Capillary Electrophoresis

Capillary electrophoresis (CE) provides a number of advantages for analysts, including high separation efficiency, short analysis times, low waste generation, and a diverse range of applications. Participants in this forum are Bill Ciccone, Microsolv; Martin Greiner, Agilent Technologies; and Mark Lies, Beckman Coulter Life Sciences.

Why hasn’t CE “taken off” as a general purpose analytical tool, despite the fact that it has been around for over a decade?

Ciccone: CE is a great analytical technique and for some compounds is by far the very best way to analyze them. However, the instruments suffer from issues relating to routine daily use. The difficulty in changing the capillaries and robustness are issues among others. For some, the issue is that CE cannot be “scaled up” to preparative CE the way that high performance liquid chromatography (HPLC) can be. For others, it is the issue of less sensitivity with a UV detector compared to HPLC.

Greiner: Not sure whether the question has been posed correctly. CE plays a crucial role in several applications like biological macromolecules as proteins, peptides, and DNA. Another successful field is high-resolution separation on chiral compounds or very polar small molecules down to ion analysis. Expectations by some people in the early days that CE could replace LC was definitely not very realistic, however CE can fill a defined niche. Obstacles for broader usage are still the less known and understood method parameters and different method development needs in electrophoresis (compared to the broad knowledge available on chromatography in most analytical labs). Lower sample loading capacity and restricted UV-sensitivity are a limitation for scientists with the highest sensitivity requirements.

Lies: The reason CE hasn’t taken off as a general purpose analytical tool is really because of its past and the lack of a clear understanding of the best uses for the technology. In the early days of the technology, many had high expectations and applied CE to help solve a number of analytical separations that weren’t consistent with its strengths.

CE is best applied to charged polar compounds, and has excelled in the analysis of proteins, ions, and carbohydrates. In fact, capillary electrophoresis–sodium dodecyl sulfate (CE-SDS) analysis of protein therapeutics is the standard method in the industry. There is also the perception that reproducibility and sensitivity are not adequate compared to LC; however, by applying the best practices like mobility measurements rather than migration times, any CE separation can be made as robust and reproducible as any analytical technique. At my company, we focus on CE’s strengths, putting resources into development and simplification of key application areas, and have been able to turn the momentum back in favor of the technology.

How does CE compare with microcolumn methods (as used in Jim Jorgensen’s or Milton Lee’s lab)?

Ciccone: I am not familiar with what Jim Jorgensen or Milton Lee are using but CE should not be compared to HPLC because it is an orthogonal technique to HPLC that offers a completely different mechanism of separation. With CE, the sample size is extremely small, uses simple buffers and coatings, and can separate with efficiency that HPLC cannot compare to. Sample solubility in organic solvents may be a problem for some compounds with CE.

Greiner: This question is unclear to me since it could relate to several items that Jim and Milton worked on:gas chromatography (GC)-like open tubular microcolumns (high efficiencies, fast separations, long columns just like in GC, but linked to low loading capacity and sensitivity); capillary electrochromatography (CEC); packed fused-silica columns run by electro-osmotic flow, having a mix of electro-driven chromatography based separation (isocratic chromatographic separations, rather delicate in setup and handling, fast separation but today an ultrahigh-pressure liquid chromatography (UHPLC) gradient is even faster and clearly more robust and versatile); microfluidics on chip; or miniaturized electrophoresis in microchannels (very fast, high separation capacity, but requires more sensitive detectors – for example, laser -induced fluorescence [LIF] — because of very small injection volumes and very complex method development — not as flexible as CE).

Lies: When we think of microcolumn technologies, we’re usually talking about narrow-bore or nano-LC. These techniques begin to move away from preparatory chromatography and take advantage of small volume and potentially higher efficiency separations. But at the end of the day, they’re still chromatographic technologies based on separation principles different from those of CE. This facilitates the selectivity, which is great for orthogonal analyses, but doesn’t necessarily provide better separations for an array of separations where CE excels.

What is the long-term outlook for CE?

Ciccone: The long-term outlook for CE is very hard to predict because as of right now it does not have a segment of the routine analytical laboratories that is measurable. However in the future, I hope one day CE will be recognized by the scientific community for the advantages it brings to any modern laboratory including efficiency, low cost of operation, and unique selectivity to HPLC, ion chromatography, and chiral HPLC. For untargeted analysis of samples, it should be an orthogonal technique that is used routinely.

Greiner: There is already a new trend that can be seen using CE as an orthogonal method to chromatography complementing existing complex sample analysis (for example, peptide mapping) by another high-resolution pattern with a completely different separation principle. CE is widely used to gather data for compounds that are difficult to handle by HPLC methods like ions or small charged compounds, or large native proteins. With the advent of robust CE coupled to mass spectrometry (MS) systems a typical and growing application for CE is in metabolomics analysis, where the challenge is to separate very similar or isomeric and often charged compounds like organic acids, amino acids, and nucleotides that show no or identical retention on an HPLC column. Another request for CE and CE–MS clearly comes from pharmaceutical and biopharmaceutical scientists focusing on new biological entities (NBE) like antibodies. In this case, CE provides excellent results on protein sizing, purity, heterogeneity, or complex modifications like glycans.

Lies: Looking out one, two, or even five years from now, the future of CE technology continues to look encouraging. There is an effort — both short term and long term — to expand the adoption of CE into new application areas, in addition to a number of innovations around CE in our technology pipeline. Its capability to analyze charged or polar species should continue to provide CE with more widespread usage, but education in these areas is key. It will likely become more and more important to provide solutions rather than just instruments, and also to simplify CE technology as research and analytical labs move towards increasing efficiency. Overall, the future of CE looks quite bright.