Electrophoretron: a new method for enhancing resolution in electrokinetic separations

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Abstract

Two capillaries, each of which have different surface preparations on their inside walls, are joined together to form a closed loop, and electrodes are placed inside the two capillaries. When the loop is filled with liquid and a potential difference is applied between the two electrodes, a circulating flow of liquid is established inside the loop because the resistance to flow is unequal in going from one electrode to another in a clockwise versus a counterclockwise direction. Consequently, a sample injected into this device, which we call an electrophoretron, repeatedly circulates between the two electrodes and the capillary separation column becomes effectively one of unlimited length. On each cycle the separation between analytes with different mobilities increases, thus enhancing resolution of analytes having nearly the same mobilities. The operation of a prototype electrophoretron is demonstrated.

Introduction

In capillary electrokinetic separations, it is desirable to increase the resolution to separate different species with nearly identical retention or migration times. For example, the resolution of capillary electrophoresis (CE) depends on several factors such as the separation voltage applied between electrodes, the electroosmotic flow velocity, the net velocity of analytes, the length of the injection volume, and the length of the separation capillary. In typical experimental arrangements for CE, two ends of a linear capillary are connected to a high-voltage power supply so that the electric field reaches a value of 100–500 V/cm. Theoretical considerations [1] indicate that the resolving power R should scale with the voltage V according to the relation of R∝(V)1/2. Consequently, a 4-fold increase in applied potential is needed to double the resolution. Theory also indicates that the best resolution is obtained when the electrophoretic mobility of one species is exactly counterbalanced by the electroosmotic flow (EOF) [1]. Heat generation and electrical breakdown, however, place practical bounds on the voltage that can be used for the separation. These considerations prevent the realization of the resolution improvement expected by simply increasing the length of the capillary without limit. In addition, precise control of EOF is difficult to achieve, posing a problem in obtaining better resolution in CE.

Several attempts have been reported in the literature to enhance the resolution in CE by controlling EOF. One of these attempts is the direct control of electroosmosis by using external (radial) electric fields [2], [3], [4], [5]. In these experiments, however, several limitations were encountered involving the dependence of the flow on pH and buffer concentration, the external voltages applied, and the diameter of the capillary.

Culbertson and Jorgensen [6] explored with great success a different approach in which a pressure-induced flow is used to counterbalance analyte migration. They applied pressure to one end of the capillary to retard, halt, or move the analytes back and forth across the detection window. In this manner, a substantial increase of both the efficiency and the resolving power of the separation were obtained by keeping the analytes of interest in the separation field much longer than the use of a simple linear CE setup. For example, they demonstrated the ability to resolve compounds with electrophoretic mobility differences as little as 1×10−7 cm2/s. Nevertheless, several drawbacks exist for this arrangement, caused primarily by the need to time the application of the pressure to the capillary end, and by the broadening caused by the pressure-induced parabolic flow.

Buggraf et al. [7] have presented a different approach for ion separations in solution utilizing the concept of repeated column switching with synchronized cyclic capillary electrophoresis on a planar microstructure [7]. Micellar electrokinetic separations were also performed with this device [8]. It was essential once again to control precisely the timing in switching the voltage between electrodes.

We present an alternative approach that does not require special timing. It is loosely based on ion cyclotron resonance mass spectrometry. In ion cyclotron mass spectrometry, gaseous ions circulate in a strong magnetic field with a frequency depending on the mass-to-charge (m/z) ratio for a given species. The application of an ac voltage having this cyclotron resonance frequency affects only ions with the same m/z ratio, causing these ions to be moved relative to the others. Here we report a related method for enhancing the resolution in CE by using what we call an electrophoretron. In this approach, we join together the two ends of a capillary to form a closed loop in which two electrodes are located so that the flow resistance in the clockwise direction differs from that in the counterclockwise direction. Upon application of a voltage between the two electrodes, electroosmosis causes the sample to circulate repeatedly inside the capillary. Provided that some separation of the analytes occurs on a single cycle, caused for example by electrophoresis or electrochromatography, then the electrophoretron acts to enhance the separation achieved by each cycle.

Section snippets

Instrumentation

Fig. 1 presents a schematic diagram of the equipment required to perform cyclic electroosmotic flow in the electrophoretron. It consisted of a closed-loop capillary column. One part of the capillary loop was a regular fused-silica column (Polymicro Technologies, Phoenix, AZ, USA) of 75 μm I.D.×365 μm O.D. The other part of the loop was completed with a capillary of the same dimensions (inside and outside diameters) that had a positively charged coating (CElect-Amine column, Supelco, Bellefonte,

Results and discussion

The essence of the electrophoretron is to generate a cyclic electroosmotic flow so that the capillary separation column becomes effectively of unlimited length. The direction of the electroosmotic flow inside the closed-loop capillary is controlled primarily by the nature of the capillary walls. The magnitude of the electroosmotic flow depends upon the buffer solution used as well as the nature of the walls. In a bare fused-silica capillary, the negatively charged surface produces

Conclusion

We have demonstrated the construction of a device we call an electrophoretron in which the sample travels around a closed loop under the action of electroosmotic flow. On each cycle, the separation between analytes increases. This feature becomes particularly important when the analytes have nearly identical mobilities. As a consequence, the electrophoretron can be used to improve the separation efficiency (resolving power). In this sense, the electrophoretron for the separation of analytes in

Acknowledgments

We acknowledge Beckman Coulter, Inc. for their support. J.-G.C. especially thanks the LG Yon-Am Foundation for financial support to make possible his sabbatical leave at Stanford.

References (8)

  • R. Kuhn et al.

    Capillary Electrophoresis: Principles and Practice

    (1993)
  • C.S. Lee et al.

    Anal. Chem.

    (1990)
  • C.S. Lee et al.

    Anal. Chem.

    (1991)
  • M.A. Hayes et al.

    Anal. Chem.

    (1992)
There are more references available in the full text version of this article.

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