Anal. Chem. 1994,66, 4105-41 11
Fluorometric Determination of AI in Seawater by Flow Injection Analysis with In-line Preconcentration Joseph A. Reslng and C. I. Measures' Department of Oceanography, School of Ocean and Earth Science and Technology, University of Hawaii at Manoa, 1000 Pope Road, Honolulu, Hawaii 96822
A highly sensitive method for the shipboard determination of A1 in seawater by flow injection analysis (FIA) has been developed. The method employs in-line preconcentrationof A1 onto a column of resin-immobilized8-hydroxyquinoline. The AI is subsequently eluted into the FIA system from the resin with acidified seawater. The eluted AI reacts with lumogallion to form a chelate, which is detected by its fluorescence. The fluorescence is enhanced -5-fold by the addition of a micelleforming detergent, Brij-35. The method has a detection limit of -0.15 nM and a precision of 1.7%at 2.4 nM. The method has a cycle time of -3 min and can be readily automated. The speed, ease of use, and relative freedom from contamination artifacts makes this method ideal for shipboard determination of AI in seawater. In oceanography, as in many other fields, the rate at which the fundamental understanding of the field advances is closely linked to the rate at which the analytical tools used to probe the system develop. In chemical oceanography, the distributions of reactive trace elements are particularly useful since their short residence times confine them to their input regions, making them valuable tracers of oceanic input processes. The distribution of A1 in the surface waters of the oceans is particularly informative in this respect. Despite being the third most abundant element in the Earth's crust (8.2%), oceanic A1 concentrations are extremely low. Thus, its surface water distribution can be used to identify the location and magnitude of inputs of continentally derived dusts to the ocean. To map these distributions on a large scale requires the development of analytical methodology that is sensitive, precise, rapid, and operable under the adverse conditions prevalent on board research vessels. We report here a method for the shipboard determination of A1 that meets these requirements and represents a significant advance over the methods currently used by ourselves and others in this field. While many techniques have been developed' for the determination of aluminum at the concentration levels prevalent in freshwaters, only three techniques have been successfully applied to the determination of A1 at the nanomolar M) levels found in seawater. The fluorometric lumogallion method of Hydes and Liss2had an original detection limit of 1.9 nM and a precision of 5% using 50-mL samples. Howard et a1.3improved the sensitivity of this method by incorporating a surfactant, Triton X- 100, to produce MacCarthy, P.; Klusman, R. W.; Cowling. S.W.; Rice, J. A. Anal. Chem. 1993, 65, 244R-292R. Hydes, D. J.; Liss, P. S . Analyst 1976, 101, 922-931. Howard, A. G.; Coxhead, A. J.; Potter, I. A.; Watt, A. P. Analyst 1986,111, 1379-1382. 0003-2700/94/0366-4105$04.50/0 0 1994 American Chemical Society
micellar-enhanced fluorescence. As a result of these and other improvements, the batch lumogallion technique currently has a reported detection limit of 0.4 nM and a precision of 3% at 10 nMe4 The batch lumogallion method has been used successfully by many researchers both at sea and in the laboratory. The A1 determination method of Measures and Edmond5 is based on the solvent extraction of the 1,lJtrifluoro-2,4-pentanedione derivative of A1 from 15-mL seawater samples and its detection by electron capture detection gas chromatography. This method has also been used successfully at sea and under recent shipboard conditions had a detection limit of 0.2 nM and a precision of 4% at 1-2 nM.6 Orians and Bruland'have achieved the lowest detection limits for A1 of -0.1 nM with a precision of 5% at 1 nM. Their shore-based analytical scheme utilizes the solvent extraction of A1 by 8-hydroxyquinoline from 250-g samples and its subsequent detection by atomic absorption spectrosCOPY.
While our previous technique using gas chromatography5 has consistently provided data of high precision and accuracy, improvements in sample throughput through automation are now limited by the availability of noncontaminating instrumentation with which to automate the solvent extraction chemistry associated with the preparation of the A1 complex. Our desire to develop an analytical scheme that is amenable to automation, yet sensitive, highly precise, and capable of rapid sample throughput with minimal sample handling, has led us to develop an FIA scheme utilizing in-line preconcentration as done by Sakamoto-Arnold and Johnsons for cobalt. We have combined the simple chemistry of the lumogallion method2 with the ease of automation of flow injection analysis and the preconcentration abilities of immobilized 8-hydroxyquinoline. We have carefully evaluated the optimal conditions for operation and have also reevaluated the work on micellarenhanced fluore~cence.~ This has resulted in a technique that permits the determination of A1 in seawater samples at subnanomolar levels in a cycle time of -3 min. Automation of the inject/load valve results in precisions of 1.7% at 2.4 nM. The minimal amount of handling involved in the flow system significantly reduces the potential for random contamination, and the simple aqueous-based chemistry obviates many of the logistical problems associated with staging oceanographic cruises out of remote ports. (4) Hydes, D. J.; Kremling, K. Continental SheljRes. 1993, 13, 1083-1101. ( 5 ) Measures, C. I.; Edmond, J. M. Anal. Chem. 1989, 61, 544-547. (6) Measures, C. I.; Edmond, J. M.; Jickells, T. D. Geochim. Cosmochim. Acta
1986, SO,
1423-1429.
(7) Orians, K. J.; Bruland, K. W . Earth Planet. Sci. Letr. 1986, 78, 397410. (8) Sakamoto-Arnold, C. M.; Johnson, K. S. Anal. Chem. 1987,S9,1789-1794.
Analytical Chemlstty, Vol. 66, No. 22, November 15, 1994 4705
-
I
Peristaltic
I
25
Carrier
06
Sample
2.5
sample wme
8-HQcolumn 8 m knitted reaction coil in heater 'A
Figure 1. FIA manifoldusedfor the preconcentration and determination of AI in seawater. The flow rates are those achieved at a pump speed of 7.5 rpm.
EXPERIMENTAL SECTION Apparatus. The flow injection manifold used for this work is depicted in Figure 1. The manifold consists of an eightchannel peristaltic pump (Rainin Rabbit), a six-port lowpressure Teflon injection valve (Hamilton Model 869 16) attached to a Thar Designs Inc. valve actuator. Pump tubing used for the reagent streams is flow-rated silicone tubing (Fisher). The remainder of the manifold is constructed from 0.062 in. outside diameter (0.d.) X 0.020 in. inside diameter (i.d.) Teflon (TFE) tubing (Upchurch Scientific). The reaction coil is constructed from 8-10 m of the same Teflon tubing that is knitted by a modification of the method of Selavka et al.9 The resultant reaction coil is -230 mm long and 3/8 in. (9.5 mm) diameter. The reaction coil is enclosed within a column heater which is fabricated by drilling a l / 2 in. (12.5 mm) diameter hole into a two-block dry bath incubator (Fisher Scientific Model No. 11-718-2). The block heater has a stated temperature accuracy of f0.5 "C. The dry bath incubator is considerably less expensive than heaters specially designed for HPLC columns. The preconcentration column is packed with 8-hydroxyquinoline (8-HQ) immobilized onto vinyl polymer gel (Supelco, TSK-Gel Toyopearl HW-65f and HW-40c) by a modificationlo to the method of Landing et a1.l The 8-HQ columns are packed as described previously.12 The detector used for these studies is a Rainin Model FL- 1 flow-through fluorescence detector with an illuminated cell volume of 3.5 pL. The excitation and emission wavelengths are set to 484 and 552 nm, respectively. The detector is run with a 5-s rise time, a lamp flash rate of 100 Hz, and a photomutilpier (PMT) voltage of 600 V. A 3-m knitted Teflon coil is attached to the exit side of the fluorometer to provide back pressure and eliminate bubble formation in the carrier stream. The effluent from the preconcentration column is passed across the pump to maintain constant back pressure in the system during valve switching. Peak detection and quantification of the results presented here were obtained using either a chart recorder, an H P 3392A integrator, or a (9) Selavka, C. M.; Jiao, K S . ; Krull, I. S . Anal. Chem. 1987, 59, 2221-2224. (10) Landing, W.M., personal communication, 1992. (11) Landing, W.M.; Haraldson, C.; Paxeus, N. Anal. Chem. 1986, 58, 30313035. (12) Resing, J. A.; Mottl, M. J. Anal. Chem. 1992, 64, 2682-2687.
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MacIntegrator I A/D and data reduction program (Rainin Instruments Inc.) running on an Apple Macintosh Model 170 (or145B) PowerBook computer. Reagents. Hydrochloric acid (6 M) and acetic acid (- 17 M) are purified by a single distillation of the reagent grade acids in a quartz finger, subboiling distillation apparatus. All water used is deionized (DW) unless noted. All plasticware is polypropylene (Nalgene) unless noted. Bottles are acidwashed by filling them with -0.5 M HCl (reagent grade), heating them at 60 "C for >18 h, in an oven, and inverting them for >18 h at room temperature. The bottles are then rinsed three times with DW. Other plasticware is cleaned by soaking in -0.5 M HCl at 60 "C for >24 h followed by three rinses with DW. Ammonium hydroxide ( 3-4 M) is prepared by isopiestic distillation of reagent grade ammonium hydroxide. Concentrated ammonium hydroxide (-2 L) is placed into the bottom of an air-tight reservoir into which are placed two 1-L bottles each containing -800 mL of DW. After 3 days, the bottles are removed and are found to be -4 M in NH40H. Lumogallion stock solution (1.7 g/L) is prepared by dissolving 50 mg of lumogallion (4-chloro-6-[(2,4-dihydroxyphenyl)azo] - 1-hydroxybenzene-2-sulfonic acid; Pfaltz & Bauer, Waterbury, CT) in 30 mL of DW in a Teflon bottle. This stock solution has been found to be stable for at least 2 months. Ammonium acetate buffer (2 M ) is prepared by adding 115 mL of 17.4 M subboiled acetic acid to 1.76 mol of NH40H. This mixture is then diluted to 1 L by adding DW and adjusted to pH 6.0 f 0.1 by further small additions of acetic acid or NH40H. Sample buffer (2M,pH5.55f 0.1) is prepared by adjusting the pH of ammonium acetate buffer (above) by the addition of acetic acid. The pH of seawater samples is then adjusted to pH 4 . 5 by the addition of 1 mL of this buffer to each 125 mL of sample. Lumogallionlbuffeerreagent is prepared by the addition of 5 mL of lumogallion stock solution to each 500 mL of 2 M ammonium acetate buffer. Brij-35 (5%) is prepared by diluting commerciallyavailable 30% Brij-35 solution (Sigma Chemical Corp). Other detergent solutions were prepared by dissolving the appropriate mass or volume of the commercially available form (Tergitol, Nonidet p-42, Triton X- 100 reduced, Triton X- 100, sodium lauryl sulfate, taurodeoxycholic acid, cetyltrimethylammonium bromide, myristylammonium chloride) in DW. Heating was used, when required, to facilitate dissolution. Clean seawater is prepared in 1-L batches by pumping seawater, whose pH was adjusted to 5.5 by the addition of 8 mL of sample buffer to 1 L of seawater, across a standard 8-HQ column at a rate of 100 mL/h. Carrier is prepared by adding 4 mL of 6 M HCl to 500 mL of clean seawater. Aluminum Standards. Stock standards (20 pmol of Al/ L) are prepared by dilution of a certified commercial standard (Fisher Chemical; 1000 ppm Al) into acidified (4 mL of 6 M HCl/L) DW and are stored in plastic bottles. Stock standards have been found to be stable for months to years under these conditions. Working standards are prepared by spiking
-
-
-
h ilr
.-
80
3.5
4
5 5.5 pH of effluent
4.5
6
6.5
Figure 2. Effect of reaction pH on response. (m) F I A system used here; pH was determined from the detector effluent and response was equal to peak height. (0)pH of reaction for Howard et €11.~
appropriate amounts of stock standard into samples of clean seawater (or any reasonably low-AI seawater) that has been buffered by the addition of 8 mL of sample buffer/L of sample. Fresh working standards are prepared each day. Recommended Procedure. The FIA manifold is set up as described above. The pump is run at 7.5 rpm and produces the flow rates depicted in Figure 1. The reaction coil heater is set to 50 OC. Sample buffer (1 mL) is added to a 125-mL sample of seawater in a wide-mouthed bottle and mixed. With the valve in the inject position, the sample inlet tube is placed into the sample bottle. The sample is immediately pumped through the inlet tubing into the column bypass, where it is pumped to waste. After 1 min, to allow rinsing of the inlet tubing, the inject/load valve is switched to the load position, causing the sample to flow through the 8-HQ column. After exactly 1 min the valve is switched back to the inject position. Within -2.5 min, the eluted AI is detected as a peak at the fluorometer and is quantified. During the A1 elution, the sample inlet tubing is placed into a new sample for rinsing of the inlet tubing prior to repeating the cycle.
-
RESULTS AND DISCUSSION The primary goal of this method development was to provide a technique that would be capable of producing analytical determinations within a few min of sample introduction. To achieve this goal, optimization efforts were focused on minimizing the reaction time and preconcentration time consistent with providing precise determination at the subnanomolar level. Optimization of the flow injection analysis scheme is divided into five separate parts: optimization of the chemistry of reaction between A1 and lumogallion, optimization of conditions for the uptake of A1 onto the 8-HQ columns, selection of detergent, optimization of detection parameters, and evaluation of interferences. Chemistry Optimization. Since the batch lumogallion method2J employs a reaction time of 1.5 h compared to our desired reaction time of 2-3 min, it was necessary to reoptimize the chemistry to obtain maximum response under these flow conditions. Reaction pH. The effect of varying the reaction pH between 4.4 and 5.9 was investigated by varying the ratio of acetic acid and ammonia in the buffer. The results (Figure 2) show that the optimal response appears to occur between
20
30
40 50 Temperature 'C
60
70
Figure 3. Effect of reaction temperature on response.
a pH of 5.0 and 5.5. This region of maximum response is much narrower than, and at the upper end of, the maximum pH response range 4.0-5.5 reported by Hydes and Liss2 and Howard et aL3 The narrower optimum range in the FIA system is a result of the combination of two factors, a slower reaction rate between AI and lumogallion at lower pH's, (see Extent of Chemical Reaction) and the shorter reaction time in the FIA system. Temperature. The sensitivity of the technique to temperature was investigated (Figure 3). It was found that increasing the temperature from 22 to 53 "C increased the response by a factor of 4. At temperatures above 53 OC the increase in response diminishes rapidly. In contrast, the batch methods2g3 used a development temperature of 80 OC. For the FIA method we adopted a lower temperature of 50 OC. This lower temperature was chosen to minimize the likelihood of Cas04 precipitation from seawater within the 0.5 mm i.d. Teflon tubing. At 50 OC most of the temperature-based reaction rate improvement has already been gained. Lumogallion. The response of the system to lumogallion concentration reaches a plateau between concentrations of -2.5 and 8 mg/L in thesystem effluent (thecombined sample and reagent mixture leaving the detector). This compares with a concentration of either 1 mg/L (2 mg/L for [All > 1 p M ) by Hydes and L i s 2 or 2.3 mg/L for Howard et aL3 The higher reagent concentration used here (3 mg/L in the system effluent) is again driven by the need to achievea greater extent of reaction during the shorter reaction time of the FIA method (see Extent of Chemical Reaction below). We found no increase in background noise over the entire concentration range of lumogallion investigated. Extent of Chemical Reaction. Hydes and Liss2 reported the half-time for the reaction in seawater between lumogallion and A1 (both natural and spiked standards) to be 22 min at room temperature. As pointed out above, the residence time of the lumogallion and A1 in the reaction coil of the FIA system is 2.5-3.0 min, implying only a small amount of reaction. Although quantitative absolute recovery is not a requirement for an analytical technique, good precision is usually associated with high yields. In addition a very low extent of reaction might lead to variability in sensitivity as external, uncontrolled parameters (e.g., room temperature) varied during long sample processing runs. In order to assess the degree of reaction, we examined the effect that changing pump speed (Le., reaction time) had on the sensitivity of the technique. Since our measurement techniques are based on Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
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1
--I 12
-5
10
.-
- 8
c u: .2 6 Y
3
u
f
4 2
0 l 0
, . . , . . . , . , . , . . . , . . , I0 2
4
6
8
10
Flgure 4. Effect of reaction time on sensitivity. Reaction time was calculated from the time of valve actuation to peak detection: (D) pH 5.46; (a)pH 4.81.
6
5
4
Time (min)
PH
Flgure 5. Effect of sample pH on column retention of AI. Table 1. Retentlon of AI vs Column Length
peak height rather than area we recognized that corollary effects which change the relationship between peak height and peak area (the true measure of the amount of reaction) could bias our results. As we expected elution from the preconcentration column to be particularly prone to such problems, we used instead a sample loop and a sample of carrier with elevated (240 nM) A1 concentrations for this study. The results (Figure 4) indicate that for reactions at pH 5.46 (the optimal pH region) we achieved -94% of our maximum reaction after 2.3 min (pump speed 7 . 5 rpm). At a lower pH (4.81), the reaction was much slower and was still incomplete after 8.3 min. We take these results to indicate that the reaction between A1 and lumogallion at the optimal pH (5.46) is essentially complete after -4 min under our conditions since there appears to be no increase in reaction beyond this time. We also conclude that the low recoveries seen below pH 5 in the FIA system are a result of the slower kinetics of the reaction at that pH. To underline this conclusion we also noted that doubling the concentration of lumogallion at pH 4.81 increased the rate of reaction substantially while a similar doubling of lumogallion concentration for the pH 5.46 reaction produced little effect. The discrepancy between our observation of a rapid reaction and the slower reaction rate reported by Hydes and L i s 2 is then simply a result of the lower pH (5.0) conditions used in their method. We interpret our results to indicate that under our FIA conditions the reaction between the A1 and lumogallion is almost quantitative. Column Optimization. The use of 8-hydroxyquinoline to complex A1 is well known and has been reviewed.13 Solvent extraction of A1 using 8-HQ from seawater was utilized by Orians and Brulanda7 Solvent extraction procedures generally allow for copious quantities of 8-HQ and extensive reaction time. In contrast, the effective concentration of 8-HQ on our columns is rather difficult to ascertain and the contact time of seawater with the resin-immobilized 8-HQ is short, on the order of seconds. In order to optimize the preconcentration process we have evaluated the effect of sample pH, column length, loading rate, and resin size on the extraction of A1 from seawater onto the 8-HQ resin. SamplepH. A1 uptake onto the 8-HQ columns was studied as a function of sample pH. The solvent extraction of the A1 (13) De, A. K.; Khopkar, S.M.; Chalmers, R. A. Solvent Extraction ofMernls; Van Nostrand Reinhold Co.: London, 1 9 7 0 p 77.
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column length (mm)
I 14 24
% retained
column length (mm)
% retained
44 53
32 50
85 91
75
complex of 8-hydroxyquinoline has been reported to be quantitativeover the pH range 4.5-1 1 our results (Figure 5) indicate the pH range of optimal uptake of A1 onto the 8-HQ columns is much narrower, the maximum uptake occurring between pH 5.3 and 5.7. The much narrower pH range for uptakeis again a result of kinetics. The short contact time between the 8-HQ and A1 as the sample flows through the column results in optimum recovery only over the pH range in which the kinetics are fastest. Column Diameter. We selected an inside diameter of 3.2 mm since that has been shownl2 to be the optimal inside diameter for in line preconcentration. Column Length. In order to assess the effect of column length on retention of A1 we constructed a series of 3.2 mm i.d. columns of various lengths. In each experiment a sample was passed through the column of interest and then through a 40 mm long secondary column, in line but downstream of the first. The two columns were then eluted separately. The results are shown in Table 1. Percent retention was calculated as being the peak height of the primary column divided by the sum of the peak heights of the primary and secondary column; note that these values have been adjusted for an assumed 90% retention by the second column. Precision, from replicate determinations, was better than 2% at all column lengths. The results indicate that, even for the longest column (50 mm), there is less than 100% A1 retention. Since standards and samples are determined with the same column, quantitative adsorption of the sample in the column is not necessary. We have adopted a target column length of -38 mm for this technique. This length was chosen because it was not too long to restrict sample loading, yet was long enough to retain most (-87%) of the Al. Loading Rate. The pump speed for the manifold was determined to satisfy several criteria: cycle time, reagent consumption, tubing lifetime, and system pressure. Nevertheless, by varying the size of the sample tubing on the pump, it is possible to vary the rate at which sample passes through
10
Table 2. Retention of AI on a 30mm Column as a Function of Loading Rate
loading rate (mL/min) 0.5 1.o
2.0 3.0
W retained 98 96 89 71
loading rate (mL/min)
4.1 5.5 1.2
This work
76 retained 61 56
51
the preconcentration column. In order to investigate the effects of loading rate on retention, we ran a series of experiments in which variations of the speed of an independent pump were used to vary the sample loading rate since this was easier than continually changing pump tubing. For flow rates below 2 mL/min, recoveries were calculated by using the same doublecolumn system employed in the column length experiments. At higher flow rates, where the back pressure induced by a second column would progressively influence loading speeds, single column values were used. Percent recoveries in these cases were normalized to the observed 2 mL/min value. Our results indicate (Table 2) that we can achieve close to quantitative recovery of the A1 at sample flow rates of 1 week. At flow rates above 3 mL/min, increased system pressure has deleterious effects. Other Factors. The initial resin, produced by the modified methodlo of Landing et al.,” was made from vinyl polymer gel in the size range 30-60 pM. Due to high pressures generated during sample loading, we prepared a larger size resin of 50-100 pM. We found that this resin significantly lowered the system pressure during the sample loading phase and subsequently used this material for all further studies. Detergent Selection. Howard et al.3 reported that the fluorescence of the lumogallion-A1 complex was increased by as much as 5-fold through the addition of a nonionic detergent, Triton X-100. Enhancement of fluorescence in the presence of detergents has been attributed to the localization of the fluorescing molecules within the micelles produced by detergents. Several possible mechanisms have been suggested that could be responsible for the enhanced fluorescence.Among them are the changed dielectric constant within the micelle compared to the bulk solvent, the elevated viscosity within the micelles, the decreased interaction of the fluorescing molecule with the bulk solvent, the protection of the fluorescing molecule from 0 2 in the bulk solvent, and the rigidization of the fluorescent molecule as it aligns itself within the structure of the micelle.’”l6 Since the factors affecting the lumogallion fluorescence were not understood3 and the kinetics of the FIA system are significantly different from that of the batch system, we decided to reinvestigate the detergent work of Howard et a1.3 (14) Moroi, Y. Micelles. Theoretical and Applied Aspects; Plenum Press: New York, 1992. (15) Singh, H.; Hinzc, W. L. Anal. Lerr. 1982, 15, 221-243. (16) Humphry-Baker, R.; Gratzel. M.; Stciger, R.J . Am. Chem. SOC.1980. 102, 847-848.
520
540
560
580
600
620
640
Emission wavelength (nm)
Flgure 6. Emlsslon spectrum for Trlton X-100 In the reaction flow stream wlth (0)no AI, (X) 1.2 pM AI, (0)6.5 pM AI, and (A)12 pM AI and (-) the emission spectra from Howard et aL3 using Trlton x-100. Table 3. Effect on Fluoreaceme of Varlous Detergents
detergent water Triton-X 100 Triton-X 100 reduced Nonidet p-40 Tergitol xd taurodeoxycholic acid myristylammonium chloride sodium lauryl sulfate cetylammonium bromide Brij-35 a
re1 fluores (this work) (W)
abs fluores3 (arb units)
100
24
380
80
440 420 440 440 450 400 410 510
68‘
41 13 68
Nonidet p-42.
Our initial experiments with Triton X-100 indicated that its addition to the flow stream caused a large increase in baseline fluorescence (and thus noise) at our then optimal wavelength of 584 nm. This rise in background fluorescence prompted us to carefully reevaluate the optimal excitation and emission wavelengths of our system. Our results (Figure 6) showed that on its own the Triton X-100produced a large fluorescent signal between 565 and 640 nm. Since this encompassed our emission wavelength (584 nm), we further examined the spectra of a variety of solutions containing lumogallion, Al, and Triton X-100.These results indicate that at 590 nm (the wavelength used by Howard et al.3) the signal is dominated by the Triton X- 100 background fluorescence. In contrast, at 552 nm the fluorescence is almost entirely associated with the A1 signal. Inspection of the emission spectra reported by Howard et al.3 shows that rather than splitting the lumogallion spectra as they suggest, Triton X-100 addition was producing a significant amount of background fluorescence at the wavelength that they used for monitoring AI concentrations. Further, the wavelength of maximum response for the Al-lumogallion complex has shifted from 584 to 552 nm. This shift in fluorescence is consistent with wavelength shifts seen in previous observations of micellarenhanced f l u o r e ~ c e n c e . ~The ~ J ~discovery of the Triton X- 100 background fluorescence prompted us to reevaluate the potential enhancement effects of as many of the detergents tested by Howard et al.3 as were available to us plus some others. Our results (Table 3) indicate that each of the detergents they had tested, as well as the new ones we selected, enhanced the fluorescence of the lumogallion-A1 complex by Analytical Chemistty, Vol. 66, No. 22, November 15, 1994
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a similar amount when used at their optimum concentrations, and each caused a shift in optimum fluorescence to 552 nm. We noted that adding the detergent stream prior to the heated reaction coil resulted in a significant decrease in analytical sensitivity. When detergent was added after the heated reaction, we observed a 5-fold increase in sensitivity. We interpret these results to indicate that the detergent enhances the fluorescence of the lumogallion-A1 complex by incorporation of the complex within the detergent micelle. In addition, our results suggest that uncomplexed lumogallion can also be incorporated within micelles but that uncomplexed A1 is not. As a result, in our system, the addition of Triton X-100 (or any other detergent) before the heated reaction causes a major reduction in fluorescence since it physically separates the reactants. Brij-35 was chosen for use in the FIA system since it showed the greatest enhancement in fluorescence (Table 3) and produces little or no background fluorescence over the range from 540 to 600 nm. In addition, it is simple to use, nontoxic, and inexpensive and is used by us in other methodologies. While the other detergents showed significant improvements in performance, they also suffer from other drawbacks such as cost (reduced Triton-X 100, taurodeoxycholic acid), difficulty of preparation (cetylammonium bromide, sodium lauryl sulfate, tergitol xd), toxicity (myristylammonium bromide), and background fluorescence (nonidet p-40, Triton-X 100). A study of the effect of increasing the Brij-35 concentration showed that the fluorescence response increased and reached a plateau between concentrations of 0.5% and 1.5% in the effluent stream; we adopted a concentration of 0.58% Brij-35 in the FIA effluent, which is achieved from a 5% solution of Brij-35 pumped at the flow rate indicated in Figure 1. We evaluated the emission characteristics of the Brij-35-lumogallion-A1 system and selected an optimal emission wavelength of 552 nm. We note that the maximum emission wavelength is shifted -30 nm from that of the lumogallion-A1 system, consistent with previous observations of micellar-enhanced fluorescence.14,15 Detector Optimization. In the course of the development of this technique, we had the opportunity to test seven different fluorometric detectors for optimum excitation and emission wavelengths. We noted during this testing that the optimal wavelengths for each machine were significantly different, in some cases by as much as 10 nm; thus it is necessary to individually optimize the excitation and emission wavelengths of any instrument used for this technique. The detector was also optimized for lamp flash rate, rise time, and PMT voltage. The optimal settings are shown in the apparatus section. Interferences. Five elements have been reported as potential interferants in the lumogallion-A1 ~ h e m i s t r y . ~We ~ ~tested ~'~ these five elements for the system developed here and found no significant interferences (Table 4). We believe that the lack of interference exhibited in the FIA system is due to the combination of the use of the preconcentration column and the different chemical conditions used here. When samples are passed through the column, interfering elements may not be fully retained or, if retained, may separate from the A1 (17) Shigematsu, T.; Yasuharu, N.; KeizO, H.; Nagano, N. Jpn. Anal. 1969, 19, 551-554.
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Table 4. Study of Potentlal Interferlng Ions
concn of interferant (nM) for l 2 nM
cu V Ti Ga
Fe Fe
50 100 50 50 500 1100
% rec of AI in sample
100 98 99 102 96 102
interferant/av seawater concn 10 4
200 150 500 1000
during elution and thus not interfere. As we have observed no other peaks during elution and have not observed an increase in A1 recovery for interferant spiked samples, we discount the possibility of positive interferences from the column trapped interferants. Similarly, the lack of negative baseline disturbances during elution appears to rule out negative interferences. The higher pH and shorter reaction time of this method may combine to minimize slower reactions between lumogallion and interfering elements that could occur over longer reaction times. Finally, as discussed above, since we use a higher concentration of lumogallion (- 3 mg/L) in our reaction than did Hydes and competitive complexation of the lumogallion by Fe may not significantly reduce the rate or the amount of its complexation with Al. Blanks, Detection Limit, and Application. In a flow system, A1 contamination in the flowing reagents, Le., the carrier, the reagent buffer, etc., only contribute to the analytical signal by raising the baseline. Blanks that contribute to the analyte signal arise from materials that are preconcentrated on the 8-HQ column and result in peaks when they are eluted. For this method the only potential source of reagent contamination is the sample buffer. To evaluate the blanks associated with sample buffer, samples are singly and doubly spiked with sample buffer, which does not alter the pH of the sample. We find that the difference in signal between singly and doubly spiked samples is analytically indistinguishable from zero. Since we have not observed any peaks arising from valve switching, contaminated columns, or other baseline disturbances, we conclude that there are no system blanks associated with the manifold. The limit of detection of the system was determined from the standard deviation of the lowest A1 water available to us. A series of 10 replicates of a 2.4 nM seawater sample yielded a standard deviation (1 CT)of *0.05 nM, indicating a limit of detection (3 a) of 0.15 nM. The ultimate test of an on-board analytical scheme in oceanography is its ability to produce coherent interpretable data. The data in Figure 7 were produced by using the recommended procedure on board an oceanographic research vessel. All samples were determined within a few hours of their collection. CONCLUSIONS We have presented a method for the ship-board determination of A1 in seawater. The advantages of this method include its high sensitivity, ease of use, speed of analysis, and amenability to automation. With a detection limit of 0.15 nM, this method is among the most sensitivemethods developed to date and with a cycle time of 3 min, it is also the most rapid method for A1 determination at nanomolar concentrations.
complexation of the A1 with both the 8-hydroxyquinoline and the lumogallion. Our results indicate that shorter times involved have significantly reduced the range of optimal conditions and apparently have eliminated interference problems reported by others. We have also made a significant improvement in the use of micellar-enhanced fluorescence for detection of the Allumogallion complex. Our reevaluation of the workof Howard et ale3is not only important for the work discussed here but also for those employing the lumogallion batch methods.2.3
AI (nM)
Flgure 7. Vertical distribution of Ai in the central gyre of the North Pacific at 22'45' N, 158' W.
The use of FIA allows for the elimination of contamination due to sample handling and facilitates the complete automation of this method. Because of the short contact time of the seawater with the resin and the short residence time of the aluminum in the system, it was important to redefine the optimal areas for the
ACKNOWLEDGMENT The authors thank Karen Selph and Rebecca Reitmeyer for scientific and editorial contributions. The authors also acknowledge reviews of this work by two anonymous reviewers. This work was funded by the Office of Naval Research Grants NOOO14-92-5-1485 to C.I.M. and N100014-90-J-1805 to Francis J. Sansone. This work was presented in part at the 1994 Winter Conference on Flow Injection Analysis in San Diego, CA, and also at the 1994 Ocean Sciences meeting in San Diego, CA, and is the School of Ocean and Earth Science and Technology Contribution 3627. Received for review May 27, 1994. Accepted August 1, 1994." Abstract published in Aduance ACS Absrracrs, September 15, 1994.
Analj.tical Chemistry, Vol. 66, No. 22, November 15, 1994
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