Anal. Chem. 1986, 58, 1449-1451
1449
Determination of Traces of Zinc in Biological Materials, Wine, and Alloys by Fluorometry Jose M. Cano Pavbn,* M. Encarnaci6n Ureiia Pozo, and Amparo Garcia de Torres Department of Analytical Chemistry, Faculty of Sciences, The University of Mdaga, 29071 Milaga, Spain
A slmpie, rapid, and selective method for the fluorometrlc determlnatlon of zinc has been developed based upon the formatlon of the salicylaldehyde thiocarbohydrazone (SATCH-Zn( 11)) complex. The reactlon is carried out at pH 4.6-4.9 In an aqueous-ethanol medium (52% v/v ethanol). The detection limit is 10 ng/mL and the relatlve standard deviations are f1.65% (15-100 ppb rlnc), f1.70% (100-500 ppb zinc), and f2.22% (500-1000 ppb zlnc). The effect of Interferences was studied. The method has been appiled to the determlnatlon of zlnc In blologlcal samples (prlor to destruction of the organic matter by using a "0,H,O, mixture), wine, and alloys.
During recent decades, great progress in analytical methodology has provided sensitive techniques for trace-element research in man. Much interest has centered on plasma or serum levels in heath and disease. Abnormally low serum zinc concentrations are encountered in a wide variety of clinical conditions in which no deficiency of zinc is suspected (1).The determination of zinc in biological fluids is a routine measurement in many laboratories; information obtained from these analysis may be of value in the diagnosis of certain biochemical abnormalities or nutritional deficiences (2). Serum zinc is commonly determined by atomic absorption spectrophotometry (AAS) (3-9). Neutron activation analysis (NAA) is a very sensitive technique (10-15) for the determination of this metal ion in biological fluids, but its use is restricted to laboratories that have access to a nuclear reactor. Other analytical techniques such as spectrophotometry (16), colorimetry ( 17), emission spectrometry (18-21), and X-ray fluorescence spectrometry (22) have been described also. In this work, detailed studies on the complexation equilibria of SATCH (salicylaldehyde thiocarbohydrazone) with Zn(I1) in 52% ethanol were carried out. Aspects of the chemistry of the reaction have been investigated, and a procedure for the fluorometric determination of zinc is proposed. The results show that SATCH can be used for the simple, rapid, and selective fluorometric determination of this metal ion. The method is suitable for measuring low levels of zinc in biological samples, wine, and alloys. EXPERIMENTAL S E C T I O N Apparatus. This work was performed on a Perkin-Elmer fluorescence spectrophotometer, Model MPF-43 A, equipped with an Osram XBO 150-W Xenon lamp, excitation and emission grating monochromators, R-777 photomultiplier, and a PerkinElmer 023 recorder. An ultrathermostatic water bath circulator, Frigiterm 5-382, was used for temperature control. A Crison Digit-501 pH meter was used for the pH measurements (throughout this paper pH is used to denote pH meter reading and not the actual concentration of hydrogen ions in solution). Reagents. All chemicals were of analytical reagent grade or better. Glass-distilledand deionized water was used throughout. Stock standard Zn(I1) solution (4.8378 g.L-l) was prepared by dissolving zinc sulfate heptahydrate (Merck) in distilled water and diluting the solution to 1L. This solutiod was standardized by EDTA titration. The working solutions were made by suitable dilution of this standardized stock solution. A 1X lo4 M SATCH 0003-2700/86/0358-1449$01.50/0
solution in ethanol was prepared by dissolving solid reagent samples prepared and purified by the authors (23) (this solution was prepared daily). A buffer solution of pH 4.0 was prepared by dissolving 500 mL of acetic acid (0.2 M) and 110 mL of sodium acetate (0.2 M) and diluting to 1 L with distilled water. Recommended Procedure. Into a 25-mL volumetric flask transfer a suitable aliquot of sample solution containing 0.375-32.500 pg of zinc and 4 mL of pH 4.0 acetic acid-sodium M SATCH solution acetate buffer solution. Add 3 mL of 1 X in ethanol and 10 mL of ethanol. Dilute to the mark with deionized water and mix well. Allow the solution to stand about 10 min. Make the fluorometric measurements using an excitation wavelength of 392 nm and a emission wavelength of 467 nm. Use a calibration graph or empirical equation to convert the fluorescence intensity into concentration. If the samples contain high amounts of other metal ions, add a suitable amount of masking agent prior to addition of the reagent. Determination of Zinc in Biological Materials. Biological samples (1 g or 10 mL of human urine) were digested with a mixture of nitric acid (10 mL) and HzOz (3 mL) in a reflux apparatus. After digestion, samples were evapored to a small volume and neutralized with sodium hydroxide; finally dilute with deionized water to 50 mL in a standard flask. A suitable aliquot of this sample solution was pipetted, and the zinc content was determined by the recommended procedure. Fluoride and thiosulfate were used as masking agents. Microamounts of zinc in blood serum were determined by the following procedure. Transfer 3 mL of serum into a 10-mL conical centrifuge tube; add 2 drops of thioglycolic acid and mix. Add, mixing after each addition, 3 mL of hydrochloric acid (2 N) and 0.8 mL of trichloroaceticacid (40%). Stir vigorously with a glass rod for about 45 s and centrifuge for 10 min at 3000 rpm. A suitable aliquot of the supernatant fluid was pipetted, and the zinc content was determined by the recommended procedure using a method of standard additions. Determination of Zinc in Wine. The destruction of the organic matter was carried out in the same manner as the biological samples. Determination of Zinc in Alloys. Microamounts of zinc in standard aluminum alloy, white metal, and lead concentrate were determined by the following procedure. Weigh accurately 0.1 g of the sample. Dissolve aluminum alloy and white metal in 10 mL of aqua regia; evaporate the resulting solution to dryness and add 1mL of hydrochloric acid (1+ 1);then dilute with distilled water to 250 mL. Lead concentrate was treated first with a mixture of concentrated nitric acid and HzOzand then with dilute nitric acid (1+ 1)and boiled to ensure complete dissolution. Upon cooling, the solution was filtered and carefully washed into a 250-mL volumetric flask. Recovery determinations with these alloys were carried out as described under Recommended Procedure. Aluminum alloy 20b had the following certificate composition (%): Al, 91.45; Cu, 4.10; Ni, 1.93; Fe, 0.43; Mn, 0.19; Si, 0.29; and Mg, 1.61. The standard composition of the leadconcentrate, X, is (%) Pb, 63.77; Zn, 7.73; Cu, 3.28; Si, 0.03; Sb, 0.16; As, 0.06; SiOz, 2.44; Fe, 3.70; S, 18.25; F, 0.0024; Ag, 0.0475; and Au, 0.000 17. White metal 8e had the following certificate composition (%): Cu, 4.57; Pb, 3.13; Sb, 9.50; Zn, 0.04; Cd, 0.14; and Sn, 82.62. RESULTS AND DISCUSSION The reagent SATCH reacts instantaneously with zinc(I1) ion to form a yellow complex having a feeble absorption spectrum in the same region as the reagent alone. This 0 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
Table 11. Determination of Zinc in Biological Samples
IF/ 601
amt of Zn found. U P of Zn/e of samde by SATCH method by AAS
sample
I
liver kidney brains
A
82.20 87.25 153.00
80.00 86.00
150.00
Table 111. Determination of Zinc in Biological Fluids sample
amt of Zn found, yg/mL by SATCH method by AAS
human urine human serum-1 human serum-2 human serum-3 I
4
6
I
8
I
10
I
I
Table I. Tolerance of Foreign Ions on the Determination of 25 ng/mL
50.00
25.00 2.50 1.25
2.700
1.400
12
Figure 1. (A) Influence of pH in the fluorescence of Zn-SATCH complex: [Zn] = 1.53 X M, [SATCH] = 8 X M. (B) Reagent alone.
100.00
1.100 2.900
,
P"
tolerance, rgbL
1.083 2.700 2.720 1.420
ion or species
Na(I), K(I), Ba(II), Sr(II),Mg(II),Mn(II), Ca(II), Rb(I), Cs(I), Pb(II), Y(III), Li(I), bromide, chloride, nitrate, fluoride, acetate, iodate, and iodide Tl(I), Al(III), sulfate, sulfide, phosphate, perchlorate, bromate, carbonate, chlorate, and borate Cd(II), In(III), Zr(IV),Cr(III), As(III), arsenate, and thiocyanate W(VI),Be(I1) U02(II),Th(IV), Fe(III), Cu(II), V(V), Ag(I), Mo(VI), Hg(II), Ni(II), and Co(1I)
complex has an intense fluorescence; the maximum emission occurs a t X = 467 nm with excitation at X = 392 nm. Effect of pH and Order of Addition. The influence of p H was studied over the range 2-12. The emission was maximum and constant in the alkaline region and showed two possible optimum pH ranges of 7.5-8.3 and 4.6-4.9 (Figure 1). All subsequent studies were carried out at pH 4.7 (under these conditions, the emission of the reagent blank at 467 nm was 1% , thus promoting excellent analytical conditions for the determination). An acetic acid-sodium acetate buffer (pH 4.0) was selected for the procedure. The volume of this buffer added (3-8 mL) had no effect. Increasing ionic strength produced no significant changes in the fluorescence. The order of reagent addition was immaterial. Influence of Reagent Concentration, Stability, and Temperature. The fluorescence intensity remains constant with the concentration of the reagent above &fold molar M reagent solution in a excess; therefore, 3 mL of 1 X final volume of 25 mL is sufficient for the analytical procedure. The optimum amount of ethanol in the final solution was 4448% (v/v); a medium containing 52% ethanol was selected. In this medium, the fluorescence intensity remains constant, after 10 min, for at least 2 h, and the apparent pH was 4.7 f 0.1. The fluorescence was not affected by temperature in the range 10-50 OC. All fluorescence measurements were made at 25 "C. Composition of the Complex. The continuous variation and mole ratio methods were applied at pH 4.7. Both methods showed that the composition of the complex was 1:l (zinc to SATCH).
Table IV. Determination of Zinc in Wines sample
amt of Zn found. ualmL by SATCH method by AAS
sweet I (PX-Moscatel) sweet I1 (Lagrima Calidad) sweet I11 (natural) dry I (Mollina 1984) dry I1 (alcoholizado) dry I11 (Mollina 1983) Old Dry
0.583 1.500 0.319 0.300
0.580 1.480 0.300 0.300
0.600 1.070
0.600 1.100
3.800
3.940
Table V. Determination of Zinc in Alloys % Zn
sample aluminum alloy (20b) lead concentrate (X) white metal (8e)
found by SATCH method 0.052 7.630 0.039
certificate compn
added 0.050
7.730 0.040
Calibration, Range, Sensitivity, and Precision. There was a linear relationship between zinc concentration and fluorescence intensity over the range 15-1300 ~g.L-l(2.3 X to 2.0 X M) zinc. The optimum working range as evaluated by the W. J. Youden method (24) is 15-1300 ppb. The detection limit is 10 ~g.L-l. The relative standard deviations are *1.65% (15-100 ppb), &1.70% (100-500 ppb), and f2.22 (500-1000 ppb). Effect of Foreign Ions. In the determination of 25 ng. mL-l zinc, foreign ions can be tolerated at the levels given in Table I. For these studies, different amounts of the ionic species were added to 25 ng/mL zinc by first testing a 4000-fold m/m ratio of interferent to zinc, and if interference occurred, the ratio was progressively reduced until interference ceased. The criterion for an interference was a fluorescence intensity value varying by more than *5% from the expected value for zinc alone. The tolerance level for some metal ions can be increased by addition of fluoride, thiosulfate, dimethylglioxime, EDTA, and iodide. Thus 10 ppm fluoride will mask 2.5 ppm Fe(II), Ga(III), or Mo(V1); 10 ppm thiosulfate will mask 2.5 ppm Cu(I1); 10 ppm dimethylglyoxime will mask 2.5 ppm Ni(I1); 1.25 ppm EDTA will mask 2.5 ppm Co(I1); and 10 ppm iodide will mask 2.5 ppm Hg(I1) or Ag(1). Applications. The recommended procedure for the determination of zinc was applied in a variety of situations to evaluate its effectiveness. For this purpose, diverse samples (bovine liver, kidney, brains, human urine, human serum, wine, white metal, lead concentrate, and aluminum alloy) were analyzed. This method was compared with a determination
Anal. Chem. lB86, 58, 1451-1453
by using AAS, with good agreement. The results obtained are depicted in Tables 11-V. A preliminary step in the determination of zinc in biological samples is the destruction of the organic matter. We have chosen the wet oxidation method described by Bajo (25),in which a mixture of concentrated nitric acid and HzOzis used. Registry No. Zn, 7440-66-6; salicylaldehyde thiocarbohydrazone, 41361-11-9;aluminum alloy (20b), 101248-16-2;lead concentrate (X), 101315-79-1;white metal @e), 101315-80-4.
LITERATURE CITED (1) Falchuk, K. H. N. N. Engl. J . M e d . 1977, 296, 1129. (2) Trace Elements ln Human Health and Disease; Prasad, A. S., Oberlease, D., Eds.; Academlc Press: London, 1976; Vol. 1, Zinc and Copper. (3) Giroux, E. L.; Durleux, M.; Schechter, P. J. Bloinorg. Chem. 1978, 5 , 211. (4) Lafargue, P.; Couture, J. C.; Monteil, R.; Guilband, J.; Saliou, L. Clln. Chlm. Acta 1978, 66, 181. (5) Allah, P.; Foussard, E.; Boyer, J. Clin. Chlm. Acta 1977, 7 8 , 183. (6) Foote, J. W.; Delves, H. T. Analyst(London) 1982, 707, 121. (7) Foote, J. W.; Delves, H. T. Analyst(London) 1982, 707, 1229. (8) Brown, A. A,; Taylor, A. Analyst (London) 1984, 709, 1455. (9) Gardiner, P. F.; Ottaway, J. M. Anal. Chim. Acta 1981, 124, 281. (IO) Parr, R. M.; Taylor, D. M. Blocbem. J. 1984, 97. 424.
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(11) Magglore Glovannetti, Q.;Malvano, R. I n Nuclear Actlvation Techniques In the Life Sciences: International Atomic Energy Agency: VIenna, 1967; p 511. (12) Frltze, K.; Robertson, R. J. Radloanal. Cbem. 1988, 7 , 463. (13) de la Cruz, B. I n Trace Elements in Relatlon to Cardiovascular Disease s ; International Atomic Enery Agency: Vienna, 1973; p 19. (14) . . Versieck. J.; Barbier, F.; Speecke, A,; Hoste, J. Clin. Chem. (Winston-Salem, N.C.)1974, 2 0 , 141. (15) Behne, D.; Jurgensen, H. J. Radioanal. Chem. 1978, 42. 447. (16) Vallee, B. L.; Glbson, J. G.J. Blol. Cbem. 1948, 776, 445. (17) Koch, H. J.; Smith, E. R.; Shimp, N. F.; Connor, J. Cancer 1958, 9, 499. (18) Heirlng, W. 6.; Leavell, B. S.; Paixao, L. M.; Yoe, J. H. Am. J. Ciin. Nutr. 1980, 8 , 846. (19) Palxao, L. M.; Yoe, J. H. Clln. Cblm. Acta 1959, 4 , 507. (20) Monacelli, R.; Tanaka H.; Yoe, J. H. Clin. Chim. Acta 1958, 7 , 577. (21) Nledermeier, W.; Crlggs, J. H. J. Chronic Dis. 1971, 23, 527. (22) Stump, I. 0.: Carruthers, J.; D'Auria, J. M.; Applegarth, D. A.; Davidson, A. G. F. Clln. Biochem. 1977, 70, 127. (23) MontafiaGonzBlez, M. T.; Gomez Ariza, J. L.; Gar& de Torres, A. Anal. Quim. 1984, 8 0 , 129. (24) Youden, W. J.; Statistical Methods for Chemist; Wiley: New York, 1951. (25) Bajo, S.; Suter, U.; Aeschllman, B. Anal. Chim. Acta 1983, 749, 321.
RECEIVED for review November 4, 1985. Accepted February 5 , 1986.
Extractive Spectrophotometric Method for the Determination of Vanadium(V) in Steels and Titanium Base Alloy Anjaneyulu Yerramilli,* C. S. Kavipurapu, R. R. Manda, and C. M. Pillutla
Department of Chemistry, Nagarjuna University, Nagarjunanagar, Andhra Pradesh, India 522 510
Vanadium(V) forms enionlc chelates with 4-(2-pyrldylazo)resorcinol (PAR) at pH 5.0-7.8, which can be quantltatlvely extracted Into nltrobensene as an ion pair with xylometaroionium cation (XMH). The ternary system has an absorption maximum at 540 nm and obeys Beer's law in the range 0-1.8 pg of vanadlum/mL with a molar absorptivity 4.56 X I O 4 L mol-' cm-'. The Job's method of contlnuous variations Indicated a composition of 1:1:1 for vanadium: PAR:XMH for the extractlng species. I n the presence of 1,2-dlaminocyciohexanetetraacetic acid as a masking agent, the extraction becomes highly selective, and this method can be applied for the determination of vanadlum(V), in the presence of varlous metal ions in synthetlc mixtures, in steels, and In tltanium base alloy.
Vanadium(V) reacts with 4- (2-pyridylaza)resorcinol (PAR) producing intensely colored water-soluble anionic complexes (1-7), which serves as a basis for the spectrophotometric determination of vanadium. The extraction of [4-(2pyridylazo)resorcinolato]vanadium(V) (V02L-) anionic complex paired with quaternary ammonium cations like tetraphenylphosphonium chloride, tetraphenylarsonium chloride, tetradecyldimethylbenzylammonium chloride, trioctylmethylammonium chloride, and with trioctylphosphine oxide has been ivestigated by several workers (8-12). However, in many of these extraction spectrophotometric procedures difficulties arise due to the delay in the separation of the two phases and significant absorbance for the reagent blank solution, particularly when concentrated solutions of reagents are used. 0003-2700/86/0358-145 1$01.50/0
In our investigations we have observed that the 1:l vanadium-PAR complex can be selectively extracted with xylometazolonium cation (XMH) in the presence of 1,2-diaminocyclohexanetetraacetic acid (CyDTA), which has been exploited for rapid and selective spectrophotometric methods for the determination of vanadium in steels and titanium base alloy, and these results are presented in this paper. The present method has many advantages over the other spectrophotometric methods for vanadium. It has high sensitivity, and the interference from many diverse ions can be completely masked with CyDTA. The molar absorptivity of the present method is found to be 4.56 X lo4 L mol-l cm-l, which is higher than those reported earlier for vanadium with phosphotungstic acid ( E = 2000) (13),xylenol orange (e = 13000) (14-16),pyrocatechol violet ( t = 36800) (In,142pyridylazo)-2-naphthol (PAN) in CHC13 (e = 16900) (18),PAN and ~-[(6-methyl-2-pyridyl)azo]-2-hydroxynaphthalene Noxide in CHC13 ( t = 16000) (19),PAR in aqueous medium ( E = 36 OOO) (20), PAR and benzyldimethyltetradecylammonium cation (t = 33500),or trioctylphosphine oxide ( 6 = 14000) (12) in CHCIS. Further, with XMH as the countercation, clear separation of the phases can be achieved immediately and there is no difficulty in the separation of phases even with high reagent concentrations. The reagent blank solutions do not show any absorbance, and the system can be best extracted over a wide pH range.
EXPERIMENTAL SECTION Apparatus. Absorbance measurements are made with a Systronics digital spectrophotometer Model 106 MK (11). pH measurements are made on an Elico expanded pH meter Model 335. 0 1986 American Chemical Society
