Anal. Chem. 1985, 5 7 , 2309-2311
2309
Determination of Traces of Gallium in Biological Materials by Fluorometry Encarnacidn Urefia, Amparo Garcia de Torres, Jose M. Cano Pavdn, and Jose L. Gdmez Ariza Department of Analytical Chemistry, Faculty of Sciences, The University of Malaga, 29006 Malaga, Spain
The method presented Is based on the reactlon of gallium with salicylaldehyde thiocarbohydrarone (SATCH). An analytical method has been developed for the fluorometric determinatlon of microgram amounts of gallium In solutlon. The reaction is carried out at pH 2.3-3.4, in aqueous-ethanol medium (52 % v/v ethanol). The influence of reaction variables is discussed. The detectlon limlt is 2 ng/mL, and the range of application is 3-30 ng/mL. The error of the method Is f1.7%. The advantages of the proposed method Include high sensitivity, selectivity, and slmpilcity of determination. Applications of this method In the determination of gallium in biological samples have been discussed.
Analytical methods capable of measuring nanogram levels of gallium are required for studying the physiological distribution of gallium in biological systems. Such studies are becoming interesting because some gallium compounds have exhibited antitumor activities (1-3). The mechanism of gallium uptake by tumors has not been fully elucidated, but the antitumor activity of gallium has been demonstrated. The pharmacokinetics of gallium, as well as gallium toxicity, have been studied extensively ( 4 , 5 ) . These studies have created the need for a specific as well as an efficient means of determining gallium in tissues and biological fluids. The increasing use of gallium compounds in the electronics industry also requires sensitive methods of determining this element in different types of samples. Several methods for determination of gallium involving the use of NAA (6, 7),AAS (8),and AES (9) techniques have been described. Several reagents, for example, lumogallion (IO),methyl-8hydroxiquinoline (11),2-(2-pyridyl)benzimidazole (12),and rhodamine B (13), are available for the fluorometric determination of gallium. In general, these methods are tedious as they involve several extraction steps. In this work, detailed studies on the complexation equilibria of SATCH (salicylaldehyde thiocarbohydrazone) with Ga(II1) in 52% ethanol were carried out, the aim being to establish the equilibria existing in solution and to determine the characteristics of the complexes formed. The optimum conditions favoring the fluorometric determination of gallium using SATCH were investigated. The method is sensitive and selective and is suitable for measuring low levels of gallium in biological samples.
EXPERIMENTAL SECTION Instrumentation. This work was performed on a PerkinElmer fluorescence spectrophotometer, Model MPF-43 A, equipped with an Osram XBO 150-W xenon lamp, excitation and emission grating monochromators, a R-777 photomultiplier, and a Perkin-Elmer 023 recorder. All experiments were performed in rectangular 1X 1cm quartz cells. An ultrathermostatic water bath circulator, Frigiterm S-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 used were of reagent grade. Standard gallium solution (0.9997 g/L) was prepared by dissolving Ga(N03)3 in 100 mL of acid solution and diluting to 1L with deionized water. This solution was standardized by EDTA titration. The working solutions were made by suitable dilution of this standardized stock solution. A pH 2.3 buffer was prepared by combining 45.8 mL of 0.1 M hydrochloric acid with 50.0 mL of 0.1 M potassium hydrogen phthalate and diluting to 1 L with deionized water. Salicylaldehydethiocarbohydrazone (SATCH) was synthesized in the usual way for hydrazones (14). The reagent was characterized by its infrared spectrum and purity was confirmed by M) were prepared elemental analysis. Reagent solutions ( daily in ethanol. General Procedure. Into a 25-mL calibrated flask, transfer a volume of sample solution containing 75-750 hg of gallium and M of SATCH solution in ethanol. Add 4 mL 3 mL of 1 X of pH 2.3 buffer and 10 mL of ethanol. Mix well and allow the solution to stand for 25 min at room temperature for complete formation of the chelate (unless other metal ions are present, see below). Dilute to the mark with deionized water. Make the fluorometric measurements using an excitation wavelength of 395 nm and an emission wavelength of 445 nm. Use a calibration graph or empirical equation to convert the fluorescence intensity to concentration. Procedure for Biological Materials. Biological samples (1 g or 4 mL of human urine) were digested with a mixture of nitric acid (10 mL) and HzOz(3 mL) in a reflux apparatus similar to that described by Bethge (15). The effectiveness of this wet-ashing method for digesting trace metals in organic materials has been discussed in a recent paper by Bajo et al. (16). After digestion, samples were evaporated to small volume, neutralized with sodium hydroxide, and finally diluted with deionized water to 50 mL in a standard flask. Three identical aliquots of each were finally taken for determining gallium. The determinationsof these metal ions were carried out fluorometrically, by means of the procedure described previously for the determination of gallium. RESULTS AND DISCUSSION Analytical Properties of the Reagent. The solubilities in several solvents were determined by the Wittenberger method at 25 "C. The reagent is soluble in ethanol and dimethylformamide and slightly soluble in water. The dissociation constants were determined spectrophotometrically from the variation of absorbance with the pH following the method of Stenstron and Goldsmith in aqueous solution with 44% v/v ethanol at 25 OC and 0.01 ionic strength. The average pK values for seven determinations were 8.30 f 0.02 for pK,, 11.5 f 0.05 for pKz, and 14.0 f 0.05 for pK3. The reagent shows an appreciable fluorescence in aqueous solutions which is much influenced by pH. The graph of fluorescence vs. p H (Figure 1)shows that the fluorescence is appreciable at pH values greater than 6. The reactions of the reagent with 40 metal ions at various pH values in a medium containing 52% v/v of ethanol to avoid the precipitation of reagent were investigated. The reagent acts as a nonselective chelating agent toward many metal ions. Colored reaction produds are formed with iron(I1) and -(HI), cobalt(II), nickel(II), copper (11), zinc(II), mercury( 11), tin(II), and palladium(II1. Fluorescence is exhibited by the gallium and zinc complexes in acid and basic media.
0003-2700/85/0357-2309$01.50/00 1985 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
!
n
IF
80 c
Table I. Elimination of Interferences by Addition of Masking Agents tolerance limit, ng/mL foreign without with ion masking agent masking agent Cu(I1) Fe(I1)
75 75
750 150
Fe(II1) Mo(V1)
150 75
750 150
masking agents, wg/mL thiosulfate, 15 permanganate, 2.25 fluoride, 0.15 fluoride, 0.15 fluoride, 0.75
Table 11. Determination of Gallium in Biological Samples organ 201
l a
0 n
2
n
6
8
10
12
PH
0.000 0.045 0.060 0.075 0.090
0.000 0.044 0.064 0.077 0.090
100 98 106 103 100
kidney
0.000 0.060 0.075 0.090
0.000 0.065 0.075 0.095
100 108 100 108
brains
0.000 0.045 0.060 0.075 0.090
0.000 0.045 0.062 0.078
100 100 103 104
0.091
101
Flgure 1. (A) Influence of pH in the fluorescence of Ga-SATCH complex: (Ga)= 1.45 X lo-' M, (SATCH) = 8 X M. (6) Reagent
alone.
Study of the Ga(II1)-SATCH Complex. Gallium forms a yellow complex with SATCH; the complex exhibits a feeble absorption spectrum in the same region as the reagent alone. The complex has an intense blue fluorescence, the maximal emission occurs at X = 440 nm with excitation at h = 378 nm. The influence of pH 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 2.3-3.4 and 11.0-11.5 (Figure 1). All subsequent studies were carried out a t pH 2.3 (the reagent, under similar conditions, showed no appreciable emission around this pH, thus promoting excellent analytical conditions for the determination). The effect of SATCH concentration was investigated by measuring the emission a t 440 nm of solutions containing 20 pg/L gallium and varying amounts of SATCH. A 558-fold molar excess of the reagent over gallium was required for maximum fluorescence. So 3.0 mL of 1 X M reagent solution in a final volume of 25 mL sufficed for less that 20 pg of gallium. The fluorescence was not affected by temperature in the range 15-30 "C. All fluorescence measurements were made at 25 "C. Order of addition of reactants was not critical. The variation of fluorescence intensity with time was studied in different ethanol percentage; the most suitable concentration was 50-52 %. The fluorescence intensity remains constant, after 20-25 min, for at least 4 h. The composition of gallium-SATCH complex was studied by continuous variations and mole ratio methods. These methods showed the formation of a 1:3 complex between the metal ion and the reagent. Calibration, Range, Sensitivity, and Precision. There was a linear relationship between gallium concentration and fluorescence intensity over the range 3-30 pg/L (4.3 X to 4.3 X M) of gallium. A negative deviation from linearity was observed at higher gallium concentrations. The optimum working range as evaluated by the Yonden method is 3-30 ppb. The detection limit is 2 pg/L. The precision of the method was studied by analyzing solutions containing known amounts of gallium. The results of determination of 15 to 20 pg/L of gallium showed a maximum relative error of f1.7%. Interference Study. In order to assess the possible analytical applications of the reaction, the effects of other ionic
recovery, %
liver
-
4
amt of Ga, pg/g added found
species on the determination of gallium by the proposed method were examined. For these studies, different amounts of the ionic species were added to 15 pg/L of gallium by first testing a 5000-fold m / m ratio of interferent to gallium, and if interference occurred, the ratio was progressively reduced until interference ceased. The following amounts ( m / m ratio) of foreign ions were found to give less than 5% error in the determination of gallium: 5000, Na(I), K(I), Ba(II), Sr(II), Mg(II), Mn(II), nitrate, chloride; 3000, Li(I), Al(III), thiosulfate; 1000, Be(II), Cr(III), Ag(I), Zn(II), Cd(II), Th(IV), Pb(II), Tl(I), Zr(IV), Bi(III), Y(III), UI1O2,acetate, chlorate, sulfate, iodide, carbonate, sulfide, sulfite,; 500, Ni(II), periodate, phosphate,; 100, W(VI), Sc(III), In(III), arsenate, iodate, thiocyanate, nitrite; 50, Hg(I), Hg(II), Sn(II),Au(III), Tl(III), As(III), Sb(III), cyanide, permanganate; 10, Ce(IV), Fe(III), La(III), fluoride; 5 Cu(II), Fe(II), Mo(VI), V(V). The pH of the solution was applied. Some cations interfere because they form nonfluorescent complexes with SATCH. Interferences caused by copper(I1) can be eliminated by means of thiosulfate and fluoride can be used in the presence of molybdenum and iron (Table I), 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 Gallium in Biological Samples. The recommended procedure for the determination of gallium was applied in a variety of situations to evaluate its effectiveness. With this purpose, diverse spiked samples of bovine liver, kidney, and brains were analyzed; the samples must be previously treated for the destruction of the organic metter. The results obtained for the analysis of different aliquots of the prepared solutions are shown in Table 11, these data demonstrate the reliability of the proposed method for the determination of gallium in these samples. Also, a series of recovery experiments were carried out by adding standard pure gallium solutions to aliquots of human urine samples treated as indicated in the Experimental Section (Table 111). The results obtained indicate that the method
Anal. Chem. 1985, 57, 2311-2314
Table 111 Determination of Gallium in Human Urine amt of Ga, ~ l g added found
recovery, %
0.000 0.043
0.000
0.045 0.060
0.061 0.092
0.090
100
96 101 103
would be effective for the analysis of samples of similar complexity. Registry No. Ga, 7440-55-3; o-HOC6H4CH=NNHC(S)NHN=CHC8H,0H-o, 41361-11-9; Cu, 7440-50-8; Fe, 7439-89-6; Mo, 7439-98-7; S20?-, 14383-50-7; Mn04-, 14333-13-2; F-, 16984-48-8. LITERATURE C I T E D (1) Hart, M. M.; Adamson, R. H. Proc. Natl. Acad. Sci. U . S . A . 1971, 68, 1823.
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Hayes, R. C. J . Nucl. Med. 1970, 18, 740. Zweidinger, R. A.; Barnett, L. Anal. Chem. 1973, 4 5 , 1563. Newman, R. A.; Brody, A. R.; Krakoff, I . H. Cancer 1979, 4 4 , 1728. Kelsen, D. P.: Alcock, N.; Yeh, S.; Brown, J.; Young, C. Cancer 1980, 46, 2009. Nakamura, K.; Fufimori, M.; Tsuchiya, H. Anal. Chlm. Acta 1982, 738,129-136. Argollo, R. M.; Schilling, J. G. Anal. Chim. Acta 1978, 9 6 , 117. Yu, J. C.; Wai C. M. Anal. Chem. 1984, 5 6 , 1889-1691. Caroli, S.;Alimonti, A.; Delle Femmine, P.; Shukle, S. K. Anal. Chim. Acta 1982, 136, 225-231. Lebed, N. B.; Pantaler, R. P. Zh.Anal. Khlm. 1965, 2 0 , 59. Shigematsu, T. Jpn. Anal. 1958, 787. Bark, L. S.; Rixon, A. Anal. Chim. Acta 1969, 4 5 , 425. Lypka, G. N.; Chow, A. Anal. Chim. Acta 1972, 6 0 , 65. Monta'ia Gonzaiez, M. T.; Golirez Ariza, J. L.; Garcia de Torres, A. An. Quim. 1984, 80, 129. Bethge, P. 0. Anal. Chlm. Acta 1954, IO, 137. Bajo, S.: Suter, U.; Aeschliman, B. Anal. Chim. Acta 1983, 149, 321.
for review December 1 3 9 1984* Resubmitted May 20, 1985. Accepted June 6, 1985.
Hydride Generation-Atomic Absorption Determination of Antimony in Seawater with in Situ Concentration in a Graphite Furnace R. E. Sturgeon,* S. N. Willie, a n d S. 5.B e r m a n Division of Chemistry, National Research Council of Canada,l Ottawa, Ontario K1A OR9, Canada
A method is descrlbed for the determination of total Sb In seawater lnvoivlng generation of SbH, uslng NaBH,. The stibine is trapped on the activated carbon surface in a graphtte furnace at 460 K and atomized at 2000 OC. Calibration is achleved wlth a slmpie aqueous working curve. An absolute detection limit (3a) of 0.2 ng and a concentratlon detection iimlt of 0.04 pg/L are obtalned using 5-mL sample volumes. Precision at the 0.2 pg/L level Is 5 % relative standard deviation. Analytical results are reported for Sampies of near-shore and open-ocean seawater.
Production of voltaile covalent hydrides of a number of elements (i.e., As, Bi, Ge, Sn, Se, Sb, Te, and Pb) for determination by atomic absorption has proved extremely useful because it serves to separate the metal from other (potentially interfering) matrix components in the sample and may also be used as a method of concentration (1,2). Significant improvements in sensitivity and detection limit over conventional AAS solution nebulization-flame or electrothermal atomization techniques are realized with hydride generation (1). The majority of workers utilize direct introduction of the hydride into an Ar-H, flame (e.g., ref 3 and 4) or hydride transfer to resistance- or flame-heated silica tubes (5-3, quartz cuvette burners ( 8 ) ,or preheated electrothermal atomizers (9-12). Improved detection limits may be realized by collection and concentration of the hydride prior to its introduction into the atomization cell. Such techniques have included cryogenic condensation in a U-tube immersed in liquid N2 (12, 13) or trapping in solutions of KI-I2 (14) or pyri'NRCC No. 24650. 0003-2700/85/0357-2311$01.50/0
dine-silver diethyldithiocarbamate (15). A considerably simpler and more elegant approach to this problem is the use of the graphite furnace as both the hydride trapping medium and atomization cell. Such an arrangement was proposed by Drasch et al. in 1980 (16) for the determination of As in biological samples. The setup was rather cumbersome and used a Perkin-Elmer HGA 72 furnace. No data were reported. Lee (17) utilized this technique to determine ultratrace levels of Bi in environmental samples. The hydride was generated in a reaction cell, stripped from solution and purged from the cell in a He carrier gas, and collected in situ in a modified carbon rod atomizer a t 350 "C. The collected Bi was subsequently atomized at 1850 "C. More recently, Brovko et al. (18)reported on the determination of Sb and Bi in plant and water samples using hydride formation and in situ preconcentration in a graphite tube furnace. Collection of SbH3 and BiH3 by dissociation at 300 and 250 "C, followed by atomization at 2200 and 2000 "C, respectively, resulted in detection limits of 1pg/L for S b and 0.5 pg/L for Bi. This study reports on the application of in situ metal trapping to the determination of total Sb in seawater by hydride generation-graphite furnace AAS. EXPERIMENTAL SECTION Apparatus. Atomic absorption measurements were made with a modified Varian spectrometer, Model AA-5, equipped with simultaneous deuterium hollow cathode lamp background correction and a Perkin-Elmer Model HGA-2200 furnace. The time constant of the detection system was 10 ms. Signals were recorded on a digital storage oscilloscope (Gould Advance, Model OS4100) and fast response strip-chart recorder (Esterline Corp.). An antimony hollow cathode lamp was run at 8 mA and detected at 217.6 nm. A nominal spectral band-pass of 3.3 nm was used. These conditions contributed to the short linear range of
Publlshed 1985 by the American Chemlcal Society
