24 Capillary Electrophoresis-based Separations*

CE-based separations often have 10⁵–10⁶ theoretical plates. These plate numbers compare with 10⁴ for HPLC and 10⁵ for UPLC. Two main factors contribute to the sharp peaks and high-resolution separations possible with CE: the nature of the EOF and a phenomenon called stacking.

For the pressure-driven flow in HPLC/UPLC columns, the velocity of the mobile phase varies parabolically across the diameter of the column, with peak velocity in the center of the column. In contrast, EOF generates a relatively uniform profile of velocities across the capillary diameter. The result is less broadening of analyte bands from flow dispersion.

Stacking arises from dependence of local electric field strength on the local conductivity of the solution. Electrolyte solutions with lower conductivity (i.e. higher electrical resistance) have higher local field strengths. Recall that ions migrate faster at higher field strengths. If a plug of hydrodynamically injected sample has a much lower conductivity than the surrounding background electrolyte (BGE; i.e. buffer filling the capillary) then stacking occurs. The electric field is stronger in the plug than in the BGE, such that sample ions migrate toward the boundaries of sample plug then slow down upon reaching the BGE. Ions from the center of sample plug eventually catch up to those that were nearer the periphery. Cations thus accumulate or “stack” at one end of the sample plug and anions do the same at the other end. Stacking stops once the conductivity of the stacked zones match that of the BGE.

Separation Mechanisms

Stacked bands of analyte ions and molecules are separated using one of several possible modes:

Capillary Zone Electrophoresis (CZE). This method is the conventional mode of separation by CE. It utilizes a constant buffer composition within the capillary and separation is based on the different mobilities of different ions. Buffer pH, solution additives (e.g. cationic surfactants), and/or covalent modifications are used to control the charge on the silica and thus the rate (or even direction) of electroosmosis. CZE is useful for the separation of ions but does not separate neutral molecules.

Capillary Gel Electrophoresis (CGE). The capillary is filled with a gel, which has a sieving effect on analytes and enhances the role of size in their separation. CGE is an effective method for the separation of many classes of biomolecules, with the advantage of higher resolution versus slab gels.

Isoelectric Focusing. The capillary is filled with a pH gradient of buffer. Analytes are separated by their pI (isoelectric point): they electrophoretically migrate until they reach the point in the capillary at which pH = pI, at which point they become neutral and only travel with the EOF.

Micellar Electrokinetic Chromatography (MEKC). The capillary is filled with a buffer containing a surfactant above its critical micelle concentration. For the common example of sodium dodecyl sulfate as a surfactant, the result is highly negatively charged micelles that migrate slower than the EOF. The interior of the micelle is hydrophobic and thus serves as a pseudo-stationary phase for partition chromatography. Different neutral molecules have different partition coefficients toward the micelles, will therefore spend a different fraction of time in the micelles versus the buffer phase, and thus will be separated with different migration times. Charged molecules may also interact with the micelles via hydrophobic or electrostatic interactions and have their migration time altered.

Capillary Electrochromatography. The capillary is filled with a true stationary phase and the EOF is used to drive buffer flow. Similar to GC, the stationary phase may be a coating (e.g. C18) on the inner wall of the capillary or, similar to LC, on packed particles or a monolith.

Connections

• In CZE and MEKC, EOF (Ch. 22) substitutes for the pressure-driven flow of a mobile phase in LC (Ch. 20) or GC (Ch. 21).
• In MEKC, the micelles substitute for the stationary phase (Ch. 18) used in LC.
• In CZE, electrophoretic mobility has the same role as a partition coefficient (Ch. 19) for LC or GC.

Post-Reading Questions

1. What CE methods are capable of separating neutral molecules?
2. What CE method separates ions based on their pI?
3. What CE method has a sieving medium and what purpose does it serve?
4. What CE method has a stationary phase?

Topic Learning Objectives

The chapter is a primer for the following learning objectives, which will be covered in lecture and/or with additional assigned reading:

• Explain why CE separations are high resolution.
• Recognize when a separation scenario is best suited to LC, CE, or either method.
• Understand the principles, strengths, and limitations of CZE, CGE, isoelectric focusing, MEKC, and capillary electrochromatography.