Anal. Chem. 1QQO. 62. 1737-1746
retro-aldol reactions, assumed to take place prior to ionization, in this case by attachment of an alkali-metal ion to desorbed neutral species. This mechanism can account for most of the observed fragmentation of sugars and their natural derivatives. The model can also be used to explain the observed reduction of fragmentation for permethylated and peracetylated sugars and for 1 1 linked sugars, since both involve the absence of a free hydroxyl group on the anomeric carbon and prevent the type of ring opening what would lead to additional ring fragmentation via a retro-aldol mechanism. Differences in fragmentation have been observed for different isomers of oligosaccharides. 1 -.6 linked pyranoses can be distinguished from their 1-.4-linked isomers by an additional fragment ion. fl-homers, on the other hand, show a distinctive loss of water from the parent molecule and specific fragments, while the a-anomers in general do not. IR laser desorption mass spectrometry provides a distinctively different and complementary approach to the structural analysis of carbohydrates and glycoconjugates, since fragmentation occurs primarily in the sugar rings rather than at the glycosidic bond. It has been an invaluable tool for the analysis of glycolipids such as lipid A, where ring fragmentation can be used to reveal the specific locations of fatty acyl groups. While such fragmentation has been reliable and predictable, the current study helps to establish with some confidence the mechanisms that give rise to the cleavages observed. An understanding of these mechanisms can, in turn, be applied to unsubstituted oligosaccharides and will be invaluable for the elucidation of the structures and linkages of complex carbohydrates containing several branches.
mann. C. J., McGinnis. G. D., Eds.; CRC Press; Boca Raton, FL, 1989; pp 27-41. Carpita, N. C.; Shea, E. M. I n Analysis of Carbohydrates by GLC and MS; Biermann, C. J., McGinnis, G. D., Eds.; CRC Press: Boca Raton, FL. 1989;pp 157-216. Reinhold, V. N.; Carr, S. A. Mass Spectrom. Rev. 1983, 2, 153-221. Dell, A.; Tiller, P. R. Biochem. Biophys. Res. Commun. 1988, 135, 1 126-1 134. Cai: S. A.;Reinhold, V. N.; Green. E. N.; Haas, J. R. B/omed. Mass Spectrom. 1985, 12,288-295. Coates, M. L.; Wilkins, C. L. Biomsd. Mass Spectrom. 1985, 12,
-
LITERATURE CITED (1) Hakamori, S.-I. J. Biochem. (Tokyo) 1984, 55,205-208. (2) Biermann, C. J. I n Analysls of Carbohydrates by OLC and Ms; Bier-
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424-428.
Coates. M. L.; Wilkins. C. L. Anal. Chem. 1987, 59. 197-200. Takayama, K.; Qureshi, N.; Hyver, K.; Honovich, J.; Cotter, R. J.; M a 5 cagni, P.; Schneider, H. J. Bid. Chem. 1988, 261, 10624-10631. Cotter. R. J.; Honovich. J.; Qureshi, N.; Takayama, K. Blomed. Envlron. MSS Spectrom. 1987, 14,591-598. Lam, 2.; Comisarow, M. E.; Dutton, G. 0. S.; Weil, D. A.; Bjarnason, A. Rapid Commun. Mass Spectrom. 1987, I , 83-87. Lam, 2.; Comisarow, M. B.; Dutton, G. S. Anal. Chem. 1988, 6 0 ,
2304-2306. Qureshi, N.; Honovich, J. P.; Hara, H.; Cotter, R. J.; Takayama, K. J. Biol. Chem. 1988, 262,5502-5504. Martin, W. B.; Silty, L.; Murphy, C. M.; Raley, T. J., Jr.; Cotter, R. J.; Bean, M. F. Int. J. Mass Spectrom. l o n Processes 1989, 92,
243-265. VanBreemen, R. B.; Snow, M.; Cotter, R. J. I n t . J . Mass Spectrom. Ion Phys. 1983, 49,35-50. Otthoff, J. K.; Lys, I.; Demirev, P.; Cotter, R. J. Anal. Instrum. 1987,
16,93-115. Domon, B.; Costello, C. E. GlycoconjugateJ. 1988, 5 . 397-409. Van der Peyi, G. J. Q.; Isa, K.; Haverkamp, J.; Kistemaker, P. 0. Org. Mass Spectrom. 1981, 16, 416-420. Tabet. J.-C.; Cotter, R. J. Anal. Chem. 1984, 56, 1662-1667. Koell, P.; Steinweg, E.; Meyer, B.; Metzger, J. Llebigs Ann. Chem. 1982, 6 , 1039-1051.
RECEIVED for Review January 4, 1990. Accepted April 16, 1990. This work was supported by a grant (BBS 86-10589) from the National Science Foundation. B.S. was supported by a Deutsche Forschungsgemeinschaft Fellowship. Research was conducted a t the Middle Atlantic Mass Spectrometry Laboratory, an NSF-supported Regional Instrumentation Facility.
Determination of Complexing Ability of Natural Ligands in Seawater for Various Metal Ions Using Ion Selective Electrodes Takashi Midorikawa,*Eiichiro Tanoue, and Y ukio Sugimura Geochemical Laboratory, Meteorological Research Institute, Nagamine 1 - 2 , Tsukuba, Ibaraki 305, Japan
A newly developed method Is presented for the measurement of the aMllty of organic ligands In seawater to form complexes wlth metal ions. Organlc ligands (nominal molecular weight 2 1000) are concentrated from seawater and desalted by lyophlllzatlon and dialysis. The concentrated solution of natural ligands Is electrodialyzed wlth ethylenediaminetetraacetic acid to remove metal Ions. The demetallzed ligands obtained In thts way are tltrated wlth a metal ion to determine the complexhg abliky of the natural ligands. The advantages of this method are as follows: the formation of a complex between organic ligands and a speclflc metal Ion can be studied wlthout consideratlon of simultaneous side-reactlons; samples from dlfferent sources (sallne and freshwater, blologlcal, sedimentary, etc.) can be compared on the same basis; repeated measurements can be made wlth the same sample afler removal of the exogenously added metal ions. The abiilty of natural ligands In seawater to form complexes with copper( 11) and cadmium( 11) is discussed.
INTRODUCTION During the past 3 decades, increased attention has been paid to metal-organic associations in natural aquatic systems. The ability of dissolved organic matter to form complexes with metal ions in natural water is of interest because of the associated biological implications, such as the bioavailabdity and toxicity of metals to living organisms (1,2), and because of its relevance to efforts at understanding geochemical cycles of metals in the environment (3). In marine and, especially, in oceanic environments, the concentrations of metals and of organic complexing ligands are extremely low. Moreover, seawater contains high levels of inorganic salts with a complex composition. Therefore, analytical difficulties are inherent in efforts at measuring the ability of dissolved organic matter to form complexes with metals in natural seawater. Two general techniques have been used in attempts to determine the complexing ability of natural ligands in sea-
0003-2700/90/0362-1737$02.50/0@ 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
water. The first approach involves the separation of organic and inorganic forms of metals, for example, by means of liquid-liquid extraction or adsorption on synthetic resin or some other adsorbent. Such methods have been reviewed in detail (4-7). This approach is convenient for isolation of metalorganic complexes from relatively large volumes of seawater. However, shifts of prevailing equilibria, induced by the separation and concentration steps, may occur and they may be difficult to quantify (5). Thus, there is no general agreement whether the metal-organic complexes separated from seawater are indigenous or artifactual. Such procedures also do not circumvent the risk of chemical and biological denaturation of the isolated ligands during the process of separation. The second approach involves the characterization of natural ligands by determining their concentrations and stability constants for formation of complexes with metals, usually by electrochemical techniques. Although improved voltammetric techniques have been developed (8, 9),the sensitivity of many electrochemical techniques is not sufficient for the determination of electroactive metal ions that occur at the low levels in nature, especially in the presence of strong chelators or of relatively high concentrations of chelators. Even if such a technique, utilizing the second approach, has adequate sensitivity, the stability constants measured for the natural ligands are essentially "conditional" (10). When the direct metal-titration method is applied to samples of seawater, numerous side-reactions need to be considered. First, the complexation of metal ions with inorganic anions occurs and should be taken into account by using numerical values from literature. In this case, a certain degree of uncertainty always remains ( I I , 1 2 ) . Second, in the case of organic ligands, except for those species that are protonated, two major types of side-reactions with coexistent ions may occur: complexation with major cations, such as Ca(I1) and Mg(II), which can bind ligands weakly but whose concentrations are so high that their side-reactions may be significant; and complexation with minor heavy metal ions to form stable complexes. Both sets of side-reactions involving organic ligands lower the apparent stability constants, and these effects cannot be excluded by manipulation of data alone because of the lack of information about these complexes. In particular, the latter type of side-reaction may further complicate the interpretation of the measured stability constant for the following reasons: ligands that have been already masked by ambient metal ions in seawater will not be detected; and the metal-exchange reaction between exogenously added metal ions and other precomplexed metal ions will occur (13-15) when larger amounts of metal ion are added than the amount present in situ in seawater. In the case of freshwater from various sources, the measurement of the complexing ability of organic ligands by direct metal titration is also affected by anions, heavy metal ions, and cations, such as Ca(I1) and Mg(II), which are present at varying concentrations in rivers and lakes (16-18). Thus, the measured values cannot be attributed exclusively to the properties of the natural ligands themselves; the values represent the contribution of analytical conditions, such as temperature, ionic strength and pH, as well as that of ions already present in the sampled water, as mentioned above. Therefore, it is hard to compare directly values obtained from different environmental samples and/or under different analytical conditions. Equilibrium dialysis has been applied to the determination of the complexing capacity of commercially available humic acid (19), soil-derived fulvic acid (20, 21), and freshwater samples (17). Although, in this method, chemical equilibria are not disturbed, the results are still influenced by the coexisting ions (17).
Table I. Characteristics of Seawater Samples siten
depth, temp, salinity, oxygen, oxygen m 'C k mL/L saturation, % pH
S
0
P
0 191
J
0
523 1071
16.9 18.9 4.1 20.9 0.46 0.20
34.59 32.95 33.77 33.57 34.06 34.06
5.6 4.7 5.2 5.5 4.9
105 64 102 69 61
8.18 7.76 -
'Site S: at 34'56' N, 138'41' E on Apr 26, 1988. Site P: at 41'32' N, 147'00' E on Aug 13, 1987. Site J: at 44'15' N, 130O58' E on Aug 26, 1987. Previous studies on metal-organic ligand interactions in natural waters have focused almost entirely on the complexation of copper. Little information is available on the interactions between natural ligands and other biologically and geochemically important metals. This is especially true for studies of ocean waters (22-24). The reason for this paucity of data is that it is difficult to determine the ability of natural organic ligands to form complexes with metal ions. For example, Cd(I1) forms organic complexes with low stability, whereas it interacts strongly with chloride ions that are present at high levels in seawater. Furthermore, the interactions of Cd(I1) with some ligands are hindered by other metals that bind the ligands more strongly. We report in this paper a newly developed method for the determination of the ability of natural ligands in waters from various environments to form complexes with various metal ions. The procedures are designed to exclude any interference by ambient and contaminating metal ions or side-reactions. The results obtained from an examination of the interactions of natural ligands in seawater with Cu(I1) and Cd(I1) are discussed.
EXPERIMENTAL SECTION Materials and Reagents. Seawater samples were collected at four locations as follows: during the cruise of M.S. Kofu-Maru, from the Hakodate Marine Observatory, in the western North Pacific (site P: 41'32' N, 147'00' E) and in the Japan Sea (site J: 44'15' N, 130'58' E) in August 1987; during the cruise of M.S. Ryofu-Maru,from the Japan Meteorological Agency, in Surgua Bay (site S: 34'56' N, 138'41' E) and in the western North Pacific (site B: 31'35' N, 145'25' E) in April 1988. Seawater was collected with a 30-L, nonmetallic Niskin sampler and filtered through a membrane filter (Millipore HA, 0.45 pm pore size) immediately after sampling. The samples were frozen (ca. -20 "C) and stored until use. Characteristicsof seawater samples are listed in Table I. Analytical grade reagents were used unless otherwise noted. All solutions were prepared with water that had been deionized by passage through a Millipore Milli R/Q water purification system. The solution of metal ions for metal titration was prepared from the nitrate of each metal and standardized against a 0.01 M solution of disodium EDTA (ethylenediaminetetraacetic acid). Contamination by metal ions caused by the addition of reagents during the preparation of the sample solution is a serious problem in the measurement of complexing ability. Four kinds of commercially available KNO, (high grade) were tested. In the case of the titration of Cu(1I) to a solution of KN09, using a Cu(I1) ISE (ion selective electrode),KNO,, Merck Suprapur grade, gave the lowest electrode-potential reading at low levels of Cu(II) and Nernstian response down to the lowest level of Cu(I1) in the calibration curve. KN03was further purified by recrystallization to eliminate a possible difference among manufactured lots. It is expected that other purification techniques, such as solvent extraction, ion exchange, adsorption, etc., would introduce other contaminants during the procedures. We consider that further purification of the salt is not necessary provided that ISEs are used as detectors in the metal-titration method because consistently good performance of ISEs was obtained, as defined in
ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990 SEAWATER
1 I
Filtration
Millipore HA filter ( 0.45 prn )
Lyophilization Spectrapor 6 ( MWCO
1000 )
CONCENTRATED soln.
1-
ML+Y *L+MY
1
Dialysis Electrodlalysis 1000 v
1 Removal 1
Of
MY
DEMETALLIZED soln.
-
CI
11
-
wl1
The complexing ability of the demetalized solution was determined by metal titration using the ISEs. The titration was carried out in a buffer that consisted of 0.7 M KNOBand 0.02 M EPPS to maintain a constant ionic strength and a pH of 8.15 f 0.05 at a temperature 25.0 0.1 "C. The EPPS buffer system was used for the titration for'the following two reasons. The protonation constant, log KHA(pK,) of EPPS is 8.0 (similar to a pH of seawater). The complexing ability of EPPS with heavy metals is negligible. The well-known buffers with pK, values near 8.0, such as Tris and triethanolamine, are not suitable for complexation measurements because they have the ability to complex with heavy metals to some extent and would complicate the interpretation of the measurements. The demetalized solution was diluted to 10 mL with the buffer solution and titrated with the solution of metal ions, dispensed with Gilson Pipetman variable micropipets. In the copper titrations, Cu(I1) was added incrementally, typically starting at a concentration of M and continuing to M. The range of concentrations of added Cd(I1) was from to lo4 M. Potentiometric measurements were made with an Orion voltmeter, Model 811, equipped with an Orion Model 94-29 Cu(I1) ISE and a Cd(I1) ISE (Denki Kagaku Keiki Co.; DKK type 7120), respectively. The reference electrode was a double-junction electrode (Orion Model 90-02) with 10% KN09 in the outer chamber. Prior to every experiment, the ISEs were conditioned as follows: they were polished with Orion polishing strips on the day before the measurement; they were rinsed in deionized water, with stirring, up to the time of measurement; and the potential of a solution of 0.7 M KNOBwas checked. Calibration of the ISE was carried out in standard solutions of the metal immediately after every measurement. During the titration, the membrane of the ISE was not coated with any deposits as evidenced by the fact that the Nernstian response was obtained reproducibly, even without polishing, after measurement of sample. This reproducibility indicates that organic ligands in the sample solution apparently did not adsorb on to the surface of the membranes of the ISEs. The response time of the selective electrode was usually between 20 min and 2 h, as checked by the time taken to reach equilibrium. During measurements, solutions were stirred with a magnetic bar coated with Teflon, at a constant rate under constant illumination. Tank nitrogen, passed through solutions of both KOH and H$04, was used to purge COz from the sample solution. The measurements of pH were made with an Orion voltameter, Model 811, equipped with an Orion Model 91-02 combined pH electrode. Calculations. We applied the discrete-ligands model of Dzombak et al. (28),based on a 1:l stoichiometry in the metalligand complexation, and used the linear transformation method of Ruzic (29)to calculate the total concentrations (C,) of natural ligands and the conditional stability constants (KIML)of the complexes with metal ions. For each metal titration, the total concentration of metal ion, CM,was calculated and the concentration of free metal ion, [MI, was obtained from the measured electrode potential ( E ) of the ISE. The negative logarithms to the base 10 of the metal concentrations are expressed as pCM and pM, respectively. The concentration of metal bound to ligand, [ML], was obtained by mass balance, i.e.
*
FILTPATE
Dialysis
1739
I
ionlc strength : 0.7 M pH 8.15. 25.C
Metal Titratlon ion Selective Electrode
Flgure 1. Schematic representation of the procedure. For abbrevi-
ations, see text.
the manuals of the ISEs (25,26). In the case of EPPS (N-(2-hydroxyethyl)piperazine-N'-3propanesulfonic acid; log = 8.0), removal of impurities that might complex with metal ions was rather more important than the possible presence of other impurities, such as metals. Four kinds of commercially available EPPS were tested. EPPS, purchased from Aldrich Chemical Co., gave the best result when EPPS was purified twice by repeated precipitations from a mixture of water and ethanol. After repeated purifications, the Nernstian response, which was similar to that of the nonbuffered solution, was also obtained at pH 8.15, as mentioned below. All glassware was siliconized to prevent the possible adsorption of organic ligands onto the glass. Spectrapor 6 dialysis tubing with a 1000 molecular weight cut-off (mwco) (Spectrum Medicine Industries, Inc., Los Angeles, CA) was used for both the desalting and the demetalization steps. The dialysis tubing was cleaned prior to use by a modification of the procedure of Richmond et al. (27). The tubes were rinsed and soaked in deionized water to remove the preservatives. They solution of disodium ethylenediwere treated with a 3 X aminetetraacetate for 60 min at room temperature, treated with 0.05% (w/v) sodium carbonate twice for 30 min at 60 "C, soaked in warm deionized water, washed with 95% ethanol for 30 min at 60 "C, soaked in 0.1 N H2S04for 5 min at room temperature, and rinsed well with deionized water. After each washing step, the tubes were washed with deionized water. They were also rinsed with deionized water just before use. Procedures. Our technique involves three separate steps (Figure 1): (1)concentration and desalting of natural ligands from seawater; (2)removal of metals bound to isolated natural ligands; and (3) measurement of complexing ability. Isolation of natural ligands in seawater was carried out by lyophilization and desalting in dialysis tubing. Approximately 5 L of the seawater sample was divided into five portions and then each portion of seawater (1L) was lyophilized to reduce the volume to 200-300mL. After addition of diethyl pyrocarbonate (to a final concentration of 0.1%) as disinfectant, the concentrated solution was dialyzed for 4 days at 4 "C against 5 L of deionized water with two changes. After a second lyophilization, the concentrated solution of each aliquot was combined and was further dialyzed against deionized water. Five liters of seawater was concentrated to a final volume of a few milliliters by this procedure. Removal of metals bound to natural ligands in the concentrated solution was performed by the ligand-exchange reaction using excess EDTA. After addition of 2 mL of 0.1 M EDTA (adjusted to pH 8 by addition of KOH) and 10 pL of diethyl pyrocarbonate, the solution was equilibrated by stirring for 24 h at 4 "C. The equilibrated solution, the volume of which was typically 6-7 mL, was dialyzed for 1day at 4 "C against 5 L of deionized water, with one change, and then subjected to electrodialysis at 1000 V,for 2 days at 4 "C, against 1L of deionized water with three changes.
where (YM is the side-reaction coefficient for the metal ion, determined as mentioned below. The total concentration of the natural ligand and the conditional stability constant of its metal complex were obtained by plotting [M]/[ML] as a function of [MI. Data limited to the range of pCMover which the Nernstian response was obtained for the buffered solution (pH 8.15, EPPS) were used for the analysis of metal titrations. Chemical Analyses. For examination of the reproducibility and extents of desalting and demetalization, the concentrations of some metals in the solution inside the dialysis bag were monitored at each step during the procedure. In these experiments, 500 mL of coastal seawater (site S) was typically used. At the first dialysis step, about 50 mL of concentrated seawater was dialyzed against 5 L of deionized water, with seven changes over 25 days, in Spectrapor 6 dialysis tubing with a flat width of 45 mm. At the second dialysis step, 6 mL of concentrated solution was dialyzed against 5 L of deionized water, with nine changes
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over 15 days, in dialysis tubing with a flat width of 12 mm. Electrodialysis and demetalization were performed, as mentioned above, in dialysis tubing with a flat width of 12 mm. For the measurements of alkaline metals, the entire solution inside the dialysis bag was concentrated to 1 mL and analyzed by flame spectrophotometry (Hitachi, Model 170-50A). For the measurements of other metals, the entire solution in the bag was digested with HNOB and HClO,, diluted to 0.2-0.5 mL, and subjected to analysis by atomic absorption spectrophotometry (Hitachi, Model 170-50A). Quantification of total carbohydrate and amino acids in the dialyzed samples was performed. The fluorometric method (30) was used for determination of amino acids after hydrolysis by 6 N HCl for 24 h at 105 OC. For determination of total carbohydrate, samples were added with H$04 (72% (v/v)) and allowed to stand for 3 h at room temperature. The solution in sulfuric acid was first diluted to 1 N by the addition of deionized water and then incubated for 24 h at 105 OC. An aliquot of the hydrolysate was used for determining total carbohydrate by the phenol sulfuric acid method (31).
-2
change of external sotutlon
0 J
e e
0
- 3 8
4
0
12
Time o f Dialysis
16 (
hr )
Flgure 2. Time course of changes in the concentration of EDTA (Cy) inside the dialysis bag. The values (0)are calculated from concentrations of EDTA in the external water, as determined with an ISE. The
direct determinations of concentrations of EDTA in the bag (0) agree well with calculated values.
RESULTS AND DISCUSSION The demetaliition of natural ligands isolated from seawater can be described by the following equation:
ML
+ Y _f
