tassium iodide and potassium cyanide, respectively. Concentrated solutions of complexon 111,potassium iodide, and potassium cyanide were used as masking agents. For eliminating the cobalt interference, a chloroform solution of PDTT was used to extract the mercury(I1) content. The extract is washed with dilute NaOH solution to remove the excess PDTT.
Confidence limits were calculated to be 66.3 f 0.67 and 227 f 2.6 pg Hg2+ at 65.5 and 230 pg Hg2+,respectively.
LITERATURE CITED Sandell, "Colorimetric Determination of Traces of Metals," 3rd ed., interscience, New York, N.Y., 1965, p 621. (2) H. Imai, Nippon Kagaka Zasshi (J. Chem. SOC.Jap.),90, 275 (1969). (3) S. Komatsu and T. Nomura, Nippon Kagaku Zasshi (J. Chem. SOC. (1) E. B.
Jao.1. 88. 542 (19671. (4) S komaku, T Nomura, and M Saito, Nppon Kagaku Zassh) ( J Chem SOC Jao ), 1124 (1967) (5) K. Kotsbji,'.6ull. Cbem. SOC.Jap.. 38, 402 (1965). (6) Y. Yamamoto, S. Kikuchi, Y. Hayashi, and T. Kumamaru. 6unsekiKagaku (Jalap.Analyst), 16, 931 (1967). (7) M. Tsubouchi, Anal. Chem., 42, 1087 (1970). (8) I. Lalezari and H. Goigolab, J. Heterocycl. Chem., 7 , 689 (1970). (9) M. Edrissi, A. Massommi, and I. Lalezari, Talanta, 19, 814 (1972). (IO) R. Maghssoudi and A . Fawzi, unpublished work. (11) J. H. Yoe and A. L. Jones, Ind. Eng. Chem., Anal. Ed., 16, 111 (1944). (12) P. Job, Ann. Chim., 9, 133 (1928); 16, 97 (1936).
PRECISION AND ACCURACY A set of six identical samples, each with a final mercury(I1) concentration of 65.5 pg Hg2+ were treated according to the recommended procedure, and their absorbances were measured. This was repeated with a concentration of 230 pg Hg2+for another set of six samples. The percentage accuracy varied from 1.11%at 65.5 to 1.31% at 230 p g Hg2+. The percentage of the relative standard deviation varied from 1.34% a t 65.5 to 1.21% at 230 wg Hg2+.
RECEIVEDfor review August 12, 1974. Accepted November 18, 1974.
Spectrophotometric Method for Copper and Mercury Determination in the Same Food Sample Using Dithizone and Lead Diethyldithiocarbamate Michal Nabrzyski Department of Bromatology, Faculty of Pharmacy, Medical Academy, 80-4 16 Gdansk, Poland
Current interest in the effect of trace quantities of various elements on the metabolism of living organisms, coupled with widespread concern over the possible accumulation of toxic substances in the environment, has created a need for the determination of ever lower concentrations of metals in different kinds of foods (1-4). Some of these-such as zinc, copper, cobalt, and manganese-play an important role in a number of essential biological processes having a beneficial effect, while otherssuch as mercury, lead, and cadmium-may be harmful. Sophisticated techniques such as spark source mass spectrometry, neutron activation analysis, and even atomic absorption spectrophotometry are usually used in well equipped laboratories. Moreover, there is still great interest in the classical analytical methods employing dithizone and similar complexing reagents, especially for estimation of the residual heavy metals in food. This paper presents a procedure for the determination of two elements, copper and mercury, in the same sample of food. The developed procedure is complementary to one previously reported ( 5 ) . As pointed out in the preceding paper, mercury may be determined selectively in a strong acid medium at pH O--i.e., sulfuric acid with a small admixture of nitric acid. Copper present even a t a level of 1 mg does not interfere when a diluted chloroformic dithizone solution is used.
EXPERIMENTAL In the method presented here, the sample is decomposed by a wet oxidation procedure developed by Gorsuch ( 6 ) and modified by Hordyriska et al. (7). Mercury is determined first in a strongly acid sample ( 5 ) . After mercury extraction has been completed, both parts of the remaining sample solution are quantitatively 552
transferred to a graduated 500-ml cylinder and diluted with water up to a volume of 300 ml. The procedure to be followed involves three steps: 1) Adjusting p H of the sample solution to 3 and extraction of copper with a 0.01% chloroformic dithizone solution. 2) Decomposition of extracts by sodium nitrite and copper reversion into a 0.1N sulfuric acid. 3) Adjusting p H of the new solution to 6, followed by colorimetric determination of copper using a chloroformic solution of lead diethyldithiocarbamate (8). The sensitivity of the developed procedure corresponds to 1 pg of copper. Reagents. All reagents were of Analytical Reagent quality, free of copper impurities. Water was copper free, distilled from a glass apparatus. Dithizone stock solution was prepared according to the AMC method (9). From this solution, a 0.01% (w/v) one was prepared by diluting 1 volume thereof with 4 volumes of chloroform. A 0.04% ( w h ) chloroformic solution of lead diethyldithiocarbamate, Pb(SCSNEt&, was prepared as follows: 0.05 gram of Pb(N0,dz was dissolved in a few milliliters of water and placed in a separatory funnel. Then 5 ml of a 5% solution of NaK tartrate and 0.1 g of NaSCSNEtZ dissolved in a few milliliters of water were added. The mixture was then gently shaken until a precipitate of Pb(SCSNEt2)P appeared. It was extracted once with 250 ml of chloroform for 3-5 min, and the aqueous phase was discarded. Then the extract was washed twice with water and filtered through a dry ashless filter paper into a dark-glass bottle. NaK tartrate--5% (w/v) solution-was purified from copper with a few milliliters of 0.04% Pb(SCSNEtZ),. Standard copper solution, containing 1 p g Cu per ml, was prepared from CuS04.5H20 according to Sandell ( 1 0 ) . Potassium cyanide-10% (w/v) solution-was prepared by dissolving 10 g of KCN in 50 ml of water, and filtered into a 100-ml dropping bottle. The solution was diluted with water up to 100 ml. Procedure. Place 50 ml of the sample solution containing up t o 15 pg of copper in a 100-ml beaker. Adjust p H to 3 by addition of a few drops of concentrated ammonia under pH-meter control. If too much ammonia has been added, correct the p H with 2N HzS04.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
Table I. Checking of the Procedure Developed by Means of a Radiochemical Method Using 64Cu
Table 11. Results of Spectrophotometric Copper Determinations a n d the Recovery Test in Fish Meat a n d Milk Powder
Copper recovered in form Material
Tench Tench Bream Bream
of h4Cu(SCS\Et2)2,
C u present, ug
I
No. of analyses
96.5 i 2 92.5 + 5 97.0 I 2 90.8 5
Min.
Max.
C u added, u g hlin.
Max.
C u recovered,
\tin.
-.
Max.
Fisha
*
32
2.6
13.9
2
30.0
36.0
2
30.0
36.0
2.0
5.0
87
104
101
104
91
96
Cephalopods (Flying Squid)
Transfer the sample solution into a 250-ml separatory funnel and extract copper with 1-ml portions of 0.01% dithizone, until the last portion remains green. Transfer combined extracts into a 250-ml beaker containing 50 ml of 0.1N H2S04, destroy the cupric complex with 20-30 mg of NaN02. Then gently heat the solution a t about 75 O C and stir to evaporate chloroform and HNOz completely. After cooling, add 5 ml of 5% NaK tartrate solution and adjust pH to 6 with a few drops of 2N NH40H under the pH-meter control. Transfer the solution into another 150-ml separatory funnel, saturate it with 2 ml of chloroform, and discard the chloroformic layer. Extract the remaining aqueous layer twice with a 0.04% chloroformic Pb(SCSNEtz), solution, using a total volume of 5 ml of it. Observe a pale yellow color of Cu(SCSNEt2)z developing in the organic layer if Cu2+ ions are present. After phase separation, run the organic phase through a dry ashless filter paper into a 20-ml glass-stoppered test tube. Measure the absorbance in a 1-cm spectrophotometric cell a t 440 nm us. the blank. Prepare a calibration graph by employing a series of standard copper solutions covering the range 0-20 fig of Cu and treat them as described. Make the final volume of the standard identical to that of the sample. T o check the Bi interference, add one drop of a 10% solution of KCN to the chloroformic Cu(SCSNEt2)2 extract, and to the blank, to decompose the cupric complex and make it colorless. Measure the residual absorbance us. the decolorized blank, and subtract the reading from that recorded previously.
DISCUSSION Many spectrophotometric procedures for the selective determination of trace amounts of copper, using various complexing agents, have been reported by Sandell ( I O ) , Kolthoff and Elving ( I I ) , and others (12). The method presented in this work differs largely from those cited, as copper may be assessed along with mercury in one food sample. Mercury is determined first in a strongly acid medium with dithizone, and copper does not coextract under these conditions as has been shown in radiochemical experiments with 64Cu ( 5 ) . Results of this work have been supported by calculations made using equations reported in Sandell's monograph ( I O ) . Accordingly, almost 98% of the primary copper dithizonate complex is dissociated in the strongly acid medium in which mercury is determined. Both the radiochemical studies and the calculations made, provided the basis for the development of the procedure described. It is to be emphasized that copper levels usually encountered in food sample are about 100 times lower than those taken for the experiment. Hence, copper may be still more effectively separated from mercury. The effect of other metals is inhibited by preliminary extraction with dithizone and by employment of Pb(SCSNEt& in chloroform, as well as by use of KCN.
3.0
Shrimps (Peneus so.)
3.0 J M i k powder
14 1.5 2.0 2.0 5.0 87 100 a Analyses of fish included white bream, bream, tench, pike, perch, zanthe, salmon, Baltic cod and sprat, tuna, false albacore, and horse mackerel.
The procedure was also examined radiochemically with labeled copper as indicator. It was added to the sample in the amount 2 X 103-1.5 X IO4 cpm (counts per minute) of 64Cu in the form of 64CuS04. The radioactivity was measured by a well-type monochannel gamma scintillation counter with NaI/Tl crystal. Recovery of copper in the form of 64Cu(SCSNEt2)2 shown in Table I ranged from 90.8 to 97%. Results of the spectrophotometric determination of copper in 50 samples of fish meat and some other marine food as well as in milk powder are presented in Table 11. The weight of most samples of fish and marine food was 50 g, with a few exceptions like perch (30 g), Baltic sprat (25 g), one sample of tuna fish (40 g), and false albacore (40 8). Milk powder samples weighed 15 g. For copper determination, YS part of each sample was used. To each sample, a few micrograms of copper were added at the beginning of the analytical procedure. Recovery of the added copper varied from 87 to 104% and the mean value was 95.4% with the standard deviation of f4.2%. A comparison of these results with those obtained radiochemically shows a satisfactory agreement. LITERATURE CITED (1)R . Nuzzi, Nature (London), 237, 38 (1972). (2)S.Hernberg, WorldHealth Organ. Chron., 27, 192 (1973). (3)Anonymous, World Health Organ. Chron., 27, 534 (1973). (4)F. C. Lu, World Health Organ. Chron., 28, 8 (1974). (5)M. Nabrzyski, Anal. Chem., 45,2438 (1973). (6)T. T. Gorsuch, Analyst (London), 84, 135 (1959). (7)S.Hordynska, B. Legatowa, and I. Bernstein, Chem. Anal. (Warsaw),7 , 567 (1962). (8) V. Sedivec and V. Vaiak, Chem. Listy, 45,435 (1951). (9)Analytical Method Committee, The Society for Analytical Chemistry, Analyst (London),90,515 (1965). (10)E. B. Sandell, "Colorimetric Determination of Traces of Metals," 3rd ed., lnterscience Publishers, New York and London, 1959,pp 148, 152, 447-449,454,457,622. (11) I. M. Kolthoff and P. J. Elving, "Treatise on Analytical Chemistry," Part 11, Vol. 3,lnterscience Publishers, New York-London, 1961,p 31. (12)R . F. Milton and J. L. Hoskins, Analyst (London), 72, 6 (1947).
RECEIVEDfor review April 22, 1974. Accepted October 15, 1974.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
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