Anal. Chem. 2000, 72, 1941-1943
Stacking Neutral Analytes in Capillary Electrokinetic Chromatography with High-Salt Sample Matrixes James Palmer and James P. Landers*
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22901
We recently described a method to stack neutral analytes in electrokinetic chromatography dependent upon using high-salt sample matrixes (Palmer et al. Anal. Chem. 1999, 71, 1679-1687) The use of a high-mobility coion in the sample matrix at a higher concentration than the like-charged electrokinetic vector (e.g., charged micelle) in the separation buffer as we previously suggested induces a single-boundary, discontinuous buffer system which subsequently stacks neutral analytes. Under these conditions, it is possible to electrokinetically inject neutral analytes directly from a high-salt sample matrix via electroosmotic flow, with stacking initiated simultaneously with injection. The correspondence by Quirino et al. (Anal. Chem., previous paper in this issue) suggests the effects of the high-salt sample matrix on stacking of micelles is obviated by a destacking of the micelle zone as it exits the sample zone. They reevaluate the high-salt stacking mode in terms of the γ function. Based on the present data, it is our interpretation that the stacking efficiency is not accurately represented by the γ function and that neither micelle zone nor neutral analyte destacking occurs under conditions we previously described. Low stacking efficiency when low-conductivity sample matrixes are used indicates that the high-salt stacking effect cannot be explained simply as a function of the k value of an analyte/micelle system.
DISCUSSION We introduced a mode of on-line sample stacking for neutral analytes in electrokinetic chromatography (EKC) dependent on a high-salt sample matrix.1 This mode of stacking is attractive because biological sample matrixes often have a high salt content. Our study utilized an EKC system with an anionic micelle in the separation buffer, pressure injection of a high-salt sample matrix containing the neutral analytes, with separation under normal polarity (the inlet was anodic) in the presence of electroosmotic flow (EOF). Under these conditions, neutral analytes have a minimum velocity toward the detector when complexed by a micelle, and a maximum velocity in free solution (the velocity of the electroosmotic flow). In the presence of increased micelle concentration, the velocity of neutral analytes is decreased further. (1) Palmer, J.; Munro, N. J.; Landers, J. P. Anal. Chem. 1999, 71, 1679-1687. 10.1021/ac0000269 CCC: $19.00 Published on Web 03/16/2000
© 2000 American Chemical Society
We suggest two requirements for sample stacking in this mode.1 First, the sample matrix must have a conductivity higher than the separation buffer containing the electrokinetic vector. This should induce a field amplification of the separation buffer zone. This, in turn, should cause an increase in the electrophoretic velocity of the electrokinetic vector in the separation buffer zone. Second, the sample matrix must contain a co-ion (ion with the same-sign charge as the electrokinetic vector in the separation buffer) with a higher intrinsic electrophoretic mobility than the electrokinetic vector. The second condition should cause the formation of a pseudo-steady-state boundary between the micelle and co-ion component of the sample matrix.3 It has been demonstrated that this will cause a stacking of the electrokinetic vector against the sample co-ion interface.1,2 While we correctly stated the conditions necessary for this to occur in our original paper,1 Quirino et al.2 have properly ascribed the origin of this second concept to workers whom demonstrated this phenomenon with nonmicellar species.3 The correspondence by Quirino et al.2 has adopted a unique approach to interpret the high-salt stacking phenomenon in terms of the γ function. They suggest the micelle zone, once stacked, transits the sample zone, stacking analytes, and then destacks upon exiting the high-conductivity sample zone. The destacking of the micelles is inferred to cause a concomitant destacking of neutral analytes contained within, thereby obviating the high-salt effect. In our original work, we demonstrated an experiment in which a chromophoric species was added to the micelle solution to allow visualization of the micelle concentrations during highsalt stacking. In this experiment, no destacking was evident (Figure 8 in ref 1). However, the results of the experiment depicted in Figure 3 of the Quirino et al. correspondence2 suggest that, while a micelle zone stacks upon encountering a high-conductivity region, it then destacks upon exiting that zone. The destacking of the micelles they observed may have been caused by a failure to meet the criteria necessary for the formation of a single-boundary discontinuous buffer system. First, the buffer system in their experiment is continuous (the background electrolyte is ammonium phosphate in all matrixes). Second, Quirino et al.2 utilized separation vials with solutions that did not contain micelles. The initial separation conditions of Quirino et al.2 therefore have four separate zones, (2) Quirino, J. P.; Terabe, S.; Bocek, P. Anal. Chem. 2000, previous paper in this issue. (3) Mosher, R. A.; Thormann, W. Electrophoresis 1985, 6, 477-482.
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Figure 1. Neutral analyte stacking effect with electrokinetic injection of varied salt concentration sample matrixes. All samples were electrokinetically injected by electroosmotic flow at 30 kV for 40 s. Analogous results are found with pressure-injected high-salt matrixes.1 Separation conditions: Separation buffer was 80 mM sodium cholate, 10% ethanol, 5 mM sodium tetraborate. Capillary was 49 µm i.d. by 48.5 cm in length, bare fused silica. Sample matrix was sodium chloride at concentrations shown in the figure. Peak order, 1-5, respectively: cortisone, cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, and progesterone. Sample plug length ∼10 cm.
respectively, from injection to detection end of the capillary: background electrolyte, high-concentration background electrolyte, background electrolyte with charged micelles, and background electrolyte. This system is not expected to behave according to conditions described previously.3,1 When conditions described previously1 are used, the data in the Quirino et al. correspondence2 in fact corroborate our previous findings. Figure 2 of the Quirino et al. correspondence2 depicts use of a high-salt sample matrix injection of a neutral analyte (progesterone) in MEKC mode, with two electropherograms and a current trace to determine migration velocities and zone profiles of the critical components. The upper electropherogram uses detection at 200 nm to characterize the chloride component of the sample matrix. The middle electropherogram uses detection at 247 nm to characterize the neutral analyte (progesterone). A current trace of the electrophoresis is included at the bottom of that figure. Our interpretation of this figure is consistent with the formation of a pseudo-steady-state boundary between the sample matrix chloride component and the separation buffer micelle component, as well as the separation of the neutral analyte from the stacked interface without concomitant destacking, as described below. The correspondence by Quirino et al.2 suggests the equilibration of the current trace indicates that the high-conductivity sample matrix has exited the capillary by 12-13 min, yet the chloride component of the sample matrix is shown at the detector at ∼26 min (electropherogram with detection at 200 nm). The equilibration of the system current simply indicates when the sample plug neutral component has completely transited the capillary; i.e., it is an electroosmotic flow indicator.4 However, the chloride zone has subsequently moved out of the sample zone and into the (4) Gebauer, P.; Thormann, W.; Bocek, P. J Chromatogr. 1992, 608, 47-57.
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separation buffer. Note that the chloride zone has an extremely flat face on the cathodic side (left), with decreasing concentration toward the anode, as suggested in our previous study.1 We propose this represents the chloride side of a pseudo-steady-state boundary. Also note the peak in the electropherogram with detection at 247 nm that aligns perfectly against the flat face of the chloride peak (time ∼26 min). This “system peak” has an extremely flat face on the anodic side. Our previous study1 suggests this system peak might actually be representative of stacked micelles, clearly devoid of the neutral analyte progesterone. We propose this system peak represents the micelle side of the pseudo-steady-state boundary. The visualization of these peaks is consistent with the formation of a pseudo-steady-state boundary at the interface of the chloride and cholate zones. While isotachophoretic assortment of like-charged species in a sample zone has been previously reported,3,5 we believe the Palmer et al. study1 was the first to utilize this phenomenon to effect the stacking of neutral analytes in EKC. In addition, the progesterone peak exhibited in the electropherogram with detection at 247 nm of the Quirino et al. correspondence2 does not exhibit a fronting asymmetry, suggesting destacking. The progesterone has clearly passed from the injection side of the system peak to the detection side (i.e., it reaches the detector before the system peak, which putatively originated on the detector side of the sample matrix in which the progesterone was contained). An interesting consequence of the discontinuous system we describe is that it operates with essentially a single boundary; i.e., the sharp demarcation of the boundary is at the chloride/micelle interface, while the anodic side of the chloride zone and the cathodic side of the micelle zone do not manifest sharp interfaces. (5) Huang, X.; Ohms, J. I. J. Chromatogr. 1990, 516, 233-240.
As such, it is possible to maintain the internal pseudo-steady-state boundary even if the inlet buffer solution is switched during the electrophoretic process. This allows for electrokinetic injection of the high-salt sample matrix directly into the capillary by electroosmotic flow. In this case, it is postulated that the pseudosteady-state boundary would be initiated at the capillary inlet during injection, with its velocity a function of electroosmotic flow velocity, opposed by the intrinsic anodic mobility of the charged species and the relative fields they experience. Neutral analytes should move into the capillary approximately at the rate of the electroosmotic flow, with subsequent stacking occurring at the pseudo-steady-state boundary. To demonstrate stacking of neutral analytes with electrokinetic injection, a 48.5-cm capillary (50 µm i.d.) was prepared for normal MEKC1 and then flushed with the micellar separation solution. The capillary inlet was then placed in the high-salt sample matrix containing the neutral analytes, and the outlet was placed in the micellar solution. A voltage of 30 kV was applied for 40 s for sample injection. The inlet was then switched back to the separation buffer vial for separation at 30 kV. The separation conditions and results can be seen in Figure 1. Minimal stacking is observed at 0, 25, and 50 mM sample matrix salt concentrations (50 mM sodium chloride has approximately the same conductivity as the separation buffer), consistent with the postulate that a pseudo-steady-state boundary cannot been established unless the co-ion in the sample matrix is at a higher concentration than the electrokinetic vector in the separation zone.1,3 As observed previously with pressure injections,1 high-salt stacking with electrokinetic injection occurs when the conductivity of the sample matrix is substantially higher than the separation buffer (75 mM and higher). Note the peak for progesterone with the 150 mM sodium chloride sample matrix is ∼9-fold higher than the peak with 50 mM sodium chloride. The result of these and previous experiments1 clearly shows the effect of salt concentration in the sample matrix on subsequent stacking of neutral analytes in EKC. Figure 1 illustrates that the critical transition to stacking occurs when the conductivity of the sample matrix exceeds that of the separation buffer, with maximum stacking at ∼3 times the conductivity of the separation buffer. Higher salt concentrations do not effect a significant improvement in stacking efficiency, as was stated in our original
study.1 This is corroborated by the Quirino et al. correspondence2 experiments in the γ value range of 2 and higher (Figures 4A,B and 5). CONCLUSIONS In capillary electrokinetic chromatography, neutral analytes must interact with a charged carrier (electrokinetic vector) to achieve either stacking or separation. Any stacking mode in MEKC is dependent upon the k value of the electrokinetic vector/ analyte system. As such, the term in the equation proffered by Quirino et al.,6 1/(1 + k), provides a dictum for stacking efficiency, but is perhaps valid only under continuous buffer conditions. This term may not be directly applicable to predict stacking efficiency under discontinuous buffer conditions. Our interpretations indicate that the stacking efficiency with high-salt sample matrixes does not relate directly to either a single k value or a γ function. The ability to manipulate a pseudo-steady-state boundary between a sample matrix co-ion and an electrokinetic vector to afford neutral analyte stacking will be further elucidated in a forthcoming publication. There is little doubt that the “sweeping” concept6 developed by Terabe and co-workers represents an elegant and interesting mechanism that may have wide applicability as a stacking method for continuous buffer systems. However, to invoke stacking of neutral analytes with the EKC systems that we have examined (sodium cholate and sulfated γ-cyclodextrin), it appears that it is necessary to provide a higher-conductivity sample matrix with a co-ion that has a higher intrinsic mobility than the electrokinetic vector. It is clear that further study of this mechanism will be required to comprehensively understand the intricasies of the phenomena occurring in the capillary with discontinuous buffer conditions, and how they may be controlled to optimize stacking. ACKNOWLEDGMENT The authors thank Hewlett-Packard, Waldbron, Germany, for use of the HP 3D-CE capillary electrophoresis apparatus used in these experiments. Received for review January 6, 2000. Accepted January 14, 2000. AC0000269 (6) Quirino, J. P.; Terabe, S. Science 1998, 282, 465-468.
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