Introduction

Capillary Electrophoresis has been around for the past 40 years. It is a universal and ultra-high efficiency separation of low to high molecular weight substances. Manifested by different operation modes, these are all executed in cylindrical (capillary) or rectangular (chip) conduits.

The market perspectives of CE as an instrumental analysis tool in the eighties were immense. But has this perspective become a reality? This question is difficult to answer. Many commercial instruments use capillary electrophoresis as the primary separation method of the sample, but they are not designated as CE instruments.

Therefore, I felt it appropriate to map the "Capillary Electrophoresis" field according to methodologies, instrumentation, and areas of application (markets). The overview is based on publicly accessible information, my background, and experiences with CE during my professional career in Hewlett-Packard and Agilent Technologies, and inferences I made from that.

*A redacted version of this manuscript can be found in the July issue of "The Analytical Scientist"  via the following link.

Capillary Electrophoresis on "Standard" HPCE equipment.

Capillary Zone Electrophoresis (CZE) is how CE began in the mid-eighties, followed by market introductions of instruments by several manufacturers at the end of the eighties/early nineties.

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

Traditional application fields have been and still are the separation and quantitation of low MW, ionizable molecules, enantiomeric molecules, and inorganic ions.

CZE, coupled with mass spectrometry (MS), is well-positioned in biomarker discovery, metabolomics, clinical research markets, and academia (details on MS coupling see later paragraph). Recently, interest in using CE to separate intact proteins followed by MS detection is growing.

Despite the ultra-high separation efficiency of CZE, the ability to modify selectivity and optimize a separation by CZE is limited. Parameters like pH, ion strength, buffer type, temperature and field strength affect the solute's migration time. Systematic variation of setpoints is required—alternatively, CE simulation tools like Peakmaster (Gas et al., Charles University, Prague) have proven useful.

Interaction of the solutes with the inner surface of the separation capillary (fused silica) must be minimal or under control. With permanent hydrophilic coatings, the electro-osmotic flow (EOF) and solute surface interactions on the inside capillary wall capillaries are suppressed. Commercially available wall-coated capillaries are expensive, however. Dynamic coatings like hydroxymethyl cellulose have been used alternatively. Deposition of multiple layers of charged polymers like polybrene and dextran sulfate allows control of the EOF's direction and minimizes solute-surface wall interactions and seem to have become a preferred method.

Capillary Isotachophoresis (CITP)

In contrast to CZE in CITP, a discontinuous buffer system is used. 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. The electro-osmotic flow is absent.

CITF is much less used for analyses, but its principle is valuable for sample pre-concentration (see later).

CE with modifiers in the background electrolytes on "standard" HPCE equipment

The limited ability of CZE to optimize selectivity and adaptation of slab gel electrophoresis in a capillary has led to several modes of CE.

Capillary Gel Electrophoresis (CGE)

CGE has provided the base technology for DNA sequencing since the early nineties. Initially executed on a standard CZE instrument, it has evolved toward multiple capillary electrophoresis systems (discussed later). Gels used are polyacrylamide or agarose—the gel in the capillary acts as a sieve. DNA, RNA, and polynucleotides are HMW molecules with a constant mass to charge ratio. They do not separate by electrophoretic mobility but by size.

1. SDS-Gel Capillary Electrophoresis of proteins 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. Separation in the gel is by size (sieving gel).
2. 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) non-polar stationary phase. The EOF is employed to propagate the solutes through the interacting medium. MEKC and CEC are supported by standard HPCE equipment and add the versatility of usage to these systems.
3. Capillary Isoelectric Focusing (CIEF)
   CIEF is an odd method out to the above. Conventional CE instruments are designed so that a sample is introduced at a time t_o 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).

How to do this in a capillary 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 mobilizing the focused bands beyond the PoD. The whole separation capillary is filled with a mixture of ampholytes, markers, the protein sample, and additives, after which the voltage is switched on.

I have written two tutorials dealing with CIEF. (https://doi.org/10.1002/9780470027318.a9731, and https://www.agilent.com/cs/library/primers/public/5991-1660EN.pdf.

Imaged Capillary Isoelectric Focusing (iCIEF)

iCIEF was invented by Prof. Janusz Pawliszin of Waterloo University in the early nineties. Here, the CIEF separation occurs in a capillary tube, 50x0.1 mm. The separation capillary is whlly filled with the sample mixture containing ampholytes, markers, and sample proteins. The whole capillary is illuminated. A CMOS camera is behind the capillary and images the separation. The camera observes the zone focusing according to their pI to a fixed location along the capillary.

Determination of purity and charge heterogeneity in R&D and manufacturing of biopharma therapeutics is the main application. iCIEF is an instrumental analysis method brought to market by Convergent Bioscience in the mid-nineties. Convergent was acquired by Cellbiosciences (now Protein Simple, part of Botechne) in 2010. Since then, Protein Simple has dominated and monopolized the market. The systems were marketed under the brand name ICE2 and ICE3 and now Maurice.

Advanced Electrophoresis Solution is a start-up by former Convergent employees in 2011. They entered the market in 2016 with a very similar system called CEInfinite. AES has proprietary ampholytes, pI markers, and other consumables for CIEF. AES developed a micropreparative option to process further collected samples and differentiate CEInfinite from ICE and Maurice.

Multi Capillary Electrophoresis

The high sample load for DNA sequencing in the Human Genome Project (HGP) in the nineties drove the development of multi capillary CE instruments in the early nineties. DNA sequencing instrumentation is regarded a market of its own and not part of the CE instrumentation market.

In-tube-gel electrophoresis, developed by LAB901 of Edinburgh, UK, is an intermediate method between multi-capillary gel and microchip electrophoresis (described below). 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. The blister package (ScreenTape) contains 16 tubes. All are prefabricated and ready to use. Sample preparation is manually and delivered to dedicated vials in the device. The instrument with the tubes (screens) has been commercialized by Agilent and branded TapeStation. Agilent has kits for DNA and RNA analyses. More details can be found in this manuscript.

But since the human genome sequencing finished, the need for analyses staggered in genomics research. DNA sequencers became increasingly used for other purposes. E.g. so-called fragment analysis has become an essential tool in genetic research and development. With the current Corona-virus pandemic, the market for genetic fragment analysis has accelerated. To my knowledge, Agilent Technologies has taken the lead and prominent position in this market with its acquisition of Advanced Analytical Technologies, Inc., Ankeny, Iowa, in 2018. Thermo Scientific plays on this market with the ABI 3500 series DNA Sequencer for DNA fragment analysis. 

Protein Analysis by CE

In routine clinical analysis, multi capillary CE is used by Sebia (France). I have been involved with them during my Agilent time and know this is significant business. Sebia uses fused silica capillaries with 25 µm i.d. Sebia CE-instruments are fully compliant with clinical laboratory regulatory requirements. Sebia CE instruments are disregarded in the CE Market.

Micro Chip Electrophoresis (MCE)

Agilent Technologies has been committed to MCE early on through its joint venture with Caliper Technologies in the late nineties. I was personally involved in that endeavor and given the task to assess the potential use of the Caliper chips as a successor for CE. The outcome was easy. The separation zone on the Caliper chip is too short (about 13 mm) to be useful for routine CE. Eventually, the BioAnalyzer came to the market in 1999 with kits for DNA, RNA and SDS-protein separations and is a successful product for Agilent.

Most recently, 908 Devices introduced the ZipChip to market. It is not a standalone MCE device but an inlet to various commercial MS systems. There are two versions of the chip. One version for fast, and one for high-resolution separation are available. This last one has a 29 cm length of the separation channel.

Intabio intended to market a chip-based, imaged-CIEF system. About a year ago, they were acquired by Sciex. This page on the Sciex website shows their intention to have an intimate coupling with MS. Details of the technology can be found on the Sciex website here (check the technical notes on this page for more information).

Detection UV-Vis and others

Initially, UV-Vis spectrophotometry, with diode arrays, was the primary detection method in CE. It was claimed to be less "sensitive" than UV-Vis detection in HPLC. This statement has been a constant irritation to me since what is meant with "sensitivity" is unclear.

The short optical detection path length and the cylindrical shape of the detection zone are fundamental limits (Lambert-Beer law) for optical detection. In spectrophotometric detection, the response is concentration sensitive. The concentration sensitivity of UV-Vis diode array detections in CE is excellent compared to HPLC, given the very high plate number and considering the small sample volumes used (nanoliter vs. µL in HPLC).

Extension of the optical path length with a bubble in the capillary or a cuvette type flow-through cell as introduced by Agilent improves the sensitivity (higher peaks) by a factor of 3-10. Also, electrokinetic sample pre-concentration methods can offset this limitation (see next).

Fluorescence is the primary mode of detection in the capillary gel electrophoresis devices described before. Laser-Induced Fluorescence detection for "standard" HPCE instrumentation is available as a proprietary solution from Beckman/Sciex. Likewise, solutions for conductivity detection with standard HPCE instruments are available. 

CE-MS

In coupling CE with MS, one is facing severe practical obstacles. There are two high voltage sources on one "conductor." It is mandatory to separate these electrical fields to avoid disturbance of the CE separation and the electrospray process. Additives to the run buffer that help the CE separation are not compliant with vacuum detection like mass spectrometry. E.g. inorganic buffers or SDS etc. should be avoided. An ultra-low zone dispersion capillary connection is required to preserve the high-resolution CE separation when the zones enter the MS. 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. Solutions for other atmospheric pressure ionization methods like photoionization (APPI) and chemical ionization (APCI) are available but barely have a role in CE-MS interfacing.

Engineers at Hewlett-Packard in the early nineties embarked on an idea by Smith c.s. to add a sheath liquid concentrically to the spraying tip. They adapted the HP LC-MS ESI sprayer accordingly. The ES-voltage on Agilent (and Bruker) MS is applied from the MS 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. Therefore this CE-MS is commonly called the "triple tube" CE-MS interface.

Consequently, the CE and ESI voltage share a common ground and are decoupled in contrast to Thermo and Sciex, which require additional resistance circuits to decouple the CE and ES voltages.

The triple tube CE-ESI-MS interface was introduced by Hewlett-Packard in 1995 and has proven robustness, ease of use, reliability, and is used routinely. Slight adaptions in the methodology like in-spray chemistry increased its versatility. Standard dimension FS silica capillaries can be used.

Adequate sensitivity in pharmaceutical applications has been demonstrated. The sheath solvent flow rate, which is 5-50x higher than the electro-osmotic flow, dilutes the sample zone accordingly.

The need for higher sensitivity in biopharmaceutical research led to Beckman's (now Sciex Separations) commercialization of a sheathless ESI interface. The so-called porous tip was invented and proposed by Moini (Texas A&M University) and exclusively licensed to Beckman. It gives 10-50x higher sensitivity in CE-MS at the cost of robustness loss and mandates expert users. Capillaries are 30 µm i.d., wall-coated, and very expensive.

A small start-up, CMP-Scientific, has commercialized a sheathless CE ESI interfaced based on work by Dovichi (Notre Dame University IL). Meanwhile, CMP has entered the market with a proprietary CE-MS system.

Sample pre-concentration techniques in CE

Like 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 caus significant zone compression. Breadmore and Sänger prepared an excellent overview of these methods (LCGC NORTH AMERICA VOLUME 32 NUMBER 3 MARCH 2014, page 174). High competence and understanding of electro-driven phenomena and practical experience by the user are mandatory. 

Free Flow Electrophoresis (FFE)

Free-flow electrophoresis (FFE) is a commercially available, efficient protein separation methodology that collects constituents of biological samples like proteins, organelles, cells, or blood constituents. FFE Service GmbH offers the system.

The separation device consists of two parallel plates spaced 0.175 – 0.4 mm apart, 10 cm wide. A laminar flow of buffer solution through the channel is established. The channel is flanked by two electrodes, generating a high voltage electric field perpendicular to the laminar flow. The sample is continuously injected. The sample components are separated by CZE or IEF (when ampholytes are present in the buffer and a pH gradient is established before the sample is injected). The eluate is collected in standard 96-well microplates. More details can be found here.

FFE is a preparative separation technique allowing the collection of milligrams of sample constituents for further characterization methods that require a higher amount of protein than available through analytical separation methods.

There has been academic research by several groups to miniaturize FFE. But to my knowledge, there are no products on the market.

Summa summarum

From this map on capillary electrophoretic separation techniques, one can conclude that the promise from the mid-eighties that CE would become a dominating technique and lead to a commercial instrumentation business is met. Deciphering the full CE-based instrumentation and solution business market would be a daunting task and beyond the scope of this work. In important application fields in proteomics, biopharmaceutical R&D and genomics, CE methods are to sole tool for a solution.