Anal. Chem. 2006, 78, 156-163
Chemical Speciation of Iron in Seawater by Cathodic Stripping Voltammetry with Dihydroxynaphthalene Constant M. G. van den Berg*
Department of Earth and Ocean Sciences, University of Liverpool, Liverpool, L69 3GP, U.K.
The chemical speciation of iron in seawater is determined by cathodic stripping voltammetry using 2,3-dihydroxynaphthalene (DHN) as adsorptive and competing ligand. The optimized conditions include a DHN concentration of 0.5-1 µM, seawater at its original pH of 8, and equilibration overnight. The r-coefficient for DHN ()[FeDHN]/[Fe′]) was calibrated against EDTA giving values of 166 for 0.5 µM DHN and 366 at 1 µM DHN and a value of 8.51 ( 0.07 for log K′Fe′DHN. The dissociation of the natural iron species FeL was found to have a characteristic reaction time of 50 min, indicating that titrations should be equilibrated overnight rather than the shorter periods sometimes used onboard ship. The method was applied to samples from the Pacific giving ligand concentrations of 1.1 and 1.6 nM for deep and surface waters, respectively, with an average value for log K′FeL of 11.9 ( 0.3 compared to a value of 11.5 for the siderophore deferoxamine. The results are similar to those obtained previously for similar samples, but the new method has much greater sensitivity for iron than previous methods, leading to lower limits of detection and shorter analysis time. Several studies have confirmed that, in equilibrium conditions, iron occurs complexed with organic matter in the oceanic water column.1-3 This is important as organic complexation is partially responsible (with low overall iron concentrations) for the limitation by iron of primary productivity in the high-nutrient, low-chlorophyll regions of the oceans.4 All studies make use of cathodic stripping voltammetry (CSV) to determine the reactive iron concentration taking advantage of ligand competition between the added ligand used for the voltammetric detection and the natural ligands. Ligands that have been used successfully for these studies are nitrosonaphthol (NN),1 2-(2-thiazolylazo)-p-cresol (TAC),5 and salicylaldoxime (SA).3 The existing methods are not without difficulties as they are all working at their limit of detection. The NN method suffers from an underlying peak at iron concentrations below 0.5 nM,2 and the SA method requires very long adsorption * E-mail:
[email protected]. (1) Gledhill, M.; van den Berg, C. M. G. Mar. Chem. 1994, 47, 41-54. (2) Boye, M.; van den Berg, C. M. G.; de Jong, J. T. M.; Leach, H.; Croot, P.; de Baar, H. J. W. Deep-Sea Res., Part I 2001, 48, 1477-1497. (3) Rue, E. L.; Bruland, K. W. Mar. Chem. 1995, 50, 117-138. (4) Martin, J. H.; Fitzwater, S. E. Nature 1988, 331, 341-343. (5) Croot, P. L.; Johansson, M. Electroanalysis 2000, 12, 565-576.
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times (10 min) and apparently works only with a large mercury drop electrode as the sensitivity with our electrode was poor. The TAC method suffers from a prepeak of the ligand, which affects the limit of detection. A more recently developed CSV method for iron makes use of 2,3-dihydroxynaphthalene (DHN, Fluka)6 and takes advantage of the catalytic effect of iron on the reduction of bromate to achieve significantly better sensitivity than the next best ligand, TAC, achieving a limit of detection of ∼10 pM iron with a 60-s deposition time. Although DHN was used 32 y ago as indicator for complexometric titrations of EDTA with iron,7 its use for the determination of iron complexing ligands in natural waters has not been reported. In this work, it is demonstrated how CSV with DHN can be used to determine iron complexation in seawater from equilibrium conditions. The method is calibrated against EDTA and is applied to samples from the Pacific onboard ship and to a siderophore added to seawater as model compound. The main advantage of the new method is its much better sensitivity for iron, which means that a much shorter analysis time is achieved (a tenth of that with salicylaldoxime) and which should lead to a lower limit of detection of complexing ligands. METHODS AND MATERIALS Equipment. Electroanalytical equipment was a µAutolab III potentiostat from Ecochemie/Metrohm, connected to a VA663 electrode stand with a mercury drop electrode, a glassy carbon counter electrode, a double junction Ag/AgCl, and a 3 M KCl reference electrode. The instrument was controlled by a laptop PC running GPES software using Project mode to automate the measurements. The reference electrode bridge consisted of a PTFE sleeve and a ceramic frit. The KCl filling solution had been purified (see below) to remove contaminating iron. During shipboard measurements, the bridge was filled with seawater and the reference electrode compartment was refilled weekly with KCl. Voltammetric cells were made from either quartz or PTFE suitable to use with sample volumes of 10-20 mL. UV-digested seawater (UVSW) was prepared freshly using a home-built system with a 100-W high-pressure mercury vapor lamp in 30-mL quartz tubes with a 1-h irradiation time. The lamp was tested with 5 ppm humic acid in seawater: using a scanning spectrophotometer, it was verified that this organic matter was >95% digested by 30min digestion. (6) Obata, H.; van den Berg, C. M. G. Anal. Chem. 2001, 73, 2522-2528. (7) Manku, G. S. Fresenius Z. Anal. Chem. 1973, 263, 335-335. 10.1021/ac051441+ CCC: $33.50
© 2006 American Chemical Society Published on Web 12/02/2005
Reagents, Sample Bottles, and Clean Facilities. Water purified using a Milli-Q/UV ion-exchange system (MQ-water) preceded by a Millipore Elix deionizer was used for reagent preparation and bottle cleaning. Methanol and hydrochloric acid were sub-boiling distilled with a quartz condenser. A stock solution of 0.01 M DHN was prepared from DHN in methanol and diluted to 1 mM with methanol. A mixed buffer/oxidant solution contained 0.1 M HEPPS (3-(4-(2-hydroxyethyl)-1-piperazinyl)propansulfonic acid, VWR), ∼0.05 M ammonia, and 0.4 M bromate. The pH of this mixture was adjusted to reach a final pH of 8 after addition of 0.5 mL to 10 mL of seawater. Contaminating iron in the HEPPS/bromate solution was removed by overnight equilibration (2×) with 100 µM MnO2 followed by filtration (0.2-µm cellulose nitrate, Whatman). A second HEPPS/ammonia/bromate solution was prepared with additional ammonia for iron analyses in acidified seawater. A stock solution of deferoxamine (DFO) contained 10 mM deferoxamine mesilate (Sigma-Aldrich) and is also called desferal or desferrioxamine B mesylate. The KCl filling solution of the reference electrode contained 3 M KCl. This was cleaned by equilibration with 100 µM MnO2 after buffering the pH to ∼7.4 with 2 mM NaHCO3, followed by filtration. The MnO2 was prepared approximately as before8 by mixing Mn2+, MnO4-, and OH- according to the following equation:
3Mn2+ + 2MnO4- + 4OH- f 5MnO2 + 2H2O
Equal volumes of 0.03 M MnCl2 and 0.02 M KMnO4/0.04 M NaOH were mixed using a magnetic stirrer, and the pH was adjusted to neutral by adding more NaOH by pipet. The brown MnO2 was centrifuged three times, taken up in MQ, and finally made to a final concentration of ∼0.05 M MnO2. Sample bottles were cleaned by soaking several days in 0.1% detergent in hot tap water, then in 4 M HCl, and finally in 1 M HCl; they were left partially filled with MQ acidified to pH 2 with HCl. All sample handling was in a laminar flow hood, either inside a “bubble” with filtered air onboard ship or in a laboratory flooded with HEPPA filtered air in the Liverpool laboratory. Sample Collection. Samples were collected during a cruise (SAFe cruise) with the RV Melville, October 15-November 8, 2004, from Hawaii to San Diego. Bottle samples were collected by Go-Flo bottles on an alloy rosette, whereas surface water was collected by hose and a pump. Filtration was through filter cartridges with a final cutoff of 0.1 µm. The quality of the water collection (lack of iron contamination) was confirmed by iron analyses onboard ship. Procedure To Determine Dissolved Iron. Α ∼30-mL sample of filtered seawater was poured into a 30 mL HDPE bottle; 30 µL of 50% HCl was added giving pH 2.4 and 30 µL of 1 mM DHN to a final concentration of 1 µM DHN. This was left for several hours or overnight before analysis. A 10-mL sample of this acidified seawater was pipetted into a quartz voltammetric cell, and 0.5 mL of the ammonia-rich HEPPS/bromate solution was added. Voltammetric parameters were approximately as before:6 5-min nitrogen (8) van den Berg, C. M. G.; Kramer, J. R. Anal. Chim. Acta 1979, 106, 113120.
purge, 90-s adsorption at -0.1 V, 8-s equilibration, and a scan using sampled dc, step size 4 mV, frequency 10 s-1. Procedure To Determine the Concentration of Organic Iron Complexing Ligands. The shipboard titrations were carried out in 30-mL HDPE bottles, whereas PTFE voltammetric cells were used in Liverpool. The ∼125 mL of seawater was poured in a 125-mL Teflon bottle, and DHN was added to a final concentration of either 0.5 or 1.0 µM. Iron was added to the titration vessels in a series of concentrations of 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 5, and 8 nM, from iron stock solutions of 0.2 and 1 µM iron, which had been acidified to pH 2.4. Then 10 mL of the seawater containing DHN was pipetted into each titration vessel. The equilibration time was overnight (∼17 h) except where indicated. Then each PTFE titration vessel was placed on the electrode stand, purging was initiated, 0.5 mL of the bromate/HEPPS mixture was added, and the reactive iron concentration was determined by CSV in order from low to high iron. The voltammetric parameters were as for dissolved iron. Onboard ship, the sequence was different: iron was added in the same concentration range to 30-mL HDPE bottles; 10 mL of seawater was added and allowed to equilibrate for typically 3 h. Then the DHN was added, and 15 min later, the bottle contents were poured into the quartz voltammetric cell. Purging was initiated, 0.5 mL of the bromate/buffer mixture was added, and the labile iron concentration was determined by CSV. The measurement sequence was from low to high iron to maximize cell conditioning. The shipboard titrations were in duplicate to minimize adsorption problems on the voltammetric cell. All PTFE cells and HDPE bottles had been conditioned by setting in titrations prior to first use. They were briefly rinsed with MQ between titrations. Each major change in concentrations of iron or DHN required new conditioning. Data Treatment. Data were fitted to an equation of the Langmuir type:8-10
[Felabile]/[FeL] ) [Felabile]/CL + (1 + RFeDHN)/(CLK′FeL) (1) This equation differs in the way it has been used before for iron1 in that the stability constant for the iron complexes with DHN and the unknown ligands L is based on inorganic iron [Fe′] rather than on the free metal ion concentration [Fe3+]:
K′FeL ) [FeL]/([Fe′][L′]) This method was selected because the free iron ion concentration in seawater is vanishingly low at ∼10-23 M, meaning that there are statistically ∼10 Fe3+ ions L-1 and there might not actually be any in some of the 10-mL aliquots used for the titrations. At such low concentrations, it is more likely that any of the inorganic iron species (predominantly Fe(OH)30 and Fe(OH)4-) reacts with the organic ligands. For this reason, it is more realistic to consider the iron complexation as a reaction between Fe′ and L′ rather than as Fe3+ with L′.11 As before, for reasons of comparison, an (9) van den Berg, C. M. G. Mar. Chem. 1982, 11, 307-322. (10) Ruzic, I. Anal. Chim. Acta 1982, 140, 99-113. (11) van den Berg, C. M. G. Environ. Chem. 2005, 2, 88-89.
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R-coefficient of 1010 was selected for the ratio of inorganic iron complexation (RFe ) [Fe′]/[Fe3+]),1,12 which ignores Fe(OH)30 species.12 If the Fe(OH)30 species were included, a value nearer 1011 would be obtained. Comparative calculations showed that the calculated values for K′FeL were not significantly affected by the choice of value for RFe. To convert the K′FeL values from this work back to constants based on Fe3+ (K′Fe3+L), it is essential to use the same value for RFe as used here: log K′Fe3+L ) log K′FeL+10. Values for CL and K′FeL were calculated by linear least-squares regression of [Felabile]/[FeL] as a function of [Felabile]. Labile iron concentrations ([Felabile]), comprising inorganic iron and iron bound by DHN, were calculated from the peak height (ip) and the sensitivity S (nA nM-1): [Felabile] ) ipS. A first estimate for S was obtained from dip/dCFe for the highest three points of the titration and was subsequently refined by correction of the total iron concentration for the fraction of iron still bound by L at each step of the titration: i.e., the corrected slope was obtained from the highest three points of a plot of ip versus [Fe′] + [FeDHN], which was calculated from
[Fe′] + [FeDHN] ) CFe - [FeLmodeled]
where [FeLmodeled] was calculated from the first estimates for K′FeL and CL obtained using eq 1. S was then iteratively refined until no further change, which invariably caused an increase in S from that estimated initially. Previously S was estimated by visually correcting the slope of a plot of ip versus CFe1 as the final sensitivity is reached asymptotically, a method that is somewhat prone to errors of judgment. Correction of S by modeling has been made before13,14 and is implemented here with a relatively simple spreadsheet calculation for 1:1 species of FeL. Plots according to eq 1 were straight, indicating that there was no need to attempt this for more than one FeL species. Thus three types of plots were used for each titration: (1) a plot of peak current (ip) as a function of dissolved iron to obtain first estimates of S; (2) a plot of [Felabile]/[FeL] as function of [Felabile] to obtain values for CL and K′FeL; and (3) a plot of ip as a function of [CFe] - [FeLmodeled] to correct S. Calibration of K′FeDHN. The stability of the FeDHN species (RFeDHN) was calibrated by titration of UVSW containing 2 nM Fe and either 0.25, 0.5, or 1 µM DHN, with EDTA. The R-coefficient for complexation of iron with EDTA, RFe3+EDTA, was evaluated using the constants from the IUPAC database,15 which includes mixed hydroxide species: log BFeL/FeOHL.H ) 7.49, log BFeOHL/Fe(OH)2L.H ) 9.41 (where L ) EDTA) and log KFe3+EDTA ) 25, all valid for an ionic strength of 0.1. No correction was made for the ionic strength as this correction is questionable for high ionic strengths and because the corrections for the iron and major ion species tend to cancel out. The side-reaction coefficient of EDTA with the major ions in seawater of salinity 35 and pH 8, log REDTA, was 8.63, calculated from the free major cation concentrations obtained from an ion-pairing model. (12) Hudson, R. J. M.; Covault, D. T.; Morel, F. M. M. Mar. Chem. 1992, 38, 209-235. (13) Turoczy, N. J.; Sherwood, J. E. Anal. Chim. Acta 1997, 354, 15-21. (14) Hudson, R. J. M.; Rue, E. L.; Bruland, K. W. Environ. Sci. Technol. 2003, 37, 1553-1562. (15) Pettit, L. D.; Powell, H. K. J. Stability constants database, IUPAC, Version 2.68 ed.; Academic Software: Otley, Yorkshire, U.K., 1997.
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Values for RFeDHN were calculated from the ratio of the current in the presence and absence of EDTA, analogous to that done before:1
X ) ip/ip0 ) RFeDHN/(RFeDHN + RFeEDTA)
(2)
Individual values for RFeDHN were calculated for each value of X, falling between 0.2 and 0.8, from which values for K′Fe3+DHN and B′Fe3+DHN2 were calculated and converted to K′FeDHN and B′FeDHN2 on the Fe′ scale. RESULTS AND DISCUSSION Preliminary Experiments. Two peaks are obtained by CSV under the conditions used in this work (pH 8, deposition at -0.2 V): a peak at -0.55 V for iron and a second peak at ∼-0.87 V. The second peak had not been identified before and was identified here as due to vanadium. The vanadium concentration in seawater is relatively constant at ∼30 nM, much greater than that of iron, and gives a much larger peak than obtained for iron. However, the iron peak is large relative to its low concentration because it is enhanced due to its catalytic effect on the reduction of bromate while the vanadium peak is not affected by the bromate. Repeated measurements of iron in either MQ, seawater, or UVSW showed that the iron peak gradually increased at a rate of several picomolar per minute. The cause was traced down to the KCl in the salt bridge of the reference electrode. For this reason, the KCl was replaced by seawater onboard ship and by MnO2purified KCl/2 mM NaHCO3 in the laboratory. The glass frit of the standard glass salt bridge was found to cause contamination when acidified samples were added to the cell and was replaced by a PTFE sleeve with a ceramic frit. The voltammetric cells, whether glass, silica, or PTFE, were all found to adsorb iron, which was minimized by conditioning the cells by repeating measurements (refilling the cell) and running the parts of titrations enhanced iron levels in duplicate or by using a separate voltammetric cell for each iron addition of a titration. DHN was added to pH 8 seawater at levels of 0.5, 1, and 2 µM and left overnight and for 3 days in one instance to test for stability. Scans for iron after addition of the bromate/buffer mixture showed the usual peak shape, peak potential, and sensitivity for iron, indicating that the DHN was stable for this period. Higher levels (10-20 µM) of DHN were stable for shorter periods at pH 8 (several hours) but showed a deterioration of the peaks at longer equilibration time, the iron peak shifting in a negative direction and becoming a shoulder of the peak for vanadium, presumably as a result of oxidation by ambient dissolved oxygen. The 10-20 µM DHN in acidified (pH 2) seawater was stable for at least 1 week while it tended to become oxidized relatively rapidly, in minutes, when the pH was raised above ∼8.5. Preliminary titrations of pH 8 seawater with iron and detection of the reactive iron concentration by CSV at low (0.25-1 µM) levels of added DHN, after several hours of equilibration, showed a curved response typical for complexing ligand titrations. Curvature was not obtained when the titration was performed inside the voltammetric cell with measurement either immediately or even after several minutes (up to 30 min tested) after each iron addition, indicating that slow kinetics played a role. For this reason, the reaction kinetics were determined initially to establish the required reaction time.
Figure 1. Kinetics of the reaction between iron bound by ligands in surface seawater and 1 µM DHN. The background iron concentration was 0.56 nM, and the natural ligand concentration 1.4 nM.
Reaction Kinetics. Buffer and bromate (0.75 mL) were added to 15 mL of surface seawater from the Pacific in a PTFE voltammetric cell that was purged to remove dissolved oxygen. Then DHN was added to a final concentration of 1 µM, and reactive iron measurements were initiated immediately. The adsorption time on the electrode was 100 s, and the first scan was obtained after 2 min, whereafter the measurements were continued automatically for several hours with 15-s purging between scans. The peak height for iron was found to increase gradually, reaching a plateau after about 150-200 min (Figure 1A). The mechanism for the reaction can be several, including a first-order reaction in which residual Fe′ is bound by the added DHN followed by a release of iron from FeL, or a second-order reversible ligand exchange reaction in which L on FeL is exchanged for DHN in FeDHN, e.g.:16
FeL + DHN S FeDHN + L′
Although a peak was obtained quickly after the DHN addition, the reaction reached final equilibrium slowly, which was not in agreement with a simple first-order reaction. More likely is a reversible ligand-exchange option or a mixture of the two reactions. A plot of the natural logarithm (ln) of (1 + FeDHN/ FeL) was straight (Figure 1B) in agreement with a pseudo-secondorder, reversible, mechanism with a characteristic time of 50 min. In view of this slow reaction time, titrations were normally equilibrated overnight (∼17 h), except onboard ship where the equilibration time between the seawater equilibrated with iron and the added DHN was 15 min. Calibration of the Complex Stability of Fe-DHN against EDTA. Only a single estimate is available for the stability constants between DHN and iron, with a value of 20.9 for log KFe3+DHN and of 36.2 for log BFe3+DHN2,15 without correction for the side reactions with the major cations in seawater. For this reason, it was necessary to calibrate the complex stability in seawater against a ligand (EDTA) of which the complex stability with iron is well known. Preliminary titrations with EDTA using a 3-h equilibration time showed that the peak height was suppressed as expected due to EDTA competition with DHN. Extension of the equilibration time from 3 h to overnight caused the suppression to increase and caused good separation between different concentrations of DHN indicating that equilibration between iron and EDTA requires a long time interval, such as for the natural ligands. Titrations of UVSW containing 2 nM Fe and 0.25, 0.5, or (16) Morel, F. M. M.; Hering, J. G. Principles and applications of aquatic chemistry; J. Wiley & Sons: New York, 1993.
Figure 2. Calibration of the complex stability of iron with DHN by titration of UVSW containing 0.25, 0.5, and 1.0 µM DHN with EDTA. Equilibration was overnight; X is the ratio of the peakheight in the presence and absence of the EDTA. Table 1. Calibration of rFe3+DHN and K′FeDHN in Seawatera DHN (µM)
RFe3+DHN
RFeDHN
K′FeDHN
0.25 0.5 1.0
6.9 × 1.7 × 1012 3.66 × 1012
69 166 366
2.75 × 108 3.31 × 108 3.66 × 108
1011
a The correction of R Fe3+DHN to RFeDHN, and that of K′Fe3+DHN to K′FeDHN, was made by division by RFe ) 1010. Average log K′Fe3+DHN ) 18.51; log K′Fe′DHN ) 8.51 (0.07.
1 µM DHN, with EDTA, are shown in Figure 2. The EDTA additions caused the peak height to decrease because of the competition between the EDTA and the DHN for iron. When the peak height is diminished by half (X ) 0.5), the complex stability of iron with the EDTA equals that with the DHN and thus the DHN complex stability can be estimated. Values for the complex stability, RFeDHN, were calculated using eq 2 and are given in Table 1. The complex stability was found to increase in a predictable manner with the DHN concentration from a value of 69 for RFeDHN at 0.25 µM DHN to 166 at 0.5 µM DHN and 366 at 1.0 µM DHN (Table 1). The data were found to best fit the predominant presence of a 1:1 FeDHN complex with a value for log K′FeDHN of 8.51 ( 0.07. Values for B′FeDHN2 were found to increase with the DHN concentration, indicating that this complex did not describe the iron complexation accurately at the tested DHN concentrations. The value of 18.51 for log K′Fe3+DHN in seawater is 1.6 units Analytical Chemistry, Vol. 78, No. 1, January 1, 2006
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Table 2. Concentrations and Conditional Stability Constants for Iron Complexing Ligands in Samples from the Pacific (Station 5 at 30°N/140°W, Coastal at 33° 09.81N/117° 24.77W)a Fe (nM)
DHN (µM)
CL (nM)
log K′FeL
log RFeL
SW tank SW tank Surf SW St.5 30 m, St.5 110 m St.5 1000 m St.5, Nov 1 1000 m St.5, Nov 1, 3 h eq coastal surface Nov 7 DFO 1.8 nM UVSW
0.51 0.51 0.35 0.13 0.08 0.75 0.75 1.6 0.6 0.2
Laboratory-Based Titrations 0.5 1.38 ( 0.09 0.5 1.43 ( 0.06 1 1.64 ( 0.06 0.5 1.17 ( 0.02 0.5 1.56 ( 0.06 0.5 1.08 ( 0.01 0.5 1.39 ( 0.04 0.5 2.14 ( 0.08 1.0 1.90 ( 0.13 0.5 0
11.42 ( 0.14 11.55 ( 0.12 11.80 ( 0.08 11.6 ( 0.06 11.45 ( 0.08 12.3 ( 0.3 12.0 ( 0.2 12.0 ( 0.3 11.54 ( 0.11 -
2.56 2.71 3.01 2.67 2.65 3.4 3.1 3.3 2.82 -
1000 m Oct 30 1000 m, Nov 1 1000 m, Nov 2 1000 m, Nov 5 coastal surface Nov 7
0.7 0.75 0.75 0.75 1.6
Shipboard Titrations 3 h Equilibrated 0.5 1.25 ( 0.05 1.0 0.86 ( 0.05 0.5 1.26 ( 0.05 0.5 1.02 ( 0.17 0.5 2.45 ( 0.11
11.75 ( 0.11 11.82 ( 0.08 12.20 ( 0.27 11.46 ( 0.22 11.61 ( 0.15
2.9 2.76 3.3 2.5 3.0
a The iron concentrations have been corrected for a reagent blank of 50 pM iron. The laboratory-based titrations were in samples that had been stored frozen except for the “SW tank” water, which was stored at 4 °C. The equilibration time was overnight except where indicated.
Figure 3. Titration with iron of surface seawater before and after UV digestion in the presence of 1 µM DHN. The ligand concentration in the untreated seawater was 1.4 nM.
less than the literature value of 20.9 for pure electrolyte; however, the overall complex stability in seawater is much weaker than that calculated from a value of 36.1 for log BFeDHN2,15 indicating that the side reactions of DHN with the major cations in seawater could be strong. A concentration of 0.5 µM DHN was selected to determine iron complexing ligands in seawater as it binds ∼99.5% of inorganic iron (RFeDHN ) 166), but 1 µM DHN was used for some experiments. Titrations of UV-SW and DFO. Preliminary experiments were carried out in UV-digested seawater to check for unknown interferences and to see whether ligand-free seawater was produced. The titration in UVSW was straight (Figure 3) compared to curved for the untreated seawater, indicating that all ligands had been destroyed and that the method did not give false 160
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positives such as due to contaminants in the buffer (any such effects were anyhow minimized as the buffer/bromate mixture was added to the voltammetric cell at the onset of purging only 5 min prior to the first scan). DFO (1.8 nM) was added to UVSW to act as a known complexing ligand with properties similar to the natural ligands occurring in seawater. The detected ligand concentration was 1.91 ( 0.13 nM (Table 2), equal (within measurement error) to the expected ligand concentration. The found complex stability, log K′Fe′L ) 11.54, is identical to that found previously (11.6 after correction of log K′Fe3+L ) 21.6 to the Fe′-based constants using log RFe ) 10) by CSV in competition against NN.17 That measurement had also been calibrated against EDTA. The result indicates that good agreement is obtained for iron speciation between different laboratories using different competing ligands but similar methodologies. The conditional stability constant for DFO for seawater is much smaller than that uncorrected for side reactions of the ligand: log K*Fe3+L)30.618 equivalent to 20.6 on the Fe′ scale, indicating that the side reaction coefficient for DFO with the major ions in seawater, RDFO(SW), should be ∼109. Titrations of Seawater Samples. Several seawater samples were collected from various depths in the Eastern Equatorial Pacific (30°N/140°W) and from a station nearer to land (33° 09.81N/117° 24.77W). Titrations of these samples showed curvature indicating an excess of iron complexing ligands. A titration of surface seawater and its data treatment is shown in Figure 4. The plot according to eq 1 (Figure 4B and C) was straight, indicating that a single ligand dominated the iron speciation in this range of labile (0.1-6.9 nM) and inorganic iron (0.3-21 pM) levels. This was the case for all titrations. A plot of the scans of a titration of a 1000-m sample is shown in Figure 5. The X-axis of the plot was truncated at -0.8 V although the scans were continued to -1.2 V to optimize the plot (17) Witter, A. E.; Hutchins, D. A.; Butler, A.; Luther, G. W. Mar. Chem. 2000, 69, 1-17. (18) Neilands, J. B. Annu. Rev. Biochem. 1981, 20, 715-731.
Figure 5. Voltammetric scans for the titration of a deep Pacific sample with iron.
∼10× greater than the peak height obtained for the final, 8 nM, addition of iron in this titration. Although the iron peak was small at the initial iron concentration, the peak was nevertheless measurable (0.8 nA for this sample). Ligand concentrations in the open ocean water were between 1.1 (deep water) and 1.6 nM (110 m depth) and higher (2.1 nM) for the coastal surface water. The standard deviation of the ligand concentrations was between 0.01 and 0.09 nM, in the worst case
