High-Voltage Focusing for Field-Amplified Sample Stacking in Capillary Electrophoresis
Capillary Electrophoresis (CE) is renowned for its high separation efficiency but often suffers from poor concentration sensitivity due to the extremely small injection volumes. Field-Amplified Sample Stacking (FASS) is a powerful on-line preconcentration technique that addresses this by creating a sharp boundary between a low-conductivity sample zone and a high-conductivity background electrolyte. The efficiency of this stacking process is governed by the precise application and switching of high voltages, requiring a power supply capable of sophisticated sequencing and rapid transition control.
The fundamental principle of FASS relies on the difference in electric field strength. The sample is prepared in a buffer of lower ionic strength (higher resistivity) than the background electrolyte. When a high voltage is applied, Ohm's law dictates that the electric field is stronger in the low-conductivity sample zone. This causes ions in the sample zone to migrate rapidly until they reach the boundary with the high-conductivity buffer, where the field strength drops abruptly. The ions slow down and stack into a narrow, concentrated band at this interface. The role of the high-voltage power supply is to orchestrate the phases of this process: injection, stacking, and separation, each requiring different voltage configurations and timing.
In a typical procedure, the capillary is first filled with the high-conductivity background electrolyte. The sample, in low-conductivity matrix, is then introduced at the injection end. For stacking to occur efficiently, the initial application of the separation voltage must be performed in a specific manner. A common method is to apply a high voltage with the correct polarity for a short period while the injection end is immersed in the sample vial. This draws ions into the capillary and initiates the stacking at the boundary. The timing and voltage of this step are critical; too short or too low a voltage results in poor stacking, while too long or too high can cause overheating or hydrodynamic instability.
After the stacking phase, the power supply must swiftly switch the configuration. The injection end is moved from the sample vial to a vial containing the background electrolyte. The voltage must be re-applied or maintained to begin the separation of the now-concentrated bands. Any delay or voltage dip during this switch can cause band broadening, undoing the benefits of stacking. Therefore, the power supply must have a low output capacitance and fast settling time to ensure the field is re-established instantaneously. Some advanced methods, like field-amplified sample injection (FASI), involve a voltage reversal step, requiring a bipolar or rapidly switchable high-voltage output.
The demands on the power supply extend beyond simple switching. During the stacking phase, the current is initially very high because the resistance of the capillary is dominated by the low-conductivity sample plug. The power supply must have a robust current compliance limit to safely handle this surge without tripping, while also providing the necessary voltage. As the sample ions stack and the high-conductivity buffer enters the capillary, the resistance changes dynamically. A supply with excellent load regulation is needed to maintain a constant electric field strength throughout this transition to ensure consistent migration velocities.
For maximum sensitivity gains, techniques like stacking with matrix removal or multiple stacking steps are used. These involve even more complex voltage programs, with sequences of different voltages applied to various capillaries or electrodes in a microchip format. This necessitates a multi-channel high-voltage power supply with independently programmable outputs and precise inter-channel timing synchronization. The control software must allow for the creation and storage of these intricate pulse sequences.
Integration with automated sample handling and detection is the final piece. The high-voltage power supply's control interface must be seamlessly integrated into the CE instrument's software. The detection system, often a UV or fluorescence detector, must be triggered at the correct time relative to the voltage program to capture the sharp, concentrated peaks. By providing this level of programmable, high-speed, and stable high-voltage control, the power supply transforms from a mere source of electromotive force into the active director of the sample preparation and separation process, enabling capillary electrophoresis to detect analytes at concentrations that rival or surpass far more expensive and complex liquid chromatography-mass spectrometry systems for suitable applications.
