimicroanalytical method for bromides, based on the van der Meulen method, is describsd. Bromide can be evaluated in 0.01-mg. quantities (0.01 mg. of bromide ion) with an This method can b e error of f3%. applied to the determination of bromide in physiological fluids and preashing of the sample can b e avoided.
L
of a convenient and accurate microanalytical method has made it difficult to investigate the bromide ACK
content of plant and animal fluid$, such as blood, urine, and sweat, when only small quantities of these substances are available. Several publications exist on bromide distribution in living matter, but few deal with normal diet conditions; most are concerned with bromide distribution in the body after bromide administration. The methods in use are inconvenient for the determination of the bromide present under normal conditions. Procedures recommended by certain authors are
rejected by others (6, 8-10) and in a critical survey of methods up to 1936, Neufeld (6) states that only some are reliable. When bromide is determined in living matter, an initial destruction of the organic material is necessary. This is usually accomplished by an alkaline ashing process, even if the bromide ion content is of interest, and not the organically bound bromide; also the micromethod recommended by Hunter (1, 2) provides for an initial alkaline ashing treatment. VOL. 30, NO. 10, OCTOBER 1958
0
1703
An accurate, reliable method 6-hich is especially adapted to organic materials is presented for determining small amounts of bromides. It does not require an ashing process, is convenient, and allows a differentiation between bromide ion and organically bound bromide. van der Meulen (4)recommended a method based on the oxidation of bromides to bromate by sodium hypochlorite, according to the equation: 3C10Br- = BrOs3C1The bromate is then titrated iodometrically. This method describes the determination of not less than 0.1 to 0.2 mg. of bromide. Even 0.01 mg. of bromide can be determined if the concentrations are suitably changed. The original volumes of the reagents and the solutions to be analyzed w r e reduced to one fourth; likewise, volumetric solutions of 0.0004Y or 0.0002.V sodium thiosulfate were used, instead of a 0.002.V solution, for the titration of bromate. The end points obtained under these conditions were very distinct (vanishing of the blue iodinestarch color).
+
+
MODIFIED VAN DER MEULEN METHOD
In a 100-ml. Erlenmeyer flask, treat 5 ml. of potassium bromide solution, containing less than 0.01 mg. of bromide, with 5 ml. of boric acid (470), and 2.5 ml. of sodium hypochlorite solution (approximately 0.3.V). Place the sample on a sand bath a t 80" to 90' C. for 15 minutes, add 0.5 ml. of formic acid (%YO), and cool to room temperature. Add 2 ml. of potassium iodide solution (5%), 4 ml. of 2N hydrochloric acid, and 0.5 ml. of starch solution (0.25%). Titrate the mixture after 3 minutes with 0.0002N or 0.0004-1; sodium thiosulfate. Perform a blank test, using water instead of potassium bromide solution. Each milliliter of 0.0004V sodium thiosulfate is equal t o 0.005328 mg. of bromide, and each milliliter of 0.0002N sodium thiosulfate is equal to 0.002664 mg. of bromide. This modification of the van der Meulen procedure was applied to three dilute solutions of potassium bromide (Table I). The blank titration values in these determinations varied from 1.46 to 2.0 ml. of sodium thiosulfate. Because this use of the van der Meulen method determines 10 y of bromide with a maximum deviation of 3yo, 1 to 2 ml. of body fluids containing bromide of the order of 0.01 mg. of bromide ion (or mg. Yo) can be studied. DETERMINATION IN URINE AND BLOOD
Methods based on the oxidation of bromides to bromine or bromate require an initial separation from the organic matter. There is no evidence that bromides in biological fluids are organically bound (3, 7 , 9); also, the 1704
ANALYTICAL CHEMISTY
bromide ion content is of great interest itself. Instead of using the ashing procedure, the authors separated the halides from the organic material by precipitation with silver nitrate in the presence of nitric acid. If protein is contained in the organic materiale.g., blood-the halide precipitation is preceded by a protein separation with tungstic acid. The silver halides are transferred to zinc halides and the zinc bromide is finally determined by the modified van der Meulen method. Before analyzing physiological fluids, this method n-as checked with solutions of known bromide content. Several determinations of about 0.15 mg. of bromide showed deviations of +0.9 to - 1.S%. Reagents. Nitric acid, %yo. Silver nitrate solution, 15%. Hydrochloric acid, 2N (containing not more than 0.7 mg. of bromide per liter). Zinc metal, granulated. Sodium hypochlorite solution, approximately 0.3K (containing not more than 0.5 mg. of bromide per liter). Boric acid solution, 4y0. Formic acid, Potassium iodide solution, 5%. Starch solution, 0.2570. Sodium thiosulfate solutions, 0.002N and 0.0004N. Sodium chloride solutions, 0.5 and 10% (containing not, more than 0.0002% bromide) Sodium tungstate solution, 10%. Sulfuric acid, 0.66N. Procedure for Urine. Dilute 60 to 80 ml. of protein-free urine with about the same quantity of distilled r a t e r . -4dd 5 ml. of 35% nitric acid and allow a small excess of a 15% silver nitrate solution to flow slowly into the urine. After intensive mixing with a glass rod for 5 minutes, keep the mixture in a dark place. When the supernatant solution is clear, filter the silver halide precipitate, and wash with water of 25' to 30" C. until the filtrate is free of nitric acid (if the filtrate is turbid, wash it with a 0.5% sodium chloride solution). Transfer the precipitate t o a 100-ml. Erlenmeyer flask with a small amount of water, add 10 ml. of 2N hydrochloric acid and 2.0 grams of zinc granules, and stir for 5 minutes. After 1 to 2 hours filter the precipitated metallic silver, collect the filtrate, together with the wash water, in a 100-ml. volumetric flask, and dilute t o the mark. Analyze 20 ml. for bromide according to the original method of van der Xeulen, using for titration a 0.002N sodium thiosulfate solution. If less urine is available, the determination can be carried out with 3 to 5 ml.; if the chlorine content is low, add sodium chloride and follow the procedure for blood or serum. Procedure for Blood or Serum. To pretreat the sample for the separation of proteins, mix the following reagents intensively in a test tube or small Erlenmeyer flask, and filter the protein precipitate: I
Blood, Serum, 2 h11. 2 "I. Distilled water, ml. 10% sodium tungstate, ml. 0.66N sulfuric acid, ml.
14
16
2 2
1 1
To 10 ml. of the filtraw (equivalent to 1 ml. of blood or serum) in a 20-ml. test tube, add 1 ml. of sodium chloride solution, lo%, 2 ml. of silver nitrate solution, 15%, and 0.5 nil. of nitric acid, %yo. After intensive stirring, allow the mixture to strnd in the dark until the supernatant fluid is clear. Separate the silver halide precipitate by centrifugation (2000 to 3000 r.p.m., for 2 minutes) and draw off the liquid. Repeat the centrifugation 5 or 6 times, each time mixing the precipitate with 4 to 5 ml. of distilled water a t 25' to 30' C. with a glass rod. T h e n the nitric acid has thus been removed, mix 0.10 g. of zinc (analytical purity) and 1 ml. of 2Ar sulfuric acid with the precipitate. After 1 hour, separate the silver formed by centrifugation. Collect the supernatant liquid together with about five portions of wash mater (using
Table 1. Determination of Bromide in Dilute Solutions of Potassium Bromide
Bromide Found, Mg. Deviation, 7' 0 038 hfg. of Bromide, O.OOO&V Sa2S203 0 0380 0 0 0 0376 -1 0 -1 0 0 0376 0 0386 +1 6 0 0387 +I 8 0.019 Mg. of Bromide, 0.0004Y Sa2S2Oa 0 0187 -1 3 0 0193 +I 5 0 0185 -2 6 0 0193 +I 8 0 0188 -1 1 0.0095 hlg. of Bromide, 0.0002-V Na2SzOa 0.00954 0.00927 0.0098 0.0097 0.00927
+0.4 -2.4 $3.1 +2.1 -2.4
Determination of Bromide in Serum
Table II.
Mg. of Bromide per 100 311
.4
B
1 38
1.37 1.40 1.42
1.38 1.36 1.41
111.
Table
Duplicate
Determinations
of Bromide in Urine and Serum (Mg. of bromide per 100 ml.)
Urine 1.27 0.59 1.70 0.78 0.79 0.37 0.76 0.67 0.090 0.51
1.29 0.62 1.71 0.76 0.77 0.38 0.74 0.68 0.093 0.53
Serum 1.26 1.42 1.58 0.72 0.69 0.75 0.98 1.66 0.85 0.45
1.26 1.47 1.55 0.71 0.74 0.77 1.00 1.69 0.85 0.42
1 ml. of distilled water followed by centrifugation) in a 100-ml. Erlenmeyer flask. Determine the bromide present according t o the modified van der lleulen method. Perform a blank test simultaneously.
Table I1 gives the results obtained for this procedure in four parallel determinations (A) compared with three parallel determinations, starting from 4 ml. of serum (B). The blank titration values for these determinations n-ere constantly 1.3 ml. of sodium thiosulfate. A maximum deviation of 1.8% was obtained with this procedure for five determinations using a solution known to contain 0.038 nig. of bromide instead of serum. Duplicate bromide determinations n-ere carried out on several samples of urine and serum (Table 111).
+
OTHER PHYSIOLOGICAL FLUIDS
Bromide can also be evaluated in sweat, saliva, or cerebrospinal fluid. Cerebrospinal fluid is treated in the same way as serum (using 2 ml. for protein precipitation). Because sweat and saliva are almost protein free, 0.5 to 2 ml. may be treated directly with silver nitrate, after adding 0.1 gram of sodium chloride. ACKNOWLEDGMENT
The authors are indebted t o hI. R. Bloch, ~ h suggested o this work, for his help and advice throughout this investigation.
(2) Hunter, G., Goldspink, A. A, Analyst 79, 467 (1954). (3), Xason, hl. F., J . Biol. Chenz. 113, 61-73 (1936). (4'1 Meulen. J. H. van der. Chem. Weekblad 28,' 82, 238 (1931); 31, 558 (1934). (5) Sagv, hl., Straub, J., Z. Seurol. Psych." 153, 215-21 (1935). 16) Seufeld. -4.H.. Can. J . Research 14 B, 160-94 (1936): (7'1 Pincussen. L.. Roman. W..Biochem. Z. 216, 361 (1929). (8)Weir, E. G., Hastinge, A. B., J . Biol. C'hem. 129, 547-58 (1939). (9) Kikoff, H. L., Bame, E., Brandt, \
,
~
\
,
,
AI..
J
(1939).
I
Lab. Clin. M e d .
24. 427
(10) Zondek, H., Tvipi, G., I . Pisani, Giorn. patol. neruosa e mentale 58
(1938).
LITERATURE CITED
(1) Hunter, G., Biochem.
(1955).
J . 60, 261
RECEIVEDfor reviev August 1, 1957. Accepted December 7, 1957.
Microestimation of Intact Phenylmercury Compounds in Animal Tissue V. L. MILLER, DONNA LILLIS, and ELIZABETH CSONKA Western Washington Experiment Station, Puyallup, Wash. F A procedure for the estimation of approximately 5 to 20 y of phenylmercury acetate per gram of animal tissue or 5 ml. of urine i s presented. The sample is saponified and oxidized with alkaline permanganate. Excess oxidant i s destroyed b y hydroxylamine and ammonium sulfamate. Following acidification with hydrochloric acid, the phenylmercury i s extracted as chloride with chloroform. The analysis is completed b y the dithizone reaction for RHgX compounds.
I
and agriculture, organic compounds of mercury are now used more than inorganic. However, almost all biochemical knowledge of mercury, whether organic or inorganic, is based on analysis of the material as inorganic mercury. Because of the many types of organic compounds of mercury with varying biological properties, knowledge of the intact organic mercurial compound in the body is desirable. In the field of toxicology, Schoeller and Schrauth (6) reported that some organic mercurials are sufficiently stable to be excreted unchanged. More recently Sivennson (7) and Hagen ( 2 ) investigated ethylmercury, niethj-lmercury, and phenylmercury compounds used as fungicides. They each reported that the symptoms produced by injection or feeding of these materials are different from the symptoms from mercuric chloride or acetate, and that each produces distinguishable N MEDICINE
symptoms. Fitzhugh et al. (1) reported that larger amounts of mercury are stored in the liver following feeding of phenylmercury acetate, than when mercuric acetate is fed. Lundgren and Swennson (3) reported that alkyl mercury compounds are stored to a much greater extent in nervous tissue than inorganic mercury, folloI\-ing exposure of workmen to these materials. All the results except those of Schoeller and Schrauth (6) are based on analysis of tissue or excretion products for inorganic mercury. Because inorganic mercury and phenylmercury react differently with the dithizone reagent (4, 5 ) , it appeared feasible to determine this organic mercurial in its original form in urine or tissue. This paper reports a method suitable for the determination of from 5 to 20 y of phenylrnercury acetate in urine, liver, kidney, spleen, muscle, or brain. REAGENTS
Although micro amounts of inorganic mercury are a common contaminant of reagents, phenylmercury is an unlikely one. Therefore rigorous purification of reagents, except for chloroform, is unnecessary. All reagents are S.C.S. unless otherwise indicated. Extract chloroform, U.S.P. or A.C.S. grade, with 2% of its volume of sulfuric acid. Then extract three times with 30% sodium chloride. Shake the chloroform with lime and anhydrous calcium chloride, filter, and distill. Add 1%ethyl alcohol as preservative. Used chloroform may be recovered by first distill-
ing over lime, then following the procedure outlined. Test all chloroform for satisfactory performance by preparing a standard curve before use. Dissolve hydroxylamine sulfate, Commercial Solvents technical grade, 30% weight per volume in water, and filter. Dissolve ammonium sulfamate, Eastman Kodak. yellow label grade, 30% weight per volume in water, and filter. Dissolve Eastman Kodak white label dithizone in purified chloroform a t the rate of 1 mg. per ml. For daily use, dilute a t the rate of 1 t o 30 ml. of chloroform. This concentration gives a working range of from 0 to 30 y of phenylmercury acetate. Absorption Columns. Prepare the columns for all tissue except brain by sealing 7 5 mm. of 0.5-mm. inside diameter glass capillary tubing t o 85 mm. of 10-mm. inside diameter tubing with a reservoir of 7 5 mm. of 18-mm. inside diameter tubing a t the top. Prepare the columns for brain by sealing 7 5 mm. of 0.5-mm, inside diameter capillary tubing to 200 mm. of 13-mm. inside diameter tubing. The adsorbant is Hyflo Supercel, which has been treated with 1N hydrochloric acid 3 to 4 times on the steam bath until the acid gives only a faint test for iron with thiocyanate. TTash the Supercel free from acid and oven dry a t approximately looo c. Prepare the small columns for use by placing a piece of glass fiber filter paper in the bottom of the column, and add and pack the Supercel t o a depth of 55 mm. Pack 5 mm. of calcium carbonate on the top. Pack the large columns for brain analysis to a depth of VOL. 30, NO. 10, OCTOBER 1958
1705
