Anal. Chem. 1999, 71, 1679-1687
A Universal Concept for Stacking Neutral Analytes in Micellar Capillary Electrophoresis James Palmer, Nicole J. Munro, and James P. Landers*
Analytical Division, Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Unlike recent studies that have depended on manipulation of separation buffer parameters to facilitate stacking of neutral analytes in micellar capillary electrophoresis (MCE) mode, we have developed a method of stacking based simply on manipulation of the sample matrix. Many solutions for sample stacking in MCE are based on strict control of pH, micelle type, electroosmotic flow (EOF) rate, and separation-mode polarity. However, a universal solution to sample stacking in MCE should allow for free manipulation of separation buffer parameters without substantially affecting separation of analytes. Analogous to sample stacking in capillary zone electrophoresis by invoking field amplification of charged analytes in a lowconductivity sample matrix, the proposed method utilizes a high-conductivity sample matrix to transfer field amplification from the sample zone to the separation buffer. This causes the micellar carrier in the separation buffer to stack before it enters the sample zone. Neutral analytes moving out of the sample zone with EOF are efficiently concentrated at the micelle front. Micelle stacking is induced by simply adding salt to the sample matrix to increase the conductivity 2-3-fold higher than the separation buffer. This solution allows free optimization of separation buffer parameters such as micelle concentration, organic modifiers, and pH, providing a method that may complement virtually any existing MCE protocol without restricting the separation method.
Capillary electrophoresis (CE) has been established as a powerful, multimode technique and shown effective as an analytical tool for a variety of analytes ranging in size and character.1-8 The small dimensions of the capillaries (20-100-µm i.d.) allow for high applied fields and fast analysis times with diminished * Corresponding author: (412) 624-1955 (0ffice); (412) 624-8363 (Lab 1); (412) 624-9699 (Lab 2);
[email protected] (e-mail). (1) Liu, J.; Shirota, O.; Wiesler, D.; Novotny, M. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 2302-2306. (2) Rush, R. S.; Cohen, A. S.; Karger, B. L. Anal. Chem. 1991, 63, 13461350. (3) Guttman, A.; Horvath, J.; Cooke, N. Anal. Chem. 1993, 65, 199-203. (4) Landers, J. P.; Oda, R. P.; Madden, B. J.; Spelsberg, T. C. Anal. Biochem. 1992, 205, 115-124. (5) Mazzeo, J. R.; Krull, I. S. BioTechniques 1991, 10, 638-645. (6) Heiger, D. N.; Cohen, A. S.; Karger, B. L. J. Chromatogr. 1990, 516, 3348. (7) Ulfelder, K. J.; Schwartz, H. E.; Hall, J. M.; Sunzeri, F. J. Anal. Biochem. 1992, 200, 260-267. 10.1021/ac981302a CCC: $18.00 Published on Web 04/01/1999
© 1999 American Chemical Society
adverse effects from Joule heating.9 However, these capillary dimensions are problematic as a result of the capillary inner diameter being the path length for detection and the need for small injections of sample (low nanoliters) to avoid overloading. Consequently, detection sensitivity is compromised substantially in comparison with other techniques such as high-pressure liquid chromatography. The small injection volume problem has been circumvented by the development of sample stacking techniques which allow for large sample plugs to be injected into the capillary without adverse effects on peak shape and resolution. The purpose of stacking is to reduce the axial distribution of analytes within the sample matrix to as small a value as possible before separation is initiated. If the sample matrix does not affect distribution of the analytes prior to separation, the analyte distribution will equal the injected plug length, and this length will become the minimum peak width at detection. Analogous to a method described by Hjerte`n for zone sharpening of proteins with paper electrophoresis,10 Chien and Burgi11 introduced field-amplified stacking of charged analytes for capillary zone electrophoresis (CZE). With this mode of stacking, charged analytes are injected into the capillary in a low-conductivity matrix. When separation voltage is initiated, the charged analytes experience enhanced velocity in the lower conductivity (amplified field) sample zone and are stacked at the sample matrix/separation buffer interface, where they experience a decrease in velocity due to the higher conductivity of the separation buffer. Stacking is roughly proportional to the difference in ionic strength between the sample matrix and separation buffer. Unfortunately, standard field-amplified stacking approaches are not applicable to all modes of capillary electrophoresis. Such is the case with micellar capillary electrophoresis (MCE), more commonly known as micellar electrokinetic chromatography (MEKC), which is a mode of capillary electrophoresis shown to provide powerful separations of neutral (uncharged) analytes.12 Separation with this mode is accomplished by imparting electrophoretic mobility to the analytes via complexation with a charged micellar carrier. With the use of a buffer system containing anionic micelles under normal polarity conditions, the micellar electrophoretic mobility is opposite that of the electroosmotic flow (EOF); (8) Guszczynski, T.; Chrambach, A. Biophys. Res. Commun. 1991, 179, 482486. (9) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298 (10) Hjerte`n, S. Biochim. Biophys. Acta 1959, 32, 531-534. (11) Chien, R. L.; Burgi, D. S. J. Chromatogr. 1991, 559, 141. (12) Terabe, S.; Otsuka, U.; Ichihara, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111-113.
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however, bulk capillary flow of all components is toward the detector due to EOF. The affinity of analytes for micelles in aqueous media is typically described as due to hydrophobic interactions. Neutral analytes with a high affinity for the micellar carrier (i.e., very hydrophobic) spend a greater amount of time complexed with the micelles and hence have a velocity lower than EOF, while those with less affinity (i.e., less hydrophobic) have velocity dependent more on EOF. Most analytes fall between these extremes, with their velocity a function of affinity for the micelles, micelle velocity, and EOF velocity. The fact that most analytes separated by this technique are uncharged precludes the use of standard field-amplified stacking since the neutral analytes have no intrinsic electrophoretic mobility. However, the ability of micellar phases to confer electrophoretic mobility to neutral analytes has been exploited for increasing the length of sample plug injection. A variety of solutions for stacking in MCE based on field amplification have been described.13-18 We have previously suggested that, contrary to conventional field-amplified stacking, high-conductivity sample matrixes may be key to sample stacking in MCE.19,20 In the present report, we describe a novel, robust, and potentially universally applicable stacking approach for extant MCE methods and discuss the mechanism through which this is accomplished. As a result of simply raising the conductivity of the sample matrix above that of the separation buffer, field amplification within the separation buffer zone leads to stacking of the charged micelles (not the neutral analytes) at the detector side of the sample matrix. The stacked micelles complex with analytes efficiently because of their high zonal concentration. Although counterintuitive in concept, this mechanism is invoked by simply adding salt to the sample matrix and can yield a several hundredfold increase in sensitivity. This mechanism differs remarkably from previous efforts in that in principle it can be applied to any MCE protocol without constraining separation conditions. EXPERIMENTAL SECTION Materials. Sodium tetraborate, sodium hydroxide, sodium chloride, methanol, ethanol, 1-propanol, 1-butanol, sodium fluoride, chloride, bromide, iodide, and chlorides of lithium, sodium, potassium, rubidium, and cesium, as well as potassium sulfate were obtained from Sigma (St. Louis, MO). Cholic acid, sodium salt hydrate, was obtained from Aldrich Chemical Co. (Milwaukee, WI). Highly sulfated γ-cyclodextrin was obtained from Beckman Coulter (Brea, CA). Corticosteroids were obtained from Steraloids, Inc. (Newport, RI). Apparatus. Experiments were conducted using Beckman P/ACE System 2020 or P/ACE 5510 machines interfaced with an IBM Value Point 486 computer utilizing System Gold software (13) Nielsen, K. R.; Foley, J. P. J. Chromatogr., A 1994, 686, 283-291. (14) Liu, Z.; Sam, P.; Sirimanne, S. R.; McClure, P. C.; Grainger, J.; Patterson, D. G. J. Chromatogr., A 1994, 673, 125-132. (15) Quirino, J. P.; Terabe, S. J. Chromatogr., A 1997, 781, 119-128. (16) Quirino, J. P.; Terabe, S. J. Chromatogr., A 1997, 791, 255-267. (17) Quirino, J. P.; Terabe, S. Anal. Chem. 1998, 70, 149-157. (18) Quirino, J. P.; Terabe, S. Anal. Chem. 1998, 70, 1893-1901. (19) Palmer, J.; Oda, R. P.; Stalcup, A. M.; Strausbauch, M. A.; Landers, J. P. Proceedings, Symposium on High Performance Capillary Electrophoresis (HPCE′97), Anaheim, CA, 1997. (20) Munro, N. J.; Palmer, J.; Hu ¨ hmer, A. F. R.; Oda, R. P.; Stalcup, A. M.; Strausbauch, M. A.; Landers, J. P. Proceedings, Symposium on High Performance Capillary Electrophoresis (HPCE′98), Orlando, FL, 1998.
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(V. 8.1, Beckman Instruments) for control of the instrument and data collection. The capillaries were 47.6 cm by 20, 50, or 75 µm i.d. as indicated (375 µm o.d.) bare-silica polyimide-surfaced capillary tubing purchased from Polymicro Technologies, Inc. (Phoenix, AZ). The sample end (inlet) was anodic. The capillary temperature was maintained at 20 °C, and detection was by UV absorbance at 254 nm. The new bare-silica capillaries were conditioned by rinsing with 1.0 M sodium hydroxide, water, and separation buffer, in order. Buffer and Sample Preparation. Separation buffers were prepared with sodium cholate at stated concentrations and 10 mM tetraborate, dissolved in Milli-Q water (Millipore, Bedford, MA), and were not adjusted for pH (range 8.9-9.1). Buffers were degassed by manual decompression in a syringe and passed through a 0.2-µm filter before use. Alcohols were included at stated concentrations. Sample matrixes were prepared with sodium chloride or other salts as indicated, at stated concentrations, dissolved in Milli-Q water (Millipore, Bedford, MA), with 0.5 mM dibasic sodium phosphate for background pH control. Crystalline corticosteroids were dissolved in punctilious ethanol at 1.00 µM (316-362 µg/mL). Standard aliquots were dried and resuspended in the various sample matrixes to stated dilutions. Standards were stored at 4 °C when not in use. Analyte concentrations were 3.5 µg/mL for all experiments except where noted. Capillary Electrophoresis Separation Conditions. For all separations, the capillary was first rinsed by high pressure (20 psi) with five capillary volumes of fresh separation buffer (1-2 min). Sample matrix was introduced by low-pressure injection (0.5 psi), to produce a sample plug at the rate of 0.22 cm/s for the 75-µm capillary, 0.07 cm/s for the 50-µm capillary. Separation was carried out at constant voltage, 12 kV with the 75-µm capillary, 30 kV with the 50-µm capillary. The electrophoresis buffer was replenished after no more than 2 h total running time, and the capillaries were reconditioned daily by rinsing with 1.0 M sodium hydroxide and Milli-Q water. Separation buffer for all matrix experiments (except where stated) was 80 mM cholate/10 mM tetraborate/10% ethanol. The sample matrix for all separation buffer experiments was 150 mM sodium chloride except where stated. Detection was by UV absorbance at 254 nm. Sample Matrix Parameters. Sodium chloride and sodium phosphate were tested as separation buffers at 100 mM concentration to confirm they did not impart electrophoretic mobility to analytes. Borate has been previously demonstrated to impart mobility to some of the analytes,21 and was thus excluded from the sample matrix in all experiments. The conductivity of solutions was gauged by purging the capillary with stated solutions and recording current required to maintain constant voltage; i.e., sodium chloride solutions from 0 to 250 mM were tested at 10, 20, and 30 kV, compared to other solutions as stated. The effects of salt concentration in the sample matrix and sample injection length were investigated in sample matrixes from 0 to 250 mM sodium chloride at injection lengths from 0.88 to 14 cm. The effect of pH of the sample matrix was examined from 3 to 12 with 150 mM sodium chloride. The effect of organic modifier in the sample matrix was examined by adding ethanol from 10 to 30% to 150 mM sodium chloride. Observation of the analytes stacking at (21) Palmer, J., Atkinson, S., Yoshida, W. Y., Stalcup, A. M. and Landers, J. P. Electrophoresis 1998, 19, 3045-3051.
various times during electrophoresis was made by injecting analytes in a 4.4-cm plug of 150 mM sodium chloride in a separation buffer of 80 mM cholate with 10 mM tetraborate. The plug was pushed sequentially closer to the detector with subsequent injections of separation buffer (0.5 psi). Observation of zonal micelle concentration during electrophoresis was made by injecting a blank 150 mM salt sample matrix (no analytes) into a separation buffer saturated with progesterone. Peaks and valleys from baseline UV absorbance correlate positively with micelle concentration at the detector. The blank matrix was pushed sequentially closer to the detector with injections of separation buffer analogous to the previous experiment. MCE Parameters. The effect of cholate concentration on stacking was examined with cholate buffers at 40, 80, and 160 mM with 10 mM tetraborate in conjunction with sample matrixes containing 0-250 mM sodium chloride with analytes as stated. The effect of organic modifiers was examined by including the homologous series of alcohol, methanol, ethanol, 1-propanol, and 1-butanol at 10% concentration in 80 mM cholate separation buffer with 10 mM tetraborate. The effect of pH of the separation buffer was examined from 7.0 to 11.0. RESULTS AND DISCUSSION The effects of a high-salt sample matrix on stacking in MCE were examined at normal polarity with a separation buffer consisting of 80 mM sodium cholate/10 mM tetraborate/10% ethanol, pH 9.0. Cholate has a critical micellar concentration around 10 mM, with an aggregation number of two to four, and a single negative charge per molecule at this pH.22 The 17hydroxycorticosteroids were chosen as model analytes (Figure 1A). These compounds share the same chromophore, a 3-conjugated ketone, and are identical except for various degrees of oxidation. The degrees of oxidations provide a range of hydrophobicities, gauged by partitioning in water/hexane, of virtually 100% in hexane (progesterone) to virtually 100% in water (cortisone and cortisol). The result of electrophoresis of a mixture containing cortisone, cortisol, 11-deoxycortisol, 17R-hydroxyprogesterone, and progesterone under these conditions is shown in Figure 1B. The velocities of the analytes with this cholate-based MCE system can be succinctly categorized by two parameters: degree of oxidation of the analyte and response of each analyte to micelle concentration. The degree of oxidation correlates inversely with affinity for the cholate micelle. This is evidenced by the fact that the least oxidized analyte, progesterone, has the lowest velocity during electrophoresis, while the higher velocities of the sequentially more oxidized analytes result more from EOF and less from opposing mobility due to micelle complexation (Figure 1C). The analytes also exhibit a logarithmic decrease in velocity due to increased micelle complexation with increasing cholate concentration (data not shown). Therefore, the micelle-complexed mobility of each analyte is directly correlated to the micelle concentration that it experiences. Effect of High-Conductivity Sample Matrixes on Stacking. Since our earlier studies19,20 showed that high ionic strength sample matrixes play a role in MCE stacking, varied salt concentration sample matrixes were examined in conjunction with (22) Hinze, W. L. In Ordered Media in Chemical Separations; Hinze, W. L., Armstrong, D. W., Eds.; ACS Symposium Series 342; American Chemical Society: Washington, DC, 1987; pp 2-82.
Figure 1. Separation of corticosteroid analytes and the relationship between electrophoretic mobility and hydrophobicity. (A) Corticosteroid structures in reverse order of biosynthesis. Progesterone precursor is hydroxylated at the 17 position to become 17-hydroxyprogesterone. Subsequent oxidations at the 21 and 11 positions produce 11-deoxycortisol and cortisol. Cortisol can be oxidized at the 11 position to form cortisone. (B) Typical electropherogram of analytes using 80 mM sodium cholate/10 mM tetraborate/10% ethanol separation buffer, 50 µm × 47 cm capillary, 30 kV, normal polarity (as described in the Experimental Section). Time scale is 10 min, and peaks 1-5 are identified as indicated. (C) Plot of apparent capacity factor versus number of oxidations on the analyte (a proxy of hydrophobicity). Other natural corticosteroids are included.
the 80 mM cholate/10 mM tetraborate/10% ethanol, pH 9.0 separation buffer. Figure 2 shows the electrophoretic results when the corticosteroids (3.5 µg/mL each) were injected in sample matrixes with sodium chloride at concentrations from 0 to 250 mM. The sample plug was 3.6 cm (7.5% of the capillary length). As shown in Figure 2, the broad, poorly shaped peaks expected with such large injections were observed with sample matrixes at low salt concentrations (0-50 mM). However, there was a dramatic improvement in peak shape and detectability with sodium chloride concentrations from 100 to 250 mM. The role of conductivity in this phenomenon was investigated. Conductivity of the sodium chloride sample matrixes was determined under electrophoretic conditions as previously described. Current required to maintain constant voltage with 50 mM sodium chloride coincided with the separation buffer (∼70 µA). Sodium chloride concentration was linear with amperage required to maintain voltage; e.g., 100 mM pulled ∼140 µA. Linearity was observed throughout the sample matrix concentration range. When the sample matrix conductivity is at or below (0-50 mM sodium chloride) the conductivity of the separation buffer, stacking is clearly not evident. However, stacking is starkly Analytical Chemistry, Vol. 71, No. 9, May 1, 1999
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Figure 2. Effect of sample matrix salt concentration on corticosteroid stacking. Separation buffer, migration order, and separation conditions same as in Figure 1. Analytes injected in sodium chloride sample matrix at concentrations shown, 3.6-cm sample plug (50 s). Electrophoresis conditions were as described in the Experimental Section.
apparent when the conductivity of the sample matrix is raised substantially above (100-250 mM sodium chloride) the separation buffer. Thus, it appears that the sharp increase in efficiency with all analytes that is obvious in Figure 2 is associated with a sample matrix that has a higher conductivity than the separation buffer. Since zones with different conductivities influence the relative fields experienced by ionic components, a mechanism of stacking is described which explains the necessity of a high-conductivity sample matrix in this mode. A Proposed Mechanism of Stacking. It is presumed chloride has a higher intrinsic electrophoretic mobility than cholate. However, their actual velocities are determined by the electrophoretic mobility times the electric field. The salt concentration in the sample matrix can be used to manipulate the relative fields in the sample matrix and separation buffer and, hence, the actual velocities of the components in these zones. The reason for failure of stacking with low-conductivity sample matrixes is discussed first. When the sample matrix has a lower conductivity than the separation buffer, it is well-known that the sample matrix constituents experience an enhanced field upon initiation of separation voltage, and the cholate (anionic) on the detector side of the sample plug would be expected to electrophorese into the sample zone. However, because of the amplified field in the sample matrix, the cholate enters and accelerates through the sample matrix. It will accumulate at the opposite interface with the separation buffer, 1682 Analytical Chemistry, Vol. 71, No. 9, May 1, 1999
never reaching a higher concentration than the background separation buffer (80 mM). The zonal cholate concentration entering the sample matrix is decreased by the low-ionic strength sample matrix, and therefore, cholate complexation of analytes is decreased and no stacking is observed (Figure 3A). At equivalent sample matrix (50 mM sodium chloride) and separation buffer conductivities, cholate enters the zone without any component of field amplification, yet does not exhibit a sufficient concentration for stacking of the analytes (as seen in Figure 2). However, when the conductivity of the sample matrix exceeds that of the separation buffer, field amplification is transferred from the sample zone to the separation buffer. The micelles within the separation buffer experiencing the enhanced field electrophorese rapidly to the detector side of the sample matrix interface, where the highconductivity sample matrix induces a stacking of the cholate micelles at the interface (Figure 3B, t ) 1). Analytes experience a reduction in velocity upon encountering the stacked micelle/ sample zone interface (t ) 2). This is due to the different velocities of analytes in the sample zone (pure EOF velocity) versus the velocities of analytes in the stacked micelle zone (greatly enhanced counter-EOF mobility and reduced velocity due to a high local micelle concentration). This is consistent with the observation that simply increasing the concentration of micelles in the separation buffer leads to increased peak height with normal sample matrixes in MCE.23 Separation mode commences when the micelle front has exited the high-salt matrix and the chloride component has diffused to separation buffer conductivity. Thus, increasing the field in the separation buffer by increasing conductivity in the sample zone will cause an increase in the anodic mobility of cholate in the separation buffer and a decrease in anodic mobility of cholate at the sample matrix/separation buffer interface. This, in combination with the maximum velocity of analytes in the sample matrix versus decreased velocity in the concentrated micelles at the sample matrix/separation buffer interface, causes analyte stacking. The relative velocities of the anionic components of the sample matrix and separation buffer may also explain an aspect of the high-salt stacking mechanism. If a sample matrix of highconcentration, high-intrinsic anodic mobility chloride is injected adjacent to a separation buffer of lower concentration, lower intrinsic anodic mobility cholate, diffusion by migration of the highconductivity chloride will occur from the anodic (injection, left) end of the matrix (Figure 3B, t ) 1). The detector end of the matrix will maintain a concentrated component of high-intrinsic mobility anions “frozen” in the high-conductivity matrix, while field amplification will cause accumulation of the anionic micelles at the detector side of the matrix. As the injector side of the sample matrix diffuses, trailing cholate micelles will approach the zone (t ) 2). At the detector side interface of the separation buffer and the sample matrix, cholate micelles will accumulate, yet they are completely excluded from the chloride zone due to their lower intrinsic anodic mobility. This should produce two effects. First, the interface will be sharpened, with cholate stacked flatly against the higher concentration chloride. Second, the cholate concentrated against the detector side of the matrix would be “locked” into place until the last portion of the sample matrix chloride has (23) Shihabi, Z. K. In Handbook of Capillary Electrophoresis; Landers, J. P., Ed.; CRC Press, Boca Raton, FL, 1996, p 466.
Figure 3. Proposed mechanism of high-salt stacking. (A) Conventional field-amplified stacking11 with a low ionic strength sample matrix. (B) High-salt stacking with a sample matrix of ionic strength greater than the separation buffer.
diffusedsthe portion immediately flanking the stacked micelles (t ) 3). This would result in a neat interface of the sample matrix with the stacking vector (micelles), and maintenance of the interface during analyte stacking. Interrogating the Mechanism. Differential Analyte Response to Stacking. Since the corticosteroids have a spectrum of affinities for the cholate micelle, it was possible to compare their individual stacking in response to increasing salt concentration within the sample matrix. Theoretical plate number (N) was used to gauge the efficiency associated with stacking of each analyte under the varied conditions. It was reasoned that a higher salt concentration in the sample matrix would induce a higher micelle concentration at the micelle front. Since stacking of the analytes is dependent upon concentration of the incoming micelles, as well as the affinity of analytes for the micelle, it is logical that less-hydrophobic analytes should require a higher micelle concentration for
maximum stacking. Theoretical plate number was examined in response to the use of different sample matrix salt concentrations in 100-s (7-cm) injections using 80 mM cholate/10 mM tetraborate separation buffer without organic modifier. A maximum efficiency is observed with a 100 mM sodium chloride sample matrix for progesterone, while that for 17-hydroxyprogesterone was found to be in the 100-150 mM sodium chloride range and at 150 mM for 11-deoxycortisol (Figure 4). With the separation buffer modified with 10% ethanol, plate numbers for each analyte increase with increasing sample matrix salt concentration up to 200 mM sodium chloride (data not shown). Effect of Sample Plug Length on Stacking Efficiency. A sample matrix of 150 mM sodium chloride was chosen to evaluate the effect of sample injection plug length on efficiency of analyte stacking. Injection of sample plugs up to 14 cm in length (200 s at 0.5 psi, 30% capillary length) were made (Figure 5A). It is clear Analytical Chemistry, Vol. 71, No. 9, May 1, 1999
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Figure 4. Theoretical plate numbers for analytes stacked using different salt concentrations in the sample matrix. Electrophoresis conditions were as described in the Experimental Section, without ethanol included in the separation buffer.
that a maximum peak height was reached at different injection lengths for the different analytes, again, in accordance with their micelle affinity. The less hydrophobic analytes, cortisol and cortisone, exhibit peak broadening at smaller injections, while progesterone, the most hydrophobic analyte, exhibits continued peak height increase up to a sample plug length of 7 cm (100 s at 0.5 psi; 15% capillary filled), with a broadened but still sharp peak associated with a 14-cm plug length. Using these data, the plate number for each of the corticosteroids was calculated and plotted against sample plug injection length (Figure 5B). Analytes exhibit a sigmoidal decrease in plate number with injected plug length from 0.88 to 14 cm. Progesterone exhibits a virtually equivalent number of plates with 3.5- and 7.0-cm injections, and a substantial plate number is even maintained with a 14-cm injection. Since progesterone is the most highly micelle-complexed analyte, this result indicates that sample stacking with this method is dependent upon strong analyte/micelle complexation within the sample zone. Organic Modification of the Sample Matrix and Separation Buffer. In addition to the importance of maintaining ionic strength of the sample matrix well above that of the separation buffer, micelle/analyte interaction must be maximized within the sample zone to afford efficient analyte complexation by the micelle front. In MCE, organic solvents can be added to the separation buffer to afford separation of analytes by altering the affinity of the analyte for the micelle phase. Organic additives alter hydrophobic analyte/ micelle interactions by displacing the analytes from the micelle, by offering alternative hydrophobic binding sites, or by decreasing the surface tension of the separation buffer.24 Thus, the addition of organic modifiers to the sample matrix or the separation buffer provides a means of interrogating the dependence of the stacking mechanism on analyte/micelle complexation in the two zones. Figure 6A shows the effect of inclusion of 10, 20, and 30% ethanol (v/v) in the sample matrix (150 mM sodium chloride) (24) Gorse, J., Balchmus, A. T., Swaile, D. F., Sepaniak, M. J. HRC & CC, J. High Resolut. Chromatogr. Chromatogr. 1988, 11, 554-558.
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Figure 5. Effects of injection length with a high-salt matrix. (A) Electropherograms resulting from injection of corticosteroids in 150 mM sodium chloride sample matrix at plug lengths from 0.88 to 14 cm. (B) Theoretical plate numbers associated with analyte stacking at the varied plug lengths used in (A) Electrophoresis conditions were as described in the Experimental Section. Peak order and identification as in Figure 1.
when electrophoresis is carried out in 80 mM cholate/10 mM tetraborate/10% ethanol separation buffer. Since organic modifiers decrease analyte/micelle hydrophobic interaction, it was expected that addition of ethanol to the sample matrix would interfere with the sample stacking. This was observed. Presumably, the stacking of micelles at the separation buffer/sample zone interface still occurs, but the presence of ethanol in the sample matrix decreases the affinity of the analytes for the micelles (despite the higher micelle concentration), thus reducing the efficiency stacking of analytes by the cholate front. It is interesting that organic modifiers can be included in the sample matrix at levels found in the separation buffer (10% in this case) without substantially affecting stacking. In cases where more hydrophobic analytes are used, organic modifier can be included in the sample matrix at or below the concentration of organic modifier in the separation buffer. Having established the importance of micelle/analyte complexation in the sample zone by showing the disruption of this phenomenon by inclusion of ethanol in the sample matrix, the
Figure 6. Effect of organic modifiers on corticosteroid stacking. (A) Addition of ethanol to the sample matrix at different concentrations as shown. (B) Effect of various alcohols as organic modifiers in the separation buffer. Electrophoresis conditions were as described in the Experimental Section. Peak order and identification as in Figure 1.
effects of organic modifiers in the separation buffer were evaluated. Electrophoresis of the corticosteroids in a 150 mM sodium chloride sample matrix was carried out with separation buffer including methanol, ethanol, propanol, or 1-butanol at a concentration of 10% (Figure 6B). While increasing ethanol concentration in the sample matrix diminished stacking of the corticosteroids, its presence in the separation buffer affords an increase in the peak efficiency relative to the unmodified separation buffer. Alcohols contribute to repression of EOF25 to varying degrees as illustrated in the Figure 6B. This can contribute to an initial increase in analysis times for analytes. However, as the hydrophobic character of the alcohol is increased (longer carbon chain), the effect of decreasing the surface tension of the separation buffer and likewise decreasing the affinity of the analyte for the micellar phase occurs. These results indicate that organic solvent modification of the separation buffer does not interfere with analyte/micelle interaction in the sample zone. The organic phase in the separation buffer on the detector side of the sample matrix presumably moves out of the stacking area with EOF and does not enter the sample zone during the stacking process. Thus, the analyte environment at the sample zone/micelle interface is determined by the components of the sample matrix, not the separation buffer. However, once the sample zone has diffused, the solvent component of the separation buffer determines separation characteristics. Interrogating Stacking in the Sample Zone. Analyte Stacking. To corroborate that stacking of micelles at the detector end of the sample matrix is ultimately responsible for the stacking analytes, observation of corticosteroids in the sample zone during the stacking process was made. This was accomplished by placing (25) Chen, N.; Terabe, S.; Nakagawa, T. Electrophoresis 1995, 16, 1457-62
Figure 7. Observing the corticosteroid stacking process. A 4.4-cm sample plug consisting of 150 mM NaCl was placed various distances from the detector window by hydrostatically injecting 80 mM cholate/ 10 mM borate separation buffer behind the sample plug. The electropherograms represent the response observed as a result of electrophoresis over the indicated distance (in cm) following the initiation of separation voltage. Arrows: a, the detector side of the matrix; b, the stacked analytes; c, subsequent separation; d, system peak, possibly representative of the stacked micelles. Electrophoresis conditions were as described in the Experimental Section.
a sample plug progressively closer to the detector before initiating the separation voltage. This provided a series of “snapshots” of the detector response to the chromophore-bearing analytes within the matrix during early formation of the stacked zone. Utilizing a larger capillary inner diameter (75 µm) reduced the injection times required to produce the 40-cm plug length required for these experiments by 3-fold (9.5 min required with a 50-µm capillary). Sample matrix was injected (4.4-cm plug length) into the capillary followed by injection of separation buffer to position the sample at desired distances from the detector, before initiating separation voltage. The longest “push” injection placed the 4.4-cm sample plug adjacent to the injector side of the detector window, while each subsequent push injection placed it 2.2 cm farther from the detector window. As shown in Figure 7, the analytes in the sample matrix appear to be concentrated from the left side (detector side in an electropherogram), based on the observation that the left face appears sharpest during early formation (Figure 7a). The attenuated zone appears maximum at ∼2 min of migration time (Figure 7b), and consequently, the separation of the various component peaks is observed at later times (Figure 7c). However, it was not possible to rule out any analyte stacking effect from the injector side of the matrix. Therefore, an analogous “push” experiment was carried out to allow observation of the stacking phenomenon from a different perspective. Micelle Stacking. Since the primary event involved in the proposed stacking mechanism is the formation of a stacked micelle front at the detector side of the sample matrix/separation buffer interface, an experiment was carried out to directly observe the micelle concentration in the separation buffer. To enable this, the micelles were rendered “visible” by complexing them with the high-affinity chromophore-bearing analyte, progesterone. Excess Analytical Chemistry, Vol. 71, No. 9, May 1, 1999
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Figure 8. Observing the micelle stacking process with progesterone-saturated micelles. This experiment is directly analogous to that observed in Figure 7 with the exception that micelles now possess a chromophore-bearing analyte (progesterone) and are visualized, while the 4.4-cm sample plug (NaCl, no analytes) is devoid of chromophoric species. Electrophoresis conditions were as described in the Experimental Section.
progesterone was sonicated with a solution of 80 mM cholate/10 mM borate separation buffer for 10 min. Normal electrophoresis was carried out using this separation buffer, with the 150 mM sodium chloride sample matrix injected neat (without analytes). In this case, UV absorption should correlate directly with micellar concentration. Analogous to the previous experiment, a 4.4-cm sample matrix plug (150 mM sodium chloride, no analytes) was pushed progressively closer to the detector to monitor the development of the micellar regions around the sample matrix. In Figure 8A, the trough in the baseline identifies the chomophore-deficient sample plug (distance from a to b). The inlet side of the trough (b) shows the difference in absorbance associated with progesteronesaturated 80 mM cholate versus the sample zone, while the detector side of the sample plug (a) shows the genesis of a stacked micelle peak. In Figure 8B, the stacking of micelles (c) is obvious on the detector side of the sample zone. The sample zone has begun to be compressed at this point (d), and there is no apparent change in the micelle concentration on the injector side of the matrix. A rough estimate of the extent of stacking can be made from the intensity of the absorbance during the stacking process. Using the magnitude of (b) in Figure 8A, a transient cholate concentration on the detector side of the sample matrix (in Figure 8b) can be estimated to be in excess of 1500 mM. A comparison of the electropherograms obtained in Figure 7 indicates that the analytes have passed out of the micelle front by 2 min (Figure 8C). An additional peak is seen electrophoresing later than all analytes (e) and may represent a zone of cholate that is free of progesterone. Since it is not possible with this method to directly observe these general phenomena without the burden of progesterone included as a chromophore, this experiment represents only a first attempt at quantitatively defining the exact processes occurring. Stacking is complete when the chloride component at the sample matrix/separation buffer interface has diffused to background (separation buffer) levels, and analytes are transferred to the separation buffer micelles. It is apparent from these 1686 Analytical Chemistry, Vol. 71, No. 9, May 1, 1999
Figure 9. Application of the high-salt stacking method to a different electrokinetic chromatography system. Separation of three corticosteroids with a buffer containing 10 mM sulfated γ-cyclodextrin (∼13 degrees of substitution)/10 mM tetraborate/10% acetonitrile, pH 9.0; 50 µm by 47 cm capillary; 30 kV. (A) Injection (100 s) of analytes in the separation buffer. (B) Injection (100 s) of analytes in 100 mM K2SO4.
experiments that micelle stacking occurs at the detector side of the sample plug. Applicability of High-Salt Stacking in MCE Mode. If the high-salt stacking approach is universally applicable to extant MCE protocols, the stacking must not be affected by variation of the separation parameters. Consequently, the robustness of the stacking method under different separation conditions was examined. This stacking approach was found to be unaffected by separation buffer pH from 7 to 11. The pH of the sample matrix was varied from 3 to 12, again, with no deleterious effect on stacking. The concentration of the cholate buffer was adjusted between 40 and 160 mM, and other than the requirement of a concomitantly higher salt concentration in the sample matrix to invoke stacking, no deleterious effects were noted. The chlorides of lithium, sodium, potassium, rubidium, and cesium as well as the sodium salts of fluorine, chlorine, bromine, and iodine were utilized at a concentration of 160 mM to elevate sample matrix conductivity, and again, there were no substantial or trending differences between the salt used and the effectiveness of the sample stacking. However, when a higher mobility salt was used, such as potassium sulfate, the concentration of salt required to invoke the stacking mechanism was concomitantly less, consistent with the postulate that it is the high conductivity of the sample matrix that effects the stacking. The efficiency of the stacking mechanism was also tested using a nonmicellar electrokinetic chromatography system. A separation buffer consisting of 10 mM highly sulfated γ-CD (13 sulfates/molecule) with 10 mM borate/ 10% acetonitrile was used to separate cortisol, 11-deoxycortisol, and progesterone. The analyte mixture was dissolved in 100 mM potassium sulfate or separation buffer, and a 7-cm sample plug was injected into a 47-cm by 50-µm capillary containing this separation buffer. The results are given in Figure 9, where it is clear that stacking is manifested for the high-salt matrix and not for the injection of analytes in separation buffer. It was not possible
Table 1. Percent Relative Standard Deviation for Peak Area and Analysis Timesa analyte
analysis time % RSD
peak area % RSD
cortisone cortisol 11-deoxycortisol 17-hydroxyprogesterone progesterone
2.07 1.53 1.41 1.38 1.83
4.23 5.60 9.80 9.58 7.07
a
Separation conditions as stated. Analyte concentration, ∼55 ng/mL.
to induce stacking with sodium chloride, consistent with the hypothesis that the sample matrix anionic mobility must be greater than the separation buffer anionic mobility. Limits of Detection and Reproducibility. With respect to limits of detection, a 7-cm (100-s) injection of 11-deoxycortisol, 17-hydroxyprogesterone, and progesterone at a concentration of 50 ng/mL in 150 mM sodium chloride showed well-resolved peaks with a signal-to-noise ratio of 5. Cortisol and cortisone were identifiable but not baseline-resolved. With 3% sulfated β-CD as the high-ionic strength sample matrix component and using estrogens as analytes, injections of up to 60% of the capillary can be made with 20-µm-i.d. capillaries. The concentration effect is somewhat reduced in larger capillaries, with functional injection lengths of approximately 10-30% of the capillary length, depending on the affinity of the analyte for the micellar carrier. The results infer that Joule heating due to varied conductivity in different zones is a limiting factor; thus, smaller capillaries allow for more efficient sample stacking. The effect of the stacking method on reproducibility was evaluated by carrying out replicate 100-s injections (n ) 10) of corticosteroid analytes at a concentration of 50 ng/mL each in sodium chloride at 150 mM. Separation buffer was 80 mM cholate/10 mM tetraborate/10% ethanol. The coefficient of variation (CV) for migration times ranged from 1.4 to 2.1% with an average of 1.6% over 10 runs. As expected, higher CV values were observed with the peak area for each of these compounds, ranging from 4.2 to 9.6% with an average of 7.2% (Table 1). Comparison of the High-Salt Stacking Method with Existing Techniques. At the same time that this work was to be submitted for publication, Quirino and Terabe26 reported an elegant stacking method different in nature but similar in many results to those described here. Their conditions include a relatively constant field (equal conductivities in the sample matrix and separation buffer), and negligible electroosmotic flow. This contrasts the data in the present work, where we show the dependence of stacking on a sample matrix conductivity that is higher than the separation buffer and do not see dependence of stacking on EOF or lack thereof. While the Quirino and Terabe study prescribes the control of numerous separation buffer parameters for effecting stacking by their mechanism, this is clearly not a requirement with the mechanism described in the present work, which provides the flexibility for MCE stacking over (26) Quirino, J. P.; Terabe, S. Science 1998, 282, 465-468.
a wide range of separation parameters, and with unmodified EOF. This will be key when translating stacking mechanisms to the microfabricated chip format, particularly in view of the fact that EOF is the standard mechanism of mobilizing components in this mode. An expected parallel between our works is the higher efficiency of stacking with more highly retained analytes13 (high k 26 or k′ 12 values). While they believe an analyte zone is narrowed by a factor of 1/(1 + k), and use their subsequent peak height to determine k values, we illustrate how low-k′ analytes are concentrated by increasing micelle concentration during stacking (higher effective k′ value). However, while the analytes they used had k values in the hundreds to over 5000 range, our high-conductivity mechanism works with low-k′ analytes (0.2-7 range) and should work considerably better with higher k′ analytes. CONCLUSIONS The addition of salt to sample matrixes is indicated for stacking analytes in MCE as well as other modes of electrokinetic chromatography. It is postulated that maintaining sample matrix conductivity 2-3-fold above that of the separation buffer is a fundamental aspect of maximizing peak efficiency and detection limits in these modes. We have postulated the physical mechanism that is responsible for sample stacking. It is activated by transfer of field amplification from the sample matrix to the separation buffer. This novel approach invokes, in a sense, reversed field amplification since it involves first stacking of micelles at the sample matrix/separation buffer interface which are subsequently responsible for stacking the analytes. It is also logical that the anion mobility of the sample matrix (e.g., chloride) be greater than that of the anionic micellar carrier (e.g., cholate) to avoid intrusion of the micelles into the sample zone during stacking. The sample stacking method efficiency is high as a result of the micelles being stacked before they enter the sample zone. While it is critical to maintain the highest possible surface tension in the sample matrix to maximize hydrophobic interactions with neutral analytes, separation buffer parameters can be manipulated in concentration, pH, ionic strength, and organic modifier extensively without compromising the sample stacking method. The bare simplicity of this method should make it easily exportable to other MCE separation protocols and eventually facilitate the more widespread use of MCE to applications that require greater column efficiency and lower limits of detection. While this method should find great utility for MCE in capillaries, it clearly holds promise for improving sensitivity on microfabricated electrophoretic chips and other EKC applications. ACKNOWLEDGMENT The authors thank Dr. Apryll Stalcup, Department of Chemistry, University of Cincinnati, and Dr. Shannon Atkinson, Department of Oceanography, and the Department of Chemistry at the University of Hawaii at Mauoa, for support and assistance, and Mark Flocco at Beckman Coulter for equipment. Received for review November 24, 1998. Accepted February 18, 1999. AC981302A
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