Sweeping of Neutral Analytes in Electrokinetic Chromatography with

Anal. Chem. 2000, 72, 1934-1940

Correspondence

Sweeping of Neutral Analytes in Electrokinetic Chromatography with High-Salt-Containing Matrixes Joselito P. Quirino,*,† Shigeru Terabe,† and Petr Bocek‡

Faculty of Science, Himeji Institute of Technology, Kamigori, Hyogo, Japan 678-1297, and Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Brno, Czech Republic CZ-61142

The concept of sweeping neutral analytes using a highconductivity matrix or under a reduced electric field in micellar electrokinetic chromatography (MEKC) using anionic micelles and in the presence of electroosmotic flow is presented. Three important processes are identified. First, stacking of the micelles at the cathodic interface between the sample solution (S) and background solution (BGS) zones is identified. This is then followed by the sweeping of analyte molecules by the stacked micelles that enter the S zone. Finally, the destacking of the stacked micelles at the anodic interface between the S and BGS zones occurs. The stacking of the micelles improves the focusing effect of sweeping by a factor approximately equal to the ratio of conductivities between the S and BGS zones (ratio ) enhancement factor ) γ′). However, the destacking of the stacked micelles broadens the swept zones by a factor approximately equal to 1/γ′. In effect, the focusing effect of sweeping using a matrix with equal or higher conductivity compared to the BGS will be roughly the same. The micelle stacking and destacking mechanisms are verified experimentally. This paper also provides comments on the mechanism of neutral analyte focusing under similar conditions proposed by another group (Palmer, J.; Munro, N. J.; Landers, J. P. Anal. Chem. 1999, 71, 1679-87). According to a recent study by Palmer and co-workers, addition of salt to sample matrixes (e.g., sodium chloride) that will maintain sample matrix conductivity 2-3-fold above that of the separation solution is indicated for effective concentration of analytes in micellar electrokinetic chromatography (MEKC).1 The high-salt analyte stacking mechanism they postulated in normal migration MEKC (electrophoretic velocity of the anionic micelle is less than the electroosmotic flow (EOF) velocity) is given below. Micelles from the separation buffer stack at the cathodic side interface between the separation buffer and sample matrix. “Analytes * Corresponding author (e-mail: [email protected]). Current address: Department of Chemistry, Stanford University, Stanford CA 94305-5080. † Himeji Institute of Technology. ‡ Academy of Sciences of the Czech Republic. (1) Palmer, J.; Munro, N. J.; Landers, J. P. Anal. Chem. 1999, 71, 1679-87.

1934 Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

experience a reduction in velocity upon encountering the stacked micelle/sample zone interface.” Increasing the conductivity of the sample zone increases the electrophoretic velocity of micelles in the separation buffer and decreases the electrophoretic velocity of micelles at the sample matrix/separation buffer interface. “This, in combination with the maximum velocity of analytes in the sample matrix (pure EOF) versus decreased velocity in the concentrated micelles at the sample matrix/separation buffer interface (high local micelle concentration), causes analyte stacking.” Separation mode was then suggested to commence when the micelle front has exited the high-salt matrix and the anionic component of the sample matrix (e.g., chloride) has diffused to the separation buffer conductivity. This form of focusing is different from that of sample stacking in capillary zone electrophoresis (CZE) or MEKC.2,3 In CZE and MEKC sample stacking, focusing basically relies on the change in electrophoretic velocities at the interface between the high electric field sample zone and low electric field separation zone. In this high-salt analyte stacking mechanism, focusing relies on the change in analyte migration velocity at the interface between the stacked micelle and sample zone. Note that migration velocity includes EOF velocity (veof). The ratio of analyte migration velocity in the sample zone (veof) to that in the stacked micelle zone (sum of the analyte effective electrophoretic velocity [vep*(a)] and veof) is then directly proportional to the extent of focusing (EF) (eq 1).

EF )

veof vep*(a) + veof

(1)

This ratio would then be large only when the veof and vep*(a) balances. Note that veof and vep*(a) have different signs. Therefore, the proposed mechanism does not explain quantitatively the enhancements in sensitivity that they obtained. Sweeping in MEKC is defined as the picking and accumulating of analyte molecules by the micelles that fill the sample region,

(2) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A-96A. (3) Quirino, J. P.; Terabe, S. J. Capillary Electrophor. 1997, 4, 233-45. 10.1021/ac990566+ CCC: $19.00

© 2000 American Chemical Society Published on Web 03/17/2000

and causes a unique focusing effect.4 The initial report on sweeping utilized high-salt-containing sample matrixes with a conductivity (measured with a conductivity meter) equal to that of the background solution (BGS) that does not contain micelles. Theoretically, sweeping predicts an almost unlimited increase in detection sensitivity for very hydrophobic analytes (high retention factor (k) analytes are focused more effectively than low k analytes). The resulting length of the injected analyte zone (lsweep), whether charged or uncharged, is dictated by the k and the length of the initial zone (linj) and is independent from the magnitude of the EOF (eq 2).4,5

1 lsweep ) linj 1+k

(2)

Depending on the analytical problem, additives can be employed in the sample matrix (organic solvent or nonionic surfactant), pH of sample and BGS buffer can be chosen, and a coated capillary can be used to improve the performance of sweeping.5 However, Palmer and co-workers did not observe efficient focusing by sweeping when the sample matrix conductivity was similar to that of the BGS conductivity under high EOF conditions.1 Sweeping was also found to work better under suppressed EOF compared to high EOF conditions.5 In this investigation, therefore, we expound upon the effect of a high-conductivity matrix compared to the background solution on sweeping as an on-line concentration method. We will show a model that would implicitly show the evolution of the zones in the presence of EOF. Aside from the stacking of micelles1 and sweeping of analyte molecules4,5 described above, which will be described in more detail, the destacking of the micelle will be considered. Sweeping in a Reduced Electric Field. Length of Injected Neutral Analyte Zones after Sweeping under a Reduced Electric Field. Figure 1 depicts a theoretical model for the sweeping of a neutral analyte (a) in the presence of EOF but with a reduced electric field in the sample solution (S) region. Movement of all pertinent boundaries upon application of voltage is illustrated. All electrokinetic velocities or electrophoretic mobilities are positive and negative when movement is directed toward the cathode and anode, respectively. The starting situation (Figure 1A) is injection of S having a greater conductivity compared to the BGS. The capillary was conditioned with the BGS before injection. Areas with diagonal lines depict the high-conductivity region. The ac and aa are the neutral analyte molecules found near the interface between S and BGS zones at the cathodic and anodic ends, respectively. The mcc and mca are the micelles at the cathodic and anodic ends of the S zone, respectively. The arrows in Figure 1A show the magnitude of electrokinetic velocities when a voltage is applied. Note that the electrokinetic velocity of mcc is lower compared to mca due to the reduced electric field in the S region, which is a difference between the present model and the one with homogeneous electric fields.5 The broken lines between Figures 1A, B, and C indicate the starting position of ac and mcc. Upon application of voltage (Figure 1B), micelles at the cathodic side of the S zone that migrate toward the cathode will (4) Quirino, J. P.; Terabe, S. Science 1998, 282, 465-8. (5) Quirino, J. P.; Terabe, S. Anal. Chem. 1999, 71, 1638-44.

stack at the S and BGS interface (Ic) and sweep a (dark shaded area). Furthermore, stacking of micelles occurs provided that the mobility of the micelle (µmc) is less than the mobility of the coion (µco-ion) of the salt present in the sample matrix (µmc < µco-ion). Here, the co-ion means the ion with the same sign as those of the micelles; in our case both are anions. The principle of the above-mentioned relationship between mobilities in order to reach stacking is well known and it is in fact the principle of the selfsharpening effect of isotachophoretic boundaries and of adjustment of concentration in accord with the Kohlrausch regulation function. Detailed theory and explanation may be found elsewhere.6,7 In cases where the sample contains inorganic salts, e.g., NaCl, such a condition (µmc < µCl) is always fulfilled. The area with squares and area with dark shading depict the zone with high concentration of micelles compared to the BGS; however, the area with squares contains no a molecules. The areas with diagonal lines, dark shading, and with squares now depict the high-conductivity zone. Also, the concentration of a in the shaded area is greater than that in the area with diagonal lines. The ac will be incorporated into the micelle, thus the migration velocity is equal to that in MEKC or higher than that of the micelle. However, the migration velocity of aa is equal to the average EOF because it will not be incorporated into any micelle until the time when aa reaches mcc. The length of a zone after sweeping (lsweep) is then the difference between the distances traveled by ac [d(ac)] and mcc [d(mcc)], assumed to be when aa reaches mcc (Figure 1C). For simplicity, lsweep in a reduced electric field is given by eq 2, where k is the retention factor in the S region (eq 3).8

k ) Kυ j [Cmc(S)]

(3)

K is the distribution coefficient, υj is specific volume of the micelle, and Cmc(S) is the concentration of the micelle in the S region. Cmc(S) is also the concentration of the micelle emanating from the BGS after focusing by stacking at Ic, and is given by eq 4.2

Cmc(S) ) Cmc(BGS)γ′

(4)

Cmc(BGS) is the concentration of the micelle in the BGS and γ′ is the enhancement factor (γ′ ) conductivity of S/conductivity of BGS). In another way, the concentration of the micelle adjusts to the higher Kohlrausch value (sum of C/µ where C is the concentration and µ is the mobility) of the S zone, and the adjusted zone of the micelles acts as the isotachophoretic terminator for the co-ion in the sample zone (e.g., chloride). A fronting concentration profile of the micelle can also be imagined if the micelles are faster (µmc > µco-ion) than the sample salt co-ion; however, such a case is rare. On the other hand, the sample co-ion (e.g., chloride) shows a fronting profile when it migrates into the BGS toward the anode. The concentration of the sample co-ion at the anodic side of the S zone adjusts to the low Kohlrausch value of the BGS zone. The co-ion zone migrates to the cathode if the EOF velocity is greater (6) Bocek, P.; Deml, M.; Gebauer, P.; Dolnik, V. Analytical Isotachophoresis; VCH Verlag GmbH: Weinheim, Germany, 1988; p 43. (7) Mosher, A.; Thormann, W. Electrophoresis 1985, 6, 477-82. (8) Otsuka, K.; Terabe, S. Bull. Chem. Soc. Jpn. 1998, 71, 2465-81.

Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

1935

Figure 1. Evolution of micelles and neutral analyte molecules during sweeping in the presence of high electroosmotic flow and a high-saltconcentration matrix. (A) Starting situation: injection of S prepared in a matrix having a conductivity greater than that of the BGS; (B) application of voltage at positive polarity, micelles emanating from the cathodic side stack at the interface between S and BGS (Ic) and sweep the analyte molecules; (C) the injected analyte zone is assumed to be completely swept; (D) the stacked micelles leave the S zone and destack at (Ia) while swept analyte zones broaden; (E) swept analyte zones completely leave the S zone; other symbols and explanations appear in the text.

than the sample co-ion velocity. In any case, if the conductivity of the sample matrix is three times that of the BGS (γ′ ) 3), the retention factor may be increased by a factor of 3. Consequently, sweeping three times more narrows analyte zones. However, the nature of the analyte, related to the K value, has a greater impact on the sweeping of zones. Length of Injected Neutral Analyte Zones after Sweeping and Broadening Caused by Destacking of Micelles. There1936 Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

after, the stacked micellar zone and swept analyte zones separate from the high-conductivity region (Figure 1D and E), which causes the destacking of micelles and the broadening of swept analyte zones at the other interface between the S and BGS zones (Ia) (Figure 1D). The stacked zone, having high concentrations, leave the high-conductivity S zone enter the region of the BGS and adjust again to the low value of the Kohlrausch function in the original BGS. This means that their concentrations decreases

they dilute (broaden). Moreover, it is necessary for the swept analyte zones to leave the S zone in order to attain separation. Note that the time or migration length required for the destacking of the analytes is proportional to the length of the sample pulse as well as to the concentration of the salt added to the sample. Obviously, too high a concentration of the salt and/or too long a sample pulse counteracts destacking and subsequent separation.9,10 The area between aa and ac (with dots) depicts the broadened analyte molecules in Figures 1D and E. The degree of destacking or broadening of micelles and analyte zones is caused by the differences in electrophoretic velocities in the S and BGS zones, somewhat similar to the destacking process observed in partialfilling MEKC that is caused by the differences in velocities in the micellar and nonmicellar regions.11 Remember that electrophoretic velocity in the S zone is lower than in the BGS zone due to higher electric field in the BGS zone. For example, the effective electrophoretic velocity of aa after passing Ia [a(BGS)] is greater than that of ac that is still inside S [a(S)] (see Figure 1D). The concentration of the stacked micelles adjusts to that of the BGS according to eq 4, and the velocity of mcc and mca becomes similar and the distances between them is the vacancy of the micelle. The zones at the anodic side of Ia are assumed to have conductivity similar to the BGS. An inverse relationship between γ′ and extent of micelle destacking is then obvious. Here, a direct relationship between the extent of micelle destacking and extent of swept analyte zones broadening is assumed. Therefore, the resulting length of the injected zone after all the swept analyte molecules exit the S zone (lfinal, Figure 1E) can be simply described by eq 2 (lfinal ) lsweep (eq 2)), where k is computed in the usual manner. In general, preparing the sample in a matrix having a higher or equal conductivity compared to the BGS should show similar focusing effects. Finally, the resulting analyte zones are eventually detected by virtue of the strong EOF. Note also that the high-conductivity region having low electric field strength travels toward the detector with the average velocity of the EOF and remains inside the capillary until it exits to the outlet vial. Some Comments on Palmer and Co-Workers’ High-Salt Analyte Stacking Mechanism. First, Palmer and co-workers suggested that the sample matrix would maintain a concentrated component of high-intrinsic mobility anions “frozen” at the cathodic end of the high-conductivity matrix. On one hand, the authors suggested that diffusion by migration of the anionic component of the sample matrix occurs at the anodic end. This infers that the anions at different parts of the sample matrix are selectively affected by the electric field. Second, it was suggested that the stacked micelle “locked” into place until the last portion of the sample matrix anionic component had diffused. This infers that the conductivity of the sample matrix is only dependent on the anionic component of the sample matrix that diffuses from the anodic end of the sample zone. Also, this infers that the highconductivity zone is narrowed from the anodic to the cathodic side of the sample matrix zone or the high-conductivity zone disappears with time, and that the whole high-conductivity zone does not migrate as a plug. (9) Gebauer, P.; Thormann, W.; Bocek, P. J. Chromatogr. 1992, 608, 47-57. (10) Gebauer, P.; Thormann, W.; Bocek, P. Electrophoresis 1995, 16, 2039-50. (11) Muijselaar, P. G.; Otsuka, K.; Terabe, S. J. Chromatogr. A 1998, 802, 3-15.

Third, the micelles at the anodic end of the sample zone were made to approach the stacked micelles from the cathodic end of the sample zone. This infers that the migration velocity of the micelles at the anodic end of the sample zone is greater than the migration velocity of the stacked micelles from the cathodic end of the sample zone. In reality, the migration velocity of the micelles at the anodic end is lower than the migration velocity of the stacked micelles because of the enhanced field in the separation solution zone. Fourth, although the sweeping phenomenon was implied because the analyte molecules were made to accumulate at the stacked micelle zone, the term sweeping was not used. Finally, the destacking of the micelles and its consequences were not taken into consideration. EXPERIMENTAL SECTION Apparatus. All capillary electropherograms were obtained with a Hewlett-Packard 3D Capillary Electrophoresis System (Waldbronn, Germany). Electrophoresis and sweeping experiments were performed in fused silica capillaries of 50 µm i.d. and 375 µm o.d. obtained from Polymicro Technologies (Phoenix, AZ). Capillaries were 56.5 cm to the detector (65 cm total length) and thermostated at 20 °C. An optimum detection wavelength was selected for each analyte based on the spectra recorded by a diode array detector. Conductivities were measured with a Horiba ES12 conductivity meter (Kyoto, Japan). The pH of solutions was adjusted and measured with the aid of a Beckman Φ 34 pH meter. Water was purified with a Milli-Q system (Millipore, Bedford, MA). Reagents and Solutions. All reagents were purchased in the highest grade possible from Nacalai Tesque (Kyoto, Japan) unless otherwise stated. Stock solutions of 0.5 M sodium dodecyl sulfate (SDS), 0.5 M sodium cholate (SC), 0.5 M sodium hydrogenphosphate, 0.5 M ammonium hydrogenphosphate (Wako Pure Chemicals, Osaka, Japan), and 1 M sodium chloride were prepared every week in purified water. Solution of 0.02 M borate buffer (pH 9.3) was obtained from Hewlett-Packard (Waldbronn, Germany). SDS BGS’s were prepared (each day) by dilution of the SDS stock solution in appropriate phosphate or borate buffers. SC BGSs were prepared (each day) by weighing an appropriate amount of SC and dilution in appropriate phosphate or borate buffer. A weighed amount of glycyrrhizic acid monoammonium salt (Wako Pure Chemicals, Osaka, Japan) was dissolved in ammonium phosphate buffer to make the micellar BGS described in Figure 3. Stock solutions of alkyl phenyl ketones (hexanophenone, butyrophenone, propiophenone, valerophenone, and acetophenone) were prepared in methanol to concentrations that ranged between 5200 and 6100 ppm. Stock solution of around 3000 ppm progesterone was prepared in 50% methanol. Portions of alkyl phenyl ketones stock solutions were combined and diluted with aqueous methanol to yield comparable peak heights. Final dilutions (concentrations in the figures) were done using the sample matrixes. The sample matrixes were prepared by appropriate dilution (and combination) of ammonium hydrogenphosphate, and sodium chloride stock solutions. The sample matrixes contain no pseudostationary phase or any additive that might impart electrophoretic mobility to the analytes. All solutions were filtered through 0.45 µm filters (Toyo Roshi, Japan) prior to capillary electrophoresis experiments. General Electrophoresis Procedure. The capillary was conditioned prior to use with 1 M NaOH (20 min), followed by Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

1937

methanol (20 min), 0.1 M NaOH (20 min), purified water (20 min), and finally with the BGS (10 min). The sample solution (S) was then injected into the capillary at end farthest from the detector using pressure (50 mbar). The velocity of a liquid at 50-mbar pressure was determined to approximate the lengths of the zones injected at different intervals (0.06 cm/s). Injecting an UVabsorbing sample into the capillary and then applying 50-mbar pressure until a response was obtained accomplished this. From the velocity that was obtained, the lengths of the zones were computed given the injection time. Voltage was then applied at positive polarity with the BGS at both sides of the capillary, until all peaks were detected. The capillary was flushed, between consecutive analyses to ensure repeatability, with methanol (1 min), followed by 0.1 M NaOH (1 min), purified water (1 min), and finally with the BGS (2-5 min). Other conditions are specified in the text or figures. RESULTS AND DISCUSSION The High-Conductivity Zone. Figure 2 shows a typical electropherogram obtained with sweeping using a high-conductivity sample matrix (γ′ ) 2.2) and high EOF (similar to the model above). The anionic component of the sample matrix (chloride) is detected as a negative peak at 200 nm and shows a tailing profile toward the cathode. Also, the test analyte (progesterone) that was swept and detected at 247 nm eluted before the chloride peak. The current plot (trace below the electropherograms) and the negative peak before 10 min in the electropherograms produced by the sample matrix clearly show that the sample matrix or highconductivity zone remains inside the capillary (a) until it exits and creates a stable current (b). This is consistent with the model and discussion earlier. A stable current indicates a capillary filled with the BGS only. The conductivity of the sample matrix changes as it moves across the capillary as shown by the unstable current observed (a), which is probably caused by the mixing of zones due to local EOF mismatch.3 The Stacking and Destacking of the Micelle. Figure 3A illustrates the experimental design used to study the stacking and destacking of the micelle when the sample matrix is a highconductivity solution. First, a nonmicellar BGS (1) is introduced to condition the capillary. Then a micellar BGS having conductivity similar to the nonmicellar BGS (1) is injected, where the micelle used is not transparent against UV light. This is followed by injection of a nonmicellar BGS (2) zone having conductivity greater than the nonmicellar BGS (1). Voltage was then applied (positive polarity) at different intervals with the nonmicellar BGS (1) at both ends of the capillary to capture the stacking and destacking processes. Pressure (50 mbar) was then applied to mobilize the zones to the detector. A UV active micelle (glycyrrhizic acid) over a non-UV active micelle and a marker was used in order to depict the concentration of the micelles accurately.12 Figure 3BI shows the concentration profile of the injected micelles without application of voltage. Figure 3BII shows the injected micelles that were not stacked and the injected micelles that were stacked when voltage was applied for 120 s. The micelles stack at the interface between the micellar BGS zone and the highconductivity nonmicellar BGS (2) zone. The stacked micelles will traverse the high-conductivity zone and eventually occupy a larger (12) Ishihama, Y.; Terabe, S. J. Liq. Chromatogr. 1993, 16, 933-44.

1938 Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

Figure 2. Typical sweeping MEKC electropherogram using a sample matrix with a higher conductivity compared to the separation solution. Conditions: BGS, 80 mM sodium cholate in 10 mM borate buffer (measured BGS pH 9.4) (7.1 mS/cm); injection, 4.8 cm of ∼2.8 ppm progesterone in 150 mM sodium chloride (15.8 mS/cm); applied voltage, 17 kV; the lower trace is the current plot where (a) shows the time when the capillary is filled with the high-conductivity sample solution and BGS, and (b) shows the time when the capillary is filled with the BGS only since the high-conductivity sample solution exited.

fraction of the high-conductivity zone if voltage was applied for a longer time. Figure 3BIII shows the injected micelles that were stacked, and injected micelles that were stacked and destacked when voltage was applied for 300 s. The stacked micelles destack at the interface between the nonmicellar BGS (2) zone (now filled with stacked micelles) and the nonmicellar BGS (1) zone that entered the capillary from the anodic end by virtue of EOF. Note that the concentration of the stacked micelles based on peak height is approximately two times higher compared to that of the injected micelles (Figures 3BI or 3BII) and destacked micelles (Figure 3BIII). Also, the γ′ is equal to 2.1. These results are all consistent with the present model and discussion above. The uneven concentration profiles of the micelles is probably due to electrophoretic dispersion. With experiments using a separation buffer saturated by progesterone, Palmer and co-workers reported more than an 18-fold increase in the concentration of micelles where γ′ is around 2-3.1 Effect of Sample Matrix Conductivity on Peak Responses. Figure 4 shows plots of peak heights obtained with sweeping using

Figure 4. Effect of enhancement factor on peak heights under high EOF. Conditions: BGS, 50 mM SDS in 20 mM borate buffer (pH 9.3) (A), 60 mM sodium cholate in 20 mM ammonium hydrogenphosphate (pH 8.3) (B); injection, 3.6 cm of ∼5 ppm test analytes in different sample matrix; sample matrix, different concentrations of ammonium hydrogenphosphate (I) and sodium chloride (II); applied voltage, 25 kV (A), 21 kV (B); peak identification, 1 (acetophenone), 2 (propiophenone), 3 (butyrophenone), 4 (valerophenone); 5 (hexanophenone); other information appears in the text.

Figure 3. The stacking and destacking of the micelle obtained from pressure-driven mobilization of zone. Schematic diagram of the experimental setup (A) (discussion found in text); concentration profiles obtained (B). Conditions in B: nonmicellar BGS (1), ammonium phosphate buffer at pH 8.8 (5.9 mS/cm); nonmicellar BGS (2), ammonium phosphate buffer at pH 8.8 (15.2 mS/cm); micellar BGS, 15 mM glycyrrhizic acid in 12.5 mM ammonium phosphate at pH 8.8 (5.9 mS/cm); length of pertinent zones, nonmicellar BGS (2), ∼6.6 cm, micellar BGS, ∼13.2 cm; time for application of voltage at 17 kV, 0 s (I), 120 s (II), 300 s (III); applied pressure for mobilization of zones, 50 mbar toward the detector.

different sample matrixes (increasing conductivity) versus γ′. Alkyl phenyl ketones were used as test analytes and two surfactants were used as micelle-forming agents, SDS (A) and SC (B). The k value increases from analyte 1 to 5. Two sample matrixes were used: ammonium hydrogenphosphate (I, 50-400 mM) and sodium chloride (II, 50-400 mM) solutions. Ammonium hydrogenphosphate was used because initial studies with sodium hydrogenphosphate incurred very high currents when used as buffer to dissolve SC. No general trend on the effect of enhance-

ment factor on the peak heights can be generated based on all of the plots; this result is consistent with our predictions. The compositions of the sample matrix and micellar solution and nature of the analyte seem to affect the results obtained in the plots in Figure 4. For example, slight increases in peak heights for some test analytes (especially those with lower k) are observed for γ′ values to around 3 (Figure 4AI and BII) or 5 (Figure 4BI). For greater γ′ values (>3 or 5 for Figure 4AI or BI, respectively), peak heights show a decreasing trend. Palmer and co-workers obtained similar results when higher-conductivity sample matrixes were used.1 Moreover, for the SC system (Figure 4BI and II), the peak of hexanophenone disappeared after an enhancement factor value of around 3. Disappearance might be caused by adsorption of the analytes into the container or capillary walls. In both systems, corrected peak areas gradually decreased with the increase in enhancement factor, which is again caused by adsorption. The effect of systems with γ′ values >3 is shown in Figure 5. Slightly better peak shapes (sharper) are observed with the increase in conductivity of the sample matrix (Figure 5B). However, as stated earlier, peak heights did not show any improvement and the last two peaks show a decrease in peak heights and corrected peak areas. More studies are essential Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

1939

Figure 5. Effect of increasing the conductivity of the sample matrix on peak shapes under high EOF. Conditions: BGS, same as in Figure 4B (7.7 mS/cm); sample matrix, ammonium hydrogenphosphate 100 mM (14.7 mS/cm) (A), 200 mM (25.8 mS/cm) (B); other information is similar to that found in Figure 4B.

regarding the use of very high conductivity matrixes. CONCLUSION The theoretical study on the focusing of analyte zones in MEKC with a matrix having a conductivity greater than the BGS and void of the micelle showed three important processes, namely the stacking of the micelle, the sweeping of analyte molecules, and the destacking of micelle. Stacking and destacking occurs at the interfaces between the high- and low-conductivity zones. The stacking of micelles provides analytes with higher k because of the increase in the concentration of micelles, and thus results in narrower peak widths after sweeping. However, the highconcentration micelles eventually destack at the other interface, because of an increase in electrophoretic velocity and decrease in Kohlrausch value. This is suggested to cause a broadening mechanism for the swept analyte zones. The cumulative effect of the two processes on the focusing of analyte zones by sweeping will produce zones that are alike compared to when the sample (13) Quirino, J. P.; Terabe, S. Anal. Chem. 1999, 72, 1023-30.

1940

Analytical Chemistry, Vol. 72, No. 8, April 15, 2000

matrix has conductivity equal to that of the BGS. This was verified experimentally under high EOF conditions, where no significant increase in detection sensitivity was observed with the increase in the conductivity of the sample matrix. A similar result was obtained for cationic analytes under suppressed EOF condition and thus similar result is expected for anionic analytes.13 Therefore, use of sample matrixes having equal or high conductivity compared to the separation solution has similar value. However, focusing results may vary depending on the analytical conditions (e.g., type of micelle and salts present in the separation solution and sample matrix, respectively) and the nature of the analyte (e.g., for low k analytes, the use of highconductivity matrixes (3 > γ′ > 1) seems to be beneficial in slightly improving peak symmetry; and high k analytes tend to adsorb more into the capillary walls with the increase in the conductivity of the matrix). Moreover, in the experimental results described here, higher-conductivity matrixes (γ′ > 3) are also found to be detrimental. With this in mind, the conductivity of the sample matrix should be kept below three times the conductivity of the background solution. In general, standards for calibration should be adjusted to the conductivity of the sample matrix (high or equal conductivity) for more precise measurements. Finally, the destacking phenomenon discussed provides further insight on the evolution of zones in a nonhomogeneous system consisting of liquids inside the capillary having different conductivity. ACKNOWLEDGMENT J.P.Q. and S.T. are thankful to Drs. K. Otsuka and N. Matsubara for their support. This work was supported in part by a grant-in-aid for Scientific Research (No. 09304071) from the Ministry of Science, Culture, and Sports, Japan. P.B. acknowledges the support of the Ministry of Education of the Czech Republic (MSMT), Grants Nos. VS-96021 and VS-97014. J.P.Q. is also grateful to the Japanese Society for the Promotion of Science for supporting his doctoral and postdoctoral studies.

Received for review May 27, 1999. Accepted December 2, 1999. AC990566+