Determination of Zinc by Direct Extraction from Urine with Diphenylthiocarbazone JEREMIAS
H. R. KAGl and BERT L.
VALLEE
Biophysics Research laborafory, Deparfmenf of Medicine, Harvard Medical School and Pefer Bent Biigham Hospital, Bosfon, Mass. F A dithizone method for the direct extraction of zinc from urine has been developed which does not require the destruction or removal of organic material b y either ashing or precipitation prior to analysis. These procedures are lengthy and provide hazards for contamination. In the present method, the metal is extracted at pH 5.7 after emulsifying substances have been removed b y extraction with carbon tetrachloride. Comparison with ashing procedures shows complete extraction of zinc from urine. Extraneous metals d o not interfere. This technique significantly simplifies existent procedures and is sensitive and precise. A side experiment with zinc-65 demonstrated that the metal is not lost b y volatilization when urines are ashed in a muffle furnace at 550" C.
Z
is extracted directly from urine with dithizone in carbon tetrachloride, and the zinc dithizonate is then determined b y a mixed-color method. Because the technique depends on a separation of phases, eniulsifying substances in urine must be removed; this is accomplished by a preliminary carbon tetrachloride extraction. The chemical characteristics of urine necessitate some modifications in the conditions previously described as optimal for the determination of zinc (6). Complete extraction of urine zinc requires lower concentrations of complexing agents and buffers than those recommended for the analysis of ashed samples. The optimal p H for the specific extraction of zinc is 5 . i . Using this technique, a coefficient of variation of 4.57c is obtained for replicates of urine with a total mean zinc content of 2.9 y. The accuracy of the method is determined by comparing these values with those obtained after ashing, and is shown b y their excellent agreement. The limit of detection is 0.2 y of zinc in a sample of 5 ml. of urine ISC
EXPERIMENTAL
All water is obtained by passage of t a p water through a mixed cation-anion exchange resin ( 3 ) . Glassware and polyethylene are cleaned as described
buffer is repeatedly extracted with 15(6). Urine is collected in polyethylene bottles through polyethylene funnels. ml. aliquots of 0.01% dithizone until Polyethylene containers are used for the dithizone remains green. After storage of reagents whenever possible. repeated treatment with 25-ml. aliquots Reagents. STANDARD ZINC SOLU- of carbon tetrachloride, the solution is TIONS. -4 stock solution of approxistored in a well-stoppered metal-free mately 1000 y per nil. is prepared by disborosilicate glass flask. Polyethylene solving a knoivn weight of spectroscopicontainers are unsuitable for storage cally pure metallic zinc (Johnson Matof the buffer because of diffusion of they Co., Ltd., London, England) in cyanide. metal-free 6N hydrochloric acid. A zinc T a R T R A T E SOLUTIOS. Olle hundred standard solution containing 1 y per grams of potassium sodium tartrate, milliliter is prepared weekly by dilution XaKC4H406.H20 (1Iallinckrodt Chemiwith metal-free water. cal Korks, analytical grade), are disDITHIZOXE (diphenylthiocarbazone, solved in metal-free water and diluted Eastman Organic Chemicals). d stock t o 500 nil. The solution is purified soluton of 0.017, dithizone in carbon by extraction with 0.01% dithizone. tetrachloride is prepared. To remove ilarnrosra. Concentrated ammonium contaminating metals and organic imhydroxide (E. I. du Pont de Seniours 8: purities, 250 ml. of dithizone solution Co.. Inc., reagent grade) is distilled and a n equal volume of water containfrom a borosilicate glaqs still into metaling about 250 mg. of (ethylene dinitri1o)free water. K h e n the distillate is tetraacetic acid (ethylenedianiinetetrasaturated, it is diluted 1 t o 1 with water. acetic acid) are shaken in a separaIt should be prepared weekly and stored tory funnel. Concentrated ammonia is in a polyethylene bottle. then added until all of the dithizone is HYDROCHLORIC ACID, 6 S (approxitransformed t o the water-soluble, mately). Metal-free 1 2 5 hydrochloric orange-colored dithizonate ion, which is acid is prepared by bubbling hydrogen readily extracted into the TT ater phase. chloride gas (Matheson Co., Inc.) The greenish organic layer is discarded through metal-free water, until the and the aqueous dithizonate solution n-ater is saturated (3). The concenis rinsed successively m-ith two 25-ml. trated hydrochloric acid is then diluted portions of carbon tetrachloride. T o 1 to 1 with metal-free mater. re-extract the purified dithizone, 250 CHLOROPHEXOLRED I X D I C ~ ~ T O R ml. of carbon tetrachloride are placed in (Howe & French, Inc.). An aqueous t h e funnel, and sufficient 6 S hydrosolution of O.lyo chlorophenol red is chloric acid is added dropv-ise n-it11 prepared. shaking until the orange color of the aqueous phase just disappears. The PROCEDURE resultant green dithizone solution is separated and stored in the dark a t A 5-nil. aliquot of urine is transferred 4' to 6 ' C. For analyses, 0.003% to a 125-ml. separatory funnel provided dithizone is prepared b y dilution ivith nith a Teflon outlet (Fischer & Porter carbon tetrachloride. Co.) and 2 ml. of tartrate solution, CARBONTETRACHLORIDE (llerck Q 2 ml. of 6N hydrochloric acid, and 1 Co., Inc., reagent grade) is used n ithout drop of chlorophenol red indicator are further purification. It is protected added. The sample is titrated n i t h from light and stored a t 4"C. animonia until the indicator just turns SODIUMTHIOSULFATE-CYAXIDE-~~CEred. Forty milliliters of buffer are TATE BUFFER. Sodium thiosulfate, added, and the mixture is shaken 17-ith KazSz03 5Hz0 (J. T. Baker Chemical 15 ml. of carbon tetrachloride for 1 minCo., Baker analyzed reagent), 1330 ute to remove the emulsifying subgrams; sodium acetate, KaC2H3O2stances. The carbon tetrachloride and 3Hz0 (RIerck & Co., Inc., reagent water phases are allowed to separate grade), 224 grams, and 15 grams of for 5 minutes, and the cloudy organic potassium cyanide, KC?: (llallinckrodt phase is carefully separated from the Chemical Korks, reagent grade), are aqueous one and discarded. This is dissolved in about 2 liters of metal-free repeated successively n-ith 10 ml. and n-ater. The p H is adjusted to 5.7 b y twice with 5 ml. of carbon tetrachloride, the addition of glacial acetic acid (E. I. making a total of four extractions. d u Pont de Nemours & Co., Inc., reIf the last carbon tetrachloride extract agent grade), and the mixture is diluted is turbid, as occurs infrequently, addito 4 liters. The final adjustment to p H tional separations are performed until 5.7 is checked at room temperature on a it is clear. Seven milliliters of 0.003% sample of the buffer diluted tenfold. dithizone are then added to the sample T o remove all contaminating metals, the and the funnel is shaken for 2 minutes. VOL. 30, NO. 12, DECEMBER 1958
1951
The organic phase is allowed to separate and is drawn off into a 25-ml. actinic red glass volumetric flask. The procedure is repeated once with 4 ml. of 0.0015% dithizone solution and again with 3 ml. of 0.001% dithizone solution. Finally, the combined extracts are brought to volume with carbon tetrachloride. If, however, the third extract has a bluish color, indicating that the zinc has not been removed completely, further extractions with O.Q03% dithizone are necessary. I n this case, the combined dithizone extracts are brought to a final volume of 50 ml. Depending upon the relative quantities of zinc and excess dithizone present, the final color may have a blue-gray or greenish tinge. For each set of analyses, except for the preliminary extraction with carbon tetrachloride, a reagent blank and a control containing 5 y of zinc are carried through the whole procedure. If the reagent blank exceeds 0.5 y, further purification of the reagents is required.
400
500 550 600 650 WAVE LENGTH,m4 Difference spectra of zinc dithizonate in 450
Figure 1. carbon tetrachloride
Extracted from 15 ml. of urine Extracted from 25.7 y of ZnCln C a r y spectrophotometer, 1 -cm. path length cells. The reference cells contain the same concentrations of free dithirone as the solutions containing the zinc
X
COLORIMETRY
The colorimetric determinations are performed in a constant temperature room in a Becknian Model DU spectrophotometer, using acid-cleaned Corex cuvettes of 1-em. path length. Dithizone dissolved in carbon tetrachloride exhibits a n absorption spectrum m-ith a maximum a t 615 and a minimum a t 510 mp. The formation of zinc dithizonate results in the appearance of an absorption peak at 532 nip, the approximate location of the dithizone minirnuni (6). To obtain the maximum difference in absorbance between the zinc dithizonate and the ewess free dithizone, measurements are performed a t 525 mp. Using the mixed-color method ( 6 ) , readings are also obtained a t 625 mp, and the zinc content of the analyzed sample is calculated from the equation
Table I.
Effect of pH on Extractability of Zinc from Urine
Total Zn added = 5 -y; total Zn65 = 3.5 p c . ; urine volume = 5 ml. Total ZnG6 Estrac- Dithizone, Found, t ion yo pH 5.5 pH 5.7 56 7 93 B I 0.003 8 3 5.0 I1 0.0015 18 0.7 I11 0.001 Zinc remaining in aqueous phase measured after extraction I11 3 2 0 7
experiment. The decrease of the numerical value of R below 5.5 denotes deterioration of the dithizone solution, which then ought to be repurified. EXPERIMENTAL RESULTS
where absorbance a t 525 nip = absorbance a t 625 nip = ratio of absorbance of dithizone alone in carbon tetrachloride = volume of carbon tetrachloride extract (nil.) = calibration constant =
R Ti
K
The amount of zinc in a given urine sample is obtained hy subtracting the reagent blank value. The calibration constant, K , represents the ratio of zinc concentration (micrograms per milliliter) to zinc dithizoriate absorbance
(E - . a22
2)
- - in the carbon tetrachlo-
ride extract. It is calculated from a calibration curve obtained from the analyses of a standard zinc solution and should be redetermined n henever a new batch of buffer is prepared. The ratio of absorbance R is determined on a fresh O.OOl7, dithizone solution in each 1952
ANALYTICAL CHEMISTRY
The completeness of extraction of zinc from urine n a s compared with that from aqueous standard zinc solutions. The absorption spectrum of zinc dithizonatc obtained from urine is shonn in Figure 1, together with the spectrum of a corresponding amount of zinc dithizonate obtained from a standard zinc solution. The spectra are identical in the nave length range of 400 to 650 mp. Extraction of zinc from increasing volumes of a urine sample and of a standard zinc solution gave identical calibration curves, demonstrating that both systems satisfy the Beer-Lambert lan. Table I shows the influence of pH on the extraction of zinc from urine. Five micrograms of zinc. containing 3.5 pc. of zinc-65 were added to t n o 10-nil. samples of urine, which were then extracted three times with equal amounts of dithizone a t pH 5.5 and 5.7. The carbon tetrachloride layers containing
zinc-65 dithizonate were collected separately after each extraction and their zinc-6.5 content was determined by counting 1-nil. aliquots in a P-20-11' Tracerlab scintillation count'er. The relative zinc-65 contents of each extract are shown in Table I. After three extractions at p H 5 . i , zinc was completely removed, while a t p H 5.5, 3% remained in the aqueous phase. The loss of zinc in the preliminary extraction of emulsifying substances was determined in five urine samples containing a known amount of zinc-65 (Table 11). Each sample n-as treated four times Ti-ith carbon tetrachloride, as described above, and the extracts were collected in a graduat'ed cylinder. After the emulsion had separated on standing, the volume of the aqueous phase n-as measured. To detect the amount of zinc in the emulsion, t'he solvents were evaporated, the residues nere redissolved in a known volume of 6-Y hydrochloric acid, and radioactivity was counted. The loss of zinc n-as directly proportional to the loss of aqueous phase in the emulsion (Table 11). I n a scrics of urine analyses on 18 normal individuals, the mean loss of aqueous phase v a s 3.02% of t'he total sample volume n-ith a standard deviat'ion of *1.26Cc. INTERFERENCES
Table I11 lists the elements with their valence states, which xere tested for ihle interference n-ith the zinc dithizone reaction. The metals were added to ;-nil. samples of urine n-hich contained 2.90 + 0.13 y of zinc. The niaxiniuni amounts which could be present without interference are given in niicrograms. Interference xas defined ns a deriation of niore t'han ti7-o standard deviations ( 2 ~ 0 . 2 6y) from the mean zinc value of the control samples.
PRECISION
Over a period of several months, two analysts determined the zinc content of 5-ml. aliquots of a normal urine specimen and of a standard solution. For urine, the results ranged from 2.79 to 2.98 y of zinc with a mean and standard deviation of 2.90 =t 0.13 y. The zinc standards were' prepared t o contain 4.76 y of zinc by weight, and 4.i2 to 4.97 y were found with a mean and standard deviation of 4.82 =k 0.10 y (Table IV). ACCURACY
The zinc content of samples was determined both b y the present extraction method and after d r y ashing, a s described (6). The ashed samples were dissolved nit11 2 ml. of metal-free 6 S hydrochloric acid, transferred quantitatively into separator? funnels. and analyzed. As a preliminary step for the comparison of the two methods, the recovery of zinc in the ashing procedure was tested by adding a known amount of zinc-65, folloned by counting of radioactivity. K h e n a urine sample was ashed for 16 hours a t 450" C., 92y0 viere recovered as zinc-65 dithizonate. Honever, 9iyowere found x h e n the sample was ashed for 12 hours a t 550" C. The low recovery of zinc-65 in the urine sample ashed a t 450" C. n a s due to absorption of zinc on carbon particles, present as a result of insufficient ashing. The samples n-ere ashed under borosilicate evaporation covcrs to detect losses through volatilization (4). Air was passed through the cover to the outside of the furnace and bubbled through a n absorption toner containing distilled nater. After ashing, the inner surface of the cover and the water of the absorption toTyer ITere tested for radioaitiT ity; 0.27, of the isotope was found in the ton-er, 11hen the and 0 12'5. sample 11 n s ashed a t 450" C.. n hen ashcd at 550" C. 1-alues for the amount of zinc excretcd in 24 hours by humans nere measured after pre-extraction ~ i t carh bon tetrachloride and after dry ashing, and are compared In Table 5'. Correeting for the constant 3% loss of volume and zinc due to the extraction procedure (see section on evperimental results), they were identical vc-ithin the statistical limits of the method. DISCUSSION
K h e n urine is extracted n ith carbon tetrachloride a voluminous stable emulsion forms which interferes with zinc analyses. The constituents of urine responsible for the formation of this eniulsion are unknown. They are not destroyed by boiling with 6-1- hydrochloric acid and trichloroacetic acid,
Table 11.
Relationship of Zinc, Not Recovered b y Carbon Tetrachloride Preextraction, to Volume Lost by This Procedure
Urine Volume, M1.
Zn%
Sample ZnaT ZnaL VT 52 5 98,200 3160 I 52 5 95,900 2014 I1 I11 5 130,812 1399 54 I11 10 264,060 4156 54 54 15 394,872 7766 I11 ZnST = total Zn65 in urine sample, counts per minute. ZnaL = ZnG lost by pre-extraction with CCla, counts per = ratio of ZnaL to ~n65T, per cent. Zn% = total volume of aqueous phase, milliliters. VT VL = volume of aqueous phase lost by pre-extraction vL = ratio of VL to vT, per cent. VT Roman numerals indicate different urine specimens.
Table 111. Specificity of Method for Zinc in Presence of Other Metal Ions Maximum amount of each metal ion which may be present in 5 ml. of urine containing 2.9 y of zinc is shown. At these levels
interference is not observed. Metal Ion Y Fe+I_ 60
E++ Pb++ cu++ Co++ hTi t + Bi +++++
!$++
60 600 600 300 300 300 30 25 60
a procedure previously recommended t o avoid the formation of emulsions in the determination of zinc in serum ( I ) , nor by the addition of antifoam agents, such as octyl alcohol. The eniulsion in urine, therefore, cannot be attributed to degradation products of proteins, such as polypeptides and amino acids. The mechanical separation of the emulsifying substances by preliminary treatment with carbon tetrachloride obviates these difficulties and leads to only a small loss of the urine vollime (3.02 d= 1.26% of the total sample volume), which corresponds directly to a n equivalent loss of zinc (Table 11). The loss in a given urine sample is small and constant; the standard deviation of consecutive zinc analyses of a urine sample is of about the same magnitude as that of consecutive analyses on a standard zinc solution (Table IT-) which Tvas not subjected to the preliminary carbon tetrachloride treatment and, therefore, was not susceptible to th6 same errors. Honerer. in urines having an abnormally high concentration of emulsifying agents. the loss of x-olume could exceed 1070, as in patients n-ith liver disease ( 7 ) , making it advisable to collect the emulsion and to measure the loss of the aqueous phase. Because
VL
Zn66T
1.70 1.25 0.55 0.86 1.06
3.22 2.10 1.03 1.53 1.90
VL VT
3.27 2.40 1.02 1.59 1.96
minute.
with CCl,, milliliters,
Table IV. Replicate Determinationsof Zinc, in Urine and in a Standard Solution
.4zinc standard was prepared by dissolving a weighed sample of spectroscopically pure zinc metal in 6 N HCl. 5 ml. contained 4.72 y of zinc. In 5 RI1. In 5 M1. Crine Standard Soln. 2.92 2.95 2.87 2.92 2.79 2.95 2.98 2.87 2.95 2.81 2.95 2.90
Mean Standard deviation & O . 13
4.i2 4.76 4.80 4.?2 4 80 4.94 4.72 4.81 4.95 4.97 4.82 &0.10
Table V. Values in y Obtained for Daily Urinary Zinc Excretion
.4. Pre-extracting urine samples with CCl, B. Dry-ashing urine samples +A B
43 1 409
435 453
393
4.53
73 285 454
74 290 467
Mean Standard deviation i.190
& 187
such losses of zinc correspond directly to those of volunie (Table 11). a volume correction of the colorimetric data can be easily made. I n alkaline urines, zinc ions may precipitate as zinc phosphate or zinc ammonium phosphate (8); this may cause large errors in the sampling of VOL. 30, NO. 12, DECEMBER 1958
1953
urine specimens. Such urines must be acidified with metal-free acid prior to analysis. T o ensure that all the zinc is in the ionic state, hydrochloric acid is added routinely to each separatory funnel, regardless of the initial p H of the sample. The buffer, containing potassium cyanide and sodium thiosulfate, is added t o complex copper, cobalt, nickel, bismuth, mercury, cadmium, and lead which may be present, while the sodium potassium tartrate serves to complex interfering iron and manganese. The present method improves the extractability of zinc dithizonate from urine. TThile a function of pH (Table I), optimal conditions also depend on the composition of the sample as well as the buffer, the complexing agents which it contains, and the addition of any substance which combines with zinc to form a slightly dissociated complex. The amount of zinc extracted increases with pH, with increasing concentration of dithizone in the carbon tetrachloride phase, and with decreasing strength of buffer and complexing agents. The balance of these parameters is critical, and desirable changes produced in one experimental variable are readily obviated by resultant disturbances of others. It has been possible to improve the extractability of zinc from urine by a change of buffer p H to 5.7 (Table I) accompanied by the use of a solution of buffer and complexing agents which is 25% more dilute than that used previously, without altering the specificity of the method for zinc (6).
Incomplete extraction of zinc may also result from the addition of insufficient amounts of dithiaone. If much zinc is present in a sample, the concentration of free dithizone in the organic phase may fall below the critical level and prevent complete extraction. I n this case more dithizone solution (0.003%) should be added. The minimum quantities of metal ions causing interference in urine zinc determination (Table 111) are of the same magnitude as those reported for the mixed-color method (9). Because the amount of these metals which normally occur in biological samples is not likely to exceed this minimum, the method is regarded as highly specific for zinc under the conditions delineated. The good agreement of the amounts of zinc found in urine after d r y ashing and by the present technique (Table V) demonstrates the high accuracy of the method. The insignificant loss of zinc in the directly extracted urine samples may be indicated by their slightly lower mean value. The accuracy of the d r y ashing method is very dependent on the ashing temperature. Ashing of organic samples a t temperatures below 550’ C. leads to losses b y adsorption on carbon particles. Loss of zinc from urine samples by volatilization does not occur a t the temperatures used under the conditions of these experiments. This technique is considerably simpler than those previously reported for the determination of zinc in urine (9) and in spite of the fact that the technique is much more rapid, there is neither loss of
precision nor of specificity. This method was employed in studies on urinary zinc excretion in patients with postalcoholic cirrhosis of the liver (7), which required large numbers of precise replicate analyses. The principle of this method is also applicable to methods for the determination of other metals in urine which involve phase separation. LITERATURE CITED
(1) Hoch, F. L., Vallee, B. L., J . Biol. Chem. 181, 295 (1949).
(2) Sandell, E. B., L‘Colorimetric Determination of Traces of Metals.” v. 620, Interscience, New York, 1950. (3) Thiers, R. E., in “hfethods of Biochemical Analysis,” Vol. V, p. 301, ed. by D. Glick, Interscience, New York, 1957. (4) Thiers, R. E., Williams, J. F., Yoe, J. H., AXAL.C H E ~27, . 1725 (1955). (5) Vallee, B. L., ANAL. CHEM.26, 914 (1954). (6) Vallee, B. L., Gibson, J. G., 11, J. Bzol. Chem. 176, 435 (1948). ( 7 ) Vallee, B. L., Wacker, W. E. C., Bartholomay, A. F., Hoch, F. L., New Eng. J . Med. 257, 1055 (1957). (8) Weitzel, G., Fretedorff, A. M., Z. physiol. Cheni., Hoppe-Seyler’s 292, 212 (1953). (9) Wolff, H., Busse, G., Biochem. Z . 322, 154 (1951). RECEIVEDfor review April 1.5) 1958. Accepted July 10, 1958. Studies supported by a contract between the Office of Naval Research, Department of the Navy and Harvard University, Contract Nonr. 1866(04), Kr 119-277, by a Grant-inAid from The National Institutes of Health, Education, and Welfare, and by the Howard Hughes hledical Institute. J. H. R. Kagi is a fellow of the American Swiss Foundation for Scientific Exchange.
A Fractional Sublimation Technique for Separating Atmospheric Pollutants JEROME F. THOMAS, ELDON N. SANBORN, MlTSUGl MUKAI, and BERNARD D. TEBBENS Sanitary Engineering Research Laboratory, University o f California, Berkeley, Calif. Fractional sublimation offers a new possibility for separating relatively large quantities of the particulate organic material found in polluted atmospheres. A method has been developed using a mixture of condensed polynuclear aromatic hydrocarbons of known composition. A quantitative separation can be made on this mixture, or any mixture of compounds that can be sublimed. When applied to an atmospheric sample of unknown composition a resinous component interferes, and several fractionations are required to remove the resinous material from the
1954
ANALYTICAL CHEMISTRY
field. Obtaining relatively large amounts of pure material i s important both for identification purposes and from the standpoint of public health. The carcinogenic activity of many individual compounds found in polluted air has never been tested because it has been impossible to obtain pure material in gross amounts.
T
HE PARTICULATE ORGANIC K 4 T E R I A L
of polluted urban atmospheres is common to the particulate organic material of the combustion effluent of any liquid or gaseous hydrocarbon fuel
subjected to incomplete combustion (7-9). Several qualitative aspects of the particulate organic type of pollutant have been investigated, including a division of these pollutants into broad functional chemical classes, the separation of pure compounds within the classes, and the identification of some compounds ( I O ) . Approximately 100 high molecular weight organics have been separated in pure form, but only a small percentage have been positively identified. This paper deals with some of the difficulties associated with the quantita-
