High-Voltage Switching Modes in Capillary Electrophoresis for Isotachophoresis
Capillary electrophoresis has revolutionised the separation of charged analytes, from small inorganic ions to large biomolecules. Among its many operational modes, isotachophoresis holds a special place for its ability to concentrate and separate samples with high resolution. The heart of this technique lies in the precise and dynamic control of the electric field along the capillary, a task that falls squarely on the shoulders of the high-voltage power supply. After fifty years in this field, I can attest that the transition from a simple constant-voltage or constant-current supply to a programmable, fast-switching high-voltage source was the key that unlocked the full potential of isotachophoresis and its hyphenated techniques.
Isotachophoresis relies on a discontinuous buffer system, consisting of a leading electrolyte with high-mobility ions and a terminating electrolyte with low-mobility ions. When a high voltage is applied, the sample ions, introduced at the boundary, arrange themselves into contiguous zones in order of their effective mobility. Once this steady state is achieved, all zones migrate at the same velocity. The electric field strength in each zone is inversely proportional to the mobility of its constituent ions. Therefore, the field is lowest in the leading zone and highest in the terminating zone. The power supply's role is to maintain a constant current during this process, as it is the constant current that ensures the zone velocities are equal. Any deviation from constant current would disrupt this delicate balance, leading to zone broadening and loss of resolution.
The high-voltage power supply for isotachophoresis must, therefore, excel in constant-current mode operation. This demands a control loop with extremely high gain and fast response. The load presented by the capillary is not static. As the zones form and migrate, the overall resistance of the capillary changes. The power supply must detect this change instantaneously and adjust the output voltage to maintain the programmed current to within microamps of its setpoint. A slow or poorly damped control loop will cause the current to oscillate, particularly during the initial stages of zone formation, preventing the establishment of the isotachophoretic steady state. The design of the feedback network, with its compensation circuitry, must be tailored to the specific time constants of the capillary system, which can range from milliseconds for short, narrow-bore capillaries to seconds for longer, wider ones.
However, the true power of modern instrumentation is revealed when we move beyond single-mode operation to complex switching protocols. A common and powerful technique is transient isotachophoresis, used to enhance the sensitivity of capillary zone electrophoresis. In this method, the system is initially set up for isotachophoresis to focus a large volume of dilute sample into a narrow zone. Then, at a precise moment, the power supply is switched from constant-current mode to constant-voltage mode, and the buffer composition is changed to support zone electrophoresis. This transition must be seamless. If the voltage is not applied smoothly and without overshoot at the switch point, the focused sample zone can be disrupted by electrokinetic dispersion.
Implementing such mode switching requires a high-voltage power supply with a true bipolar output and the ability to switch polarities rapidly. Some separation protocols call for a reversal of the electroosmotic flow, which is achieved by reversing the polarity of the electric field. This must be done in a controlled manner to avoid generating large pressure gradients within the capillary that could blow the sample out of the column. The power supply must be able to ramp the voltage down to zero, reverse the output stage connections, and then ramp up to the new voltage of opposite polarity, all within a few seconds, without any transient spikes.
Furthermore, the integration of the power supply with the detection system is crucial. In isotachophoresis, the length of the zones is often measured by a conductivity detector. The output of this detector can be used as a feedback signal for the power supply. For example, once the zone of interest has passed the detector, the power supply can be triggered to switch to a different voltage mode to either accelerate the remaining zones or to stop the separation entirely. This level of synchronisation requires a digital interface with low latency and precise timing. The high-voltage unit is no longer a standalone instrument but a component in a tightly integrated analytical platform.
The safety and protection features of these supplies also warrant discussion. Capillary electrophoresis deals with very high electric fields and minuscule currents. A partial blockage of the capillary, or the formation of a bubble, can cause the resistance to spike. In constant-current mode, the power supply will attempt to maintain the current by increasing the voltage to its compliance limit. If unchecked, this could lead to dielectric breakdown of the capillary wall or arcing at the electrode reservoirs. Therefore, a well-designed supply must have programmable voltage and power limits, and it must be able to detect a non-ideal load condition and shut down or alert the user before damage occurs. The evolution of capillary electrophoresis from a research curiosity to a routine analytical tool is inextricably linked to the parallel evolution of the high-voltage power supply from a simple source of potential to a sophisticated, programmable, and intelligent system component.
