Alternating Voltage Capillary Electrochromatography - American

Jul 14, 2003 - ing voltage is to control the capacity factor of sample solutes. The variation of the capacity factor was studied using a mini-bed (sho...
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Anal. Chem. 2003, 75, 3512-3517

Alternating Voltage Capillary Electrochromatography Hiroyuki Nakagawa, Masahiko Sato, Shinya Kitagawa,* and Takao Tsuda

Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555, Japan

A phenomenon was found whereby the application of an alternating voltage to a capillary column can vary the capacity factor of a sample solute. The alternating voltageinduced variation in the capacity factor was studied using an anion exchange mini-bed (a short capillary column 12 mm long). The capacity factor varied according to both the amplitude and frequency of an applied alternating voltage. The variation greatly depended on the kinds of sample solutes and packing materials. A new separation mode for capillary electrochromatography using an alternating voltage, that is, alternating voltage capillary electrochromatography (AV-CEC), was proposed as an application of this phenomenon to control the retention of a sample solute. The chromatographic behavior of three organic acids (benzoic acid, phthalic acid, and salicylic acid) was studied in AV-CEC using an anion exchange column. Capillary electrochromatography is a separation method using both chromatographic and electrophoretic behaviors.1-6 In capillary electrochromatography without a pressurized flow, the solute is transferred to a column outlet by an electroosmotic flow or an electrophoretic migration of an ionic solute. Therefore, either a positive or negative voltage can transfer a sample solute to a column outlet. In pressurized flow-driven electrochromatography, when the pressurized flow velocity is sufficiently high to elute a sample solute against an electroosmotic flow and an electrophoretic migration, both negative and positive voltage can be applied to a capillary column.7-10 Therefore, pressurized flowdriven electrochromatography is suitable for studying the effect * To whom correspondence should be addressed. Fax: +81-52-735-5368. E-mail: [email protected]. (1) Tsuda, T., Ed. Electric Field Applications in Chromatography, Industrial and Chemical Processes; VCH: Weinheim, 1995. (2) Wistuba, D.; Schurig, V. J. Chromatogr. Libr. 2001, 62, 317-339. (3) Pyell, U. In Advances in Chromatography; Brown, P. R., Grushka, E., Eds.; Marcel Dekker: New York, 2001; pp 1-51. (4) Bartle, K. D.; Myers, P. J. Chromatogr., A 2001, 916, 3-23. (5) Bartle, K. D., Myers, P.; Eds. Capillary electrochromatography; RSC: Cambridge, 2001. (6) Unger, K. K.; Huber, M.; Walhagen, K.; Hennessy, T. P.; Hearn, M. T. W. Anal. Chem. 2002, 74, 200A-207A. (7) Kitagawa, S.; Tsuda, T. J. Microcolumn Sep. 1994, 6, 91-96. (8) Kitagawa, S.; Tsuji, A.; Watanabe, H.; Nakashima, M.; Tsuda, T. J. Microcolumn Sep. 1997, 9, 347-356. (9) Chaiyasut, C.; Tsuda, T.; Kitagawa, S.; Wada, H.; Monde, T.; Nakabeya, Y. J. Microcolumn Sep. 1999, 11, 590-595. (10) Sato, M.; Kitagawa, S.; Tashita, H.; Tsuda, T. Chromatography 2001, 22, 195-199.

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of an electric field on electrochromatographic behaviors. Thus, we have found interesting phenomena induced by the applied electric field. Those phenomena are variations of the capacity factor,9-12,14 conductivity of a column,8,9,13 and electroosmotic mobility.9 The phenomena suggest that the applied electric field affects the properties of a stationary phase or an interface between stationary and mobile phases. The variation of the capacity factor was first observed when indirect ultraviolet (UV) absorbance detection was performed.12 The mobile phase containing phthalic acid was continuously supplied to a capillary column packed with anion exchangemodified silica gels. When a positive high voltage was applied to the column, the UV signal, which was detected just after the packed column end, was elevated. This phenomenon indicated that applying a positive high voltage induced a release of phthalic acid from the stationary phase. The application of negative high voltage declined the UV signal; i.e., the distribution of phthalic acid to the stationary phase was accelerated. A variation of the capacity factor was also observed when the sample solute was electrically neutral.9,10 The voltage-induced capacity factor variation suggests the possibility of controlling the capacity factor with the application of a voltage. Although applications of both positive and negative voltages can induce variations of the capacity factor, the magnitude of the variations differs.12 Therefore, we speculated that the application of an alternating voltage can also vary the capacity factor. In this report, we propose a new separation mode for capillary electrochromatography using an alternating voltage, that is, alternating voltage capillary electrochromatography (AV-CEC). In AV-CEC, a pressurized flow is used to transfer a mobile phase and a sample solute to a column outlet, and an alternating voltage is applied to a capillary column. The objective of the application of an alternating voltage is to control the capacity factor of sample solutes. The variation of the capacity factor was studied using a mini-bed (short column).11 The chromatographic behavior of organic acids in AVCEC using ion exchange packing materials was also studied. EXPERIMENTAL SECTION Apparatus. Two types of apparatus were used in this experiment, as shown in Figure 1. One used a mini-bed (a short capillary column of 12 mm long),11 and the other is for AV-CEC. The mini(11) Kitagawa, S.; Tsuda, T. Anal. Sci. 1998, 14, 571-575. (12) Kitagawa, S.; Watanabe, H.; Tsuda, T. Electrophoresis 1999, 20, 9-17. (13) Chaiyasut, C.; Kitagawa, S.; Wada, H.; Tsuda, T. Anal. Sci. 2000, 16, 413416. (14) Kitagawa, S.; Tsuda, T. J. Microcolumn Sep. 2000, 12, 285-291. 10.1021/ac034064e CCC: $25.00

© 2003 American Chemical Society Published on Web 07/14/2003

Figure 1. Schematic diagram of apparatus of (I) mini-bed system and (II) alternating voltage capillary electrochromatography: A, pump; B, high voltage power supply; C, mini-bed; D, detector; E, sample solution; F, function generator; G, capillary column; H, injector; I, splitter; J, resistance tubing; K, reservoir; and L, eluent.

bed (Figure 1-I) was used to study the basic behaviors of the alternating voltage-induced variation in a capacity factor. The apparatus for AV-CEC (Figure 1-II) was similar to the arrangement of a pressurized flow-driven electrochromatography described in our previous reports7-10 and was used for attempting to control chromatographic behavior using an alternating voltage. The apparatus using a mini-bed was composed of a pump (LC6A, Shimadzu, Kyoto, Japan), a UV detector (UV-790, Jasco, Tokyo), a laboratory-made mini-bed, an alternating voltage power supply (HOPP-1B3, Matsusada Precision, Shiga, Japan), and a laboratory-made alternating voltage controller. The inner diameter and length of the laboratory-made mini-bed were 0.7 mm and 12 mm, respectively. Silica gel supports modified with anion exchange functional group (dp. 5 µm, IC-ANION-SW, kindly donated by Tosoh, Tokyo) and polymer-based anion-exchange resins (dp. 5 µm, MCI GEL SCA04, Mitsubishi, Tokyo) were used as packing materials for a mini-bed. Both ends of the mini-bed were connected with platinum tubing. The sample solution was provided to and eluted from the mini-bed through the platinum tubing, which also acted as the electrode to apply an alternating voltage. The platinum tubing at the inlet was grounded, and the alternating voltage was applied to it at the outlet end. The UV detector continuously measured the UV absorbance of the solution eluted from the minibed, and variations in the UV signal were recorded. The apparatus for AV-CEC was composed of a pump (LC-6A, Shimadzu), an injector (model 7410, Rheodyne, CA), a UV detector (UV-970, Jasco), a laboratory-made capillary column, a laboratorymade splitter (the split injection method was used), a high alternating voltage power supply (HVA 4321, NF, Yokohama, Japan), and a function generator (model 455, Kikusui Electronics, Yokohama, Japan). The inner diameter and length of the laboratory-made column were 0.32 mm and 8.8 cm, respectively, and the anion exchange supports (IC-ANION-SW) were packed. The alternating voltage was applied to the injector that also served as an electrode. The platinum tubing connected to the column outlet, that is, the end of the packed part, was always grounded. An extra platinum electrode equipped with a reservoir at the end of the capillary was always grounded; i.e., three electrodes were used in the apparatus for AV-CEC, and the UV detector was inserted

Figure 2. Typical capacity factor variation induced by application of an alternating voltage. Dark bar under UV signal denotes a period (10 minuets) during the application of an alternating voltage. Minibed: i.d. 0.7 mm, length 12 mm, packed with IC-ANION-SW. Sample solution: methanol/3 mM phthalic acid, 3 mM hexamethylenediamine, 0.15 mM HEPES (pH 5.6) (10:90). Applied voltage, Vpeak ) 1 kV, f ) 1 kHz; flow, constant pressure mode of 50 kg/cm2; detection, 210 nm.

between two grounded platinum electrodes to reduce the noise generated by the applied alternating voltage. The magnitude of an applied alternating voltage was indicated using the amplitude in a sine wave, that is, Vpeak, in both apparatuses. Reagents. Details of the sample solution and the mobile phase are described in the Figure captions. All reagents (Wako Pure Chemical Industries, Kyoto, Japan) were guaranteed grade. RESULTS AND DISCUSSION Typical Variation of the Capacity Factor Induced by the Application of Alternating Voltage. A typical capacity factor variation induced by the application of an alternating voltage is shown in Figure 2. A sample solution of the mixture of the aqueous solution (pH 5.6) containing 3 mM phthalic acid, 3 mM hexamethylenediamine, and 0.15 mM N-2-hydroxyethylpiperazineN-ethansulfonic acid (HEPES) and methanol (90:10) was continuously supplied to the mini-bed using a pump. The pump was operated at a constant pressure mode of 50 kgf/cm2. In Figure 2, the dark zone in the horizontal axis denotes the duration of the application of an alternating voltage (Vpeak ) 1 kV, f ) 1 kHz). When the alternating voltage was applied to the mini-bed, the UV signal was elevated, then the UV signal returned to its original intensity. When the applied alternating voltage was cut off, the UV signal declined and returned again to its original intensity. A similar phenomenon had been observed when the direct voltage was applied to a column in pressurized flow-driven electrochromatography.12 This variation in UV signal intensity reflected the variation of the capacity factor. Since the mini-bed was in an equilibrium condition before the alternating voltage was applied to the mini-bed, components of the sample solution supplied to and eluted from the mini-bed were the same and constant. The application of an alternating voltage intensified a UV signal. Namely, the concentration of UV absorbent reagent (phthalic acid) in the mobile phase eluted from the mini-bed was enlarged. The Analytical Chemistry, Vol. 75, No. 14, July 15, 2003

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Figure 3. Dependence of variation in UV signal on frequency of alternating voltage. Dark bar under UV signal denotes a period (30 s) during the application of alternating voltage. Applied voltage: Vpeak ) 1 kV, f ) (A) 1176, (B) 273, (C) 117, and (D) 26 Hz. Other conditions were the same as in Figure 2.

alternating voltage released phthalic acid from the stationary phase; i.e., the capacity factor was varied by the application of an alternating voltage. The continuous provision of the sample solution to the mini-bed resulted in a reequilibrium condition under the application of the alternating voltage. Since the composition of the sample solutions supplied to and eluted from the mini-bed were the same in the reequilibrium condition, and UV signal was returned to its original intensity. The varied capacity factor was returned to its original value by terminating the alternating voltage. Since phthalic acid in the mobile phase was transferred to the stationary phase, the UV signal was suppressed. When the mini-bed reached equilibrium condition, the UV signal returned to its original intensity again. These UV signal variations suggested that the application of an alternating voltage changes the capacity factor of the solute. Dependence of the Capacity Factor Variation on the Frequency of an Alternating Voltage. One of the important properties of an alternating voltage is its frequency. Therefore, the dependence of the capacity factor variation on the frequency of an applied alternating voltage was studied using the mini-bed. Figure 3 shows the variations in UV signal intensity induced by the applications of the alternating voltage (Vpeak ) 1 kV) with various frequencies. The alternating voltage was applied to the mini-bed during the period indicated by the dark zone in Figure 3. In Figure 2, the voltage was applied until the UV signal returned to its original intensity; however, the applied alternating voltage was cut off before the reequilibrium condition in Figure 3. The duration of the application of an alternating voltage was 30 s. The sample solution used in Figure 3 was the same as Figure 2; i.e., phthalic acid was the sample solute. As shown in Figure 3, the magnitude of the increment in UV signal intensity strongly depended on the frequency of an applied alternating voltage. Since the scales of four chromatograms in Figure 3 are the same, the peak height in Figure 3 reflected directly the variation of UV signal. The application of an alternating voltage of 273 Hz markedly reduced the UV signal intensity and 3514 Analytical Chemistry, Vol. 75, No. 14, July 15, 2003

Figure 4. Relationship between peak height induced by application of an alternating voltage and the frequency. Sample solutions: (O) methanol/3 mM phthalic acid, 3 mM hexamethylenediamine, 0.15 mM HEPES (pH 5.6) (10:90); (0) methanol/3 mM salicylic acid (pH 2.7) (10:90); (2) methanol/3 mM benzoic acid (pH 3.0) (10:90). Other conditions were the same as in Figure 3.

accelerated the distribution of phthalic acid to a stationary phase. The application of an alternating voltage forced the changing of the condition of the stationary phase, and the changed condition would return to the original condition after the releasing of the applied voltage. Therefore, the returning process was strongly dominated by the changed condition under the alternating electric field. Each alternating voltage-induced variation of the stationary condition is different, and it might determine the difference of the profile of the signal. The relationship between the frequency of an applied alternating voltage and its peak height (an indicator of the variation of the capacity factor) is shown in Figure 4. Since the application of alternating voltage induced an increment in the UV signal, as shown in Figure 3, the peak heights of phthalic acid in Figure 4 were positive values, except for 273 Hz. The peak height at 117 Hz was significantly smaller, as compared to those at other frequencies. In this manuscript, we call these frequencies that induce strange behavior “singular frequency”. Therefore, it seems that the frequencies of 117 and 273 Hz might be singular frequencies. The mechanism why there are two minimums for phthalic and salicylic acids is still not clear. However, we suppose that these minimums might be caused by a kind of hysteresis of the variation of the stationary phase condition. The application of alternating voltage might increase the column temperature by Joule heating, and it might induce the releasing of sample solutes from the stationary phase. When the frequency of an applied alternating voltage was increased, the electric current was also increased; i.e., the Joule heating was increased with the application of the higher frequency alternating voltage. Therefore, the Joule heating could not explain the nonlinear capacity factor variation shown in Figures 4. The alternating voltage might cause this nonlinear capacity factor variation. The phenomenon of the alternating voltage-induced capacity factor variation was further investigated using other sample solutions. Mixtures of 3 mM salicylic acid (pH 2.7) and methanol (90:10), as well as 3 mM benzoic acid (pH 3.0) and methanol

Figure 5. Effect of difference in packing material on the alternating voltage induced capacity factor variation. Mini-bed: i.d. 0.7 mm, length 12 mm, packed with IC-ANION-SW (O) and MCI-GEL SCA04 (0). Other conditions were the same as in Figure 3.

Figure 6. Alternating voltage capillary electrochromatogram of carboxylic acids. Column: i.d. 0.32 mm, length 8.8 cm, packed with IC-ANION-SW. Eluent: methanol/10 mM ctric acid, 5 mM hexamethylenediamine (pH 4.8) (10/90). Applied voltage: (A) without voltage, (B) Vpeak ) 2.5 kV, f ) 400 Hz. Flow: constant pressure mode of 150 kgf/cm2. Detection: 254 nm. Peak identification: (1) uracil, (2) benzoic acid, (3) phthalic acid, and (4) salicylic acid.

(90:10) were used for sample solutions. The application of an alternating voltage induced UV signal variations in both sample solutions, as shown in Figure 4; i.e., the capacity factor variations were induced in both sample solutions. When salicylic acid was used as a sample solute, its dependence on the frequency was similar to that of phthalic acid; i.e., the singular frequencies at 117 and 273 Hz were observed in both. As for benzoic acid, the voltage-induced peak heights were almost constant, and no singular frequency was observed. The point in common between phthalic and salicylic acids is a carboxyl group. Therefore, the frequency dependence in Figure 4 might be originated in the carboxylic group. The alternating voltage-induced capacity factor might be dominated by the property of functional groups in a sample solute. Dependence on the Kind of Stationary Phase. The variation in the capacity factor induced by the application of an alternating voltage might reflect a variation of the property of a stationary phase. Therefore, the variation might be dominated by the properties of packing material itself. The dependence of the alternating voltage-induced capacity factor variation on the kind of base material of packing supports was studied. As packing materials, IC-ANION-SW (silica gel base) and SCA04 (polymer base) were used. The phthalic acid was used as a sample solute. As shown in Figure 5, the variation in frequency using SCA04 (open square) achieved a different pattern, as compared to that using IC-ANION-SW (open circle). Although SCA04 also generated peaks induced by the application of alternating voltage, its dependence on the frequency was less remarkable, as compared to IC-ANION-SW. In particular, no singular frequencies at 117 and 273 Hz were observed. Since the peak heights of IC-ANION-SW were smaller than those of SCA04 in the range over 500 Hz, the capacity factor variation using SCA04 was sensitive as compared with that using IC-ANION-SW. One possibility is that this difference in sensitivity might come from the difference in the flexibility of the structure of the packing materials. Since the structure of SCA04 is softer than that of IC-ANION-SW, the structure of SCA04 was deformed easily by the application of the alternating voltage. The alternating voltage-induced capacity factor

variation strongly depended on the kind of packing material. Therefore, this variation might be attributed to variations in the property of a stationary phase. Typical Chromatograms of Alternating Voltage Capillary Electrochromatography. As shown in Figures 2-5, the application of an alternating voltage can vary the capacity factor of sample solutes. We applied this phenomenon to chromatography and performed alternating voltage capillary electrochromatography (AV-CEC) using an anion exchange column. Figure 6 demonstrated the separation of four sample solutes of uracil, benzoic acid, phthalic acid, and salicylic acid with and without the application of the alternating voltage (Vpeak ) 2.5 kV, f ) 400 Hz). In Figure 6, the sample solution was injected into a column using a split injection method, though such a sample injection was not performed in the experiment using the mini-bed (Figures 2 and 3). An aqueous solution composed of a mixture of 10 mM citric acid and 5 mM hexamethylenediamine (pH 4.8) and methanol (90:10) was used as an eluent. The chromatogram (A) in Figure 6 was an ordinary capillary liquid chromatogram because it was performed with no alternating voltage. Since uracil was a nonretained sample solute, its elution time was regarded as t0 to calculate a capacity factor. The application of the alternating voltage accelerated the elution of the sample solute as shown in Figure 6. Figures 2 and 3 show the application of an alternating voltage induced the release of sample solutes from packing materials, except for the singular frequency; i.e., the decrease in the capacity factor was induced. Thus, decreasing elution times of the sample solute in Figure 6 was reasonable. The reduction of the elution time of salicylic acid was significant, as compared with the two other sample solutes. The capacity factors of benzoic acid, phthalic acid, and salicylic acid without a voltage were 1.88, 4.02, and 7.02, respectively, but were reduced to 1.63, 3.55, and 5.08, respectively, by the application of the alternating voltage. All capacity factors were decreased by such applications. To evaluate the capacity factor variation of each sample solute, we used the variation ratio calculated by (kAV - k0V)/k0V where kAV and k0V are capacity factors with and without an application of an alternating voltage. The variation ratios Analytical Chemistry, Vol. 75, No. 14, July 15, 2003

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Figure 7. Relationship between magnitude of an applied voltage and variation ratio of the capacity factors of benzoic acid (2), phthalic acid (O), and salicylic acid (0). Frequency of alternating voltage: 600 Hz. Flow: constant pressure mode of 100 kgf/cm2. Other conditions were the same as in Figure 6.

Figure 8. Relationship between frequency of an applied alternating voltage and variation of the capacity factors of benzoic acid (2), phthalic acid (O), and salicylic acid (0). Applied voltage: Vpeak ) 1.5 kV. Other conditions were the same as in Figure 6.

of the capacity factors of benzoic acid, phthalic acid, and salicylic acid were -0.13, -0.12, and -0.28, respectively. Since the reduction ratios of the three sample solutes were not the same value, the application of an alternating voltage might be useful for the characteristic control of the retention behavior of sample solutes. Relationship between the Amplitude of an Applied Voltage and the Variation of the Capacity Factor. Figure 6 shows the ability of the application of an alternating voltage to control chromatographic behavior. The amplitude of an alternating voltage, that is, Vpeak, is one of the important factors for AV-CEC. The effect of Vpeak on variations of the capacity factor was studied using an alternating voltage of 600 Hz. Figure 7 shows the relationship between the amplitude of the applied alternating voltage (Vpeak) and the variation ratio of the capacity factor. When the applied Vpeak increased, all variation ratios of the three sample solutes decreased; i.e., the higher voltage accelerated the reduction of the capacity factor. In the case of the application of a direct voltage, the variation of capacity factors was also determined by the magnitude of the applied voltage.10-13 The effect of Vpeak on the variation ratio was specified by the species of sample solute. Salicylic acid is the sample solute that is most susceptible to an applied electric field. When the Vpeak was 0.75 and 1.25 kV, the variation ratio for phthalic acid was greater than that for benzoic acid. However, the variation ratios for phthalic acid and benzoic acid assumed almost the same value with the application of 1.75 kV; i.e., there was a voltage lag for benzoic acid. Figure 7 shows that the relationship between Vpeak and the capacity factor variation might conform to a kind of sigmoidal curve. Therefore, the voltage lag for benzoic acid might mean that benzoic acid requires the higher voltage to induce the reduction of the capacity factor. In this mobile phase (pH 4.8), the carboxyl group in benzoic acid (pKa ) 4.2) could not dissociate completely. However, both carboxyl groups in salicylic (pKa ) 3.0) and phthalic (pKa1 ) 3.0) acids could be ionized completely. This difference in ionization might be one of reasons for the voltage lag for benzoic acid. The amplitude of an alternating voltage is an important factor in controlling the capacity factor of the sample solute in AV-CEC.

Relationship between the Frequency of an Applied Alternating Voltage and the Capacity Factor. As shown in Figures 3 and 4, the voltage-induced sample release from packing materials depended on the frequency of an applied alternating voltage. The dependence of the capacity factor variation in AV-CEC on the frequency of an applied alternating voltage (Vpeak ) 1.5 kV) was studied. Benzoic acid, phthalic acid, and salicylic acid were used as the sample solutes. Uracil was used as nonretained sample solute to calculate each capacity factor. Figure 8 shows the relationship between the frequency of an alternating voltage and the variation ratio of the capacity factors of three sample solutes. All three variations had a negative value; i.e., the application of alternating voltage decreased the capacity factors of all sample solutes. All variations of the capacity factors of the three sample solutes were suppressed with the application of alternating voltages of 450 and 500 Hz. This frequency range might be a singular frequency for the capillary column used in this experiment. The mechanism of this suppression might also be a kind of hysteresis of alternating voltage-induced variation of the capacity factor. However, further investigation is required to realize this phenomenon. The application of 1000 Hz induced a relatively small variation of the capacity factor. Applied voltage forces the variation of the stationary phase property, and it would take some time; i.e., a time lag might exist. When the frequency of an alternating voltage was high enough, the applied voltage could return to 0 before the variation of the stationary property. Therefore, the decrease in the capacity factor variation around 1000 Hz might suggest that the variation speed of the property of the stationary phase might not be remarkably fast. As in Figure 7, salicylic acid is the sample solute that is most susceptible to an applied voltage at any frequency. Although the capacity factor variation of benzoic acid was almost greater than that of phthalic acid, this tendency was reversed with applications of 250, 450, 500, and 600 Hz, respectively. The frequency of the alternating voltage is an important factor in achieving selective control of the retention behavior of a sample solute.

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CONCLUSION The application of an alternating voltage to a capillary column varied the capacity factors of sample solutes, and the magnitude of that variation depended on the amplitude and frequency of the voltage applied. The capacity factor variation was governed by the kinds of sample solutes, especially the kind of functional group, and the packing materials used. Alternating voltage capillary electrochromatography was newly proposed as the application of this phenomenon to control the retention behavior of a sample solute. Although the underlying mechanism of the voltage-induced capacity factor variation is still unclear, the use of alternating voltage capillary electrochromatography might contribute to advances in the field of separation analysis.

ACKNOWLEDGMENT This research was supported in part by the Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research (B) no. 1244209, 2000 (Tsuda) and Grantin-Aid for Young Scientists (B) no. 14740402, 2002 (Kitagawa). Our thanks also to Tosoh for their kind donation of packing materials.

Received for review January 23, 2003. Accepted April 12, 2003. AC034064E

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