1162
ANALYTICAL CHEMISTRY
The initial redox values changed but little with volume of permanganate added up to about 14 ml. and then gradually reversed. The rate of drop in redox potential increased with volume of permanganate added up to 20 ml. (1 ml. over the end point). Permanganate was still being reduced after the end point was reached, indicating incomplete oxidation a t this point.
CONCLUSION S
A comparison of the results obtained brings out the highly empirical nature of the two methods examined. The fairly close agreement with the phenolic substances, isolated from fruit when tannic acid was used as reference material, was due to the mixture of different polyphenolic materials. This agreement varies from one mixture to the other and therefore from one fruit to the other. Inspection of the absorption spectra during the course of the Loewenthal titration suggests the possibility of developing a colorimetric determination of the end point to replace the present difficult visual determination. Results of the redox potential measurements show that a po-
tentiometric determination of the end point of the permanganate titration is not feasible. LITERATURE CITED
- h o c . Offic.Agr. Chern., Washington, D. C., “Official Methods of Analysis,” 7th ed., pp. 175. 225,479, 1950. Barua, D. N., and Roberts, E. -4.H.. B i o c h a . J.,34, 1524-31 (1940). Bate-Smith, E. C., and Swain, T., Chemistry & I n d u s t r y , 1953, pp. 377-8. Folin, O., and Denis, W., J . B i d . Chem., 12, 23943 (1912). Joslyn, AI. $., “Food -%nalysis,” pp. 473-80, Academic, New York, 1950. Loewenthal, J., 2. arsal. Chena., 16,33-48 (1877). hlitchell, C.A., A n a l y s t , 61, 295-300 (193G). Rlonier, AI., Compt. rend., 46, 577-9 (1858). Nierenstein, AI., “Xatural Organic Tannins,” pp. 1-20, J. and A. Churchill, London. 1934. Pro, AI. J., J . Assoc. Ofic. -4gr. Chemists. 35, 255-7 (1952). Proctor, H.R.,Che7n. S e w s , 36,58-GO (1877);37,2568 (1878). Rosenblatt, AI., and Peluso, J. V.,J . Assoc. Ofic.A g r . Chemists, 24, 170-81 (1941). Sherman, L., Luh, B. S.,and Hinreiner, E., Food Technol.. 7, 480 (1953). Williams, A. H., A W L .Rept., d g r . and Hort. Research Sta., Long Ashton, Bristol, 1952, .. DP. 219-24. RECEIVED for review Korember 9, 1954. Accepted January 20, 1955.
Rapid Microprocedure for Determination of Mercury in Biological and Mineral Materials DOROTHY POLLEY and V. L. MILLER State College o f Washington, Western Washington Experiment Station, Puyallup, Wash.
A rapid procedure to determine microamounts of mercury in soil and biological materials was needed in an investigation of organic mercurial fungicides. 4 method is described which requires only standard laboratory equipment, is specific and relatively rapid, and has an accuracy within 1 y or 5 % at the 1- to 100-y level. Common metallic ions do not interfere. Thid procedure may be used to determine mercury in small amounts of soil, plant and animal materials, and in air.
A
RAPID and specific quantitative microprocedure for mercury in soil and biological materials was needed in an investigation of organic mercurial fungicides. Previous Boil analysis for mercury was reported by Stock and Cucuel(16), who separated the mercury by pyrolysis and measured the mercury globule formed following electrolytical deposition. The determination of mercury in biological material a t the microlevel is usually based on the color reaction between mercuric mercury and diphenylthiocarbazone (dithizone) in chloroform, the colored mercury dithixonate being measured in a photometer. This reaction together with the limitations is adequately discussed by Sandell (11) and Snell and Snell (13). Klein (3, 4)has made careful studies of this procedure. Snell and Snell pointed out that numerous modifications of this reaction leave much to be desired. The proposed method has comparable accuracy and fewer steps than many of the available procedures. The sample is prepared by digestion in concentrated sulfuric acid with dropwise additions of 507, hydrogen peroxide. This digestion mixture was used in the determination of mercury by Stettbacher ( 1 4 ) ,Graham ( 1 1, and recently by Melles and de Bree (6). The analysis of the solution for mercury is based on the same chemical reaction as one of the procedures for diphenylmercury previously reported by the authors ( 7 ) . An excess of an alcoholic solution of a diorganic mercurial reacts with a weak acid solution of mercury to form two molecules of the
corresponding organic mercury compound. The resulting organic mercurial is measured by the color formed from reaction with dithizone. REAGEYTS
Sulfuric acid, concentrated and 1.85. Hydrogen peroxide, 50 and 90%. Precautions for handling this reagent are given by Shanley and Greenspan (12’). (Both peroxide samples supplied by Buffalo Electrochemical Co.) Potassium permanganate solution, 3.0%. Hydroxylamine hydrochloride solution, 20.0%. Sodium chloride, 1N. Sodium acetate, 4N. Accutint indicator paper, No. 60. Ditolylmercury solution. Twenty milligrams of ditolylmercury (Eastman Kodak No. 1448) are dissolved in 200 ml. of neutral redistilled ethyl alcohol by heating under reflux on a steam bath. This reagent is stable for a t least 6 weeks when stored in a dark bottle. Chilling is avoided to prevent formation of crystals. Acetic acid, 0 . 3 5 . Dithizone solution. Eastman Kodak white label diphenylthiocarbaxone is dissolved a t the rate of 1 mg. per ml. in chloroform and stored under refrigeration. Dilutions in chloroform are made dailv to give the desired working range. Using a 4 to 120 dilution, 0 to 8 y of mercury may be determined, while a 10 to 120 dilution gives a 17- to 27-r range. Standard mercury solution, 1000 y per ml. Mercuric chloride is dissolved at the rate of 0.1354 gram pel 100 ml. of liV sulfuric acid. Measured amounts of a 1 to 100 dilution are used to prepare a standard curve each day. The sodium chloride, sodium acetate, and hydroxylamine hydrochloride solutions are extracted with dithizone, followed by chloroform. The water and chloroform are redistilled in a glass still. PREPAR4TION OF SAMPLE
The sulfuric acid-hydrogen peroxide digestion procedure must be modified for different types of material because very rapid reactions result in loss of mercury. The modifications necessary are determined by the organic matter and moisture content, and by the physical form of the sample. Each analyst should re-
1163
V O L U M E 2 7 , NO. 7, J U L Y 1 9 5 5 cover knawn amounts of mercury from the particular t) pe of material involved. One person can conduct several digestions Eimultaneously.
Soil, -4I-grqm sample of soil, that has been passed through a ijg-mesh scree4 $a remove rocks, roots, and other extraneous material, is weighed into a 125-ml. standard-taper flask,and 5 ml. of concentrated sulfuric acid are added. For soil contaming 15% or more organic matter 10 ml. of acid are used. The flask is attached to a West condenser and is swirled until the soil is mixed completely with the acid. Hydrogen peroxide, 5070, is added dropwise through the condenser and the flask swirled vigorously after each drop. The drops are added a t a rate which keeps the mixture bubbling gently but never so there is a Iaige accumulation of peroxide. Hydrogen peroxide, 50% is added until the solution portion becomes white or pale yellow, or until 2 drops a t once cause no reaction. Mineral soils clear rapidly, but dropwise additions for 1.5 hours are sufficient for even the very high organic matter soils. The mixture is heated slowly ~ i t the h flask on an asbestos board using a microburner. The additions of 50% hydrogen peroxide are continued for 20 inutes and the mixture is boiled for approximately 2 minutes i#rFr the last addition. Following a brief air cooling, the flask p jinpersed in a cold water bath and 15 ml. of redistilled water are &ed. The solution is mixed and cooled again. Potassium 'permgqganate, 3.070, is added until the permanganate color jemajqs. The condenser is rinsed with water, and the excem permanganate removed by the addition of 6 ml. of 20% hydroxylamine hydrochloride. The condenser is washed down again. After standing for 15 minutes or longer the mixture is filtered using a medium porosity sintered-glass filter and made to 100ml. Sodium phloride lS, is used for the transfer. Plant and Animal 'hissue. A sample low in moisture and in a finely giound form is typified by fish meal. Ten milliliters of ,concentrated sulfuric acid are added to a 1-gram sample and mixed thoroughly before adding the first drop of 50% hydrogen peroxide. The first few drops must be added very slowly with much mixing to prevent a rapid reaction with visible vapor loss When the initid foaming has subsided the drops may be added fairly rapidly. Additions are mgde until the solution is pale yel'low or Tvhite. If the solution hecames cold and the peroxide is not reacting, the additions m+y be stopped when the solution is still prange. It is heated slowly on a steam bath with occasional swirling and with no additional peroxide until the excess has reacted. Drops are then added until the solution is clear and almost cslorless. The flask is removed from the steam bath and heated cautiously with a microburner. Drops of peroxide are added as the solution darkens and heating is continued until the solution remains white after heating for approximately 2 minutes. Potassium permanganate, 3.0%, and 20% hydroxylamine hydrochloride are added as with soil samples. The solution is transferred using 1-V sodium chloride and made t o 100 ml. The husk of a narcissus bulb is a sample low in moisture and in the form of scales. This type of sample is soaked approximately 0 minutes in 10 ml. of the acid and mixed thoroughly before the rst addition of 5070 hydrogen peroxide. It is then treated like (be fish meal. An example of a sample low in moisture and in the form of chuqks is kernels of wheat. A 1-gram sample is soaked 10 minutes in 5 ml. of concentrated sulfuric acid. Hydrogen peroxide, SO%, is added dropwise with swirling until approximatelv 15 drops have been added. The solution will still be black a t this point. It is placed on a steam bath and the digestion procedure follows that outlined for fiqh meal after placement on the steam bath. The meat of a bulb is an example of high moisture content and chunk form. To a sample not exceeding 3 grams of dry matter aIe added 10 ml. of concentrated sulfuric acid. The mixture is cooled before addition of 50% hydrogen peroxide. I t is then treated in the same way as the wheat kernels, except that 90% hydrogen peroxide is used after the first reaction on the steam bath is over. It will not become colorless on the steam bath and the heating with the burner is begun when the sample is light orange. As there is considerable way present, the solution must be filtered.
6
DETERMIYATION
.4n aliquot of the digest containing 0 to 8 or 17 to 27 y is placed in a 250-ml. separatorg funnel containing an equal volume of water, or with small aliquots sufficient water to make a total volume of 75 ml. When 10 ml. of concentrated sulfuric acid are used in the digestion, the water must have a volume of twice the aliquot. With fish-meal samples it is necessary to add a t this point 3 ml. of 5 5 sodium chloride for every 25 ml. of the sample
used. Mudium acetate, 4N, is added with swirling to give a pH of 3.0 tu 4.0 a8 determined by Accutint indicator paper. One milliliter of ditolylmercury solution is added and the separatory shaken 3 times. If the sample is in the 17- to 27-7 range,.2 ml. of ditolylmercury solution are used. After standing 1 minute, approximately 9.5 ml. of chloroform are added and the funnel is shaken for 1 minute. When the volume of solution in the first separatory is 150 ml. or greater the extraction with chloroform is done in two steps. The first is with 8.0 ml. of chloroform and the second with 3.0 ml. The chloroform phaEe is transferred to a second separatory funnel which contains 25 ml. of 0.3N acetic acid. One milliliter of diluted dithiaone solution, corresponding to the expected mercury content, is measured into the funnel and shaken for 30 seconds. The chloroform phase is transferred and made to volume in a 10-ml. volumetric tube. The percentage of transmittance is determined with a 620-mp filter in an Evelyn photoelectric colorimeter, As in the determination of organic mercury compounds by the dithiaone procedure (8),the unreacted dithizone color is measured. The amount of mercury present is determined by comparing with a curve prepared by using the standard mercury solution in the above procedure. For the standards the first separator\funnel contains 50 ml. of water, 12.5 ml. of 1.8&Vsulfuric acid, and 3 ml. of 20% hydroxylamine hydrochloride. The standard curve follows Beer's law in the indicated range. DISCUSSION
In the usual procedures for the determination of mercury in biological material, the digestion is accomplished by a mixture of nitric and sulfuric acids. I n the described procedure nitric acid could not be used in the digestion. Some types of samples, notably bulbs, on digestion with nitric and sulfuric acid in the apparatus described by Klein ( 4 ) form a substance which prevents the reaction between mercury and the diorganic mercurial. This subst'ance could be removed by aeration or steam distillat,ion from acid solution and collected in alkali, but it was not characterized further. High results were occasionally obtained with nitric acid digests which were unexplained. Using the small samples described in this procedure, the coinplicated apparatus of Klein (4)offered no advantage over the simple flask and condenser. Fuming sulfuric acid \vas tried in place of the concentrated acid in the digestions. I t seemed to offer no advantages, and had the disadvantage of causing a more violent reaction with the peroxide. Much of the success of the digestion is believed to be due to a gentle oxidation, avoiding sudden vigorous reaction when mercury is lost, presumably by expulsion from the condenser. If soil was digested and filtered without the addition of the chloride ion, the results were always low. With tile addition of the chloride it is believed that the complex ion between mercury and chloride forms ( 9 ) . This prevents adsorption of the mercury by the silica or other insoluble material. il large excess of chloride is necessary for the reaction. Fish iiieal requires an unusually large excess; otherwise the results are low. Three milliliters of 5 N sodium chloride per 25 nil. of solution give accurate readings and a greater excess gives no higher result,s. Mercury has been reported to be adsorbed onto the iyalls of containing vessels, especially from very dilute solution ( 1 1 ) . To test the effect of acidity and chloride on the stahilitL- of mercury solutions in glass, known amounts of niei.cury ivel'e placed in flasks in solutions of various acidity and chloride conceritration. Without chloride there was no appreciable loss of mercury with acidification below 0 . 3 N , but as acidity increased the losses became kigher reaching 18% a t 1.8X. -4bove 1.8X the adsorption decreased until with 9.V acid there was no loss. The mercury was not adsorbed from 0,001 to 9 S sulfui,ic acid in a 24-hour period xhen the solution was 0 . 3 S in chloride. The concentration of mercury was approximately 1 y per nil. of solution. Four dimercurials were found to react quantitatively in the general procedure, but required different reaction conditions. These were di-p-tolylmercury, diphenylniercury, dinitrophenylmercury, and bis-m-(or,cr,~-trifluorotolrl)niercury. The latter two were prepared by known reactions (2, ,5, 10, 1 6 ) . The reaction of
ANALYTICAL CHEMISTRY
1164 diphenylmercury with mercuric chloride required a p H of 4.3. Even a t this acidity the procedure had t o be carried out rapidly or there was some breakdown of the diphenylmercury. The di(nitropheny1)mercury required 2 to 4N sulfuric acid and the virtual absence of chloride for the reaction t o take place. ilcidity down to p H of 2.0 could be used with the bis-m-(a,a,a-trifluoroto1yl)mercury but it had the disadvantage of reacting slowly. The ditolyl compound was chosen for this procedure because itwas more stable toward acid and had a more favorable solubility ratio than the diphenylmercury, and the reaction took place more rapidly than with the fluorinated compound. It was also available commercially. Blanks showed no detectable breakdown of the ditolylmercury under the conditions of the procedure. Ditolylmercury is soluble only with difficulty in ethyl alcohol. Other solvents that were tested in the preparation of the ditolylmercury reagent were methanol, isopropanol, acetone, and ethoxyethanol. The first two solvents appeared to be satisfactory but offered no advantages over ethyl alcohol. The latter two solvents, while dissolving the ditolylmercury much more readily, seriously interfered in the determination by causing decomposition of the ditolylmercury. The spectrophotometric curve for the dithizonate resulting in this procedure has a minimum a t 480 mp, which agrees with x standard p-tolylmercuric dithizonate curve and is 10 mp shorter than the minimum for mercury. Equations 1 and 2 show that the sensitivity of the new procedure is the same as t h a t of the usual dithizone reaction indicated by Equation 3. C H r a H g a C H a
+ 2HSD, HgC12 + 2HSDz
2 C H 3 a H g C I
+ HgC12
-+
-*
-+
2CH3C>HgC1
2CH9P l H g S D , (D,S)pHg
+ 2HC1
+ 2HCl
Table 11. Recovery of Added nIercury from Soil or Biological llaterial (50 t o 100 y of Hg)
MoistureMaterial
Solubility ratio
2 6 3 2 2 3 2 3 2 5 17 6 8 5 2
M a x . Dev., 70 -4.4 -2.4
$3.6
-1.0 -4.1
-2.0
-2.0 -3.5 -3.4
101 8
9 +3.0
-3.0 -3.3 -2.8 -3 2
Av. Std. Recovery, “u
+3.0 f2.8 4-1.3
98 1 101 i 99 0 97 4 98 8 97 I 97 5 97 9 98 7 98 8
100 1 97 9 101 1
(2) Table 111. Range of Procedure ( H e added to soil)
(3)
CHtCoHpHpCl, y per hII. 13,650
12 4
CHCls reagent mixture
4.9 5.5 6.1 7.2 8.3 11 3 14.8 15.6 17.4 41.1 88.9 95.2 98.0 98.2
ples
Using the Beckman DU spectrophotometer and a 2 to 120 dilution of the dithizone reagent, amounts down to 0.5 y can be readily determined. With the Evelyn photoelectric colorimeter and using either the 4 to 120 or 10 to 120 dilutions of the dithizone, 1-gram samples of soil containing 0 to 100 y of mercury were nnnlyzed (Tahle 111). Standard added in the form of organic mercury compounds gave equally satisfactory results. Great care must be used when analyzing samples containing methyl mercury compounds due to the volatility of these materials. The accuracy of the procedure is 1 */ or 5%, whichever is the (1) larger.
Solubilities of p-Tolyl Mercuric Chloride a t Room Temperature
Solvent Chloroform Water Reagent mixture
%
Soil Lauren sandy loam h-isqually gravelly loam Puyallup sandy loam Lynden sandy loam Conroy sandy loam Salkum loam Chehalis silt loam Buckley loam Cinebar loam Peat Fish meal Bulb husks Bulb meat Wheat (kernels) Air analysis solution
Standard curves prepared by the reactions outlined in Equations 1 and 2 and Equation 3 are identical. The extraction of the p-tolyl mercuric chloride from the reagent mixture to the chloroform is accomplished by one separation, since the solubility is so great in chloroform (Table I). The two extractions with chloroform for the large volume samples are necessary because of the loss of chloroform in the larger amount of water and not because of solubility of the p-tolyl mercuric chloride.
Table I.
Free O m . No. of l l a t t e r , Sam-
= 3413
One thousand micrograms of iron, cobalt, nickel, zinc, cadmium, lead, iron, copper, manganese, and bismuth do not interfere in the determination. Silver requires an additional shake-out of the chloroform phase with a IN sodium chloride solution to prevent mechanical carry-over of the silver chloride precipitate t o the dithizone solution. This analytical procedure can be used t o determine mercury in soil, air, and material high in organic matter content, with equally satisfactory results (Table 11). The biological materials include both plant and animal matter and represent samples coarse and finely ground and high and low in moisture. Mercury standard was recovered from a reagent mixture that is used in the absorption of mercury from air (11).
.
Added
As HgCIz
Hg, y
n 5 30 50 100
24 75.7
98.3
Found Av., y H g
N ~ of. Samples
0 2 5 3 29 9 50.2 96 4 23 5 73 8 95.4
Maximum Deviation Y of Hg +0.7
-1.5 -0.5 -3.8 -0.6 -2.1 -3.1
+1.0 +1.6 +1,5
LITERATURE CITED
(1) Graham, J. J. T., J . Assoc. Ofic. -4gr. Chemists, 13, 156 (1930). (2) Hein, F., and Wagler, K., Be?., 58, 1499 (1925). (3) Klein, A. K., J . Assoc. Ofic. Agr. Chemists, 32, 351 (1949). (4) Ibid., 35, 537 (1952). ( 5 ) Kobe, K. A , , and Doumani, T. F., I n d . Eng. Chem., 33, 170 (1941). (6) Melles, J. L., and Bree, W. de, Rec. trau. chim., 72, 576 (1953). (7) hliller, V. L., and P o k y , Dorothy, A N ~ L C. H m f . , 26, 1247 (1954). (8) Miller, V. L., Polley, Dorothy, and Gould. C. J., I h i d . , 23, 1286 (1951). (9) Moeller, Therald, “Inorganic Chemistry. ;In hdvanced Textbook,” Wiley, New York, 1952. (10) Nesmejanow, A. N., Ber., 62, 110 (1929).
(11) Sandell, E. B., “Colorimetric Determination of Traces of Metal,” 2nd ed., Interscience, S e w York, 1950. (12) Shanley, E. S., and Greenspan, F. P., Ind. Eng. Chem., 3 9 , 1536 (1947).
Snell, F. D., and Snell, C. T., “Colorimetric Methods of Analysis,” 3rd ed., Van Sostrand, Xew York. 1948. (14) Stettbacher, A , , Tech. I n d . Schweiz. Chem.-Ztg., 1924, p. 242. (15) Stock, Alfred, and Cucuel, Friedrich, Sati~ricissenschuften,22, (13)
390 (1934). (16)
Whitmore, F.C., and Sobatski, R. J., J . A m . Chem. Soc., 55,
1128 (1933). RECEIVEDfor review November 20, 1954. Accepted January 21, 1965. Presented a t the Northwest Regional Meeting. AXERICANCHEYIC.%I, SOCIETY, Richland. Wash., June 11 and 12, 1954. Scientific Paper No. 1367. Washington Agricultural Experiment Station, Pullman. Project N o s . 721 and 1208.
