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CORRESPONDENCE Ion-Exchange Electrokinetic Chromatography with Polymer Ions for the Separation of Isomeric Ions Having Identical Electrophoretic Mobilities Sir: High-performance capillary electrophoresis (HPCE) is a rapidly growing separation method ( I , 2). The separation principle of HPCE is based on differences in electrophoretic mobilities, and therefore, analytes have to have different electrophoretic mobilities to be separated from each other. The electrophoretic mobility depends on the charge of the analyte, molecular size and shape, number of solvation molecules, etc. In order to increase the selectivity, we have to select conditions where the differences in electrophoretic mobilities become maximum. The pH of the separation solution is the most important factor for the separation of ionic compounds, except for completely ionized ions. The pH dependence of the ionization of some proteins has been theoretically calculated by Mosher et al. (3). The optimum pH value for the separation of analytes having very similar pK, values has been calculated to be close to the pK, values (4). The second choice to alter the selectivity is the use of organic modifiers such as methanol and acetonitrile ( 5 ) . Another choice is to add complexing ligands and metals (6). Constituents of the buffer solution and temperature may also alter the relative magnitudes of electrophoretic mobilities. It is possible that some compounds will have very similar electrophoretic mobilities over a wide pH range, in particular, this will be the case with isomers of strong acids and bases. The electrophoretic separation of this mixture of analytes is almost impossible. However, we can modify their electrophoretic mobilities by introducing a chemical equilibrium, in which the ionic analyte participates to form a dynamic complex with an additive. In other words, even if analytes have identical electrophoretic mobilities, they should have apparently different electrophoretic mobilities, provided their complex formation constants are different. Although the technique may be termed a modification of electrophoretic mobilities through complex formation, we think that this should be classified as electrokinetic chromatography (EKC), which combines the experimental technique and high efficiency of HPCE with the separation principles of chromatography (7). Micellar EKC, or MEKC, is presently a well-known technique that belongs to EKC and has been developed for the separation of electrically neutral analytes by HPCE (8, 9). The ionic micelle added to the separation solution can modify the electrophoretic mobility of the neutral substance, which is actually zero, through micellar solubilization. We have also described cyclodextrin EKC, which uses a cyclodextrin derivative having an ionic group, instead of the ionic micelle in micellar EKC, and which allows a separation based on inclusion-complex formation (7, 10).
In this paper, we describe ion-exchange EKC with polymer ions, which is developed for the separation of ionic analytes having very similar electrophoretic mobilities. In this method, a polymer ion having an opposite charge to the analyte ions is added to the separation solution as a modifier. A brief consideration of the principles and a successful application of this technique are presented.
THEORY Figure 1 shows schematically the separation principle of ion-exchange EKC. In this figure, a polymer cation and analyte anions, and their electrophoretic migrations are illustrated, but other ions, such as buffer constituents and counterions of both analyte anions and the polymer cation, are intentionally omitted. The electroosmotic flow also is not shown. The analyte anion that combines with the polymer cation, through ion-pair formation, will migrate with the same velocity as other polymer cations. That is because only a partial neutralization of the positive charge occurs on the polymer, and a small increase in molecular size will not significantly alter the electrophoretic velocity of the polymer cation. The free analytes and the bound analytes migrate in the opposite directions, as shown in Figure 1. Accordingly, the apparent velocity of the analyte anion depends on what extent the analyte forms the ion pair with the polymer ion. The velocity of the analyte solute, ug, is described as us = u,, + Ru,,(free) + (1- R)u,,(pi) (1) where u, u,,(free), and u,,(pi) are electroosmotic velocity, electrophoretic velocity of the free analyte ion, and the electrophoretic velocity of the polymer ion, respectively. The electrophoretic velocity of the bound analyte is supposed to be equal to that of the polymer ion, as mentioned above. The quantity R is the fraction of the analyte ion free from the polymer ion. Since the technique is mainly applicable to the separation of ions having very similar electrophoretic mobilities, it is reasonable to assume that ionic solutes 1 and 2 have identical electrophoretic mobilities. Therefore, the difference in migration velocities between the two solutes l and 2, Aus, is given as Au, = usl - u,2 = (R1 - R2)(ue,(free) - u,,(pi)I (2) where R1 and R2 are the R values of the solutes 1 and 2. The equilibrium reaction between the solute, S-, and the polymer ion, P+, is supposed to be
s- + p+ + s-.p+
(3)
Kip = [S-.P+]/[S-][P+]
(4)
where Kip is the ion-pair formation constant. Here, S- and P+ do not always mean singly charged species. The R value is related to Kip through R = [S-]/([S-] + [S-.P+]) = 1/(1 + Ki,[P+]) (5) The concentration of free polymer ion, [P+],is considered to be substantially equal to the total concentration of the polymer ion, [P+],,provided that the concentration of the analyte ion is low compared with [P+],. Combination of eq 5 with eq 2 gives (Kip2 - Ki,J[P+Iob,,(free) A0 =
- uep(Pi)l
(1 + KipI[P+IJ (1 + Kipz[P+lo)
(6)
where Kipl and KipZare the ion-pair formation constants of
@ 1990 American Chemical Society 0003-2700/90/0362-0650$02.50/0
ANALYTICAL CHEMISTRY, VOL. 62, NO. 6, MARCH 15, 1990
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'i Figure 1. Schematic diagram of the principle of ionsxchange electrokinetic chromatography: a polymer cation is shown; R-, analyte anion; arrows show the electrophoretic migrations. 1
the solutes 1 and 2, respectively. Equation 6 predicts that Au is proportional to the difference between the two formation constants, (Kip*- &), and also to the difference between the two electrophoretic velocities, {uep(free)- ueP(pi)). It should be noted that u,(free) and u,(pi) have different signs, because they have opposite charges. Equation 6 suggests that the relationship between Av and the concentration of the polymer ion [P+], will be complicated, depending on the sizes of the values Kip[P+l0relative to unity. However, we will not discuss the dependence of Au on [P+], further in this paper.
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Figure 2. Electropherogram of 1- and 2-naphthalenesulfonates and 1& 1,6-, 1,7-,2,6-, and 2,7-naphthalenedisulfonates: (1) five na-
phthalenedisulfonates,(2)two naphthalenesulfonates; capillary, 71 cm in total length, 46 cm to the detection point; applied voltage, 20 kV; current, 35 FA.
EXPERIMENTAL SECTION Apparatus and Procedure. An apparatus similar to that described (3,8)was employed. Fused silica capillaries of 50 jtm i.d. from two different sources (Scientific Glass Engineering, Ringwood, Victoria, Australia, and Polymicro Technologies, Phoenix, AZ) were used. Sample solutions were introduced manually into the capillary by the hydrostatic method or siphoning. The solute band migrating in the capillary was detected on-column by W absorption at 210 nm with a Jasco Uvidec-100-V (Tokyo, Japan). Electropherograms or electrokinetic chromatograms were recorded with a Shimadzu C-R6A Chromatopac (Kyoto, Japan). All experiments were carried out at ambient temperature (ca. 25 "C). Materials. Poly(diallyldimethy1ammonium chloride) (PDDAC) was purchased from Polysciences, Inc. (Warrington, PA), as 15% solids in water and (diethy1amino)ethyldextran (DEAE-dextran) was obtained from Pharmacia (Uppsala, Sweden). Hydroxypropylcellulose(HPC) was purchased from Tokyo Kasei Kogyo (Tokyo, Japan). Sodium 1- and 2-naphthalenesulfonates and disodium 1,5-,2,6-, and 2,7-naphthalenedkulfonates were purchased from Wako Pure Chemical Industries (Osaka, Japan) and disodium 1,6- and 1,7-naphthalenedisulfonateswere gifts from Sumitomo Chemical Co. (Osaka, Japan). Water and purified with a Milli-Q system (Nihon Millipore, Tokyo, Japan). All other reagents were of analytical-reagent grade, and all materials were used without further purification.
RESULTS AND DISCUSSION Figure 2 shows an electropherogram of a mixture of two isomers of naphthalenesulfonates and five isomers of naphthalenedisulfonates, which was obtained with a 50 mM phosphate buffer solution (pH 7.0) containing 0.1% HPC. Since electroosmosis was almost completely suppressed in the presence of HPC, all the analyte anions migrated toward the positive electrode. Group separation by the number of the sulfonate groups was easily brought about, as expected from the difference in charge, but isomer separation was not successful. Although 1- and 2-naphthalenesulfonic acids may have different pK, values, they are considered to be fully ionized at pH 7.0. The electrophoretic mobilities of the two naphthalenesulfonate ions must be very similar, judging from the result shown in Figure 2. The five disodium naphthalenedisulfonates employed in this study were also considered to be completely ionized a t pH 7.0, because these isomeric ions were not resolved, but migrated much faster than the monosulfonate ions toward the positive electrode. This result suggests that the isomeric
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Figure 3. Ion+xchange electrokinetic chromatograms of the analytes shown in Figure 2: (A) 1, 2-; 2, 1-naphthalenesulfonate;(B) 1, 1- and 2-naphthaienesulfonates; 2, 2,6-; 3, 2,7-; 4, 1&; 5, 1& 6, 1,7naphthalenedisulfonates. Separation solution, (A) 0.3% DDAC in 50 mM phosphate buffer (pH 7.0),(B) 2% DEAEdextran in the same buffer as in (A); capillary, 75 cm in total length, 50 cm to the detection point; applied voltage, 20 kV; current, (A) 48 pA, (B) 58 pA.
dianions seem to have very similar electrophoretic mobilities, and hence, it is very difficult to separate these isomers under fully ionized conditions. It may be possible to resolve these isomeric disulfonic acids by HPCE under acidic conditions, under which the pH should be close to the second pK, values of the disulfonic acids (4). However, we adopted another technique to resolve them, as described below. Figure 3 shows ion-exchange electrokinetic chromatograms of the same analytes employed in Figure 2. In Figure 3A, 0.3% PDDAC was added to the same 50 mM phosphate buffer solution (pH 7.0) as employed in the above HPCE experiments, and in Figure 3B, 2 % DEAE-dextran was added. Although analyte ions migrated toward the positive electrode, the monosulfonates migrated faster than the disulfonates, as shown in Figure 3B, in contrast to the result shown in Figure 2. The electroosmotic flow was very weak, but toward the positive electrode in the presence of the polymer ions.
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Although the two monosulfonates were not resolved with the 2% DEAE-dextran solution, as shown in Figure 3B, they were easily separated with the 0.3% PDDAC solution, as shown in Figure 3A. Use of the 0.3% PDDAC solution also permitted the separation of the five naphthalenedisulfonates shown in Figure 3B, but migration times were too long compared with those obtained with DEAE-dextran solutions. The reversed migration order of the monosulfonates and disulfonates between Figures 2 and 3B indicates that the disulfonates interact with the polymer cation more strongly than the monosulfonates. It is reasonable that dianions tend to form more stable ion pairs with polymer cations than monoanions, from the viewpoint of electrostatic interaction, but it seems difficult to reasonably explain the relative stability of ion pairs between the isomeric ions and with the polymer ions. The relative migration orders shown in Figure 3 suggests that the sulfonate group at the 1position of the naphthalene structure binds to the polymer cation more strongly than that a t the 2 position, because the stronger ion-pair formation causes a slower migration velocity. In conclusion, in order to increase selectivity in HPCE, electrophoretic mobilities can be manipulated through the ion-pair formation reaction of analyte ions with polymer ions. We have tried to modify mobilities in a similar manner with ionic micelles, such as dodecyltrimethylammonium chloride, instead of polymer ions, but this was not very successful. A long chain configuration of the polymer ion is probably more effective to discriminate isomers than the spherical and dynamic structure of the micelle. It may be possible to extend
this technique to the separation of polymer anions, such as oligonucleotides.
ACKNOWLEDGMENT We thank Professor S. H j e r t h for his suggestion of using DEAE-dextran for the technique described.
LITERATURE CITED Gordon, M. J.; Huang, X.; Pentoney, S. L., Jr.; Zare, R. N. Science 1988, 242, 224-220. Wailingford, R. A.; Ewing, A. G. A&. Chromtogr. 198% 3 0 , 1-76. Mosher, R. A.; Dewey, D.; Thormann, W.; Saviile, D. A.; Bier, M. Anal. Chem. 1989, 61, 362-366. Terabe, S.; Yashima, T.; Tanaka, N.; Araki, M. Anal. Chem. 1988, 60, 1673-1677. FuJiwara, S.; Honda, S. Anal. Chem. 1987, 59, 467-490. Gassmann. E.; Kuo, J. E.; Zare, R. N. Science 1985, 230. 813-814. Terabe, S . TrAC, Trends Anal. Chem. 1989, 8 , 129-134. Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A,; Ando, T. Anal. Chem. 1984. 56, 111-113. Terabe, S.: Otsuka, K.; Ando, T. Anal. Chem. 1985, 57, 834-841. Terabe, S.; Ozaki, H.: Otsuka, K.; Ando, T. J . Cbromafogr. 1985, 332, 211-217.
Shigeru Terabe* Tsuguhide Isemura Department of Industrial Chemistry Faculty of Engineering Kyoto University Sakyo-ku, Kyoto 606, Japan RECEIVED for review November 3,1989. Accepted December 21, 1989. This work is partially supported by grants from Yokogawa Electric Corp. and Shimadzu Corp.
TECHNICAL NOTES Preparative Method for Fabricating a Microelectrode Ensemble: Electrochemical Response of Microporous Aluminum Anodic OxSde Film Modified Gold Electrode Kohei Uosaki,* Kentaro Okazaki, and Hideaki Kita Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060, Japan
Hideaki Takahashi Analytical Chemistry Laboratory, Faculty of Engineering, Hokkaido University, Sapporo 060, Japan
INTRODUCTION Recently, much attention has been paid to microelectrode arrays (ensembles) because of their various advantages in many applications, including high signal to noise (S/N) ratio compared to conventional electrodes with the same electrode area (1). Various approaches have been taken to prepare microelectrode arrays. Hepel and Osteryoung constructed microelectrode arrays that are composed of disks of 0.375-pm radius, employing electron beam lithography (2). Martin and colleagues used microporous polycarbonate membranes (Nuclepore) as hosts and Pt ( r = 0.1-0.5 pm) or carbon ( r = 8 or 12 pm) as an electrode material (3, 4 ) . Morita and Shimizu prepared a microelectrode array employing platinized carbon fiber (5-7 wm) as an electrode and epoxy resin as a substrate (5). The smallest radius of an individual electrode reported is 0.1 wm. It is known that the smaller the active electrode area, the higher the S / N ratio (6). 0003-2700/90/0362-0652$02.50/0
In this paper, we propose a novel method for the preparation of a microelectrode ensemble with smallest electrode size reported by using micropores of aluminum anodic film as templates. Such oxide films, formed on A1 in acidic media, are known to possess micropores of 10-200 nm diameter normal to the surface with a barrier layer between the anodic oxide film and A1 substrate (7,8). Recently, Majda and his colleagues reported the electrochemical behavior of electroactive species confined within the micropores of the aluminum anodic oxide film (9-14). Tierney and Martin deposited “transparent” Au microcylinden within the micropores of the anodic oxide film (15). We have thought that these pores can be used as templates for microelectrode disks. Thus, after the anodic oxide film was removed from the Al substrate, gold was vacuum evaporated into the micropores of the oxide film, and the barrier layer was gradually removed. At a certain level of barrier layer removal, voltammograms of sigmoidal shape having a current independent of scan rate were observed, confirming the formation of an ensemble of microe0 1990 American Chemical Society
