1524
And. Chem. 1993, 65,1524-1528
Automated Determination of Iron in Seawater by Chelating Resin Concentration and Chemiluminescence Detection Hajime Obata Department of Chemistry, Faculty of Science, Kyoto University, Kyoto 606-01, J a p a n
Hajime Karatani Department of Polymer Science and Engineering, Kyoto Institute of Technology, Kyoto 606, J a p a n
Eiichiro Nakayama* Research Center for Instrumental Analysis, Faculty of Science, Kyoto University, Kyoto 606-01, Japan
A new automated shipboard analytical method for determining iron(II1) in seawater has been developed. The method is based on a combination of selective column extraction using chelating resin and improved chemiluminescence (CL) detection in a closed flowthrough system. In this method, Fe(II1) in an acidified sample solution is selectively collected on 8-quinolinolimmobilized chelating resin and then eluted with dilute hydrochloric acid. The resulting eluent is mixed with luminol solution, aqueous ammonia,and hydrogen peroxide solution successively, and then the mixture is introduced into the CL cell. The iron concentration is obtained from the CL intensity. The detection limit of iron(II1) is 0.05 nmol L-'when using an 18-mLseawater sample. The method was applied to ordinary oceanic waters and hydrothermal waters collected in the North and South Pacific Oceans. Worldwide marine chemists and marine biologists have focused on the behavior of iron in seawater, since Martin et al.1-5 pointed out that the phytoplankton growth in oceanic water was limited by the deficiency of iron derived from the atmosphere rather than the lack of nutrients in some oceanic regions, such as the equatorial Pacific, Gulf of Alaska, and Antarctic Ocean. This attractive hypothesis created a heated argument in various journals6-9 and spurred the geochemical study of iron. For example, Zhuang et al.l0reported recently that more than half of the iron in aeolian mineral dust existed in the form of Fe(II),resulting in the enhancement of solubility (1) Martin, J. H.; Fitzwater, S.E. Nature 1988, 331, 342-343. (2) Martin, J. H.; Gordon, R. M.; Fitzwater, S.E.: Broenkow, W. W. Deep-sea Res. 1989, 36,649-680. (3) Martin, J. H.; Gordon, R. M.; Fitzwater, S. E. Nature 1990, 345, 156-158. (4) Martin, J. H.; Fitzwater, S.E.; Gordon, R. M. Global Biogeochern. Cycles 1990, 4, 5-1. (5) Martin, J. H. Nature 1991, ,953, 123. (6) Betzer, P. R.; Carder, K. L.; Duce, R. A.; Merill, J. T.; Tinpale, M. W.; Ilematsu, M.; Costerro, D. K.; Yong, R. W.; Feely, R. A.; Breland, J. A.; Bernstein, R. E.; Greco, A. W. Nature 1988, 336, 568-571. (7) Young, R. W.: Karter, K.; Betzer, P. R.; Costerro, D. K.; Duce, R. A,; Ditullio, G. R.; Tidero, N. W.; Laws, E. A.; Uematsu, M.; Merill, J. T.; Feely, R. A. Global Biogeochern. Cycles 1991,5, 119-134. (8) Broecker, W. S. Global Biogeochern. Cycles 1990, 4, 3-4. (9) Dugdale, R. C.; Wilkerson, F. P. Global Biogeochem. Cycles 1990, 4, 13-19. (10) Zhuang, G.; Yi, Z.; Duce, R. A,; Brown, P. R. Nature 1992,355, ,531-539.
of iron in surface water. In order to verify whether or not the iron deficiency contributes to the limitation of primary production and also to clarify the chemical species of iron, an accurate and rapid analytical method for determining iron in seawater is essential. A conventional analytical method, like solvent extraction-graphite furnace atomic absorption spectrometric detection, requires a contamination-free technique. Moreover, it is time-consuming and troublesome as liters of the sample solution must be treated, because the dissolved concentration of iron in oceanic water is extremely low (1 nmol L-I = 56 ng L-I). Martin et found recently that the dissolved concentration of iron was less than 0.02 nmol L-l in the shallow water (60 m) of the equatorial Pacific. The classical chemiluminescence (CL) method using a luminol-hydrogen peroxide system11J2 is thought to be a promising method for the shipboard analysis of iron because it is highly sensitive to iron and requires only a small size detection device. However, Fe(II1) must be separated from the other heavy metal ions, such as Mn(II), Cr(III), Co(II), and Cu(II), prior to detection, since the method is not specific to Fe(II1). We have developed an automated analytical method for determining iron in seawater using a closed flow system with a combination of a chelating resin concentration and CL detection. In this method, Fe(II1) is concentrated with a MAF-8HQ (8-quinolinol-immobilized fluordie containing metal alkoxide glass) resin column from a weak acidic sample solution which separates it from the sea-salt matrix and the other heavy metals such as Mn(II), Cr(III), and Fe(11). Fe(II1) eluted with the dilute hydrochloric acid is mixed with luminol solution, aqueous ammonium, and hydrogen peroxide solution, successively. The ammonium buffer also acts as a masking agent for Co(I1). Thus, sub-nmol L-l levels of Fe(II1) can be specifically determined without interference from the other metal ions in ca. 20 mL of sample solution. In addition, Fe(I1) can be concentrated on the resin column by raising the sample pH after separating it from Fe(II1) at a low pH. On the basis of similar principle, Sakamoto-Arnold and Jonson13 developed a highly sensitive flow injection analysis CL technique for cobalt in seawater. Analogous methods for manganese and copper in seawater were also reported r e ~ e n t l y . ' ~ Elrod J~ et al.I6 reported a flow injection method for Fe(I1) using CL detection system with brilliant sulfoflavin (11) Rigin, V. I.; Blokhin, A. I. Zh. Anal. Khirn. 1977, 32, 312-316. (12) Pilipenko, A. T.; Barovakii, V. A,; Kalinichenko, I. E. Zh. Anal. Khirn. 1978, 33, 1880-1884. (13) Sakamoto-Arnold, C . M.; Johnson, K. S.Anal. Chern. 1987,59, 1789-1794. (14) Chapin, T. P.; Johnson, K. S.; Coale, K. H. Anal. Chirn. Acta 1991,249, 469-478.
0003-2700/93/0365-1524$04.00/0 0 1993 American Chemical Society
ANALYTICAL CHEMISTRY. VOL. 65. NO. 11. JUNE 1. 1993
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S SAMPLE s o w . CLEANING SOLN. (Mow)
(XEANINGSMN.(0.5M HCI)
LUMINOLSOLN.
0.4M NH&H SMN. HzOi SOLN. L--J
noUn 1.
Schsmatlcda~mofme(lowcarcsntratlonandadetectionsystemsrorautomaticdeterm1na~of Imn(1II)insenwater:P.perlstakk pump (hllhed Ikw, m a n s Inta(Ock1ng):S.sobwid vakrve (NO = m i open. NC = ncinal closed. IN = inlet): C. MAF-8HQ resin column; RC, reactkm coll; CL. CL dete*lon Wll; HV, hW-vtage supply: R. recorder: F. (lowmeter.
(BSFbhydrogen peroxide. Although thin CL system is relatively specific to Fe(ID, i t in not highly sensitive compared to the luminol-hydrogen peroxide system. Therefore, i t is though to be difficult to apply this method to the determination of iron in subO.l nmol L-I levels of sample seawater. In our method, this level of iron can be determined by prolonging the period of column extraction by a several minutes. EXPERIMENTAL
SECTION
Apparatus. The chelating resin concentration and CL systems are shown in Figure 1. The concentration column was a Teflon tube (4-mmi.d., 5-em length) packed with MAF-8HQ resin, which wan stopped with two pieen of Teflon mesh (170 mesh). The samplesolutionwassent to thecolumn by aperistaltic pump (Masterflex) which also sent the cleaning solution. The absorbed ironions wereelutedfrom thecolumn hy hack-flushing. All thestages wereswitched with three-waysolenoidvalves which were controlled by a programmable controller. The CL system was composed of a four-channel peristaltic pump for sending a carrier solution (eluent), aqueous ammonia, luminol solution, and hydrogen peroxide solution; a CL detector which had a flow cell and a photomultiplier tube (PMT) built in; a high-voltage power supply; and a recorder. The flow cell was a clear 1-mm i.d. Teflon tube coiled on an aluminum block and attached by double-faced Scotch tape. Both the concentration system and the CL system were connected with Teflon connectors and a 1.9-m reactioncoil of Teflon tubing (1-nunid.) to increasereaction time and to mix the reagents and carrier solutions sufficiently. The containers of reagent and carrier solutions and the reaction coil were put in a water bath regulated a t 27 OC to stabilize the CL reaction and to prevent generation of air bubbles in the flow system. All flow lines were 1-mm i.d. Teflon tubing. All Teflon tubes, fittings,and containerswere clenned hy heating in a mixture of concentrated nitric acid, sulfuric acid, and perchloric acid for severalhour8,foUowedhywashingwith hotpure water forseveral hours. Law-densitypolyethylenecontainers(Nargene)for sample preservation were washed with hot 0.3 M nitric acid for 12 hand hot pure water for 12 h, successively, after the usual washing with 5% Extran MA 01 (Merk) and 4 M hydrochloric acid. The tube for the peristaltic pump was a Phar Med tube (Norton), whichwasusedafterwashingwithhot5% ExtranMAOl (Merk) and hot 3 M hyrochloric acid. The tube was good for at least 4 weeks of continuous operation. (15)Coals,K.H.;Stout,P.M.:Johnson.K.S.:Sakamoto,C.M.A~l. Chim. Acta 1992.266.345-351. (16) Elrod. V. A.; Johnson. K. S.; Coale, K. H. AMI. Chem. 1991.63, 893-898.
Reagents. Luminol (Wako Pure Chemical Industries) WBB recrystallized twice from 0.6 M hydrochloric acid. Triethylenetetramine (TETA, Wako) was recrystallized twice from methanol and hydrochloric acid. Potaasium aluminum sulfate was recrystallized three times from acidic solution. Hydrochloric acidandaqueousammoniaofreagentgrade were further purified by rapid isopiesticdistillation. Formic acid and acetic acid were purified by conventional distillation. Hydrogen peroxide (CicaMerk. for atomic absorption npectrometry) and potassium carbonate (Merk, Supurapur) were used without further purification. Allsolutionswere made upwith Millipore Mi1li.Q water (MQW). Solutionsofammonium salts were prepared by mixing the purified aqueous ammonia and acids. The carrier solution (eluent) was 0.2 M hyrochloric acid. A 0.74 mM of luminol solution was made by diluting luminol with a small amount of potassium carbonate. This solution also contained 0.3 mM of TETA. A 0.7 M solution of hydrogen peroxide was used. A 0.4 M solution of aqueous ammonia was used to maintain the pH of mixed solutions at a pH around 9.5 because the carrier solution was 0.2 M hydrochloric acid. The cleaning solutions were 0.5 M hydrochloric acid (after elution) and pure water (before elution and after acid cleaning). A stack solutionof 1.8nmolofFeilll)mL-'wasmadefromthe IOOOppm FelllI) standard solution (Wako). Working standards were prepared by diluting the stack solutions with purified Seawater. Standardsolutions(1000ppm)ofCu,Co,Ni,Cr,Ph. Mo,Se.Ti, TI, Bi, Zn. Cd. AI, Sb. and M n (Wako) were used. Pure seawater for calibration was prepared from surface seawater collected by pumping at an open Ocean station. The seawaterwas purified hya coprecipitationmethod withaluminum hydroxide (20 mg of AI 1. made from purified potassium aluminum sulfate and aqueousammonia. It wasconfirmed that the aluminiumconcentration in the purifiedand filtered seawater was ca. 7 nmol L I by the fluorometric method using Lumogalion (5-chloro-3-[i2,4-dihydroxyphenyl~azol-2~hydroxy~~enesuUonic acid). When purifying with a chelating regin column, a small portion of functional group (ligand) bleeding from the solid support interfered with the CL detection by masking Fe(1ll) in cases of low iron concentration (less than 4 nmol L-'). Preparation of Chelating Resin. A resin of &quinolinol immobilized on hydrophilic vinyl polymer [TSK gel Toyopearl HW.'t;j(F), Tosoh C o . ] .TSK-BHQ, was prepared according to themethudofLandingetal.'- A reninof&quinolinnlimmobilized on silica gel, MAF-8HQ, was prepared as follows. Tetraethyl orthonilicate wan purified by distillation, followed by hydrolysis with pure hydrochloric acid and pure hydrofluoric acid (CicaMerk. Supurapur) in R mixture of MQW and ethanol ISitOC2H51i ~~
(17) Landing. W. M.; Haraldson. C.; P s ~ ~ u sN. , Anal. Chem. 1986.58, 3031-3035.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1,
c
1993
add buffer s o h . (PH 3.0)
Table I. Interference of Other Chemiluminescence metal concn intensity Cu(I1) 1 mg L-I 0.20 Co(I1) 1 mg L-' 0.11 Ni(I1) 1 mg L-' 0.22 Cr(II1) 0.1 mg L-' 2.4 Pb(I1) 1 mg L-' 0.44 MOW) 1 mg L-I 0.21 Ti(1V) 1 mg L-' 0.025 a
1--1 Washing with hydrochloric acid 90 s
[Washing with pure water 120 s
I
I Sample exchange 1 Figure 2. Flow chart of the analytical procedure for the automatic determination of iron(II1) in seawater.
HCLHF:H20:MeOH = 1:0.054:0.25:3.5:3.5 in the mole ratio). The resulting hydrogel was pulverized and dried in a vacuum oven at 80 "C. After being dried (weightconstant), hydrogel was heated at 250 "C in an electric f ~ r n a c e . ~The ~ J resulting ~ silica gel was gently ground in a ceramic mortar and sieved with a nylon mesh sifter to obtain 100-200 mesh grains. After being cleaned with diluted hydrochloric acid, the silica gel (5 g) was silanized with 3-aminopropylethoxysilane (100 mL of 5 vol 5% ) in an aqueous solution acidified with acetic acid (pH 3-4) at 85 "C for 3 h. After being rinsed with MQW and methanol, the silanized glass (2 g) was benzoylated withp-nitrobenzoyl chloride (2 g) in chloroform (95 mL) containing triethylamine (5 mL) at 60 "C for 24 h. After being rinsed with chloroform and ethanol, successively, the product was dried at 60 "C for 12 h. The product (2 g) was reduced in sodium dithionite (5 wtivol % ) at 37 "C for 3 h. After being rinsed with MQW, the product (2 g) was diazotizated in 0.2 M acetic acid containing 5 wt/vol % sodium nitrite at 0 "C for 1 h. After being rinsed with cold MQW, the product was diazo-coupled with 8-quinolinol(l g in 50 mL of ethanol). The resulting chelatingresin was washed with 1M sodium hydroxide, MQW, 1 M hydrochloric acid, and MQW, successively, and was preserved in MQW. Procedure. The analytical procedure is shown by the flow chart in Figure 2. Unfiltered seawater was adjusted at pH 3.0 with 5 M formic acid-ammonium formate buffer solution (60 pL/lOO mL of sample solution). The sample solution was filtered through a 0.22-pm Fluoropore filter (Sumitomo Electric Ind., Ltd.) in the line and was passed through the chelating resin column for 4 min at a flow rate of 4.5 mL min-'. In this stage almost all of the other heavy metals, together with the sea-salt matrix, were separated. Then the column was rinsed with the cleaning solution (MQW), and after switching the solenoid valves, (18)Karatani, H. J T N PAT. No. 1642449. (19) Oka. S.: Tahara, S.; Minakuchi, H.; Karatani, H. U.S. PAT. No. 4897468
Metals to metal Tl(1) Bi(II1) Zn(I1) Cd(I1) Al(II1) Mn(I1) Fe(I1)
concn 1 mg L-'
intensitp
10rgL-'
0.050 0.025 0.0075 0.010 0.035 3.3
0.5 pg L-'
0.49
1 mg L-' 1mg L-I 1 mg L-' 1 mg L-'
Intensities are relative to the intensity of 1 fig L-' Fe(II1).
the eluent was passed through the column at a flow rate of 1.1 mL min-' in the reverse direction of the sampling. The eluent was mixed with each of the reagent solutions pumped at a flow rate of 1.1 mL min-I, and the mixture was introduced into the CL cell through the mixing coil. The iron was determined by measurement of CL intensity. The column was washed with 0.5 M hydrochloric acid after elution and washed with MQW to remove residual hydrochloric acid.
RESULTS AND DISCUSSION Interference of Other Elements to Chemiluminescence. The ions which are thought to interfere with the luminol-hydrogen peroxide CL of Fe(II1) are Cu(II), Co(II), Cr(III), Mn(II), Ni(II),and Fe(I1). Table I shows the relative CL intensities produced by these elements at indicated concentrations. The experiment was done by using only the CL system in the Figure 1. The concentration of each ion was much higher than that in natural seawater. Except for Cr(III), Fe(II), and Mn(II), almost all of the ions hardly catalyzed the luminol-hydrogen peroxide CL system at all. Co(II),which is as sensitive in the luminol-hydrogen peroxide CL system as iron, might be masked by the aqueous ammonia because Co(I1) forms a stable amine complex. Fe(I1) showed almost equal sensitivity to Fe(II1). Therefore, Cr(III), Mn(111, and Fe(I1) should be separated prior to the chemiluminescent reaction. Selection of Chelating Resin. In order to concentrate Fe(II1) and to separate interfering ions, we have tested many kinds of chelating resins both commercially obtained and synthesized by ourselves. Muromac A- 1(Muromachi Kagaku Co., Ltd.) and DDQ resins, (7-dodecenyl-8-quinolinol-impregnated Amberlite XAD-4,20Amberlite XAD-7, octadecyl silica, and CHP-20P (Mitsubishi Chemical Ind., LTD.)) could not be adopted due to iron contamination from the resin support when eluting with hydrochloric acid. This iron contamination could not be removed even after careful acid cleaning for a long time. Sumichelate MC-76 and MC-95 (Sumitomo Chemical Ind., Ltd.) could not be used because of physical and chemical drawbacks. Chelex-100 (Bio-Rad Laboratory) was relatively clean, however, but not usable for our purposes due to the swelling and contraction of the resin itself. TSK-8HQ'; was also relatively clean and could be used when the concentration of hydrochloric acid for eluent was less than 0.07 M. However, it gave significant iron blank when the concentration of hydrochloric acid was higher than 0.1 M. Furthermore, the functional group (ligand) of TSK8HQ could bleed from the support easier than that of MAF8HQ. This made the determination of Fe(II1) of low concentration impossible due to the masking effect of the bleeding functional group. MAF-8HQ resin exhibited the best result, that is, the lowest iron contamination and ease of elution with a weak acidic solution. As can be seen from extraction curves on Figure 3, Fe(1II) is quantitatively collected at a pH around 3.0, whereas Fe(I1) begins to be ~
~~
~
(20) Issiki, K.; Tsuji, F.; Kuwamoto, T.; Nakayama, E. Anal. Chem. 1987,59, 2491-2495.
PiNALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993
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I
I ‘ “t
. A
80 loo
I
I
i
Fe(ii) Bo 0
8
40
20
0.18 nM
I
p
o Fe(ll1) e
I
1527
Figure 4. Recording chart of measurement of Fe(II1).
l o 0
0
e
1
Table 11. Comparison of Total Dissolved Iron Values Determined by This Method and Certified Values for Trace Metal Standard Seawater Solutions. Fe concn
std soh
certified
this method
CASS-2 NASS-4
21.5 f 2.2 1.88 f 0.29
23.8 f 0.6 2.29 0.01
*
a National Research Council of Canada, Marine Analytical Chemistry Standards Program. Values are in nmol L-I f 95% confidence interval.
Calibration. The relative standard deviation was 1.2 % for five replicate measurements of a seawater sample containing 0.91 nmol L-l Fe(II1). The calibration curve was linear in the concentration range of 0.09-1.8 nmol L-l for a 4-min concentration. The detection limit was 0.05 nmol L-l in the case of an 18-mL sample (4-min concentration). A typical peak of CL intensity is shown in Figure 4. The background signal was subdued at a very low level which was equivalent to the peak hight for 0.1 nmol L-I Fe(II1). The detection noise was not observed when setting the sensitivity of chart recording to a usual scale [full scale was equivalent to the CL intensity for ca. 2 nmol L-l Fe(I1I)l. Also very low blanks were achieved. The blank values for purified seawater were equivalent to those for 0.05 nmol L-I Fe(II1). The value was employed as a detection limit of the method because peaks for standard samples of such a concentration level were almost indistinguishable from those for blanks. In addition, a negative peak seen before the positive CL peak was produced by the pH change of the solution sent into the CL cell because a small amount of pure water remained in the void volume of the extraction column and was sent prior to the acidic eluent. Accuracy. Accuracy of this method was ascertained by analyzingstandard reference seawater obtained from National Research Council of Canada, Marine Analytical Chemistry Standards The results are shown in Table 11. Good agreement was found between our values and the certified values for NASS-4 (North Atlantic Surface Seawater) and CASS-2 (Coastal Atlantic Surface Seawater). Seawater Analysis. This method was applied to the analysis of seawater samples collected during the cruise of R.V. Hakuho-Maru (The University of Tokyo), KH-90-3 (Nov 24-Dec 14,1990) at the Manus Basin, Bismarck Sea, where hydrothermal activity was observed.24 It was also applied to shipboard analysis of seawater samples collected during the cruise of R.V. Sohgen-Maru, SG-91 (Oct 5-22,1991) in the northern North Pacific Ocean. The vertical distribution of Fe(II1) in the Manus Basin is shown in Figure 5. The samples were acidified with 0.05 M hydrochloric acid and were preserved for about 10 months. The vertical profile exhibits a plume of highly concentrated Fe(II1) at a depth of around ~~
(21)Wang,J. H. J.Am. Chem. SOC.1955,77,4715-4719. (22) Nakayama, E.;Issiki, K.; Sohrin, Y.; Karatani, H. Anal. Chem. 1989,61, 1392-1396.
(23) Berman, S. S.; Sturgeon, R. E., Desaulniers; J.A.H.; Mykytiuk, A. P. Mar. Pollut. Bull. 1983,14 (2) 69-73. (24) Gamo, T.; Sakai, H.; Ishibashi, J.; Nakayama, E.; Issiki, K.; Matsuura, H.; Shitashima, K.; Takeuchi, K.; Ohta, S. Submitted for publication in Deep-sea Res.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993
Concentration I nM
Concentration I nM 0
PB
0.2
0.4
0.6
0.8
I
I
I
1
1000 L
2000 E
-. E
1
I
5
4 n
f g. 3000 n 4000
zooE
2500 Flgure 5. Vertical profiles of Fe(1II)and Mn(I1) in the eastern Manus Basin. Bismark Sea. 2500 m, where the anomalyof Mn(II), reflecting hydrothermal activity, is also observed.24 It is considered that the highly concentrated Fe(II1) seen over the whole water column is driven from particulate iron which may have dissolved during the preservation. Although samples were collected with conventional 10-L Teflon-coated Niskin bottles mounted on the CTD rosette sampler, the contamination from bottle wall was thought to be negligible since Fe(II1) concentration in these samples was 1-2 orders higher than that of ordinary oceanic waters. Figure 6 shows the vertical distribution of Fe(II1) in the northern North Pacific Ocean. Unfortunately, samples were collected with conventional 10-LTeflon-coated Niskin bottles attached directly to a steel armored cable because the CTD rosette sampler did not work well on board. Therefore, the samples should have been contaminated with iron from the cable and the bottles. However, the values of Fe(II1) obtained are comparable to those reported by Martin et al. (0-1.0nmol L-l),l,zand the vertical profile is nutrientlike although it fluctuates somewhat. The values for the surface water samples, which were collected with a contam-
5000
6000 Figure 8. Vertical profile of iron(II1) in the northern North Pacific Ocean (39' N, 155' E).
ination-free bellows pump, are 0.097 and 0.054 nmol L-la Consequently, this method is valid for the on-board determination of Fe(II1) in oceanic water. In addition, Fe(I1) in seawater can be automatically determined when an extra MAF-8HQ column for removing Fe(III),a peristaltic pump for sending a buffer solution which raises the pH of sample solution to a pH around 6, and a solenoid valve are attached to the flow systems shown in Figure 1. Current work aims at building a fully automatic Fe(I1) and Fe(II1) analyzer equipped with an autosampler (Kimoto Electric Co. Ltd.) for improved shipboard analysis.
ACKNOWLEDGMENT We wish to thank the scientific group and crew of R.V. Hakuho-Maru and R.V. Sohgen-Maru for their assistance during sample collection. We are grateful to Kimoto Electric Co., Ltd., for offering some tools and materials. This research was supported in part by the Ministry of Education, Culture and Science, Japan (04232101).
RECEIVEDfor review November 17, 1992. Accepted March 8, 1993.
