Cookies

Like most websites The Analytical Scientist uses cookies. In order to deliver a personalized, responsive service and to improve the site, we remember and store information about how you use it. Learn more.
Subscribe to Newsletter
Techniques & Tools Capillary Electrophoresis

Surveying the Capillary Electrophoresis Landscape

Capillary electrophoresis (CE) – a universal and ultra-high efficiency separator of low to high molecular weight substances – has been around for almost 40 years. I remember the sky-high expectations for CE back in the 1980s: Becoming predominant in its field and being used across a wide variety of application areas. Has this lofty goal become a reality? It is difficult to say. Many commercial bio-analytical instruments use CE as their primary separation method, but they are not designated as “CE instruments.” And that raises other important questions: What does the capillary electrophoresis field look like today? Where and how are the many modes of the technique applied? Where do we go from here?

Capillary electrophoresis in HPCE equipment
 

CE began in the mid-1980s with capillary zone electrophoresis (CZE), and several manufacturers had already launched CZE-based instruments by the early 1990s. 

In “standard” (conventional) HPCE equipment, one capillary is used to separate the sample constituents. These self-contained instruments provide all the necessary functions to execute a separation from sample handling and injection, capillary pre-preparation, buffer handling, thermal control, spectrophotometric detection, instrument control, automation, and data handling. Major market players today are SCIEX (was Beckman Coulter) and Agilent Technologies. 

Traditional application areas are the separation and quantitation of low MW ionizable molecules, enantiomeric molecules, and inorganic ions. CZE coupled with mass spectrometry (MS) is well established in biomarker discovery, metabolomics, and clinical research. Recently, we’ve seen growing interest in using CE to separate intact proteins followed by MS detection. 

Despite the ultra-high separation efficiency of CZE, the ability to modify selectivity and optimize separations by CZE is limited. Some parameters, including pH, ion strength, buffer type, temperature, and field strength, affect the solute’s migration times, so systematic variation of setpoints is required – alternatively, CE simulation tools like PeakMaster (Gas et al., Charles University, Prague) have proven useful.

Another potential drawback is the interaction of the solutes with the inner surface of the separation capillary (fused silica), which must be minimal or under control. With permanent hydrophilic coatings, the electro-osmotic flow (EOF) and solute–surface interactions on the inside capillary wall are suppressed. Commercially available wall-coated capillaries are expensive, but dynamic coatings, have been used instead with success. Here, deposition of multiple layers of charged polymers, such as polybrene (cationic layer) and dextran sulfate (anionic layer), allows control of the EOF’s direction and minimizes solute–surface wall interactions – and seems to have become the preferred method. These factors, especially the limited ability of CZE to optimize selectivity, have led to several different CE modes:

i) Capillary gel electrophoresis (CGE) has been used as the standard technology for DNA sequencing since the early 1990s. Initially executed on a standard CZE instrument, it has evolved toward multiple capillary electrophoresis systems – more on this later. The gels, which act as a sieve in the capillary, are either polyacrylamide or agarose – DNA, RNA, and polynucleotides are heavy molecular weight molecules with a constant mass to charge ratio. They do not separate by electrophoretic mobility but by size.

ii) SDS-gel capillary electrophoresis is most extensively used for rapid characterization, release, and stability testing methods of therapeutic proteins in biopharmaceutical R&D and manufacturing. The gel used here is polyacrylamide. Sodium dodecyl sulfate binds to proteins in fixed ratios, rendering all with a similar mass/charge ratio. Again, separation in the gel is by size (sieving gel).

iii) Micellar electrokinetic chromatography (MEKC) and capillary electrochromatography (CEC) allow the separation of uncharged hydrophobic molecules by interaction with a non-polar, moving (micelles) or stagnant (HPLC packing material) stationary phase. The EOF is employed to propagate the solutes through the interacting medium. MEKC and CEC are supported by standard HPCE equipment and so increase their versatility.

iv) Capillary isoelectric focusing (CIEF) is the odd one out. Conventional CE instruments are designed so that a sample is introduced at a time to and its constituents detected some time t later. Isoelectric focusing (IEF), in its original inception on flat slab gels or with immobilized pH gradient (IPG) strips and sheets, delivers a separation in space (like thin-layer-chromatography). So how can we do this in a “standard” HPCE instrument? IEF on a conventional, one-capillary HPCE instrument is achieved by executing the focusing process in the capillary part before the point of detection (PoD). The focusing step is followed by a second step to mobilize the focused bands beyond the PoD. Before the run the whole separation capillary is filled with a mixture of ampholytes, markers, the protein sample, and additives, after which the voltage is switched on. You can find details in my two tutorials on dealing with CIEF (1, 2).

Other avenues 
 

iCIEF

Imaged capillary isoelectric focusing (iCIEF) is another important technique invented in the early 1990s – in this case by Janusz Pawliszyn at the University of Waterloo in Canada. Here, the CIEF separation occurs in a 50 x 0.1 mm capillary tube. The separation capillary is wholly filled with a sample mixture containing ampholytes, markers, and sample proteins. The whole capillary is illuminated, with a CMOS camera behind, which images the separation. The camera observes the zone, focusing – according to their pI – to a fixed location along the capillary. 

The main application for iCIEF is the determination of purity and charge heterogeneity in R&D and biopharmaceutical manufacturing. iCIEF was brought to market by Convergent Bioscience in the mid-1990s. Convergent was acquired by Cellbiosciences (now ProteinSimple, part of Bio- Techne) in 2010. Since then, ProteinSimple/Bio-Techne has dominated the market.

Advanced Electrophoresis Solutions (AES) was set up by former Convergent employees in 2011. They entered the market in 2016 with a very similar system called CEInfinite, as well as proprietary ampholytes, pI markers, and other consumables for CIEF. AES developed a micro preparative option to process further collected samples and differentiate CEInfinite from ProteinSimple’s offerings (ICE2 and ICE3, which are being replaced by its Maurice platform).

Multi-capillary CE

Later in the 1990s, the high sample load for DNA sequencing in the Human Genome Project (HGP) drove the development of multi-capillary CE instruments (DNA sequencing instrumentation is regarded as a market of its own and not part of the CE market). 

Since the completion of the Human Genome Project, the need for analyses staggered in genomics research and DNA sequencers became increasingly used for other purposes. For example, DNA fragment analysis has become an essential tool in genetic research and development. And the COVID-19 pandemic has accelerated the market for genetic fragment analysis. 

Agilent Technologies is a leader in the field, having acquired Advanced Analytical Technologies, Iowa, USA, in 2018. Thermo Fisher Scientific is another big player with their ABI 3500 series DNA Sequencer for DNA fragment analysis.

Microchip electrophoresis

Agilent has also been heavily involved in microchip electrophoresis (MCE) through its joint venture with Caliper Technologies in the late 90s. I was personally involved in that endeavor – one of my tasks was to assess the potential use of the Caliper chips as a successor for CE. The outcome? It turns out the separation zone on the Caliper chip is too short (about 13 mm) to be useful for routine CE – and that was that. Eventually, however, Agilent’s Bioanalyzer came to the market in 1999 with kits for DNA, RNA and SDS-protein separations and still is.

Most recently, 908 Devices introduced the ZipChip to the market. It is not a standalone MCE device but an inlet to various commercial MS systems, with two versions of the chip available: one version for fast separation, the other for high-resolution separation (the latter has a 29 cm-long separation channel).

Intabio intended to market a chip-based, imaged-CIEF system. About a year ago, they were acquired by SCIEX. Interestingly, SCIEX have publicly discussed their intention to have an intimate MS coupling of the Intabio iCIEF chip.

What about MS? 
 

As I mentioned earlier, CZE has been successfully coupled with MS and is well-established in biomarker discovery. But several practical obstacles must be overcome to couple CE techniques with MS. First, there are two high voltage sources on one “conductor” in MS that must be separated to avoid disturbance of the CE separation and the electrospray process. Second, run buffer additives that aid CE separation are not compliant with vacuum detection (inorganic buffers or SDS should be avoided, for example). Third, an ultra-low zone dispersion capillary connection is required to preserve the high-resolution CE separation when the zones enter the MS. And fourth – and most importantly – there must be an outlet vial to close the electrical current of the CE system. 

Electrospray is a proven, robust ionization method in HPLC and the dominant IF technique for CE-MS. Solutions for other atmospheric pressure ionization methods like photoionization (APPI) and chemical ionization (APCI) are available but not commonly used in CE-MS interfacing. 

Engineers at Hewlett-Packard in the early 1990s embarked on an idea by Richard D. Smith to add a sheath liquid to the spraying tip. They adapted their HPLC-MS ESI sprayer accordingly, with the ES-voltage on Agilent (and Bruker) MS instruments applied from the mass spectrometer inlet. The tube delivering the sheath solvent is grounded and becomes, in effect, the outlet vial. A nebulizing gas is delivered via an additional concentric tube for pneumatic assistance of spray formation. As a result, this is commonly referred to as the “triple tube” CE-ESI-MS interface.

The triple tube CE-ESI-MS interface was introduced by Hewlett-Packard in 1995 and has proven to be robust, easy to use, and reliable – hence its routine use. Slight adaptations in the methodology, such as in-spray chemistry, increased its versatility and, today, standard dimension FS silica capillaries are available. 

The sensitivity of triple tube CE-ESI-MS has proved adequate for many pharmaceutical applications. But the need for higher sensitivity, especially in biopharmaceutical research, led to Beckman’s (now SCIEX) commercialization of a sheathless ESI interface. The so-called porous tip was invented and proposed by Mehdi Moini (Texas A&M University) and exclusively licensed to Beckman. It gives 10–50 times higher sensitivity in CE-MS at the cost of robustness loss – expert users are mandatory, and capillaries are very expensive.

A small start-up, CMP Scientific, has commercialized a sheathless CE-ESI interface based on work by Norman Dovichi (Notre Dame University, USA).

Mission accomplished?
 

Back in the mid-1980s, we wondered whether CE would become a predominant technique with several instruments on the market. Deciphering the full CE-based instrumentation and solution business market today would be a daunting task and beyond the scope of this work. Nevertheless, for many basic analytical tasks in proteomics, biopharmaceutical R&D and genomics, CE methods are the only tool available. 

So, has CE met the lofty expectations set by its early pioneers 40 years ago? In contrast to HPLC and GC, which can be regarded as open platform systems, CE – adaptable to different analytical questions and in routine laboratory work – has become the heart of closed analytical platforms that solve specific (and important) bio-analytical questions. And given the large market for genetic analysis, and spurred by the pandemic, proteomics, metabolomics and clinical analysis, the answer is “yes.”

If I had to mention one area in particular, microchip electrophoresis when seamlessly coupled with mass spectrometers have enormous potential – provided that the high resolution obtained by CE is conserved upon entrance of the analytes into the mass spectrometer. 

However, there are some downsides associated with these developments, such as the loss of technological insights in capillary electrophoresis, which puts demands on the manufacturers and suppliers in these markets. But there are lessons to be learned here. MCE-MS should not become monopolized by manufacturers. Users need flexibility in choices and do not want to be locked into one manufacturer’s solution. Remember, the Agilent HPLC-Chip MS solution, despite being an elegant and integrated solution, did not survive in the market…

Even More Avenues… 
 

  • Capillary isotachophoresis (CITP), in contrast to CZE, requires a discontinuous buffer system. The sample is sandwiched between an electrolyte with a high mobility ion and a low mobility ion of the same charge sign as the analyte ions. After switching the voltage on, the sample ions are sorted into zones according to their mobility. Electro-osmotic flow is absent. CITF is not commonly used for analyses but can be valuable for sample pre-concentration.
     
  • Free-flow electrophoresis (FFE) is a commercially available (FFE Service GmbH), efficient protein separation methodology that collects constituents of biological samples, such as proteins, organelles, cells, or blood constituents. As a preparative separation technique, FFE allows the collection of milligrams of sample constituents for further characterization methods that require a higher amount of protein than available through analytical separation methods. Several academic groups have tried to miniaturize FFE, but – to my knowledge – there are no products on the market.
     
  • In tube gel electrophoresis, developed by LAB901 of Edinburgh, UK, is an intermediate method between multi-capillary gel and microchip electrophoresis, and has been commercialized by Agilent. The separation takes place in a tube filled with polyacrylamide gel. The tube has a length of about 2 cm and a diameter of 1 mm. The tube is contained in a blister package with connected reservoirs containing electrolyte solution and staining agent. Sample preparation is manually and delivered to dedicated vials in the device.
     
  • Protein Analysis by CE, has been used in routine clinical analyses by Sebia (France). I was involved with them during my Agilent days and know what they can do! Sebia uses fused silica capillaries with 25 µm i.d. in full compliance with clinical laboratory regulatory requirements. 
     
  • Sample pre-concentration techniques in CE are also worth mentioning. As with solid-phase extraction (SPE) for CZE, large sample zones introduced into the separation capillary can be compressed using electrokinetically driven methods. Differences in field strength or pH of the sample zone cause significant zone compression. High competence and understanding of electro-driven phenomena and practical experience by the user are mandatory.
Receive content, products, events as well as relevant industry updates from The Analytical Scientist and its sponsors.
Stay up to date with our other newsletters and sponsors information, tailored specifically to the fields you are interested in

When you click “Subscribe” we will email you a link, which you must click to verify the email address above and activate your subscription. If you do not receive this email, please contact us at [email protected].
If you wish to unsubscribe, you can update your preferences at any point.

  1. G Rozing, “Encyclopedia of Analytical Chemistry: Applications, Theory and Instrumentation Capillary Isoelectric Focusing” (2021). DOI: 10.1002/9780470027318.a9731.
  2. Agilent, “Principles and Applications of Capillary Isoelectric Focusing” (2014). Available at: https://bit.ly/3xhixi9.
About the Author
Gerard Rozing

An emeritus Agilent Research Fellow and currently is a technical consultant at Rozing.com Consulting. He is a member of the scientific advisory board member at Advanced Electrophoresis Solutions, Cambridge, ON, Canada.

Register to The Analytical Scientist

Register to access our FREE online portfolio, request the magazine in print and manage your preferences.

You will benefit from:
  • Unlimited access to ALL articles
  • News, interviews & opinions from leading industry experts
  • Receive print (and PDF) copies of The Analytical Scientist magazine

Register