Anal. Chem. 1989, 6 1 , 499-503
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termination system, subsequent chemistry needed to be performed. In the aniline determination system, the overwhelmingly large optical absorption from neat benzene and the tendency of one phase or the other to adhere to the detector window made it impossible to reliably monitor the absorbance of the aqueous segment without phase separation. However, there may be situations where immiscible solvent elution can be performed without the need to isolate the segment. For example, an aqueous sample stream can be merged with the stream of a suitable ligand solution, e.g., 8-hydroxyquinoline, and the resulting chelate taken up on a nonpolar sorbent. The sorbed metal complexes can be later eluted by a plug of an immiscible nonpolar solvent and conducted to the nebulizer of an atomic sepctrometer. The advantage, compated to that of miscible stripping reagents, is the lack of dispersion in the inevitable conduit between the preconcentration system and the atomic spectrometer.
LITERATURE CITED Flgure 4. System output for the mercaptan system. R1 is 1 M NaOH; R2 is 1 M NaHCO, (see Figure 1). A: blank. B, C, D, E: C5, C4, C3, and C2 n-alkyl mercaptans, 1 mM each.
The aniline determination system provided linear response in the range 0-10 ng/mL, with a detection limit (3 times blank noise over blank, as in ref 19) of 0.2 ng/mL and a calibration slope of 8.5 mAU/(ng/mL). Repeat injections of the benzene passed through the Catex resin showed that aniline was quantitatively taken up by the cation-exchange resin. The response tended to saturate above 10 ng/mL; it is likely that the nonpolar phase does not penetrate into the interior of the water-swollen resin beads, and thus the capacity for uptake is limited to surface sites. It is likely that a decrease in the particle size of the resin and/or an increase in the microcolumn dimensions can be used to extend the upper dynamic range. It is noteworthy, however, that the captured aniline is quantitatively eluted by the 6 5 - ~ Lslug of 0.1 M H2S04 with the present microcolumn. The process of zone sampling (20),wherein any desired portion of a dispersed sample is isolated by timed actuation of a valve, is well-known in flow injection analysis (15). In the present case, the desired zone is the immiscible liquid segment, and obviously a timed actuation of V2 can also be used. Active sensing of the segment to actuate V2 is, however, potentially advantageous. The immiscible plug containing the eluted analyte is axially inhomogeneous. In a time-based segment isolation scheme, any fluctuations in the flow rate will result in a slightly different cut of the segment being sampled by V2. Even with only 25% of the segment being sampled by V2, as in the mercaptan system, acceptable precision (typically 2-3% RSD) is obtained with the active sensing approach. On the other hand, for cases where sensitivity is a major issue, the majority of the liquid injected by V1 can be successfully isolated by V2. The difference between V1 and V2 loop volumes for the aniline system is only 15 p L and can conceivably be reduced further. Isolation of the immiscible segment was necessary in both of the cases presented in this paper. In the mercaptan de-
(1) Oisen, S.; Pessenda, L. C. R.; Ruzicka, J.: Hansen, E. H. Analyst 1983, 108, 905-913. (2) Hartenstein, S. D.; Ruzicka, J.; Christian, G. D. Anal. Chem. 1985, 57, 21-25. (3) . . Hartenstein. S. D.: Christian, G. D.: Ruzicka, J. Can. J. Spectrosc. 1986, 30,144-148. (4) Hirata, S.; Umezaki, Y.; Ikeda, M . Anal. Chem. 1988, 5 8 . 2602-261 1. (5) Maiamas. F.; Bengtsson, M.; Johansson. G. Anal. Chim. Acta 1984, 160. 1-10.
(6) Fang, Z.;Xu, S.; Zhang, S. Anal. Chim. Acta 1984, 164, 41-50. (7) Fang, 2.; Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1984, 164, 23-39. (8) Miiosavijevic, E. 8.; Ruzicka, J.; Hansen, E. H. Anal. Chlm. Acta 1985, 169, 321-324. (9) Bengtsson, M.; Maiamas, F.; Torstensson, A,; Regneii, 0.: Johansson, G. Mikrochlm. Acta 1985, I I I . 209-221. (10) Storgaard Jargensen, S.; Petersen, K. M.; Hansen, L. A. Anal. Chlm. Acta 1985, 169, 51-57. (11) Bergamin Filho, H.; Reis, B. F.; Jacintho, A. 0.; Zagatto,E. A. G. Anal. Chlm. Acta 1980, 117, 81-89. (12) Martin, G. €3.; Meyerhoff, M. E. Anal. Chim. Acta 1988, 186. 71-80. (13) Chang, Q.; Meyerhoff, M. E. Anal. Chim. Acta 1986, 186, 61-90. (14) Audunsson, G. Anal. Chem. 1986, 58, 2714-2723. (15) Ruzicka, J.; Hansen, E. H. Flow Inlection Analysis, 2nd ed.; Wiley: New York, 1988. (16) Johnson, K. S.; Petty, R. L. Anal. Chem. 1982, 5 4 , 1185-1187. (17) Ellman, 0.L. Arch. Blochem. Biophys. 1959, 82, 70-77. (18) Title 13, Section 2252, California Code of Regulations, State of California, Sacramento, CA. (19) American Chemical Society Committee on Environmental Improvement. Anal. Chem. 1980, 52. 2242-2249. (20) Reis, B. F.; Jacintho, A. 0.; Mortatti, J.; Krug, FJ.; Zagatto, E. A.O.; Bergamin Filho, H.; Pessenda, L. C. R. Anal. Chim. Acta 1981, 123, 22 1-228.
Wei Lei Purnendu K. Dasgupta* Jorge L. Lopez Department of Chemistry and Biochemistry Texas Tech University Lubbock, Texas 79409-1061
Don C. Olson Shell Development Company Houston, Texas 77251-1380 RECEIVED for review September 27,1988. Accepted November 30,1988. This work was supported by Shell Research and Development Co., Houston, TX.
Anion-Selective Electrodes Based on a Hydrophobic Vitamin B, Derivative Sic Vitamin BI2is a rather complex molecule that contains the essential trace element cobalt. In the isolated vitamin, also known as cyanocobalamin, the cobalt has a coordination
number of six. The four equatorial coordination sites are occupied by the nitrogens of the corrin ring, the axial ligands being a cyano group and the dimethylbenzimidazole ribo-
0003-2700/89/0361-0499$01.50/00 1989 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 5, MARCH 1. 1989
n
U
4
R :-CH ~ - C H ~ - C ~ H S
Figure 1. Structures of vitamin B,, derivatives: (1) vitamin B,, (cyanocobalamin);(2) dicyanocobalt(II1)g-(3-aminopropyi)imidadazoiyl-a,b ,d,e ,f-pentapropyl-c-propionohctone cobyrinate; (3) aquocyanocobalt(1I I) a ,b ,d,e ,f,g-hexamethyl-c-octadecyl cobyrinate; (4) aquocyanocobalt(1II ) hepta(2-phenyiethyi)cobyrinate.
nucleotide part (proximal base) of the corrin ring of the vitamin (Figure 1). Aside from its biochemical importance, vitamin B12has remarkable ion-exchange properties that are governed by the nature of the cobalt coordination with the axial ligand(s) and the proximal base (1-3). Rather recently, Simon and co-workers used cobyric acid derivatives (cobyrinates) to prepare ion-selective electrodes (4-6) (see structures 3 and 4 in Figure 1). Cobyric acid consists of a corrin ring system coordinated with cobalt but it lacks the nucleotide part of vitamin B12that plays an important role in dictating the properties of the vitamin. These electrodes were found to be selective for nitrite and thiocyanate. Likewise, the groups of Meyerhoff (7,8) and Simon (9) have demonstrated independently that by incorporating porphyrins into liquid polymeric membranes it is possible to develop anion-selective electrodes. The selectivity of the electrodes reported by both research groups WBS dependent on the nature of the side chains and the central metal of the porphyrins. Even though cobyrinates and porphyrins associate selectively with certain ligands, their complexation behavior is different from that of vitamin B12(1,IO). In order to take advantage of the selective complexation properties of the latter in the development of ion-selective electrodes, a hydrophobic derivative of the vitamin is necessary. Murakami and coworkers have synthesized several such compounds and demonstrated that their behavior was similar to that of the natural isolated vitamin (11-14). One of these derivatives (Figure 1, compound 2) was kindly provided to us by Y. Murakami. In this correspondence, we report the development and
evaluation of poly(viny1 chloride) liquid membrane based anion-selective electrodes using the hydrophobic vitamin B12 derivative, 2, as the ionophore. The electrodes exhibit an anion selectivity pattern that clearly deviates from the Hofmeister series (15)and from that of the previously reported cobyric acid derivatives (4-6). Indeed, they are highly selective for iodide over a variety of anions. The response is Nernstian for the preferred anions and the electrodes are stable over at least a 2-month period of time.
EXPERIMENTAL SECTION Reagents. Chromatographic grade poly(viny1chloride) (PVC) was obtained from Polyscience (Warrington, PA). Bis(1-butylpentyl) adipate (BBPA, purum), and bis(2-ethylhexyl) sebacate (DOS, purum) were purchased from Fluka (Ronkokoma, NY). Tetrahydrofuran (THF), ethanol, acetic acid, and hydrochloric acid were obtained from Fisher Scientific (Cincinnati, OH). 2-(N-Morpholino)ethanesulfonic acid (MES), tris(hydroxymethy1)aminomethane (Tris), sodium salicylate, and all the inorganic salts were from Sigma Chemical Co. (St. Louis, MO). All standard solutions and the buffers were prepared with deionized (Milli-Q, Millipore Corp., Bedford, MA) distilled water. Apparatus. Voltages were monitored with a Fisher Accumet 810 digital pH/mV meter. This potential was recorded on a Linear (Model 1200) strip-chart recorder. A Fisher saturated calomel electrode was used as reference. UV-vis experiments were carried out with a Cary/AVIV 14DS spectrophotometer interfaced with an AT&T PC6300 personal computer. The spectrophotometer was equipped with a thermostated cell compartment set at 25.5 "C. UV-vis Studies. A 2.5 X mol L-' solution of compound
ANALYTICAL CHEMISTRY, VOL. 61, NO. 5, MARCH 1, 1989
2 was prepared in ethanol. A volume of 10.0 mL of this solution was placed in a round-bottom flask and concentrated acetic acid (enough to achieve a 10% (v/v) acetic acid solution) was added. The solvent was eliminated in a rotaevaporator (Buchi, RE 111) while heating at 45 "C. The residue was redissolved in 10 mL of a 10% acetic acid-thanol solution and the procedure described above was repeated for a total of three times. After the last treatment, the red residue (the color of the compound had changed from purple to red) was dissolved in 5.0 mL of ethanol. Four 1.0-mL aliquots were taken and each of them was diluted with 1.0 mL of different pH solutions: 0.100 mol L-' HC1; 0.100 mol L-' MES-NaOH, pH 5.50; 0.100 mol L-' MES-NaOH, pH 6.50; 0.100 mol L-' Tris-HC1, pH 9.00. The spectra of the untreated and treated compound at the different pH values were taken and these data were used to estimate the pKbase-off of the derivative. Membranes and Cell Assembly. The solvent polymeric membranes were prepared by dissolving 0.78 mg of the hydrophobic vitamin Blz derivative, 2,56 pL of BBPA or 55 pL of DOS, and 26 mg of PVC in 1 mL of THF. The solution was poured into a 16 mm i.d. glass ring and the solvent was allowed to evaporate at room temperature overnight (16). The resulting membranes were cut into small diameter disks and were positioned on a IS-561 Philips electrode body. The composition of these membranes was 1% (w/w) ionophore, 66% (w/w) plasticizer, and 33% (w/w) PVC. Blank BBPA and DOS membranes contained 67% (w/w) BBPA or DOS and 33% (w/w) PVC. All potentiometric measurements were performed with the following cell assembly: SCEIIKCl (saturated)Jlsamplesolutionlmembrane11.00 X mol L-' NaC1, 1.00 X mol L-' sodium salicylatelAg-AgC1 Since this electrode responds minimally to chloride, sodium salicylate was included in the internal f i g solution to yield stable internal solution-membrane interfacial potentials. Procedure. The calibration of the electrodes was carried out by adding, while stirring, aliquots of known concentrations of the different anion standard solutions to a beaker containing 20.0 mL of buffer. The buffers used were 0.100 mol L-' MES-NaOH, pH 5.50; 0.100 mol L-'MES-NaOH, pH 6.50; 0.100 mol L-' Tris-HC1, pH 7.40; 0.100 mol L-' Tris-HC1, pH 8.40; and 0.100 mol L-' Tris-HC1, pH 9.00. The response of the electrodes was measured by the pH/mV meter and it was registered by the strip-chart recorder. In this study, response time is defined as the time elapsed to reach 95% of the steady-statesignal after each addition. All measurements were performed at room temperature. When not in use, the electrodes were stored in deionized distilled water at room temperature and protected from the ambient light to prevent any possible photodecomposition of the ionophore. This was just a precautionary measure since we have observed no evidence of such an effect. To obtain the calibration curve of the electrodes, the data were plotted as the decrease in potential, AE (with respect to the base line), vs the logarithm of the activity of anion present in the buffered solution.
RESULTS AND DISCUSSION Vitamin Blz is an important cofactor involved in many biochemical processes. Its physicochemical properties have been extensively studied and they have been the subject of numerous review articles and books (1,17,18). Such studies include the effect of the proximal base and axial ligand(s) on the ion-exchange properties of the molecule. This information suggests that the vitamin associates preferentially with certain anions. Consequently, it appears feasible to develop anionselective electrodes using vitamin B1z derivatives. The derivative chosen for this study differs from the naturally occurring vitamin BIZin the peripheral propionamide and acetamide moieties that are replaced with five carboxylic ester groups and with a lactone. However, this derivative retains a proximal base (an imidazole ring) which provides the molecule with a distinct resemblance to the original vitamin (Figure 1). Murakami and co-workers (11-14) have performed extensive studies with this type of hydrophobic compounds and concluded that their properties are compa-
\y31
00
20 20
501
l -6.0
-5.0
-4.0
-3.0
-2.0
-1.0
Flgure 2. Anion-selectivity pattern of the BBPA-based vitamin B,, electrode. AWltions were made to a 0.100 mol L-' MES, pH 6.50. A€ corresponds to the change in potential from the base line due to the presence of a certain activity of an anion in the solution. The electrode was exposed to the sodium salts of perchlorate (3),salicylate (4), chloride (9),sulfate (lo),nitrate (7), nitrite (6), bicarbonate (5), phosphate (8),and iodide (1) and to the potassium salt of thiocyanate (2).
> E
W
a
,
V"
-7
-6
-4
-5 log
-3
-2
-I
aanion
Selectivity pattern of the DOS-based electrode immersed into a 0.100 mol L-' MES, pH 5.50 buffer. For a key to the numbers associated with each calibration curve, see the legend of Figure 2. Flgure 3.
rable to those of the naturally occurring vitamin. In view of the potential applications of this new derivative in ion-selective electrodes, we prepared several PVC-based liquid membranes. Two different membranes were tested by using the plasticizers BBPA and DOS. Their response was compared to that of blank membranes containing only PVC and plasticizer. The blank membranes showed negligible response to the anions to which they were exposed. On the other hand, experiments performed with the vitamin Blz membrane electrodes demonstrated a marked preference for some anions. Specifically, the electrodes were selective for iodide over the rest of the anions tested (Figures 2 and 3). The response toward iodide was Nernstian at a considerably low concentration range. Thiocyanate, perchlorate, and salicylate were the main interferent anions. Non-Nernstian response was observed with the rest of the anions to which the membranes were exposed. The plasticizer influences somehow the response of the electrodes. For instance, it is interesting to note that when DOS was used as the plasticizer, the detection limits for salicylate were better than the ones of the BBPA-based electrode, suggesting that with the latter plasticizer the interference of salicylate can be minimized. Although these two plasticizers have similar dielectric constants and the same functional groups (i.e., both are diesters), there is a difference in their size. Indeed, in DOS the two ester groups are sepa-
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 5, MARCH 1, 1989
0.52
2
base-off
11
Kbose-off
PH Flgure 5. Effect of pH on the maximum absorbance of the y-region of the vitamin BIZ derivative in 50% ethanol, 50% buffer. base-on
Figure 4. Schematic representation of the effect of pH on the coordination properties of the vitamin derivative.
rated by eight methylenes, while only four methylene groups separate the two ester moieties of BBPA. Moreover, in a separate investigation with an electrode based on aquocyanocobalt(II1) heptapropyl cobyrinate, an ionophore with a structure similar to 4,it was demonstrated that the detection limits for salicylate were worse when BBPA was used as the plasticizer (19). Therefore, it appears that even this small difference in the structure of the plasticizer may be responsible for this effect on the response of the electrode to salicylate. Further, it is evident from these figures that when DOS was used as the plasticizer, the range where the electrode showed Nernstian response for iodide was wider than when the plasticizer was BBPA. Several conditions for the storage of the electrodes were evaluated. It was found that the best results were obtained when the electrodes were kept at room temperature, dipped in water, and protected from ambient light. Indeed, the standard deviation of the starting potential (i.e., potential of the cell before any additions of anions) was
E w
log Qsolicykte
F I g m 6. Calibration curves of the BBPA-based vitamin B,, electrode for salicylate in different pH buffers: (0)0.100 mol L-' MES-NaOH, pH 5.50; (A)0.100 mol L-' MES-NaOH, pH 6.50; (0)0.100 mol L-' Tris-HCi, pH 7.40; (0)0.100 mol L-' Tris-HCI, pH 8.40; and ).( 0.100 mol L-' Tris-HCI, pH 9.00.
branes has been well documented by Horvai et al. (22). The dicyanocobyrinate, 2, was hydrolyzed in the presence of 10% acetic acid to remove one of the cyano groups and yield the base-off derivative. This was accompanied with a shift of the absorbance band of the vitamin at 588 nm (characteristic of dicyanocobyrinates) toward lower wavelengths. Then, the spectrum of the aquocyano derivative was recorded at various pH values. A gradual decrease in the maximum absorbance of the 7-band was observed with increasing pH (Figure 5 ) . At the same time the peak at the y-region shifted from 353.5 nm at pH 0 to 357.5 nm at pH 9.00. Assuming that the absorbance at pH 0 is due solely to the base-off form and at pH 9 to the base-on vitamin, an apparent can be estimated from the midpoint of the spectral change (23);this value is around 5.7. Furthermore, the pKbasePoff can be calculated by using the absorbance values at the ,A, of the y-region of the spectra and the following equation, as suggested by Brown et al. (21):
Ax, ABH,and A B are the values of the absorbance of the compound at a given pH, at pH 0 (the base is in its acidic form), and at pH 9.00 (the base is in its basic form), respectively. By application of the above equation to both pH 5.5 and 6.5, a p K ~ , ~ was ~ . ~estimated f~ at 5.6. In a separate investigation the response of the electrode at different pH values was studied (Figure 6). From this figure, it is evident that at higher pH the starting potential shifts
ANALYTICAL CHEMISTRY, VOL. 61, NO. 5, MARCH 1, 1989
to more negative values with a concomitant decrease in the slope of the electrode response. Very little difference was observed between pH 5.5 and 6.5. The reason for both these observations is that at high pH there is an increased OHinterference along with the fact that the base is on. Having the base on is equivalent to decreasing the concentration of the ionophore in the membrane. Indeed, it was observed in our experiments that membranes that were loaded with less amount of ionophore had a more negative starting potential and worse slopes. It should be mentioned that the response of electrodes based on ionophore 3 is also being affected by pH changes (5);this effect was also explained by OH- interference. Even though the pH of the external aqueous solution should not be the same as the pH in the bulk of the membrane, the above studies indicate that there should be a pH effect at least at the membrane-external solution interface. Indeed, such a pH effect has been implicated in the studies of Umezawa et al. (24) to explain the mechanism of response of an ATPselective electrode; a macrocyclic polyamine served as the ionophore in this electrode. The hydrophobic derivatives of cobyric acid based electrodes (4-6) show a selectivity pattern quite different from the one that we are reporting here for the hydrophobic vitamin B12based electrode. While the former ones respond preferentially to thiocyanate and nitrite, we observed low nonlinear response for nitrite, especially when BBPA was used as the plasticizer. On the other hand, our electrodes demonstrated Nernstian response to thiocyanate and are more sensitive for iodide than for any of the other anions to which they respond. The different behavior between these two types of electrodes might be explained by the differences in the molecular structure of the corresponding ionophores. A proximal base and the lactone ring of ow vitamin BI2derivative are the main structural differences between this and the cobyric acid derivatives. Possibly, both factors play an important role in the physicochemical properties of the compound and, ultimately, in the behavior of the electrodes prepared with it. As previously discussed, this makes the molecule resemble more closely the naturally occurring vitamin and makes it more distant to the cobyric acid derivatives. The selectivity for iodide observed with these vitamin B12 electrodes is much higher than the one that was reported for iodide with the cobyric acid based electrodes (4-6). We have attributed this to the large size of iodide which may enable the ion to interact with both the positive centers on the ionophore. Indeed, when the vitamin is at ita base-off form, the imidazole ring is protonated. Therefore, the iodide may interact with both the positively charged nitrogen and the cobalt. The size of the iodide may be just right to afford such an interaction. In conclusion, this report demonstrates that hydrophobic derivatives of vitamin B12can, indeed, be used to prepare new
503
functional anion-selective electrodes. The response of these electrodes is due to complexation between the positively charged Co(II1) and anions when the base of the vitamin is off.
ACKNOWLEDGMENT The authors thank Yukito Murakami (Kyushu University, Japan) for the donation of the vitamin B12derivative used in this study. LITERATURE CITED (1) Pratt, J. M. Inorganic Chemishy of Viramln 8,2;Academlc Press: New York, 1972. (2) Thusius, D. J. J . Am. Chem. Soc. 1971, 93, 2629-2635. (3) Haslnoff, B. B. Can. J . Chem. 1974, 52. 910-914. (4) Schulthess, P.; Amman, D.; Slmon, W.; Caderas, C.; Step&” R.; Kriutler, B. Helv. Chim. Acta 1984, 67, 1026-1032. (5) Schulthess, P.; Amman, D.; Krautler, B.; Caderas. C.; StepPnek. R.; Simon. W. Anal. Chem. 1985. 57, 1397-1401. (6) Steplnek, R.: KGutier, E.; Schulthess, P.; Llndemann, B.; Amman. D.; Simon, W. Anal. Chlm. Acta 1988, 182. 83-90. (7) Chaniotakis. N.; Meyehff, M. E. Anal. Chem. 1988, 60, 185-188. (8) Chang. Q.; Meyerhoff. M. E. Anal. Chim. Acta 1988. 186. 81-90. (9) Amman, D.; Huser, M.; Kriutler, B.; Rusterholr, B.; Schulthess. P.; Llndemann, B.; Hakler, E.; Slmon, W. Helv. Chim. Acta 1986, 69, 849-854. - .. - - .. IO) Hambright, P. I n Porphvrns andMetallopwphyrlns; Smith, K. M., Ed.; Elsevier: New York: 1976: DO 233-278. 11) Mwakami, Y.; Hlsaeda. Y:;’Ozaki, T. Chem. Lett. 1985, 473-476. 12) Murakami, Y.; Hlsaeda, Y.; Ohno. T.; Ozaki, T. Chem. Lett. 1985, 477-480. 13) Murakaml, Y.; Hisaeda, Y.; Dzaki, T.; Ohno, T. Chem. Lett. 1985, 1711-1714. (14) Murakami, Y.; Hisaeda, Y.; Ohno, T.; Matsuda, Y. Chem. Lett. 1988. 731-734. (15) Hofmeister, F. Arch. Exp. Pathol. phermakol. 1888, 2 4 , 247, (16) Moody. G. J.; Thomas, J. D. R. I n Ion-selective ElectrodeMeihoddcgy; Covlngton, A. K., Ed.; CRC Press: Boca Raton. FL; 1980; pp 111-130. (17) hioldlng, 8. T. I n Comprehenslve Organic Chemkfry; Barton, D., Ollis, W. D.. Eds.; Pergamon Press: New York. 1979; Vol. 5, Chapter 24.4. (18) Murakaml, Y. A&. Chem. Ser. 1980, No. 791, 179-199. (19) Daunert, S.; Witkowskl, A.; Bachas, L. G., unpublishedresults, University of Kentucky, Lexington, KY. 1988. (20) Bax, A.; Marzllll, L. G.; Summers, M. F. J. Am. Chem. SOC. 1987. 109, 566-574. (21) Brown, K. L.; Hakimi. J. M.; Nuss, D. M.; Montejano. Y. D.; Jacobsen. D. W. Inorg. Chem. 1984, 23. 1463-1471. (22) Horvai, G.; GrBf, E.; T6th, K.; Pungor, E.; Buck, R. P. Anal. Chem. 1988, 58, 2735-2740. (23) hdd, J. N.; Hogenkamp, H. P. C.; Barker, H. A. J. Bioi. Chem. 1961, 296. 2114-2118. (24) Umezawa, Y.; Kataoka, M.; Takaml, W.; Kimura, E.; Koike, T.; Nada, H. Anal. Chem. 1988, 60, 2392-2396.
Sylvia Daunert Leonidas G. Bachas* Department of Chemistry and Center of Membrane Sciences University of Kentucky Lexington, Kentucky 40506-0055 RECEIVED for review August 24,1988. Accepted November 23, 1988. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of this research.
