Improvements in the Wet Oxidation-Dithizone Method for Determining Low Mercury Levels in Food Michal Nabrzyski Department of Bromatology, Faculty of Pharmacy, Medical Academy, Gdansk, Poland
Mercury in the environment is of current interest. Reports on a significant tendency of mercury to accumulate in fish have emphasized the potential hazards of entry of this element into the food chain ( I ) . To control mercury residue in food as the effect of pollution, it is necessary to recognize every level of mercury actually present in food. Special attention should be paid to fish because most of the mercury exists in the form of methylmercury compounds. Neutron activation analysis ( 2 ) and atomic absorption spectrophotometry methods (3, 4 ) allow determination of nanogram quantities of mercury. Wet oxidation-dithizone procedures developed by Gorsuch ( 5 ) ,Hordydska et al. (6, 7 ) , as well as those reported by IUPAC-1965 (8), and by the Analytical Methods Committee (AMC) (9) make it possible to assay more than 1 kg amounts of mercury per sample. The proposed procedure presented here involves the wet oxidation stage based on that reported by Gorsuch ( 5 ) and by Hordydska et al. (6, 7 ) . The improvements made refer to the dithizone extraction stage. Some items of the reverse sodium nitrite techniques recommended for mercury determination by IUPAC (8) and the AMC (9) have been utilized. However, the procedure presented as a whole differs markedly from those methods. By successively combining the dithizonate extracts obtained from two or more digested samples, it is possible to prepare one sample richer in mercury. This makes it possible to determine lower levels of mercury. Neither methods recommended by IUPAC, nor those reported by other authors quoted have been found suitable for assaying such small levels of mercury in food. The only condition is that a proper amount of material should be available for analysis.
EXPERIMENTAL Reagents. All reagents should be of Analytical Reagent Quality. If necessary, they should be purified by a procedure recommended by IUPAC. Glassware should be thoroughly cleaned with nitric acid and then washed with distilled water before each use. Digestion Procedure. The most convenient digestion apparatus proved to be that designed by Hordydska et al. (6). Step I . The procedure to be followed depends on the mercury level in the sample. If less than 1 pg of mercury is present, two or more parallel samples should be taken for wet oxidation. In this work, 50 g of a fresh fish muscle or 15 g of milk powder or rice grains were taken as a single sample for wet oxidation. The sample was placed in a 750-ml flask of the oxidation apparatus and digested by a mixture of sulfuric, perchloric, and nitric acids. The (1) G . Westoo, M. Rydalv, VlrFoeda, 7 4 , 1 7 9 , (1971) (2) K. Ishida, S. Kawamura, and M . Izawa. Anal. Chim. Acta., 50, 351 (1970). (3) G. Lindsted and I . Skare, Analyst (London), 96, 223 (1971). (4) I. Skare,Ana/yst (London) 97, 148 (1972). (5) T. T. Gorsuch, Analyst (London)84, 135 (1959). (6) S. Hordynska, 6. Legatowa, and I. Bernstein, Chem. Anal. (Wars a w ) . 7, 567 (1962). (7) S. Hordynska, 6.Legatowa, K. Kobylecka, D. Rotycka, and M. Strycharska, Rocz. Panst. Zakl. Hig., 20, 391 (1969). (8) International Union of Pure and Applied Chemistry, Pure Appl. Chem., 10, 77 (1965). (9) Analytical Methods Committee, The Society for Analytical Chemistry, Analyst (London) 90, 51 5 (1965).
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oxidation process should be carried out not too rapidly (danger of explosion!) and an excess of nitric acid should be avoided. Step 2. The contents of the oxidation flask are then quantitatively transferred through a filter paper into a 250-ml volumetric flask and diluted to the mark with water. The acidity Of the digest (normally within the range 1-1.4N) was estimated by titration of a 1-ml aliquot with a standard NaOH solution. Step 3. Divide the digested solution into two 125-ml portions and heat gently in 400-ml beakers, evaporating each portion to 50 ml. The temperature should be kept within the range 80-90 "C. Add 100 ml of water and repeat the evaporation to obtain 100 ml of solution. Then add 1 g of crystalline hydroxylamine chloride and let the sample cool. Extraction Procedure. Step 4. Transfer the samples from the beakers into the 250-ml separating funnels. Add 2 ml of 6 N acetic acid and 2 ml of chloroform. Shake vigorously for about 30 sec and discard the chloroform layer. Then extract mercury with an excess of a diluted dithizone solution in chloroform (at higher mercury levels follow Step 12). The dithizone solution should be prepared according to the AMC method. Step 5. Combine all dithizone extracts and place in a 250-ml beaker in which 25 ml of 2N sulfuric acid had been added. Step 6. Destroy dithizone extracts in the combined sample by the addition of a few crystals of sodium nitrite, and heat the solution gently on a hot plate to remove chloroform and nitrous acid. The temperature should not exceed 75 "C. Step 7. Discontinue heating, dilute the sample with 25 ml of water, add 0.2 g of hydroxylamine chloride and allow solution to cool to room temperature. Then add 1 ml of 6 N acetic acid and run the combined sample quantitatively into a separating funnel of suitable capacity (100 ml). Visual Determination. Step 8. Saturate the sample with 2 ml of chloroform, shaking the contents in the funnel for 30 sec. Allow the organic layer to separate and then discard it. Titrate the sample with 0.5 ml of a diluted dithizone solution from a 5-ml buret. Shake the mixture vigorously for 1 min. Run the dithizone extract into a 20-ml test tube with ground glass stopper. Continue the extraction with successive small portions of dithizone until the last portion of dithizone remains green. If mercury is present in the sample, salmon-colored extract is observed. All portions of these dithizone extracts should be collected in the test tube. The extraction should be continued until the color of the extract collected in the test tube becomes intermediate between the salmoncolored mercury complex and the green color of dithizone. If there is too much orange in the color, an additional portion of dithizone should be used to obtain a proper color of the mixture. Note the total quantity of the dithizone solution consumed from the buret. Step 9. In another 20-ml test tube, place 1 ml of 2N HzS04, add 1 ml of water and 1 ml of 6 N acetic acid. Run from the buret the same amount of dithizone as used in sample (Step 8) and titrate with small portions of the standard mercury solution from another 5-ml buret. (Preparation of the standard mercury solution containing 1 pg Hgz+ per ml: dissolve 0.1354 g of HgC12 in 1000 ml of 0.2M HzS04 to obtain a stock solution containing 100 pug of Hg per ml. Dilute 10 ml of the stock solution to 1 liter with 0.2M HzS04 immediately before use.) Shake the mixture in the test tube vigorously after each addition of the standard mercury solution. Continue the titration until the solution acquires the same color as that of the sample. The number of milliliters of the mercury standard solution consumed is equivalent to the mercury content in sample, expressed in micrograms. Precautions. Step 10, The extraction Steps 4-6, and visual determination Step 8, should follow one another. It is inadvisable to wait (a few hours) between Steps 4-6 and Step 8 since the dithizonate complex collected in Steps 4-6 decomposes and mercury liberated may be adsorbed on the wall of the test tube. In such cases, it is advisable to wash the test tube with an additional
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973
Table I. Efficiency of the Back-Extraction Procedure Z03Hgfound after 203Hgadded with back-extraction and dithizone extract, removing CHC13 and cpma "02. cpm Sample
Canned mackerel Canned mackerel Pike muscle Pike muscle Redfish fillet Redfish fillet a
3901 4120 3322 3653 4189 5267
f90 f 92 i a4
aa f 130 f 146
3703 4038 3119 3603 3960 5014
f 130 92 116 f 130 f 90 & 142
*
cpm = counts per minute.
portion of dithizone. Otherwise the back extraction method may give low results. Step 11. The best way for comparing the color of the titrated dithizone extracts in test tubes is looking a t them against a fluorescent light. Step 12. If the sample to be analyzed comprises more than 2 pg of mercury, there is no need to combine extracts and Steps 5-7 may be omitted. In this case, mercury may be determined according to the modified Hordyhska et al. (6, 7 ) procedure. This is as follows: The sample should be prepared for extraction as in Steps 3 and 4. Then mercury should be titrated with dithizone directly in Step 4 and visually measured as in Steps 8 and 9. Colorimetric Assay. Step 13. Besides the titration procedure, it is also possible to determine mercury colorimetrically. In this case, the dithizone extract should be diluted to 4 ml with chloroform. If there is a higher level of mercury in one sample, the determining may be performed directly as in Step 4 and Steps 5-7 may be omitted (see Step 12). Then the diluted extract is filtered through a filter paper into a 1-cm spectrophotometric cell and the absorbance is measured a t 490 nm against a dithizone solution treated with the same reagents as in Steps 3 and 4 (at higher Hg levels) or as in Steps 4-7 (at lower Hg levels). Step 14. Place into a beaker 25 ml of 2N HzS04, add 1 ml of 6N acetic acid and a few crystals of sodium nitrite. Add proper amounts of the standard mercury solution within the range 0-10 pg. Heat the solution gently to remove the nitrous acid. Let the mixture cool, then add 0.2 g of crystalline hydroxylamine chloride. Transfer the mixture to a suitable separating funnel, and then proceed as described in Steps 8 and 13. Plot the calibration curve relating absorbance to the number of micrograms of mercury.
DISCUSSION Mercury Losses. Mercury losses were also deterrhined during the wet oxidation stage with nitric, sulfuric, and perchloric acids. These tests were carried out using known amounts of labeled mercury in the form of 203HgC12 and 203Hg(CH&00)2. Labeled mercury was added to the charged oxidation flask. After the wet oxidation, 92-1059'0 of the added 203Hg was recovered, Practically no losses were found. Some variations (f5-10%) in the obtained results covered the range of the statistical counting errors. The measurements of the radioactivity were carried out with a well-type monochannel gamma-scintillation counter with a NaI/T1 crystal. Higher losses of mercury above 5-10% were found at Step 3 when the samples were boiled or heated too rapidly or too long. Therefore, it is advisable to heat the sample cautiously keeping the temperature within the range 80-90 C . The efficiency of the mercury back-extraction procedure (Steps 4-6) with sodium nitrite was also investigated. With an excess of dithizone, all mercury was extracted from the digestates and quantitatively transferred to the combined sample. The amount of 203Hg was counted as the dithizone extract before destroying with sodium nitrite and then in an aqueous solution after back-extraction
Table II. Extraction of 64Cuand Hg from Wet Digestates (1 mg of Stable Copper Added to Each Specimen) Hg Hgre64Cu Hg present, added, covered, Material pH extracted YO Pg pg Irg Baltic herring 0 0.7 1 .a 1 2.8 0 1.7 Baltic herring 0 0.1 1 .a Baltic cod 0 0.2 undetectable 1 1.4Q
Mercury previously extracted Baltic herring Milk powder 1N ti~S0.4
0 0 0
Rice Milkpowder Rice Rice Bream Tench
1 1 2 2 3 3
Q
0.2 0.4 0.2 3.8 1.a 9.0 10.0 96.5 f 1.4 97.6 f 1.4
0 0
0
0
3
0
5 5
0
0 0 0 0
5
3
5 5 o
0 4.9 3.1 5.0 4.9
x* x x x
Abnormally high recovery may be explained as due to the occurrence
of undetectable amounts of Hg in the material tested. b X 1 = difficultly
measurable.
and heating. The results are presented in Table I. No losses of mercury were Eound in the back-extraction procedure. Copper Interferences. A series of radiochemical experiments was carried out with labeled 64Cu. The 64Cu was added together with 1 mg of a stable copper isotope to a number of digestates of food samples. A single sample taken for wet oxidation involved 50 g of fresh fish muscle or 15 g of milk powder or 15 g of polished rice. The tests were carried out using one-third of the digested sample. The results are shown in Table 11. It is necessary to explain that the last nine samples shown in Table I1 were previously mercury extracted with dithizone and then known amounts of mercury were added again to the same specimens together with the mixture of radioactive and stable copper isotopes. The main purpose of this approach was the examination of mercury and copper extraction under similar conditions to those existing in samples obtained from foods in the wet oxidation procedure. The results in Table I1 show that copper added to the digestates has no effect on mercury extraction at pH 0. Hence, there are suitable conditions for the selective extraction of mercury from copper. Slight radioactivity (0.11-0.71%) present in the dithizone extracts was due to its contamination by a few drops of the radioactive aqueous phase containing G4Cu. Raising the pH to 1 causes a slight coextraction of copper (2.9-3.8%), but it was still possible to determine mercury selectively. At pH 2, the interfering effect of copper was more apparent. It was difficult to detect the end point of the mercury titration, since the last portions of dithizone used for extraction quickly became violet. At this stage, copper began to coextract in higher amounts (up to 10%). Further extraction with dithizone makes it possible to extract the total copper present in the sample, but it was a time-consuming procedure. A t pH 3, copper is coextracted with mercury very rapidly. Under these conditions, mercury is unmeasurable, because of difficulties in detecting the end point during the dithizone titration. The salmon-colored complex was strongly masked by a violet copper complex. CONCLUSIONS The proposed modification makes it possible to combine samples with very low levels of mercury into one sample
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 14, DECEMBER 1973
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richer in mercury where it can be determined quantitatively. Thus, this procedure allows us to assay low levels of mercury in many less contaminated foodstuffs and can also be employed to determine mercury in natural waters. Since mercuric ions are selectively extracted from the cupric ones a t p H 0, the procedure was shown to be particularly useful for simultaneous colorimetric determina-
tions of both these elements in one sample of food. In many samples with mercury level less than 0.05 ppm, it was impossible to assay it quantitatively without the combining and back-extraction procedure. Received for review December 7, 1972. Accepted June 6, 1973.
Optical-Crystallographic Properties of P-D-Glucose George R. Dean1 Miles Laboratories, Inc., Elkhart, Ind. 46514
Although the optical-crystallographic properties of CY-Dglucose are listed by Winchell (I), corresponding data for the P isomer have never been published. Other properties such as phase relationships (2-4) and crystal structure by X-ray diffraction (5-7) have been described, as well as methods for preparing the compound in the laboratory and in industry (8, 9). Because D-glucose is such a common and important sugar, the additional data reported herein should fill a long-standing need of the microscopist and analyst.
EXPERIMENTAL Apparatus. A 1-liter resin flask was fitted with a vacuumsealed stirrer, condenser, solution inlet, thermometer, and electric heating mantle. Reagents. D-Glucose was the commercial hydrate form of the sugar. Seed crystals of 6-o-glucose were prepared by an accepted procedure (8). Procedure. Crystals of 6-D-glucose were prepared by slowly cooling a hot, concentrated aqueous solution of D-glucose. An aqueous solution containing 90% of the sugar was prepared separately and introduced into the flask a t 90 "C. After a few seed crystals of 6-D-glucose were added, the solution was stirred gently. After crystallization began, the solution was allowed to cool gradually t o 80 "C. At the same time, the concentration was slowly decreased by admitting small amounts of a more dilute solution of D-glucose. Vacuum was adjusted so that the solution boiled gently at all times. After 1 hour, the mixture was poured into two volumes of glacial acetic acid at 100 "C, quickly filtered, washed with hot acetic acid, and dried. By deliberately holding the.yield to a low level, comparatively large, well-formed crystals which were suitable for the present study were obtained. Otherwise, the product contained too many fine crystals. Initial specific rotation showed the product to contain 90% pand 10% a-D-glucose. Anal. [ a I z 0 ~ Accepted: , +18.7", Found: lRetired. Present address, 404 N. Buchanan St., Edwardsville, Ill. 62025. (1) A. N . Winchell, "The Optical Properties of Organic Compounds," Academic Press, New York, N.Y., 1954. (2) R. F. Jackson and C. G. Silsbee, Nat. Bur. Stand. ( U S . ) , Sci. Papers, 437, 715 (1922). (3) W. B. Newkirk, Ind. Eng. Chem., 28, 760 (1936). (4) F. E. Young,J. Phys. Chem., 61, 616 (1957). (5) 0. L. Sponsler and W. H. Dore, J. Amer. Chem. SOC., 53, 1639 (1931). (6) W. G. Ferrier, Acta Crystallogr., 13, 678 (1960). (7) /bid., 16, 1023 (1963). (8) R. L. Whistler and J. N. BeMiller in "Methods in Carbohydrate Chemistry," Vol. I, Academic Press, New York, N.Y., 1962, pp 130-131. (9) G. R. Dean and J. B. Gottfried, Advan. Carbohyd. Chem., 5, 136137 (1950)
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+28.0". The accepted value (10) had been determined only after repeated fractional dissolution in ice cold water and subsequent precipitation with cold ethanol. Because larger crystals from water alone were desired, they were not further purified.
RESULTS Optical-crystallographic properties, determined by common microscopic procedures, are summarized in Table I. Figure 1 is a photomicrograph of the crystals in contact with the mother liquor. In Figure 2 are diagrams of principal views of P-D-glucose, together with crystallographic axes, profile angles, and principal optical directions.
DISCUSSION Table I shows p and y to be equal within experimental error. Actually, they differ slightly as evidenced by the observed biaxial interference figure, the latter of which indicates (-)2V = 8". Calculation shows that for this angle P and y differ by less than one in the fourth decimal place. The crystals from water uniformly show positive elongation for all orientations with c parallel to the microscope slide except that represented by the second diagram in Figure 2. Here one is looking down the acute bisectrix and birefringence is extremely low. Since such is not observed with the CY isomer, it should be easy for the microscopist to recognize this exceptional case. Otherwise, it ought to be safe to consider sign of elongation as a useful analytical property. The common method for analyzing a mixture of 6- and a-D-glucose is to observe the changing optical rotation of a freshly prepared solution and then extrapolate to the moment of dissolution ( 1 1 ) . From known initial rotations of the P and CY forms, the composition can be calculated. This method, however, can be used only with chemically pure D-glucose. Optically active impurities, especially those that exhibit mutarotation, give inconclusive results. In such cases, the microscopic method is especially useful. Large, well grown crystals of P-D-glucose can be recognized in contact with the mother liquor and distinguished by crystal form alone. Small, irregular crystals or fragments can be detected by refractive index measurement. Since for this purpose, the crystals must be isolated in (10) H. S. isbell and W. W. Pigman, J , Res. Nat. Bur. Stand.. 18, 141 (1 937). (11) C. S. Hudson and J. K. Dale. J. Amer. Chem. SOC., 39, 320 (1917).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973
