Anal. Chem. 1989, 67, 1169-1171
when the starting estimate for the single missing value is zero. It is easily proven that for any starting point there will be convergence and the rate of convergence is equal to the leverage value. However, sinice convergence for the whole class of EM methods has already been proved, there is no reason to prove it for each individual case. The procedure used for chromatographic data with a multiple wavelength detection system (8, 9) may also be viewed as a subset of the EM algorithm with the M step. But rather than using the maximum likelihood principle, the knowledge of nonnegativity is inserted in the M step to zero all negative values.
LITERATURE CITED (1) Brayden, T. H.; Poropatic, P. A,; Watanabe, J. L. Anal. Chem. 1988, 6 0 , 1154. (2) Mallnowski, E. R.; Howery, D. G. Factor Analysis in Chemistry; Wiley: New York, 1980. (3) Golub, G. H.; Van Loan, F. Matrix Computations; John Hopkins University Press: Baltimore, MD, 1983. (4) Wold, S.; Slostrom, M. J. Chemom. 1987, 1 , 243. (5) Hoaglin, D. C.; Welsch, R. E. Am. Statist. 1978, 3 2 , 17-22. (6) Weisberg, S. Applied Linear Regression: Second Edition; Wiley: New York, 1985. (7) Lorber. A. Anal. Chem. 1984, 5 6 , 1004.
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(8) Gemperline, P. J. J. Chem. Inf. Comput. Sci. 1984, 2 4 , 206. (9) Vandeginste, B. G. M.; Leyten, F.; Gerritsen, M.; Noor, J. W.; Kateman, G.;Frank, J. J. Chemom. 1987, 1 , 57. (IO) Little, R. J. A.; Rubin. D.B. Statistical Analysis with Missing Data; Wiley: New York, 1987. (11) Dempster, A. P.; Laird, N. M.; Rubin, D. B. J. R. Statist. SOC.B 1977, 39. 1. 'On leave from Nuclear Research Centre-Negev, PO Box 9001, BeerSheva, Israel.
Avraham Lorber' Bruce R. Kowalski* Center for Process Analytical Chemistry, and Laboratory for Chemometrics Department of Chemistry BG-10 University of Washington Seattle, Washington 98195 RECEIVED for review June 15,1988. Resubmitted December 9,1988. Accepted January 1, 1989. This work was supported by the Center for Process Analytical Chemistry (CPAC), a National Science Foundation Industry/University Cooperative Research Center a t the University of Washington.
Contribution of Membrane Components to the Overall Response of Anion Carrier Based Solvent Polymeric Membrane Ion-Selective Electrodes Sir: Anti-Hofmeister behavior of anion carrier based ionselective electrodes originates from strong complexation of some anions with anion carriers. In positively charged anion carriers, especially in the case of carriers having a monovalent positive charge such as metalloporphyrins and metallocorines (1-3), this complexation reduces significantly the net charge densities in membranes. On the other hand, recent studies on the nature of negative sites in neutral carrier based solvent polymeric membrane electrodes have revealed that poly(viny1 chloride) (PVC) contains ionic or ionizable impurities that function as negative sites (4-6). Thus, plasticized PVC membranes without additions of sensing materials (blank membranes) are not potentiometrically inert and respond to certain ions (blank response). Indeed, several researchers have found ideal responses of blank membranes to proton and ionic surfactants (7, 8). By considering the two facts mentioned above, one can predict a possibility that a normal anionic function originating from the positively charged anion carrier will be strongly affected by the blank response of a membrane matrix when a positively charged anion carrier complexes too strongly with a specific anion and when the membrane matrix is not free from ionic impurities as in the case of plasticized PVC membranes (4-6). During examinations of performances of solvent polymeric nitrite-selective electrode membranes based on nitrite salts of cobalt(II1) complexes of two different porphyrins, remarkable pH dependence different from that reported in similar works (1,3) was observed. In order to clarify whether this anomalous pH-dependent response can be ascribed to the complexes themselves or rather to membrane matrices, the present work was conducted.
EXPERIMENTAL SECTION Reagents. Ligands a,/3,y,b-tetrakis(4-n-octyloxyphenyl)porphyrin (TOOPP) and a,P,y,6-tetraphenylprphyrin(TPP)and a plasticizer, 2-nitrophenyl octyl ether (0-NPOE),were obtained from Dojindo Lab. PVC was purchased from Katayama Chemical, and thin-layer chromatography (TLC) plates were Silicagel 70 Plate (Wako Chemical). All other chemicals and solvents were of reagent grade. Distilled and subsequently deionized water was used throughout. To assure high purity of o-NPOE for measurements in liquid membranes where a rather large amount was required, it was prepared according to the reported procedure (9). After 10 washings of the crude product with aqueous NaOH, it was purified by distilling twice under reduced pressure. Preparation of Anion Carriers. First, Co(I1) complexes of both porphyrins were prepared according to the method of Adler et al. (IO) and oxidized into Co(II1) form as described (11). Because only a very small amount was oxidized in the case of Co(I1)-TOOPP, the above mentioned procedure was modified as follows: First the Co(I1)-TOOPP complex was dissolved in a small amount of tetrahydrofuran, and then methanol was added, resulting in a precipitation of the complex in the form of very fine particles. With this modification, oxidation proceeds in a manner similar to that for the nonalkylated complex. Both Co(II1) complexes were obtained as chloride salts. Oxidation of both complexes was checked by TLC and CHN analyses. Nitrite forms of complexes were prepared from the chloride salts by using a solvent extraction technique (12). emf Measurements of Plasticized PVC Membrane Electrodes. Plasticized PVC membranes containing 1, 2, and 3% nitrite forms of complexes of both ligands were prepared according to the reported method (13). Blank membranes without additions of sensing materials were also prepared. All membranes had almost the same matrix, which consisted of PVC and o-NPOE in weight ratios from 10:25 to 1023. A small piece of membrane was glued to a tip of an electrode body (Denki Kagaku Keiki Co., Ltd., Tokyo) having an internal silver-silver chloride electrode.
0003-2700/89/0361-1169$01.50/00 1989 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
I
EImVI
'
I '
I
I
I
1
I
200
150I
10d
, 5
6
P Figure 1. emf response of solvent polymeric membranes containing to lo-' M solutions of NaNO,. The Co(II1)-TPP-NO, ( 2 % ) in membrane matrix consists of PVC and o-NPOE in weight ratio 10:25. 4
7
8
After the tip was filled with a reference solution (0.01 M NaCl plus 0.01 M NaN02),it was fixed to the electrode body. The emf of the electrochemical cell, consisting of external reference electrode/ /sample/plasticized PVC membrane electrode, was measured by using an ion meter IOC-10 (Denki Kagaku Keiki). As the external reference electrode, a double-junctionsilversilver chloride electrode was used. Here, a 0.1 M lithium acetate solution was used as a salt bridge solution. Samples were aqueous solutions of sodium nitrite. In evaluations of responses of blank membranes, the internal reference solution was a 0.01 M sodium chloride solution and samples were 0.1 M sodium chloride solutions of different pH. emf Measurements of Liquid Membranes. In order to avoid ambiguous effects from supporting materials of liquid membranes such as porous polymer membranes, U-shaped glass cells were used in assemblies of electrochemical cells. A liquid membrane solution was prepared by dissolving the nitrite salt of Co(II1)-TPP (0.3 w t %) or Co(II1)-TOOPP (0.5 wt %) in o-NPOE. The bottom parts of the U-shaped glass cells were filled with the liquid membrane solution (8 mL). Then, aqueous solutions of sodium nitrite (6mL) were filled on each side of the liquid membrane, and the electrochemical cells, SCE/solution I/liquid membrane/solution II/SCE, were assembled by using a pair of saturated calomel electrodes (SCE). Here, solutions I and I1 correspond to samples and an internal reference solution, respectively. A concentration of nitrite in solution I1 was fixed at 0.01 M, and that in solution I was varied. In potential measurements, the whole system was shielded by a Faraday cage. Adjustment of pH. Microliter volumes of hydrochloric acid or sodium hydroxide solutions were added to sample solutions (solution I) with a micropipet in order to minimize dilution.
RESULTS AND DISCUSSION Responses of Solvent Polymeric Membrane Electrodes. Membranes containing complexes in the nitrite form showed nearly Nerstian responses to nitrite, and their potentials were stable, as long as no electrolyte except sodium nitrite was contained in samples. Remarkable p H dependence of potentials, however, was observed when the pH of samples containing sodium nitrite was changed with additions of hydrochloric acid or sodium hydroxide, as illustrated in Figure 1. The membrane responds not only to nitrite but also to protons. Then, the p H response of blank membranes was examined. Figure 2 shows potentials of a blank membrane for 0.1 M sodium chloride solutions of different pH. Potentials of the blank membrane are clearly dependent on pH. Sugimot0 e t al. have also reported an ideal p H response of PVC membranes plasticized with o-NPOE (7). These imply that the membrane matrix consisting of PVC and o-NPOE is not
Figure 2. emf response of blank membrane in 0.1 M NaCl solutions of different pH. The membrane matrix consists of PVC and o-NPOE in weight ratio 10:23.
-1ooc
5 6 7 8 9 10 PH Flgure 3. emf response of liquid membrane consisting of o-NPOE solution of Co(II1)-TOOPP-NO, (0.5%). Solution I corresponds to lo-' to lo-' M NaNO, of different pH; solution I 1 is 0.01 M NaNO,.
potentiometrically inert. Thus, the response pattern shwon in Figure 1 seems to be the superposition of an anionic function originating from the anion carrier and pH response of the membrane matrix. Responses of Liquid Membranes. First, we tried to measure potentials across a blank liquid membrane, i.e., oNPOE without an addition of any carrier. However, it was impossible to measure potentials because of extremely high impedance of the blank liquid membrane, indicating that our o-NPOE was potentiometrically inert. This makes it possible to evaluate responses of the present anion carriers to nitrite without the influence of the blank response of the membrane liquid matrix. Liquid membranes containing the nitrite form of the complexes gave satisfactory results. Figure 3 shows membrane potentials across a liquid membrane containing the nitrite form of Co(I11)-TOOPP. I t is noteworthy that potentials in weakly acidic regions are independent of p H a t relatively high concentration levels of nitrite. This is quite different from the pH-dependent response to nitrite shown in Figure 1. A response pattern similar to the one shown in Figure 3 was also observed in responses of a liquid membrane containing the nitrite form of Co(II1)-TPP, but the pH-independent region is rather narrow. The potential decreases
Anal. Chem. 1989, 6 1 , 1171-1174
in the high-pH regions shown in Figure 3 are ascribable to an interference from hydroxide. In a previous paper (12),we reported selectivity sequences for the Co(II1)-TPP anion carrier and a classical anion exchanger (trioctylmethylammonium chloride). By comparing reported selectivity coefficients of both types of solvent polymeric membranes which have almost the same membrane matrices as those in the present work, one can roughly estimate selectivity changes of thiocyanate and nitrite induced by the Co(II1)-TPP anion carrier. Since perchlorate does not readily complex with the metal center, this is selected as a reference ion in estimations of selectivity changes. For the classical anion-exchange solvent polymeric membrane, log kC104-,N02and log kC104-,SCN- are -4.6 and -1.3, respectively. In the case of the Co(II1)-TPP anion carrier based solvent polymeric membrane, log kC104-,N02- is 1.6 and log kC104-,SCN- is 2.7. Thus, selectivity changes for nitrite and thiocyanate induced by the Co(II1)-TPP anion carrier relative to those of the classical exchanger are estimated to be 6 and 4 orders of magnitude, respectively. Although detailed studies on associations of the complex cation with nitrite as well as with thiocyanate are required, it can be estimated that nitrite associates with the carrier more strongly than thiocyanate does. Consequently, complexes in the nitrite form cannot contribute as much to overall conductivities of solvent polymeric memrbanes as complexes in the thiocyanate form do. This suggests that the blank response of the membrane matrix will be reflected more strongly in the case of complexes in the nitrite form than in the case of complexes in the thiocyanate form. Indeed, solvent polymeric membranes containing the Co(II1)-TPP in the thiocyanate form showed virtually no pH dependence in response to thiocyanate (12),while ones containing complexes in the nitrite form showed the strong pH dependence in response to nitrite, as is shown in Figure 1. In contrast to the potentiometrically active plasticized PVC matrix, the anomalous pH effect shown in Figure 1 is successfully eliminated in the potentiometrically inert liquid membrane matrix (Figure 3). These results imply that the anomalous pH effect observed in responses of solvent polymeric membranes containing complexes in the nitrite form comes from the membrane matrix, although the whole mechanism of this pH-dependent response is not now clarified. As is shown in this work, there is a potential for unfavorable
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side effects from membrane matrices in responses of positively charged anion carrier based solvent polymeric membrane electrodes when anion carriers having a monovalent positive charge, like the presented complexes, interact too strongly with a specific anion like nitrite.
LITERATURE CITED (1) Schulthess, P.; Ammann, D.; Krautler, B.; Caderas, C.; Steplnek, R.; Simon, W. Anal. Chem. 1985, 5 7 , 1397-1401. (2) Ammann, D.; Huser, M.; Krautler, 8.; Rusterholz, B.; Schulthess, P.; Lindemann, B.; Hadler, E.; Simon, W. Helv. Chim. Acta 1988, 6 9 , 849-854. (3) Chaniotakis, N. A.; Chasser, A. M.; Meyerhoff, M. E.; Groves, J. T. Anal. Chem. 1988, 6 0 , 185-188. (4) Horvai, G.; Grlf, E.; T6th, K.; Pungor, E.; Buck, R. P. Anal. Chem. 1986, 58, 2735-2740. (5) T6th. K.; Grlf. E.; Horvai, G.; Pungor. E.: Buck, R. P. Anal. Chem. 1988, 5 8 , 2741-2744. (6) Van den Berg, A.; van der Wal, P. D.; Skowrofiska-Rasiirska, M.; Sudholter, E. J. R.; Reinhoudt, D. N.; Bergveld, P. Anal. Chem. 1987, 5 9 , 2827-2829. (7) Sugimoto, H.; Tooda, K.; Suzuki, K.; Shirai, T. Abstr. No. 2IVB 01, 54th National Meeting of the Chemical Society of Japan, Tokyo, April 1987. (8) Masadome, T.; Imato, T.: Ishibashi, N. Anal. Sci. 1987, 3, 121-124. (9) Allen, C. F. H.; Gates, J. W., Jr. Organic Synthesis Collective Volume 3; Hornig. E. C., Ed.; John Wiley & Sons, Inc.: New York, 1955; pp 140, 141. (10) Adler, A. D.; Longo, F. R.; Kampas. F.; Kim, J. J . Inorg , Nucl. Chem , 1970, 32, 2443-2445. (11) Sakurai, T.; Yamamoto, K.; Naffo, H.; Nakamoto, N. Bull. Chem. SOC. Jpn. 1978, 4 9 , 3042-3046. (12) Hodlnar, A.; Jyo, A. Chem. Len. 1988, 993-996. (13) Anker, P.; Wieland, E.; Ammann, D.; Dohner, R. E.; Asper, R.; Simon, W. Anal. Chem. 1981, 5 3 , 1970-1974.
Ale6 HodinHi. Laboratory for Endocrinology and Metabolism Faculty of Medicine Charles University at Prague U Nemocnice 1 Prague, CSSR
Akinori Jyo* Department of Applied Chemistry Faculty of Engineering Kumamoto University 2-39-1 Kurokami Kumamoto 860, Japan RECEIVED for review March 23,1988. Accepted February 24, 1989.
Effect of Protein Binding on the High-Performance Liquid Chromatography of Phenytoin and Imirestat in Human Serum by Direct Injection onto Internal Surface Reversed-Phase Columns Sir: Internal surface reversed-phase (ISRP) columns have been designed to facilitate the HPLC analysis of drugs in blood serum or plasma by direct injection (1). The ISRP concept consists of binding a diol-gly-phe-phe peptide partitioning phase to the internal surface of 5 pm porous silica, while rendering the external surface hydrophilic and nonabsorptive to proteins via a glycerylpropyl bonded phase. The peptide-bonded phase is removed from the external surface of the supports by enzyme cleavage (2). The final median pore diameter of the packing is 52 A, so serum proteins are size excluded from the internal regions of the packing (3). Drugs with low molecular weights (CZOOO) penetrate the ISRP packing and partition with the internal peptide-bonded phase.
Serum proteins, on the other hand, elute in the column interstitial void. The diol-gly-phe-phe internal bonded phase favors the retention of aromatic drugs and separates analytes primarily by a reversed-phase mechanism ( 4 ) . With the carboxylic acid terminal on the peptide-bonded phase, the packing exhibits a secondary cation-exchange mechanism and provides strong selective control for positively charged aromatic amines on variation of mobile phase ionic strength ( 5 ) . The ISRP columns have been used for the direct high-performance separation of a wide variety of drugs in serum or plasma (4-7); for the precolumn switching isolation of substances from serum or plasma ( 1 , 8, 9); for the determination endogenous me-
0003-2700/89/0361-1171$01.50/00 1989 American Chemical Society
