Choosing a capillary electrophoresis system: how capillary electrophoresis separates on charge to size rather than on a stationary phase, which mode suits which analyte, and the capillary conditioning that decides whether migration times are reproducible

Choosing the mode and the system

1. Understand the separation principle. Analytes move through a buffer-filled capillary under a high electric field, separating on the ratio of charge to hydrodynamic size, with bulk flow from the charged capillary wall carrying everything along. There is no stationary phase, which is why the capillary surface matters so much.
2. Pick the mode from the analyte. Zone electrophoresis in free solution for small ions and charged molecules. Gel-filled capillaries for sizing nucleic acids and for protein sizing as an alternative to a gel. Isoelectric focusing for protein charge variants, which is one of the technique's strongest applications.
3. Specify capillary dimensions and the detector together. Narrow capillaries dissipate heat better and allow higher fields, at the cost of a shorter detection path length and so less sensitivity with absorbance detection. Fifty to seventy five micrometres internal diameter covers most work, and laser induced fluorescence is what rescues sensitivity where it is needed.
4. Condition the capillary, and do it the same way every time. Migration time drift is almost always the capillary wall changing as sample components adsorb. A documented rinse sequence between runs, and a longer conditioning at the start of a sequence, is what turns an irreproducible method into a reproducible one.
5. Control temperature and injection precisely. Both migration time and peak area depend on temperature and on the injection, which is by pressure or by voltage rather than by a fixed loop. An internal standard is close to mandatory for quantitation.

Where it has become the standard method

Charge variant analysis of therapeutic proteins by isoelectric focusing in a capillary, and purity and sizing of proteins as a replacement for a gel, are both routine in biopharmaceutical quality control. Nucleic acid fragment sizing is the other established place.

In those applications the technique is not competing with chromatography, it is doing something chromatography does poorly, which is why it sits alongside rather than instead.

Consumables and running cost

Capillaries, coated capillaries, sieving gels and buffers are the running cost, and coated capillaries have a defined number of runs rather than an indefinite life. Price them per run over a realistic year.

Buffer preparation matters more than in chromatography because conductivity sets the current and therefore the heat. Prepare consistently and record it, because an inconsistent buffer moves every migration time.

Troubleshooting the common failures

No current usually means a bubble or a blocked capillary. Drifting migration means the surface is changing. Broad or split peaks often mean the sample matrix conductivity is too high relative to the buffer, which is fixed by desalting rather than by the instrument.

Keep a standard mixture and run it at the start of each sequence. Most capillary electrophoresis problems are diagnosed in one injection of a known sample, and almost none are diagnosed by inspecting the instrument.

Common questions

What does a capillary electrophoresis system do better than HPLC?

Charge variant separations, small ion analysis and nucleic acid fragment sizing, with tiny sample volumes and very high efficiency. It is not a general replacement for chromatography and it is much better at those particular jobs.

Why do my migration times drift?

The capillary surface is changing as sample components adsorb, which alters the bulk flow. A consistent rinse and conditioning sequence between runs, and a coated capillary for sticky samples, fix most of it.

Is capillary electrophoresis quantitative?

Yes, with care. Injection is by pressure or voltage rather than a fixed loop and peak area depends on migration velocity, so an internal standard and corrected peak areas are the normal practice rather than an optional refinement.

When should I use a coated capillary?

When proteins or other sticky analytes adsorb to the wall, which shows as tailing and drifting migration. Coatings suppress the bulk flow and the adsorption together, and they are consumables with a limited life.

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