Question

What are the benefits and drawbacks in CZE (Capillary Zone Electrophoresis) with other more conventional separation technique, such as HPLC (High Performance Liquid Chromatography)?

Answer 1

Capillary Zone Electrophoresis (CZE) versus High Performance Liquid Chromatography (HPLC)  

1. CE has a flat flow, compared to the pumped parabolic flow of the HPLC. The flat flow results in narrower peaks and better resolution. (Figure)
2. CE has a greater peak capacity when compared to HPLC—CE uses millions of theoretical plates.
3. HPLC is more thoroughly developed and has many mobile and stationary phases that can be implemented.
4. HPLC has more complex instrumentation, while CE is simpler for the operator.
5. HPLC has such a wide variety of column lengths and packing, whereas CE is limited to thin capillaries.
6. Both techniques use similar modes of detection.
7. Can be used complementary to one another.

Resolution benefits

The narrow tubes used in capillary electrophoresis help to give the technique good resolution. When a sample is introduced in a tube and an electric field applied — the components move at different rates leading to separation. The advantages of using capillary tubes are that lateral diffusion effects are reduced and temperature differences across the tube are reduced. The properties of the tube, and other properties set by the technician, lead to what is known as plug flow where the velocity of the fluid is considered constant across the tube’s cross-section, perpendicular to the flow. Under plug flow, axial diffusion is the only factor leading to dispersion, so the separation efficiency using CE is very high. Using narrow capillaries also helps to reduce band-broadening seen in the peaks generated in other techniques such as high performance liquid chromatography (HPLC). In CE the velocity of the liquid as it travels along the tube is uniform across the tube. In the wider tubes and using pumped flow in other techniques, the velocity is not uniform across the tube. This is known as laminar flow, the velocity is slower at the interface between tube wall and liquid, giving a velocity profile with a bulge at the centre of the tube. This leads to band-broadening and the wider peaks seen in HPLC for example.

Maintaining a balance

Small pH affects a molecules charge and flow in CE, thus small variations in pH have a greater impact in CE than in HPLC. Compared with HPLC, the control of the pH is critical in CE, and there are many factors, including temperature, that affect pH.