Anal. Chem. 1987, 59,888-890
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Separation and Determination of Parts-per-Billion Concentrations of Gallium in Biological Materials Nelson Scott and Dean E. Carter
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721 Quintus Fernando*
Department of Chemistry, University of Arizona, Tucson, Arizona 85721
Galllum Is separated from an aqueous digest of a bioioglcal matrix, by the selective extraction of the ion palr QaCI4-.R4N+, Into a hexane-methyl Isobutyl ketone mlxture (R4N+represents the methyitrlcaprylammonlum Ion). The extracted gallium species reacts wlth the Ilgand, 1,5-bls( sallcyl1dene)thlocarbohydrazone, to form a complex that fluoresces at 440 nm when the excltatlon wavelength is 395 nm. Thls fluorometric method has been used to determine 0.10-40 ppb of galHum In Mologlcal materlals with a relative precision of 7 % The principal interferences with the fluorometric method in aqueous solutions are Fe3+, Fez+, Cu2+, and Zn2+. I n the aqueous digest, the Fe3+ Is reduced to Fez+ and the Cu2+ Is masked wlth S20,2-. The Fe2+ and Zn2+ are not extracted into the organic phase and do not Interfere wRh the fluorometric method.
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Exposure to airborne particulates of gallium arsenide (GaAs) is a potential health hazard in the semiconductor industry. With the exception of the two reports that purported to show that pulmonary fibrosis was caused by GaAs (1-2), there has been no systematic study of the toxicity of GaAs. Preliminary studies that were carried out in our laboratory (3)have shown that GaAs had a substantial solubility in vitro and in vivo. After intratracheal dosing of rats with GaAs particulates, up to 10% of the dose as arsenic was determined in the blood by flame atomic absorption spectrophotometry. Gallium levels, however, were below the attainable detection limits ( E 15 pg/mL) with this technique. Analytical methodology for the determination of nanogram and subnanogram levels of gallium in blood, tissues, and other biological materials must be developed, therefore, to investigate the distribution characteristics of gallium. Neutron activation ( 4 ) and atomic emission techniques (5) are capable of determining nanogram levels of gallium. Matrix effects and interferences from elements that are commonly present in biological materials cause serious difficulties when these methods are used for the routine determination of gallium. Sensitivities of N 1 ng/mL have been attained with reagents that form fluorescent complexes with Ga3+ (6-8), but several extraction steps are required to separate Ga3+from a large number of metal ions that quench the fluorescence of the gallium complex. A promising new method for the fluorometric determination of gallium in biological materials has been reported recently (9). The method is based on the reaction of Ga3+ with 1,5bis(salicy1idene)thiocarbohydrazone (SATCH) to form a fluorescent complex in an aqueous-ethanol medium. A detection limit of 2 ng/mL and a linear dynamic range of 3-30 ng/mI, were claimed for this method. A large number of ions interfered seriously with the reported method, but it was claimed that the effect of many of these interferences could be minimized by the use of appropriate masking agents. For
example, Sz03z-masked the interference caused by copper(I1) and F- masked the iron(II1) and molybdenum interferences. We have reinvestigated this method for the determination of gallium in biological materials. Several metal ions, such as zinc(II), that are present in relatively large concentrations in biological materials interfere seriously with this method and common masking agents do not eliminate these interferences. We have modified the reported method considerably and we have eliminated many of the serious interferences by the introduction of a single-step extraction procedure. We report below the results that we have obtained with a modified method for the determination of gallium in biological materials.
EXPERIMENTAL SECTION Instrumentation. A Beckman DU-40 spectrophotometerwas used to record ultraviolet and visible absorption spectra. An Aminco Bowman SPF spectrophotofluorometer with a xenon excitation source and an RCA photomultiplier was used for the fluorescence measurements. Reagents. A stock solution of gallium, containing 1000 ppm Ga, w&s made by dissolving 99.99% Ga203(Alfa Products, Danver, MA) in the minimum volume of concentration HC1 and diluting the solution with deionized distilled water. The reagent, 1,5-bis(salicylidene)thiocarbohydrazone,SATCH, was synthesized by the condensation of salicylaldehyde (98%, Aldrich Chemical Co., Inc., Milwaukee, WI) with thiocarbohydrazide (Reagent Grade, Sigma Chemical Co., St. Louis, MO) (10). A solution containing 1 g (0.01 mol) of thiocarbohydrazide in 25 mL of hot water was added slowly and with continuous stirring to a solution containing 3.6 g (0.03 mol) of salicylaldehyde in 25 mL of ethanol. The resulting mixture was refluxed for 45 min on a water bath. The solid obtained upon cooling was separated by filtration. H
I
HN-C-NH a::=N
I SI t N=CH I HO
1.5-bis(saiicylidene) thiocarbohydrazone SATCH
The reaction product was recrystallized twice from an ethanol-water mixture, mp 195 "C (11, 12). The mass spectrum of the compound, however, had a relatively intense peak, at m / e 354, which could not be accounted for on the basis of reaction 1. After a minimum of six recrystallizations, pale yellow crystals, that had a melting point of 225 "C were obtained (10, 13). A high-resolution mass spectrum of the compound confirmed the molecular formula, C15H14N402S, for SATCH. In a 50% (v/v) ethanol-water mixture the reagent, SATCH, had a molar absorptivity of 4.0 X lo4M-' cm-' a t the absorption maximum of 345 nm: the gallium complex of SATCH had a molar
0003-2700/87/0359-0888$01.50/0 0 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987
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pletely digested, more nitric acid and hydrogen peroxide were added and the mixture was reheated until a clear solution was obtained. The solution was evaporated until all the nitric acid was expelled and dense white fumes of H2S04 were observed. The solution was cooled, neutralized by the addition of an NaOH solution, transferred quantitatively into a 25-mL volumetric flask, and diluted to 25 mL with water. One-milliliter aliquots of this solution containing a known concentrationof gallium were used in the fluorometric procedure described above.
I
!
i
Figure 1. Absorption spectra of the ligand, SATCH, and the Ga3+-
SATCH complex in a phthalic acid-hydrogen phthalate buffer (pH 2.83) in 50% (v/v) aqueous-ethanol mixture: (-) SATCH (C, = 3.73 X M); (---) Ga3+-SATCH complex (C, = 1.71 X lO-'M, CHaL= 3.73 x 10-4 M). absorptivity of 3.2 X lo4 M-' cm-l at the absorption maximum of 395 nm. As shown in Figure 1, the absorption spectrum of the reagent undergoes a red shift when complexed with gallium. This absorption spectrum of the galliumSAWH complex was obtained by subtracting the absorbance of the uncomplexed ligand from the total absorbance. As reported previously (9), the galliumSATCH complex as well as SATCH, in ethanol-water solutions, fluoresce at 440 nm when the excitation wavelength is 395 nm. The fluorescence spectrum of the gallium-SATCH complex consists of a broad featureless band with a maximum at 440 nm. The fluorescence of both the ligand and its gallium complex vary with pH; the fluorescence of the ligand is negligible between pH 2.3 and 3.4 whereas in this pH range the fluorescence of the gallium-SATCH complex is constant and at a maximum (Figure 2). In this work, therefore, the fluorescence of the galliumSATCH complex was determined in an aqueous-ethanol solution at a pH of 2.6 maintained by a phthalic acid-hydrogen phthalate buffer. Fluorometric Determination of Gallium. A standard solution of gallium (containing up to 40 ppb of gallium) was mixed with 2 mL of a 27% (w/v) aqueous solution of NaCl and 0.1 mL of 0.1% Na2S203. An aqueous solution of hydroxylamine hydrochloride (0.1 mL of 0.1%) was added and the mixture was allowed to stand for about 5 min. The GaC14- in solution was extracted as an ion pair that was formed with methyltricaprylammonium ion (Aliquat 336 obtained from Henkel Corp., Minneapolis, MN) into an organic phase consisting of a mixture of hexane (5% (v/v)) and methyl isobutyl ketone (MIBK). The organic phase containing 5% Aliquat 336 and 2 mL of the hexane-MIBK mixture was used for all the extractions. The aqueous and organic phases were stirred vigorously in a vortex mixer and the phases were allowed to separate (20-25 min). The organic phase was tTansferred quantitatively, with the aid of an additional 1mL of the hexane-MIBK mixture, into a 10-mL volumetric flask containing 1 mL of 1.0 X M ethanol solution of SATCH, 1 mL of the phthalate-hydrogen phthalate buffer (pH 2.6), and 3 mL of ethanol. The solution in the volumetric flask was diluted to 10 mL with ethanol, mixed thoroughly, and allowed to stand for 30 min. The fluorescence of the gallium-SATCH complex in this solution was recorded at 440 nm when the excitation wavelength was 395 nm. Standard solutions containing 0.1-40 ppb Ga were used to obtain a calibration curve. Blank solutions that were subjected to the above procedure in the absence of gallium showed no fluorescence. Fluorometric Determination of Gallium in Biological Materials. Standard solutions containing gallium were added to a sample of biological material (1g, kidney, liver, or lung tissue, 5 mL of blood plasma, or 25 mL of urine) and the resulting material was digested with 0.5 mL of concentrated H2S04and 5 mL of concentrated HNO, in a round-bottom flask for about 45 min. Five milliliters of H202solution (20% (v/v)) was added dropwise during the digestion procedure and the solution was concentrated by slow evaporation. If the material was incom-
RESULTS AND DISCUSSION The fluorometric determination of gallium described previously is based on the formation of a 1:3 complex of Ga3+with SATCH (9). The ligand, SATCH, has three replaceable protons (pK, = 8.3, pKz = 11.5, pK3 = 14.0) (9,lO);in the pH range 2.3-3.4, the formation of the uncharged 1:3 galliumSATCH complex can proceed to a reasonable degree of completion only in the presence of a very large excess of the ligand. It is not surprising, therefore, that a several hundredfold excess of SATCH is required to maximize the fluorescence of the gallium-SATCH complex that is formed in solutions containing parts per-billion concentrations of gallium. If the compound, SATCH (H3L), forms a 1:3 gallium-SATCH complex, Ga(HzL)3,by the displacement of three protons from three molecules of SATCH, the proton displacement constant, Kpd, is given by Kpd
= [Ga(HJJ31[H+13/[Ga3+l[H3L13
(2)
The value of Kpd can be estimated by measurement of the absorbances of solutions containing known initial concentrations of Ga3+and the ligand, H3L, in a 50% (v/v) aqueous-ethanol medium in the presence of a phthalic acid-hydrogen phthalate buffer. The concentration of the complex, Ga(H3L)3,was calculated from the measured absorbance a t 395 nm and the molar absorptivity of the complex (Figure 1). The concentrations of the uncomplexed Ga3+and free ligand, H3L, were calculated from the mass balance equations for the gallium and for the ligand, respectively. The hydrogen ion concentrations in the 50% aqueous-ethanol solutions were reeding vs. [H+], obtained from a calibration curve of, pHmeter where values of [H+] were determined from known concentrations of standard HC1 that were added to the 50% (v/v) aqueous-ethanol solutions. These calculations provide only an estimate of the proton displacement constant, Kpd, 4nd merely confirm the experimental observation that a stable gallium-SATCH complex is formed under the solution conditions that are employed in this work. The average value of the protos displacement constant, obtained when the initial ratio of SATCH:gallium was varied between 10 and 40, was 1026.
The ligand, SATCH, is not a selective reagent for Ga3+. A very large number of cations form complexes with SATCH and interfere with the fluorometric determination of Ga3+. These interferences can be masked, in principle, by the addition of an appropriate masking agent. We have found, however, that several cations that are present at parts-permillion levels in biological materials cannot be effectively masked by the addition of masking agents. For example, Fe3+, Fez+,Cu2+,and Zn2+at parts-per-million levels interfere seriously with the fluorometric method. Citric acid, ascorbic acid, and sulfide and phosphate ions were ineffective masking agents for these cations. The gallium-SATCH complex is not formed in the presence of either citric or ascorbic acid because Ga3+forms strong complexes with these acids (14). If sulfide or phosphate ions are added as masking agents, Fe3+is precipitated and Ga3+ is lost by coprecipitation or adsorption. The use of fluoride as a masking agent also causes difficulties; Fe3+as well as Ga3+form relatively strong complexes with F(15, 16) (log WaF=25.86 + and log = 6.04) and the fluorescence intensity af the gallium-SATCH complex is
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987
-Log [!it]
Figure 2. Relative fluorescence intensity of the ligand, SATCH, and the Ga3+-SATCH complex as a function of the hydrogen ion concentration in 50% (v/v) aqueous-ethanol mixture: (- -) Ga3+-SATCH complex (Cas+ = 2.9 X M, CHIL = 8.3 X M); (-) SATCH (cHJL = 8.3 x 10-5 M)
-
noticeably decreased if the gallium: F- ratio is 1:7 or greater. The interference caused by Fe3+can, however, be minimized by adding an equimolar concentration of fluoride, but this cannot be accomplished unless the concentration of Fe3+in every sample is determined prior to the addition of the masking agent. In the modified fluorometric method that we have proposed, the Ga3+is separated from parts-per-million levels of interfering cations by extraction as the ion pair, GaC1pR4N+,into an organic solvent and the fluorescent GaS+-SATCH complex is formed by the addition of a large excess of the ligand, SATCH. The stable GaC1,- species readily forms ion pairs with quaternary ammonium salts such as Aliquat 336 (17), and the ion pairs are extracted by methyl isobutyl ketone. The chloro complexes of Cu2+.Zn2+,and Fez+ are not extracted to any appreciable extent into the MIBK phase under the conditions that we have specified above. Any Fe3+ that is present in solution is reduced to Fez+ by the addition of hydroxylamine hydrochloride, before the GaClL is extracted as an ion pair into the organic phase. The concentration of NH20H.HC1 that is used for the reduction is not critical and could be varied over a wide range without affecting the fluorescence of the gallium-SATCH complex. The only precautions that are necessary are that the NH20H-HClsolution be freshly prepared and that 5-10 min be allowed for the reduction of Fe3+to Fe2+. TJnder these conditions, some Cu2+may be reduced to Cu+ and CuC12-extracted into the organic phase. Copper complexes, however, do not fluoresce and, therefore, the extraction of any copper(I1 species into the MIBK phase is of little consequence. We have found that the use of ascorbic acid as an alternative reducing agent can lead to several problems. Ascorbic acid complexes with Ga"+ in aqueous or ethanolic media and prevents the formation of the gallium-SATCH complex. The extraction of ascorbic acid, even in very low concentrations, will therefore, decrease the observed fluorescence intensity of the gallium-SATCH complex. This effect can be minimized if the ratio of ascorbic acid:Fe3+is about 1O:l and if the same concentration of ascorbic acid is added to each of the standard gallium solutions. The reduction of Fe3+to Fez+by ascorbic acid takes place in 20-30 min; this is much longer than the time required for the reduction by hydroxylamine. The only advantage that ascorbic acid has, in this analytical method, is that solutions of ascorbic acid are stable for several days if stored in the dark. In the modified method for the determination of gallium that we have proposed, the Ga:'+ is separated from interfering cations in the aqueous solution by the extraction of GaC14-.R4N+into an organic solvent consisting of 5% Aliquat 336 dissolved in a MIBK--hexane ( 5 % ( V I \ , ) ) mixture. We
have found that when the fluorescent 1:3 gallium-SATCH complex is formed in the organic phase by the disruption of the ion pair and the displacement of the chloride ions, the linear dynamic range extended from 0.10 to 40 ppb G d + . If necessary, the lower limit of 0.10 ppb can be decreased further by decreasing the volume ratio of the two phases, VOrgmic: Vaqueousr in the extraction step. The relative precision of the method is less than 7% for the determination of 5 ppb Ga3+. The theoretical detection limit is 0.041 ppb, defined as twice the standard deviation of the signal intensity of 15 blank solutions. Interferences caused by common cations that are present in biological materials have been eliminated. This modified procedure is a substantial improvement over other methods that have been reported for the routine determinat,ion of gallium in biological materials. We have evaluated the applicability of this method for the determination of trace levels of gallium in urine, blood plasma, and kidney, liver, and lung tissue. Standard solutions of gallium containing between 20 and 600 ppb of gallium were added to 25 mL of urine or 5 mL of blood plasma. The solutions were digested as described above and extracted with a 5% solution of Aliquat 336 in MIBK. The extracted gallium was determined fluorometrically and the percentage recovery was calculated. In the urine samples, the recoveries varied between 93% and 110%, and in the blood plasma samples, the recoveries varied between 88% and 115%. Standard solutions of gallium containing between 80 and 320 ppb of gallium were added to 1.0-g samples of rat liver, kidney, and lung tissue and digested with nitric acid and hydrogen peroxide until all the organic matter was destroyed. The gallium was extracted and determined fluorometrically; t,he calculated recoveries of the added gallium varied between 83% and 106%, and there was no correlation between the concentration of gallium determined and the percentage gallium recovered. The variation in the percentage of gallium recovered is attributable primarily to the errors that are incurred in the quantitative separation of 3 mL of the organic phase from 3 mL of the aqueous phase, prior to the addition of the ethanol solution of SATCH to form the fluorescent gallium-SATCH complex. A relative precision of 7 % in the fluorometric determination of gallium and an uncertainty of &15% in the separation and determination of gallium in biological matrices are sufficient for the st,udies that we propose to carry out on the pulmonary toxicity of gallium arsenide.
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RECEIVED for review September 2,1986. Accepted November 10,1986. We gratefully acknowledge the support of this work, under Contract No. F239 U, hy the IBM Corp., Poughkeepsie, NY, 12602.
