Anal. Chem. 1984, 56,1050-1052
1050
Table IV. Mean 90% Rise Times in Two-Phase Flow System Using Bacterial BL [NADH], M 1x
5x 1 x 10-5
mean 90% rise time, min
M
mean 90% rise time, min
1.5 1.9 2.0
1 x 10-4
2.1 2.6
[NADH], 5
x
Although steady-state intensities were quite stable (as illustrated in Figure 6), a gradual decrease in intensity was observed with successive measurements. Because the rate of the decrease was greater a t the higher level of apyrase, we believe that the apyrase preparation used had some protease activity which degraded luciferase. Thermal and mechanical denaturation of the enzyme may also occur. Although the luciferase reagent is stable at temperatures up to 30 OC, it may get warmer than this in the cell upon extended use.
NADH Measurement Based on Bacterial Luciferase. The measurement of NADH based on bacterial bioluminescence is an attractive method to adapt to the flow cell because NADH is an important analyte and the method does not involve expensive dialyzable substrates. The reagent phase was a stabilized commercial preparation, LUMASE, containing both bacterial luciferase and NADH-FMN oxidoreductase. It was reconstituted with 3.0 mL of 0.1 M phosphate buffer, pH 6.9, containing 4.6 pg/mL FMN and 0.017% (v/v) decanal. At first, response to NADH decreased rapidly with time. It was found that stable response could be achieved by employing separate flow lines for 16 mg/L FMN in 0.1 M phosphate buffer, 0.068% v/v decanal in 0.1 M phosphate buffer, and blank/NADH. The decanal solution had to be stirred to keep decanal in suspension, and the FMN solution was protected from light.
The signal for NADH resembles that for ATP. An interesting and unexplained feature of the response is that the response time is concentration dependent. These data are summarized in Table IV. Response is linear with NADH concentration. Relative standard deviations for replicate measurements are typically 5%. The detection limit is only slightly below 1 X lo4 M NADH. Response is constant for 3 to 4 h. It then decreases steadily with time. We attribute this to thermal denaturation of the enzymes since with extended use the solution in the reagent phase gets significantly warmed up. Accordingly, it should be possible to deal with this problem by designing the cell to allow for cooling of the reagent phase.
ACKNOWLEDGMENT The authors thank Lumac, Inc., for providing reagents and the UNH Machine Shop for their help in making the twophase cell.
LITERATURE CITED (1) Lee, Y.; Jabionski, E.; DeLuca, M. Anal. Blochem. 1977, 80, 496. (2) Jabionski, E.; DeLuca, M. Clln Chem. ( Winston-Salem, N .C,) 1979, 25, 1622. (3) Merenyi, q.; Wettermark, G.; Wiadimiroff, W. In “Proceedings of the International Symposium on Analytical Application of Bioluminescence and Chemiluminescence, Stanley, P. E., Schram, E., Eds.; State Printing and Publishing: Westlake Village, CA, 1979; p 272. (4) Freeman, T. M.; Seitz, W. R. Anal. Chem. 1981, 53, 90. (5) Pliosof, D.; Nieman, T. A. Anal. Chem. 1982, 54, 1696. (8) Seitz, W. R. CRC Crlf. Rev. Anal. Chem. 1981, 13, 1. (7) DeLuca, M. A., Ed. “Bioluminescence and Chemiiumlnescence, Methods of Enzymology”; Academic Press: New York, 1978; Vol. 57. (8) Gorus, F.; Schram, E. Clin. Chem. (Winsfon-Salem, N . C . ) 1979, 25. 512. (9) Freeman, T. M.; Seitz, W. R. Anal. Chem. 1978, 50, 1242. (IO) Cormier, M. J.; Prichard, P. M. J. Biol. Chem. 1968, 243, 4706
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RECEIVED for review December 7, 1983. Accepted January 23, 1984. Partial support for this research was provided by NSF CHE 7825192.
Determination of Aluminum, Lead, and Vanadium in North Atlantic Seawater after Coprecipitatlon with Ferric Hydroxide Clifford P. Weisel,’ Robert A. Duce, and James L. Fasching* Graduate School of Oceanography and Department of Chemistry, Center for Atmospheric Chemistry Studies, University of Rhode Island, Kingston, Rhode Island 02881 Analyses of trace metals in seawater during the last decade have demonstrated the importance of avoiding contamination and the utility of being able to measure several metals in a single sample. Correlations between the concentrations of nutrients and several trace metals in seawater have been shown to exist and trace metal concentrations in seawater can be influenced by aeolian transport and ocean floor/seawater interactions (1-4). Being able to compare the distribution of one element with an unknown source or controlling mechanism in the water column to elements whose concentrations are controlled by known processes can lead to a better understanding of the cycling of elements to and within seawater. It was proposed over 20 years ago f5,6) that coprecipitation with ferric or aluminum hydroxide could be used to separate As, Co, Cr, Mo, Mn, Ni, W, and V from seawater for subsequent chemical analysis, but due to the analytical techniques Present address: National Oceanic and Atmospheric Administration, Atlantic Oceanographic and Meteorological Laboratory, Ocean Chemistry and Division, 4301 Rickenbacker Causeway, Miami, FL 33149.
available a t that time, large quantities of seawater were required. Adaptations of ferric hydroxide coprecipitation were used in the mid 1960s (7,8) iri an attempt to determine Mn, V, and Zn in seawater. More recently, Nakashima (9, 10) has modified the coprecipitation technique with fe$c hydroxide by using bubble flotation to collect the precipitate followed by analysis for As and Se. These procedures used in excess of 1L of seawater or required several manipulations of the sample, therefore decreasing the procedures’ utility as a routine technique and increasing the possibility of contamination. Recent advances in instrumentation technology for atomic absorption spectrometry using the heated graphite furnace now make it possible to analyze for many trace metals after concentration from small volumes (50-200 mL) of seawater when the major ions are eliminated. Coprecipitation with ferric hydroxide requires few reagents and minimal sample manipulation, suggesting its use as a routine preconcentration procedure for the analysis of seawater. We describe the separation of Al, Pb, and V from small volumes of seawater
0 1984 American Chemical Society 0003-2700/84/0356-1050$01.50/0
ANALYTICAL CHEMISTRY, VOL. 56, NO. 6, MAY 1984
by this technique followed by their analysis by neutron activation or atomic absorption spectrometry. EXPERIMENTAL SECTION Reagents. All water used for standards and solutions was distilled, deionized water (D.D. HzO) with an electrical resistance >17 MR. The ferric ion solutions were prepared from Alfa Puratonic ferric nitrate and were centrifuged to remove any insoluble particles. The liquid was then transferred to an acid-cleaned polyethylene vial until needed. Nitric acid was purified from reagent grade acid in a quartz still. Ammonium hydroxide was purified from reagent grade base by placing an opened, acidcleaned Teflon beaker containing D.D. HzO into a plastic basin containing the base. A second basin, with an inner plastic lid to prevent any condeming base from dripping into the Teflon beaker, was used as a cover. The two basins were held together with a piece of slitted Tygon tubing which fit snuggly over both rims forming an essentially airtight seal. The ammonia vapor was allowed to equilibrate with the D.D. HzO in the Teflon beaker and the reagent grade base was then replaced. This procedure yielded base with pH values in excess of 11. Trace metal standards were prepared from the chloride salts of the metals. These standards were verified for accuracy by comparison with solutions prepared from commercially available standards and with EPA trace metal reference standards (No. 571) in aqueous solutions. All polyethylene bottles and vials were leached either in 4 N nitric acid at 55 "C for at least 4 days and then in 2% nitric acid at 55 "C for an additional 4 days or in 8 N nitric acid at room temperature for a t least 7 days and then in a mixture of 10% nitric and hydrochloric acid for a second week. The bottles were rinsed well with D.D. H20 and the seawater sample to be analyzed prior to being used. The 0.4-pm pore size Nuclepore filters were cleaned by sonicating them for 12 h in a solution of 5% nitric and 5% hydrofluoric acids. The filters were then washed with D.D. HzO and stored in fresh 2% nitric acid. Analytical Procedure. Two hundred milliliters of unfiltered seawater, acidified upon collection with nitric acid to pH 2, was transferred to a 250-mL, acid-cleaned,conventional polyethylene bottle. Trace metal spikes were added to some samples for coprecipitation efficiency studies. One hundred microliters of the -2 M ferric nitrate solution was added to each sample, and the bottles were shaken. The pH was then adjusted with ammonium hydroxide, and the bottles were reshaken for approximately 30 s. After a minimum of 30 min, during which the precipitate formed completely, the seawater was filtered through an acidwashed Nuclepore (0.4 pm, 47 mm) fdter placed in an acid-cleaned polycarbonate filter holder. This procedure concentrated the dissolved species of the metals which formed insoluble hydroxides and particulate trace metals trapped on the 0.4-pm Nuclepore filter. The filter holder was rinsed with dilute nitric acid and D.D. HzO between samples. The precipitate was washed twice with 25-mL aliquots of D.D. HzO, which was at the same pH as the samples. Problems due to ionic strength were not observed. This removed any sea salt adhering to the precipitate or filter. The filter containing the precipitate was then placed in an acid-cleaned 2-dram polyethylene vial and 2 mL of a 10% nitric acid solution was added. The vial was agitated for 5 min until the entire filter was wetted by the acid. The precipitate was assumed to be completely dissolved when the red color of the Fe(OH)3had disappeared. For fresh water the precipitate can be dissolved by the addition of concentrated acid, which then is diluted. After dissolution of the precipitate, the filter was removed and the solution split into two portions, one for analysis by flameless atomic absorption spectrometry (FAAS) using the heated graphite furnace (HGA) and the second for analysis by instrumental neutron activation analysis (INAA). The above procedures were performed in a Class 100 clean air room while wearing particle-free polyethylene gloves. An estimate of the inherent blank level was obtained by examining the analytical procedure used. Various aspects included the nitric acid solution used for dissolution of the precipitate, an acid leached from the filter, the ferric nitrate solution, and a total processing blank, obtained by placing a filter in the holder followed by the usual wash and treatment as if it contained a precipitate, The largest source of metals was the filter leach with a secondary contribution from the ferric nitrate solution. There were large
Table I. Efficiency of Coprecipitation of A1 and V with pHa % efficiency at the following pH 5.5 6.0 6.5 7.0 7.5 4.5 5.0 85 95 A1 95 100 100 100 100 v 95 95 100 100 100 100 100 a Values are i 10%.
1051
>10
