Troubleshooting : Capillary Electrophoresis

What is Capillary Electrophoresis (CE)?
  • CE is used to analyze and separate unknown samples based on their mobility under an applied electrical voltage. Mobility is dependent upon the molecular charge, atomic radius, and viscosity. Ions are the species that migrate to opposing ends of an applied voltage, and its motion is dependent on the applied electric field. 
  • CE is extremely useful in micro and nanofluidics and provides detection and separation of ionic species quickly and with high resolution. The method has a capillary filled with a conductive fluid, which is given a charge, and the sample ions migrate through the capillary at different speeds, causing them to divide.
  • CE offers a novel format for liquid chromatography and electrophoresis that:
  1. employs capillary tubing within which the electrophoretic separation occurs;
  2. utilizes very high electric field strengths, often higher than 500 V/cm;
  3. uses modern detector technology such that the electropherogram often resembles a chromatogram;
  4. has efficiencies on the order of capillary gas chromatography or even greater;
  5. requires minute amounts of sample;
  6. is easily automated for precise quantitative analysis and ease of use;
  7. consumes limited quantities of reagents;
  8. is applicable to a wider selection of analytes compared to other analytical separation techniques.

The main advantages of doing electrophoresis in a capillary are magnificent efficiencies and automation. Because of the small diameters of the capillary, typically in the 20–100 ┬Ám inner diameter range, the Joule heat dissipation is very efficient. This means that high voltages, usually up to 30000 V, can be applied.

Troubleshooting: Capillary Electrophoresis
  • What follows is a short list of problems, "observations" followed by a list of areas that should be investigated, as appropriate in parenthesis (), to troubleshoot common problems seen when using the analytical technique of capillary electrophoresis.
Observation (Investigate for cause):
Excessive Baseline Drifting up or down
  • Temperature is not stable (stabilize room and/or capillary temperature).
  • Fouling of capillary (replace or clean and wash capillary with fresh, filtered solution).
  • Current levels are unstable (loose connections, partial obstruction in a capillary, or running out of buffer solutions).
  • Capillary may have poorly cut ends resulting in poor connections or flow (replace capillary).

Excessive Signal Noise
  • Detector has air in flow cell (purge capillary and wash flow path).
  • Current level may be too high (reduce current).
  • Detection parameters, wavelength and bandwidth, may be inappropriate for buffer solution (select appropriate detection settings which are appropriate for the buffer used and selective for the analyte).

Loss of Signal
  • Voltage/Current has turned off (turn ON or investigate if the system is in an “alarm” state due to an error).
  • Detector parameters not selected.
  • Capillary has not been fully equilibrated (equilibrate capillary and auto-zero the scale).
  • Baseline offset may be off-scale (after equilibration, adjust scale or auto-zero).
  • Detector lamp(s) off, not ignited or due for replacement (verify lamp operation).

Signal Peak Shape Issues
  • Truncated, clipped or ‘square’ peaks (sample overload, reduce concentration 10x, shorten load time and re-evaluate).
  • Tailing peaks often result from very high current or when the concentration of buffer is too high (lower the current and/or reduce the buffer concentration, then re-evaluate).
  • The sampling rate may be too low (measure the peak width in units of time (i.e. seconds), then configure the detector to ensure that the sampling rate allows for at least 20 points to be collected per average peak width (30 points is a better target # to use).
  • No peaks observed (Many possible causes, including Partially or fully obstructed capillary, broken capillary, out of buffer, no injection, detector settings inappropriate for analysis, current too low, pressure too low. Look for a small peak from the injection along the start of the baseline to confirm that analysis was started, then troubleshoot the method and settings).

General Stability and Noise Issues
  • When the CE system has not been used in a few days, salts from the buffer solution(s) may deposit on and clog the capillary line, flow cell and/or sensors. To avoid these problems, be sure to thoroughly clean, flush and wash down the flow path before use. 
  • Take the time to prepare fresh filtered solutions (each day) and allow time for the system to equilibrate. Taking these basic steps will avoid many hours/days of frustration.

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