Anal. Chem. 2002, 74, 100-106
Spectrofluorometric Determination of Vanadium Based on the Formation of a Ternary Complex between Vanadium, Peroxides, and 2-r-Pyridylthioquinaldinamide. Application to the Determination of Hydrogen Peroxide and Peroxy Acids E. K. Paleologos, D. L. Giokas, S. M. Tzouwara-Karayanni, and M. I. Karayannis*
Department of Chemistry, University of Ioannina, Ioannina 45110, Greece
A selective and sensitive method for the determination of the total amount of vanadium in nutritional and biological substrates is proposed. The method is based on the reaction of vanadium with 2-r-pyridylthioquinaldinamide (PTQA) in the presence of H2O2. The product of this reaction emits constant fluorescence, in a sulfuric acid environment, at 490 nm, with the exciting radiation set at 340 nm. Various parameters such as acidity, flow rate, solvents, and temperature were studied. The presence of a surface-active agent was also considered in order to increase sensitivity. At the optimal conditions, a calibration curve was constructed, revealing a linear range of 2-100 µg L-1 and a detection limit as low as 0.5 µg L-1 while the RSD ranged in the area of 0.1-1.8%, depending on vanadium concentration. The method was successfully applied to the analysis of a wide variety of food samples, which are known to contribute to the dietary required amount of vanadium and to relevant biological matrixes. Reversing the conditions of the above reaction, the effect of the peroxy group on the vanadium-PTQA system was examined. The formation of a vanadyl complex was revealed which was suitable for the determination of hydrogen peroxide and peroxy acids. Linear calibration curves in the range of 0.2-50 µM for H2O2 and 0.1-2 µM for a respective peroxy acid were obtained, yielding detection limits of 0.05 and 0.03 µM, respectively. Vanadium is considered, nowadays, as an important trace element.1 It has been shown to be an essential nutrient for rats and other animals, as well as for some marine organisms, functioning as an oxygen carrier.2-4, It is regarded as an essential * Corresponding author. E-mail:
[email protected]. (1) Nielsen, F. H.; Uthus, E. O. The essentiality and metabolism of vanadium. In Vanadium in Biological Systems, Physiology and Biochemistry; Chasteen, N. D., Ed.; Kluwer Academic Publishers: Dordrecht, The Netherlands. 1990. (2) Hopkins, L. L.; Mohr, H. E. In The Biological Essentiality of Vanadium. Newer Trace Elements in Nutrition; Mertz, W., Cornatzer, W., Eds.; Marcel Decker: New York, 1971. (3) Schwartz, K.; Milne, D. B. Science 1971, 174, 426-434. (4) Mravcova, A.; Lener, J.; Babicky, A. Abstracts of the Inter. Union of Physiol. Sci. regional meeting, Prague 1995, PF 57, 105-106.
100 Analytical Chemistry, Vol. 74, No. 1, January 1, 2002
nutrient also in higher organisms, although its role in the body is not yet clearly defined and understood.5 However, its universal presence in the tissues of animals and human suggests a predominant role. It is connected with lipid metabolism, because high doses of vanadium may inhibit the synthesis of cholesterol and lower the phospholipid and cholesterol content of the blood. Vanadium is mainly found in teeth, bone, hair, and nails, and it is regarded as an inhibitor to the development of caries by stimulating mineralization of teeth.1,4 Industrial exposure to vanadium has been shown to causesin addition to eye and lung irritationsinhibition of activity to the enzyme cholinesterase, resulting in a deficiency of choline. Such a deficiency may have adverse effects, including liver and kidney damage. However, when inserted into the organism through the diet, vanadium seems to pose no danger to the human, although its toxicity on rats and small animals is well established, even at low dietary levels.2,6 Perhaps the major reason for that lack of toxicity is its poor absorption by the gastrointestinal tract.7 Its confined presence could not be interpreted otherwise, as there are foods, such as fish and oils, and food supplements, with a vanadium concentration up to 50 µg g-1.8-11 The only possible danger could originate from the uptake of vanadium by cooking devices and containers because vanadium is a main constituent of stainless steels and corrosion-resistant pots and pans. Many methods have been proposed for the determination of vanadium. A great number of them are based on the spectrophotometric analysis of its colored complexes, after reaction with various reagents,12,13 and on catalytic kinetic methods.14 Those (5) Reilly, C. Metal Contamination of Food; Applied Science Publishers Ltd.: London, 1980. (6) Nechay, B. R.; Nanninga, L. B.; Nechay, P. S. E.; Prost, R. L.; Grantham, J. J.; Macara, I. G.; Kubena, L. F.; Philips, T. D.; Nielsen, F. H. Fed. Proc. Fed. Am. Soc. Exp. Biol. 1986, 45, 123-132. (7) Nechay, B. R. Annu. Rev. Pharmacol. Toxicol. 1984, 24, 501-524. (8) Sabbioni, E.; Kueera, J.; Pietra, R.; Vesterberg, O. Sci. Total Environ. 1996, 188, 4-58. (9) Lisk, D. J. Adv. Agron. 1972, 24, 267-320. (10) Schroeder, H. A.; Nason, A. P. Clin. Chem. 1971, 17, 461-474. (11) Berrow, M. L.; Weber, J. J. Sci. Food Agric. 1972, 23, 93-100. (12) Bu-Olayan, A. H.; Al-Yakoob, S. Sci. Total Environ. 1998, 223, 81-86. 10.1021/ac0108008 CCC: $22.00
© 2002 American Chemical Society Published on Web 11/30/2001
methods, although sensitive enough, faced several problems with selectivity, especially in food matrixes, where the abundant presence of other metallic ions such as iron, copper, zinc, and molybdenum presented significant interferences. Recently a nonaqueous catalytic method was proposed, after cloud point extraction,15 yielding remarkable results. Electrochemical approaches also produced good results for the determination of vanadium in water samples,16,17 but in large, the use of element-specific techniques is usually recommended for the analysis of vanadium in food samples.5,18-20 Sophisticated techniques, such as ICPMS21 and ICP-AES22 as well as AAS23 have been used for the determination of total vanadium. However, at microgram per liter levels, these methods are only applicable after preliminary isolation and preconcentration while increasing the cost of analysis.24,25 Furthermore, GF-AAS has various memory and background effects, as well as a high detection limit.24,25 Vanadium has been reported to interact with H2O2, and peroxides in general, to form coordination compounds.26 These binary complexes show low molecular absorptivities and they are not available to sensitive determinations. On the other hand, formation of ternary complexes with organic chromophores has been extensively used as their molecular absorptivities allow for low detection limits. Of the most widely applied are pyridylazo dyes such as pyridylazoresorcinol (PAR)27 and derivatives of pyridylazophenols.28,29 Due to their high molecular absorptivities, these methods were successfully applied to the determination of either V or H2O2 at submicromolar levels. Although these methods demonstrate high sensitivity and a wide range of applications, some interference problems have to be dealt with prior to realtime analysis. The major problems stem from the nonselectivity of the pyridylazo group, which reacts, with a multitude of metals yielding intensely colored species. Furthermore, the absorptimetric nature of those approaches prohibits their application to colored or turbid samples such as brewery or winery wastes. A probable innovative approach, would be fluorometric monitoring, which although extensively used for the determination of both vanadium and peroxides, through catalytic reactions, has not, to (13) Gao, J.; Zhang, X.; Yang, W.; Zhao, B.; Hou, J.; Kang, J. Talanta 2000, 51, 447-453. (14) Yamane, T.; Osada, Y.; Suzuki, M. Talanta 2000, 45, 583-589. (15) Paleologos, E. K.; Koupparis, M. I.; Veltsistas, P. G.; Karayannis, M. I. Anal. Chem. 2001, 73, 4428-4433. (16) Shiobara, T.; Teshima, N.; Kurihara, M.; Nakano, S.; Kawashima, T. Talanta 1999, 49, 1083-1089. (17) Ni, Y.; Jin, L. Chemom. Intell. Lab. Syst. 1999, 45, 105-111. (18) Sander, S. Anal. Chim. Acta 1999, 394, 81-89. (19) Pearson, D. The Chemical Analysis of Food, 7th ed.; Churchill-Livingstone: Edinburgh, 1976. (20) Official Methods of Analysis of the Association of Official Analytical Chemists, 16th ed.; AOAC: Gaithersburg, MD, 1998. (21) Wann, C.-C.; Jiang, S.-J. Anal. Chim. Acta 1997, 357, 211-218. (22) Wuilloud, R. G.; Salonia, J. A.; Gasquez, J. A.; Olsina, R. A.; Martinez, L. D. Anal. Chim. Acta 2000, 420, 72-79. (23) Sanchez-Vinas, M.; Bagur, G. M.; Gasquez, D.; Camino, M.; Romero, R. J. Anal. Toxicol. 1999, 23, 108-112. (24) Adachi, A.; Ogawa, K.; Tsushi, Y.; Nagao, N.; Kobayashi, T. Water Res. 1997, 31, 1247-1250. (25) Ekinci, C.; Ko ¨klu ¨ , U. Spectrochim. Acta Part B 2000, 55, 1491-1495. (26) Orhanovic, M.; Wilkins, R. G. J. Am. Chem. Soc. 1967, 89, 278-282. (27) He, X.; Tubino, M.; Rossi, A. V. Anal. Chim. Acta 1999, 389, 275-280. (28) Zucchi, C.; Forneris, M.; Martinez, L.; Olsina, R.; Marchevsky, E. Fressenius J. Anal. Chem. 1998, 360, 128-130. (29) Oszwaldowski, S.; Lipka, R.; Jarosz, M. Anal. Chim. Acta 2000, 421, 3543.
Figure 1. FIA manifold employed for the measurements. For vanadium measurements: C, carrier, PTQA 2 × 10-4 M in 0.15 M H2SO4; R, reagent, H2O2 0.1 M in 0.15 M H2SO4. For peroxide measurements: C, carrier, PTQA 2 × 10-4 M in 0.15 M H2SO4; R, reagent, vanadium 50 mg L-1 in 0.15 M H2SO4.
the best of our knowledge, ever been targeted in the investigation of such a ternary complex. The aim of this work was to develop a simple, cost-effective system for the determination of vanadium in food and biological matrixes, based on its effect on the 2-R-pyridylthioquinaldinamide (PTQA)-H2O2 system and fluorometric monitoring, linked to a FIA manifold. PTQA is a well-known fluorogenic reagent, which has been reported to yield fluorescent products with several metals and strong oxidants, under strong acidic conditions (1-2 M sulfuric acid).30-34 Under mild, acidic conditions, though, no such action is observed and organometallic interactions is a prosperous area of investigation. As it appears, vanadium is the most reactive species allowing its determination at the low-microgram per liter level, while other metallic ions do not interfere even in 100:1 excess. The only interference that exceeds the tolerance limits arises from copper, due to its abundance in real samples, but proper masking can alleviate it. In a parallel alternative approach, the determination of H2O2 and peroxy acids was advanced in this work, as preliminary experiments revealed similar behavior of the system when either vanadium or peroxides were maintained at reasonable excess over one another. EXPERIMENTAL SECTION The FIA manifold for the analysis was constructed of Teflon and PVC tubing of various diameters (Figure 1). The system consisted of a four-way pneumatically actuated injection valve (type 50, Teflon; Rheodyne, Cotati, CA), an eight-channel peristaltic pump (Ismatec, Glattburg-Zurich, Switzerland), and a spectrofluorometer (RF-551; Shimadzu, Kyoto, Japan), equipped with a 12-µL flow-through measurement cell. Data collection and processing were performed by an IBMcompatible personal computer as described in previous publications.33,34 A GBC-2000 (GBC Ltd., Victoria, Australia) atomic absorption spectrometer, equipped with a graphite furnace, was (30) Ahmed, M. J.; Chakraborti, A. K. Chem. Environ. Res. 1992, 1(4), 397403. (31) Pal, B. K.; Chakraborti, A. K.; Ahmed, M. J. Mikrochim. Acta 1989, I, 393397. (32) Pal, B. K.; Chakraborti, A. K.; Ahmed, M. J. Anal. Chim. Acta 1988, 206, 345-349. (33) Ahmed, M. J.; Stalikas, C. D.; Veltsistas, P. G.; Tzouwara-Karayanni, S. M.; Karayannis, M. I. Analyst 1997, 129, 221-226. (34) Paleologos, E. K.; Lafis, S. I.; Tzouwara-Karayanni, S. M.; Karayannis, M. I. Analyst 1998, 123, 1005-1009.
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Table 1. Temperature Program and Modifier Used for GF-AAS Measurementsa program step
furnace temp (°C)
ramp time (s)
hold time (s)
nitrogen flow rate (mL min-1)
signal output
1 2 3 4 5 6 7
85 95 120 500 1100 2600 2600
5 30 20 5 5 1.2 0
5 10 20 10 12.5 4 3
300 300 300 300 300 0 300
no no no no no yes no
a Modifier used: 2.5% citric acid + 0.5% Mg(NO ) + 1.25% 3 2 (NH4)2HPO4 + 0.5% Pd(NO3)2.
used to compare the results, obtained by the proposed method. Table 1 shows the graphite furnace parameters and the program adopted for comparison analysis of the results. Reagents. All chemicals were of analytical reagent grade, free from vanadium traces. Distilled water and analytical grade ethanol, which is nonfluorescent, under UV radiation, were used throughout. A 0.5-L volume of 1 M H2SO4 working solution, was prepared by diluting 27.8 mL of concentrated H2SO4 (95-97%) (Merck) with distilled water. Standard stock solutions of vanadium(IV) and vanadium(V) were prepared by dissolving appropriate amounts of VOSO4‚5H2O and NH4VO3 (Merck) in double-distilled water. Working standard solutions in the range of 5.0-50.0 µg L-1 were daily prepared with appropriate dilution in 0.15 M sulfuric acid. A standard solution of 1 M hydrogen peroxide was prepared by diluting the concentrated solution (Riedel de Hae¨n). The solution was kept in the refrigerator. Working solutions of 0.1 M were prepared daily in a 0.15 M sulfuric acid medium. For the determination of hydrogen peroxide and peroxides, standard solutions were prepared by appropriate dilutions of the concentrated standard. tert-Butyl hydroperoxide (Aldrich) was selected as the representative organic peroxide due to its high reactivity. Triton X-100 was used, without further purification, to prepare a 10% w/v aqueous solution. The solutions of various cations and anions used for the interference study were obtained from the respective inorganic salts (Aldrich) with proper dilution in distilled water. PTQA Working Solution. The synthesis and purification procedure of PTQA has been described in detail in previous publications.33,34 Reagent stock solutions (10-2 M) were prepared by dissolving the required amount (266 mg in 100 mL) in ethanol. Working standard solutions (2 × 10-4 M) were prepared daily by appropriate dilution. Safety Precautions. Sulfuric acid, both concentrated and diluted, is extremely corrosive, while hydrogen peroxide and especially organic peroxides are flammable, corrosive, and an irritant upon contact with skin or even inhalation. Preparation of stock and working solutions must proceed with extreme caution in a ventilated area wearing proper clothing (gloves, glasses, etc.) Procedure. Standards (5-100 µg L-1 V) and sample solutions were injected into the carrier stream by means of the peristaltic pump. Reagent solution was added right after the valve, and the reaction mixture was conveyed to the measuring cell where the fluorescent intensity, proportional to vanadium concentration, was measured at 490 nm (excitation 340 nm). The reaction is fast 102 Analytical Chemistry, Vol. 74, No. 1, January 1, 2002
Table 2. Optimal FIA Parameters Selected for the Determination of Vanadium parameter size of sample loop (µL) overall flow rate (mL min-1) reagent flow rate (mL min-1) temperature (°C) concentration of reagents H2SO4, (M) 2-(R-pyridyl)thioquinaldinamide (M) H2O2, (M) TX-100, % w/v
studied range
selected value
20-500 0.5-5.0 0.25-2.5 20-70
100 2.5 1.5 25.0
0.01-2.50 10-5-10-3
0.15 2 × 10-4
10-4-2.0 0.01-1.0
0.1 0.05
enough, and the fluorescence intensity remains stable for at least 24 h. The concentration of vanadium was determined from the peak height of the signal, which was measured automatically and stored in the computer memory. A calibration graph was constructed using standard solutions. RESULTS AND DISCUSSION Optimization of the Flow Injection System. Preliminary tests were conducted using different flow assemblies in order to select the optimum manifold configuration. The configuration shown in Figure 1 was selected since it resulted in the best mixing and reaction conditions, while producing the optimal peaks with regard to height, shape, and reproducibility. To optimize the above selected manifold, the effect of hydrodynamic and chemical parameters on the peak variables were studied. The univariate method was adopted for the optimization of the system. Table 2 shows the results of optimization for 25 µg L-1 vanadium concentration. As for the instrumental adjustments of the method, a sample volume of 100 µl, an overall flow rate of 2.5 mL min-1, reagent and carrier flow rates of 1.25 mL min-1, a residence time of ∼45 s, and normal, room temperature (20-25 °C) were selected for subsequent experiments. With regard to the reaction, the following parameters influenced the analytical performance of the proposed method and, therefore, they were thoroughly investigated and optimized: acidity, solvents, reagent concentration, H2O2 concentration, and the presence of a surface-active agent. Based on the results of previous studies,31-34 it was expected that the reaction system would perform better in an acidic environment. Of the various mineral acids studied (sulfuric, nitric, hydrochloric), sulfuric acid was found to be the most suitable, as it was anticipated, in accordance with previous reports using PTQA.33,34 Different concentrations of sulfuric acid were tested, and it was found that the reaction is promoted at a range of 0.10.2 M H2SO4. More acidic conditions favor the redox reaction, as H2O2 becomes a powerful oxidant (Figure 2.) At 1-2 M H2SO4 concentrations, it has been reported to act as a significant interference to the determination of manganese, selenium, and chromium with PTQA.32-34 The H2O2 concentration was kept at 0.1 M as at lower values the reaction was too slow to meet with the FIA requirements while at larger quantities it promoted the redox reaction to a greater extent than the complex formation, thus increasing the background signal.
Figure 2. Effect of H2SO4 concentration on the fluorescence signal of 25 µg L-1 vanadium. [PTQA] ) 2 × 10-4 M; [H2O2] ) 0.1 M. (O f with vanadium; b f without vanadium).
As PTQA shows poor solvability in a purely aqueous phase, different solvents and mixtures were examined. Among methanol, ethanol, 2-propanol, and butanol, which showed fluorescence, the signal increased in the following order: methanol, ethanol decreasing again in 2-propanol and butanol. Aqueous solutions of ethanol were tested, to obtain the optimum performance. A 20: 80 ethanol/water solution was selected for exhibiting maximum and constant fluorescence. To enhance solubility, addition of a surface-active agent was attempted. The following surfactants were examined: the nonionic Triton X-114 and Triton X-100, the cationic cetyltrimethylammonium bromide (CTAB), and the anionic sodium dodecyl sulfate (SDS). The choice of these surfactants was based on their commercial availability and frequent use in the literature.35,36 It came as no surprise that the presence of surfactant not only increased miscibility but it also enhanced the fluorescent signal by a factor of 2.5, thus improving the sensitivity of the method. As expected, the surfactant assemblies acted as light-harvesting units focusing much of the exciting radiation on the fluorescent system.37,38 A 0.05% w/v solution of Triton X-100 was selected to produce the optimum results. The reaction leading to the fluorescent product becomes very fast in the presence of vanadium. Constant and maximum fluorescence is attained in less than 1 min and remains stable for 24 h. Different concentrations of PTQA solutions were tested in the ranges shown in Table 2. A reagent concentration of 2 × 10-4 M was selected as the optimum value. Evaluation of the Method. The reproducibility of the procedure and sample throughput was evaluated by repeated injections of a 25 µg L-1 standard vanadium solution. The relative standard deviation was found to be 0.1-1.8% (n ) 5) with decreasing concentration of vanadium. This indicates that the method is precise and reproducible over a wide range of concentration and can be safely applied even at the microgram per liter level. (35) Sanz-Medel, A.; Fernandez de la Campa, M. R.; Gonzalez, E. B.; FernandezSanchez, M. L. Spectrochim. Acta, Part B 1999, 54, 251-287. (36) Lienado, R.; Neubecker, T. A. Anal. Chem. 1983, 55, 93R-102R. (37) Non Ionic Surfactants; Schick, M. J., Ed.; Marcel Dekker: New York, 1987. (38) Solution Chemistry of Surfactants; Mittal, K. L., Ed.; Plenum: New York, 1979.
A linear working curve in the range of 2-100 µg L-1 vanadium was obtained by measuring the peak height. The respective regression equation was y ) 5.30 +1.47 (V, µg L-1) with a correlation coefficient r ) 0.9995. The detection limit, defined as 3 times the signal-to-noise ratio (LOD ) 3σ/a) was 0.5 µg L-1 l. The sample throughput was 45 measurements/h provided that autosampling is available. The differentiation between V(IV) and V(V) was also examined. It was found that both oxidation states exhibit identical activity. This observation can be explained by the fact that V(IV) is readily oxidized to V(V) under acidic conditions forming mono- or diperoxovanadate complexes.39,27 Mechanistic Studies. The vanadium-PTQA complex was synthesized by mixing excess of PTQA with ammonium metavanadate in ethanol. The mixture was gently heated at 50 °C and refluxed overnight. The obtained light blue-green compound was recrystallized from hot water, and its IR spectrum was obtained. Comparison of the spectrum with that of pure PTQA revealed significant interaction of the PTQA donor groups with vanadium. Vanadium’s coordination in the sphere of PTQA is evidenced by the appearance of a sharp peak (995 cm-1) indicating the presence of VdO bonds. Furthermore, translocation of characteristic peaks (1550-1650 cm-1) to greater wavenumbers and alleviation of several peaks in medium wavenumbers (1200-1350 and 14001550 cm-1) suggest rigidity of the system stemming from the direct complexation of sulfur and nitrogen groups. This rigidity combined with the extended resonance of the induced double bonds is probably the cause for the fluorescence activity. This observation is further supported by the enhanced sensitivity of the system after incorporation of peroxy acids, which introduce another carbonyl group, thus expanding the resonance. Further support to the suggestion for complex formation rather than redox reaction is given by cyclic voltammetry (CV), which, under the working conditions, revealed no oxidation peaks. Intereferences. The interference of several anions and cations that are abundant in food and biological samples and can influence the performance of the system was studied by using a solution containing 25 µg L-1 vanadium and adding various concentrations of the potential interferences. The criterion upon which a substance could be characterized as an interference was a 5% impact on the fluorescence response, compared to that obtained with the vanadium solution. The results of Table 3 reveal that the only food constituent abundant enough to interfere with the determination of vanadium is copper andsto a´ smaller degrees iron. These interferences can easily be alleviated by adding citrate or EDTA to the sample before its injection to the FIA stream. Applications. The proposed method was applied to the determination of total vanadium in various vanadium-rich food matrixes, to a food supplement available in the Greek market with known vanadium content, and to nails and hair that are the body parts that accumulate vanadium. (a) Procedure for the Determination of Vanadium in Real Samples. Foods that are high in vanadium concentrations are seafood and vegetable oils with a vanadium content up to 50 mg L-1, cereals up to 6 mg L-1, and nuts up to 2 mg L-1.5,13,40,41 The (39) Brooks, H. B.; Sicilio, F. Inorg. Chem. 1971, 10, 2530-2534. (40) Sperling, K. R.; Bahr, B.; Ott, J. Fresenius J. Anal. Chem. 2000, 366, 132136. (41) Seiler, H. G.; Sigel, A.; Sigel, H. Metals in Clinical and Analytical Chemistry; Marcel Dekker: New York, 1994.
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Table 3. Effect of Interfering Ions on the Determination of 25 µg L-1 Vanadium
interfering ion EDTA, nitrate, citrate, tartrate chloride, phosphate, oxalate sodium, potassium, ammonium, zinc, calcium, magnesium platinum, palladium, iridium, bismuth, cadmium, chromium(III) chromium(VI), arsenic, cobalt, nickel, mercury, arsenic tin, lead, molybdenum ironb copperb
maximum permissible ratioa (CI/CV) 20000 10000 2000 1000 500 250 50 5
a A 5% criterion is adopted. b Removable interference with EDTA, or citrate. CI, concentration of the interfering agent;, CV, concentration of vanadium.
Table 4. Analysis of Real Samples and Comparison with GF-AAS V found, µg g-1 sample
proposed method
GF-AAS
flour (whole grain) walnuts almonds hazelnuts sun oil olive oil corn oil fried oil tuna (canned) mackerel (canned) nails hair tablet
2.25 0.58T 0.42 0.75 12.3 14.2 11.1 24.8 5.9 7.3 0.007 0.005 0.020
2.20 0.60 0.45 0.77 12.3 14.2 11.1 25.3 5.7 7.3 0.007 0.005 0.020
Table 5. Recovery Studies of Spiked Samples
only commercial food supplement with vanadium readily available in the Greek market is Centrum. Analysis of those samples proceeds as described below. Ten grams of each food sample, 1 g each of nail and hair, and one tablet of the supplement were thoroughly homogenized with a hand mill. Then they were acid digested, and the remaining was diluted to 100 mL with a 0.15 M sulfuric acid solution, containing sodium citrate 10 mg L-1. The sample was filtered with a Whatman No. 40 filter, to remove any substance that could not be dissolved and injected to the FIA stream. Hair and nail samples were dissolved in 10 mL after digestion to ensure that their concentrations are within detection limits. Whenever the measured signal exceeded that of the linear working range, the sample was further diluted until reaching measurable values. Fish samples (canned) were dried from oils and heated to dryness by gentle heating in a furnace before weighing so that the outcome would refer to dry weight. For comparison of the results, the samples were analyzed with a GF-AAS program, operating at 314.8 nm with a lamp current of 20 mA. (b) Performance Characteristics in the Analysis of Real Samples. The method was applied for the determination of vanadium in several real samples and the results, along with the outcome of the comparison with a reference method (GF-AAS), are gathered in Table 4. As a part of the quality assurance/quality control (QA/QC) protocol, several spiked samples were also analyzed and the results are presented in Table 5. As can be seen, both recovery and comparison studies gave satisfactory results, which indicates that the proposed method can be reliably used for the determination of total vanadium content in a wide variety of different matrixes. Application to the Determination of Hydrogen Peroxide and Peroxides. The feasibility of the described reaction scheme for the determination of H2O2 or peroxides, by exploiting their effect on the PTQA-vanadium system, was also investigated. From the implementation of the preceding study, it was revealed that the most important parameters affecting the performance of the system was the PTQA and H2SO4 concentrations. The catalytic action of vanadium concentration on the proposed reaction was utilized for the determination of H2O2 (and organic peroxides). An optimization method based on a central composite design (CCD) procedure was chosen for the investigation of the optimum 104 Analytical Chemistry, Vol. 74, No. 1, January 1, 2002
sample distilled water tap water table water
V added (µg L-1)
V found (µg L-1)
recovery (%)
5.0 10.0 50.0 5.0 10.0 50.0 5.0 10.0 50.0
5.0 10.2 49.0 4.8 9.7 49.0 4.9 9.5 48.0
100 102 98 96 97 98 98 95 96
experimental conditions for this application. The experimental design procedure was selected as means of optimization of the experimental conditions, because it offers distinct advantages over the univariate optimization approach, the most important being that it allows for the identification of interactions between variables.42,43 The CCD chosen in this study allows for all three selected variables to be investigated individually as squared terms and considers two-component interaction effects. The results of the CCD were evaluated by multivariate regression analysis using the STATISTICA 5.0 automated algorithm. Each variable was assigned limits that depended upon their effect on the PTQA-vanadium fluorescence as resulting from the implementation of the preceding study. The assigned limits as coded levels -2, -1, 0, +1, +2 are given in Table 6. The corresponding design matrix was then constructed for a H2O2 concentration of 5 µM. The multiple regression and ANOVA results are gathered in Table 7. At the 95% confidence level, certain variables are statistically significant while for others a weaker significance is observed. The quadratic term of vanadium concentration was not significant while the lack of significance of the cross-product terms of sulfuric acidvanadium and vanadium-PTQA suggests nonsignificant interactions between these variables at the zone studied. From the available data, a reduced model was set forth that takes into account only the significant contributions. The intercept is the response at zero coded levels of the parameters. As illustrated in Table 7, the main effect (coefficient) of sulfuric acid (42) Morgan, E. Chemometrics: Experimental Design; Wiley: Chichester, U.K., 1991. (43) Otto, M. Chemometrics: Experimental Design; Wiley: Weinheim, Germany, 1999.
Figure 3. Response surfaces of the multivariate optimization procedure for the determination of H2O2 and peroxides: fluorescence signal as a function of (a) H2SO4 and PTQA concentration, (b) H2SO4 and vanadium concentration, and (c) vanadium and PTQA concentration. Table 6. Coded Levels of the Central Composite Design coded levels -2 -1 0 +1 +2
[H2SO4] (M)
[PTQA] (M)
10-2
10-5
5 × 10-2 10-1 5 × 10-1 1
5 × 10-5 10-4 2 × 10-4 5 × 10-4
Table 7. Summary of Multiple Regression and ANOVA Results for Peroxide Fluorescence Signala
[vanadium] (mg L-1) 5 10 25 50 80
has a negative sign, which indicates that the fluorescence signal is deteriorated as the H2SO4 concentration increases. This behavior can be attributed to the enhancement of the redox activity of H2O2 under strong acidic conditions, as previously discussed. On the other hand, the main effects of vanadium and PTQA concentration have a positive sign, which signifies the fact that higher fluorescence is emitted by respective increase in their concentrations. The fluorescence signal as a function of the two studied variables was investigated by keeping the rest of the variables constant at the medium level (0 coded level) of the experimental domain. The 3-D response surfaces are depicted in Figure 3. As can be seen, optimum experimental conditions correspond to the
term
coefficient
intercept H2SO4 vanadium PTQA H2SO4‚PTQA H2SO4‚H2SO4 PTQA‚PTQA
140 -25.94 16.81 19.68 -19.37 -19.15 -29.15
regression linear square residual total a
sum of squares
46946.7 4241.8
df
p-value
1 1 1 1 1 1 1
0.00051 0.0085 0.0033 0.024 0.0012 4.87 × 10-5
6 3 3 10 16
R ) 0.957; R2 ) 0.917.
following uncoded values: [H2SO4] ) 0.85 × 10-1 M, [PTQA] ) 2 × 10-4 M, and [vanadium] ) 50 mg L-1. The highest signal is obtained when [H2SO4] is maintained at low levels (-1 uncoded value and below) and PTQA and vanadium concentration at high levels (1 uncoded values and above). However, no improvement is obtained when either H2SO4 is further reduced down to the Analytical Chemistry, Vol. 74, No. 1, January 1, 2002
105
Table 8. Results from the Analysis of Real and Spiked Samples sample
brewery wastewater
measured (µM)*
added (µM)
a
recovery (%)
peroxides
H2O2
peroxides
H2O2
peroxides
H2O2
peroxides
nda
nda
0.80
0.50
0.76
0.47
95
96
0.80 0
0 0.50
0.81 0
0 0.51
101
duration of cleaning activity (h) contact lenses cleaning fluid
found (µM)
H2O2
102
H2O2 (spiked) (µM)
H2O2 (found) (µM)b
recovery, %
0 0 0 0 0.50 1.20
8.8 × 2.8 × 104 27 0.90 0.50 1.25
100 104
0 1 3 4 8 8
105
nd, below the detection limit. b Average value of three measurements.
minimum value of the CCD (-2 uncoded value) or PTQA or vanadium is increased up to the value assigned as the maximum in the CCD (+2 uncoded value) (Figure 3b and c, respectively). The PTQA fluorescence signal pattern exhibits a decrease at both lower and higher values from the optimum, possibly due to insufficient reagent or autoquenching behavior of the excess quantity of PTQA at high concentrations, respectively (Figure 3a and c). Furthermore, fluctuations of vanadium concentration have the same effect on the PTQA fluorescence signal pattern at any vanadium concentration (Figure 3c). The optimum experimental conditions found above were employed for obtaining the calibration curves for either H2O2 or peroxides. In the case of H2O2, the calibration curve was rectilinear in the range of 0.2-50 µM, according to the expression, y ) 120.3 [H2O2, µM] +30.0. The correlation coefficient was r ) 0.998 and the detection limit 0.05 µM. For peroxides, the equation y )152.7 [C, µM] - 7.5 (r ) 0.999) corresponded to a linear range of 0.1-2 µM, yielding a detection limit of 0.03 µM. The relative standard deviation (RSD) for H2O2 (n) 3, C ) 1 µM) was 2.4% and for peroxides (n ) 3, C ) 0.5 µM), 2%. The interfering effects of several cations and anions on the determination of H2O2 and peroxides was also assessed by means of spiking known amounts of the potential interferences in standard solutions of the target analytes. The species examined were identical to those reported previously in the interfering study of vanadium. Neither cations nor anions present in solution were found to pose any adverse effects on the analytical signal since the abundance of vanadium can compensate for any side reaction(s). The analytical applicability, accuracy, precision, and potential interference effects of the method were assessed by monitoring the H2O2 content of a contact lens cleaning fluid during an overnight cleaning process. Also, recovery experiments were
106 Analytical Chemistry, Vol. 74, No. 1, January 1, 2002
performed, by spiking the same fluid and brewery wastewater with H2O2 and peroxides. The samples were directly analyzed, after appropriate dilutions have been made, without any other pretreatment. When organic peroxides were determined, H2O2 was removed by filtration of the sample through manganese oxide. As given in Table 8, the concentrations of H2O2 and peroxides obtained with the proposed method were in good agreement with the expected values. CONCLUSIONS A novel FI spectrofluorometric procedure is proposed for the determination of total vanadium in food and biological samples. The method is based on the formation of a novel ternary complex of vanadium with PTQA and peroxides, under mild acidic conditions. The sensitivity of the method was enhanced by the use of a mixed ethanolic-aquatic media and the presence of a surfaceactive agent allowing for determination of vanadium at the milligram per liter level. The method was successfully applied to the analysis of various samples after eliminating the interference imposed by copper with proper masking. In a different function, the effect of H2O2 or peroxides on the V-PTQA system was found to be an efficient method for the determination of these compounds at the sub-micromolar levels even in complex matrixes. The proposed method generates the possibility of a simultaneous evaluation of vanadium and peroxy compounds through kinetic stopped-flow measurements, while offering a unique opportunity for pre- or postcolumn chromatographic separation of peroxides with selective noncatalytic fluorometric detection. Contemporary research is oriented toward these possibilities. Received for review July 17, 2001. Accepted August 31, 2001. AC0108008
