In 1989, Beckman Instruments introduced the first fully automated capillary electrophoresis instrument (P/ACE™ 2000) to the scientific community. At that time, CE demonstrated exceptional resolving of selected compounds, but the new technology lacked a track record of applications. The subsequent application of automated CE to real-world separation problems has propelled the advancement of this technology to the robust, dedicated analyzers in use today.

• Separation Technology 
• Putting CE to Work--Applying the Technology to Real World Applications 
• Rationale for Selecting CE as a Bioseparation Technique
   

Capillary electrophoresis (CE) encompasses a family of related separation techniques that use narrow-bore fused-silica capillaries to separate a complex array of large and small molecules. High electric field strengths are used to separate molecules based on differences in charge, size and hydrophobicity. Sample introduction is accomplished by immersing the end of the capillary into a sample vial and applying pressure, vacuum or voltage. Depending on the types of capillary and electrolytes used, the technology of CE can be segmented into several separation techniques. Examples of these include:

• Capillary Zone Electrophoresis (CZE), also known as free-solution CE (FSCE), is the simplest form of CE. The separation mechanism is based on differences in the charge-to-mass ratio of the analytes. Fundamental to CZE are homogeneity of the buffer solution and constant field strength throughout the length of the capillary. The separation relies principally on the pH controlled dissociation of acidic groups on the solute or the protonation of basic functions on the solute.

• Capillary Gel Electrophoresis (CGE) is the adaptation of traditional gel electrophoresis into the capillary using polymers in solution to create a molecular sieve also known as replaceable physical gel. This allows analytes having similar charge-to-mass ratios to be resolved by size. This technique is commonly employed in SDS-Gel molecular weight analysis of proteins and the sizing of applications of DNA sequencing and genotyping.

• Capillary Isoelectric Focusing (CIEF) allows amphoteric molecules, such as proteins, to be separated by electrophoresis in a pH gradient generated between the cathode and anode. A solute will migrate to a point where its net charge is zero. At the solutes isoelectric point (pI), migration stops and the sample is focused into a tight zone. In CIEF, once a solute has focused at its pI, the zone is mobilized past the detector by either pressure or chemical means. This technique is commonly employed in protein characterization as a mechanism to determine a protein's isoelectric point.

• Isotachophoresis (ITP) is a focusing technique based on the migration of the sample components between leading and terminating electrolytes. Solutes having mobilities intermediate to those of the leading and terminating electrolytes stack into sharp, focused zones. Although it is used as a mode of separation, transient ITP has been used primarily as a sample concentration technique.

• Electrokinetic Chromatography (EKC) is a family of electrophoresis techniques named after electrokinetic phenomena, which include electroosmosis, electrophoresis and chromatography. A key example of this is seen with cyclodextrin-mediated EKC. Here the differential interaction of enantiomers with the cyclodextrins allows for the separation of chiral compounds. This approach to enantiomer analysis has made significant impact on the pharmaceutical industry's approach to assessing drugs containing enantiomers.

• Micellar Electrokinetic Capillary Chromatography (MECC OR MEKC) is a mode of electrokinetic chromatography in which surfactants are added to the buffer solution at concentrations that form micelles. The separation principle of MEKC is based on a differential partition between the micelle and the solvent. This principle can be employed with charged or neutral solutes and may involve stationary or mobile micelles. MEKC has great utility in separating mixtures that contain both ionic and neutral species, and has become valuable in the separation of very hydrophobic pharmaceuticals from their very polar metabolites.

• Micro Emulsion Electrokinetic Chromatography (MEEKC) is a CE technique in which solutes partition with moving oil droplets in buffer. The microemulsion droplets are usually formed by sonicating immicible heptane or octane with water. SDS is added at relatively high concentrations to stabilize the emulsion. This allows the separation of both aqueous and water-insoluble compounds, and is used effectively by the pharmaceutical industry as generic methodology to analyze a broad spectrum of pharmaceuticals.

• Non-Aqueous Capillary Electrophoresis (NACE) involves the separation of analytes in a medium composed of organic solvents. The viscosity and dielectric constants of organic solvents affect both sample ion mobility and the level of electroosmotic flow. The use of non-aqueous medium allows additional selectivity options in methods development and is also valuable for the separation of water-insoluble compounds.

• Capillary Electrochromatography (CEC) is a hybrid separation method that couples the high separation efficiency of CZE with HPLC and uses an electric field rather than hydraulic pressure to propel the mobile phase through a packed bed. Because there is minimal backpressure, it is possible to use small-diameter packings and achieve very high efficiencies. Its most useful application appears to be in the form of on-line analyte concentration that can be used to concentrate a given sample prior to separation by CZE.

Valuable applications of CE include:

• Genetic analysis
• Analysis of pharmaceuticals (containing nitrogenous bases)
• Pharmaceuticals with chiral centers (enantiomers)
• Counter-ion analysis in drug discovery
• Therapeutic Protein Characterization
• Protein characterization
• Carbohydrate analysis for the determination of post translational modifications

Genetic Analysis

Since the first publication of DNA's double helical structure by Watson and Crick in 1953, electrophoresis has been a standard among the analytical tools used in modern biochemistry. CE's automation and quantitation capabilities made it a natural successor to replace the slab-gel format for genetic analysis. By introducing replaceable physical gels (polymers in solution) into a capillary, a molecular sieve is created that readily resolves molecules of DNA and RNA by size. The automation capability of this format has enabled significant advances in genetic analysis, accelerating the discovery of new genomic information.

The Beckman Coulter GenomeLab™ GeXP Genetic Analysis System is a fully automated genetic analysis system that employs an array of coated capillaries, novel infrared dyes, an optimized linear polyacrylamide gel (LPA) and comprehensive informatics to fully automate the processes of DNA sequencing, gene expression and fragment analysis. Plate bar coding and linkage to Beckman Coulter's Biomek® liquid handlers provide efficient automation and sample tracking generating highly reproducible results. CE's simplicity of operation has moved the formerly complex genetic analysis tasks from the hands of a specialized few into the hands of many, enabling scientists to focus on the biology of interest rather than the technology.

CE technology is now in routine use for the purity analysis of oligonucleotides and siRNAs. If not pure, synthesized oligonucleotides can cause problems in hybridization reactions, so good quality assurance can save significant time and money. This rigorous characterization is particularly essential in the development of nucleic acid-based therapeutics. Beckman Coulter offers this assay within the automated P/ACE™ MDQ platform using the ssDNA 100 R chemistry. This system provides n-1 resolution of oligonucleotides with a high level of quantitative precision and accuracy.

Pools of messenger RNA (mRNA) are widely used for the creation of cDNA libraries, the development of expressed sequence tag (EST) databases and for gene expression profiling. Gene expression arrays and real-time sequence detection systems have improved the means by which gene expression studies can be carried out. However, the quality of the mRNA is an important consideration because these molecules are highly susceptible to degradation by naturally occurring RNAses. CGE separation of RNA molecules can be performed rapidly and in an automated manner, allowing the user to quickly assess the quality of the RNA. The P/ACE™ MDQ with laser-induced fluorescence detection is ideal for rapid, unattended profiling of up to 96 RNA samples per session with cycle times of less than five minutes per sample.

Pharmaceutical Analysis (Bases)

The highly polar nature of pharmaceuticals containing basic amine functional groups makes the use of chromatography quite complex. Ion pairing reagents and stringent column regeneration is often necessary to reduce nonspecific ionic interactions that occur with reverse-phase chromatography. With CE, these highly functional amines are favored and may be exploited to provide extraordinary resolution. The most common and simplified format of operation is to use bare-fused silica capillaries at a well defined acidic pH. Under these conditions the capillary surface is essentially non-reactive while an analyte's amine functional groups are maximally ionized, rendering a simplified robust assay for the analysis of basic drugs. The P/ACE™ MDQ, coupled with photo-diode array detection, is being used for this application, and is being applied effectively in the pharmaceutical industry for the analysis of basic drugs, pharmokinetic profiling, bioavailability determinations, plasma protein binding studies and drug activity level determination.

One of the challenges of drug discovery is in developing analytical methods for the pharmacokinetic (PK) profiling of new drug candidates. Important to this process is the development of rapid, generic methods that allow the screening of large numbers of compounds isolated from complex sample matrices. CE is being used routinely to quantify drugs in blood plasma, as well as in brain, kidney and heart tissue. The high efficiency and the lack of interference from the matrix make CE a fast and easy analytical tool for PK screening. A derivative of this application is also being used for screening drugs of toxicological interest, enabled by combining an analytes spectral signature with its electrophoretic mobility to provide highly reproducible identification.

Enantiomer Analysis (pharmaceuticals with chiral centers)

Pharmaceuticals with asymmetric carbons that exist as enantiomers provide a significant challenge. As these stereoisomers are physically and chemically identical, one must construct chiral environments to facilitate their separation. One of the attributes of an in-solution technique such as CE is the ease with which one can define experimental conditions. The capillary provides an ideal format for creating a chiral environment, as chiral reagents in solution are easily introduced by the simple application of pressure. The P/ACE™ MDQ methods development system is the primary system for this work. The most effective chiral reagents have been the highly sulfated cyclodextrins. Using this approach, the separation of racemic mixtures yielding resolution values of five or greater is common, and the best separations produce values greater than 20. The consequence of such good resolution is the ability to detect enantiomeric impurities rapidly at levels well below 0.1%. This approach is being used in a large number of pharmaceutical companies and is rapidly becoming the primary methodology for developing assay methods on new enantiomer containing drugs.

Ion analysis

By nature, ions are highly charged polar species that lend themselves well to the CE format. The most routine ion analysis uses bare fused-silica capillaries with simple buffer systems that carry a cationic surfactant to reverse and modulate electroosmotic flow. The primary mode of detection for this work has been indirect UV absorption, in which a UV-absorbing ion is added as a background electrolyte. The displacement of this background ion by the ion being analyzed provides the basis for detection. In the discovery phase of pharmaceutical development, concentration-dependent biological assays are used to test the efficacy of compounds to fight disease. One of the most important pieces of data when performing these assays is the correct assignment of molecular mass to the compound to be tested. The validity of the results from these assays cannot be certain without it, and the formula weight of most drugs can not be accurately determined without first quantifying the drug counterion. The P/ACE™ MDQ methods development system has proven ideal for this work.

Therapeutic Protein Characterization

Protein-based drugs have widespread roles in prevention and treatment of disease. Among these, monoclonal antibodies, vaccines, and recombinant proteins have been shown to be effective in combating various cancers and immune diseases, and make up a growing cross-section of drugs gaining approval for the treatment of a variety of these clinically significant diseases. Careful characterization of the molecules in question is critical to ensure safety and efficacy. CE technology plays a key role in the characterization process as it lends its strengths to the determination of protein purity, heterogeneity, and identity prior to their use in the clinic.

Protein Characterization

CE technology has been valuable for the comprehensive characterization of macromolecules used both as biologics and in proteomic study. The analysis of proteins at a proteome level involves not only the characterization and quantification of proteins, but also the study of interactions between proteins with other proteins, nucleic acids and small molecules.

• SDS-Gel Molecular Weight Analysis. CE has become an effective replacement for manual slab gel electrophoresis due to its automation, quantitation, speed and high efficiency. Many biomolecules, such as proteins, carbohydrates and nucleic acids are separated by molecular sieving electrophoresis using gel matrices, a technique referred to as capillary gel electrophoresis (CGE). For polypeptides and proteins, it is necessary to denature the sample in the presence of SDS, an anionic detergent that binds the proteins in a constant ratio of 1:1.4 of protein. The constant mass-to-charge property of the SDS-bound proteins allows separation according to differences in protein molecular size.
• Isoelectric Focusing Analysis. Isoelectric focusing (cIEF) has been widely used for the separation of proteins based on differences in their isoelectric points. The cIEF method is accomplished by electrophoresis of proteins or peptides through a stable pH gradient until they reach the pH equal to their isoelectric point (pI), at which time the net charge and mobility are zero. The pI of an unknown protein can be interpolated from a pI calibration plot generated from a series of protein standards with known isoelectric points. Therefore, in addition to providing separation with high resolution, this method can be used for wide-range (pI 3-10) screening and pI identification of proteins and peptides.
• Carbohydrate Analysis. Carbohydrate analysis is essential to fully characterize a glycoprotein yet has long been considered a challenging and formidable task. CE has been effectively employed to separate and quantify oligosaccharides released from glycoproteins. As most carbohydrates lack readily ionizable functional groups and the ability to either absorb or fluoresce, this procedure usually requires a reductive amination reaction step using reagents like 1-aminopyrene-3,6,8-trisulfonate (APTS) to provide specificity, ample charge and strong fluorescence to the oligosaccharides. APTS has been used successfully for the derivatization of both oligosaccharides and monosaccharides and coupled with CE-LIF, provides high sensitivity quantitative information with increased resolving power.
• Peptide Mapping. The electrophoretic separation of peptides generated by enzymatic digestion of proteins is a commonly used technique for protein identification. Different proteins will generate different peptides after digestion with proteolytic enzymes and separation of these peptides using electrophoresis produces a characteristic "map" or "fingerprint" of that protein. The proteolytic enzyme trypsin is commonly used for this purpose. CE is an ideal tool for studying peptide maps of proteins owing to the speed, resolution and reproducibility of separations that can be achieved. This technology can be used in a standalone format with either UV or fluorescence detection or can be interfaced with mass spectrometry (MS) for more comprehensive protein identification.

The PA 800 plus Pharmaceutical Analysis System has been developed to manage the comprehensive range of protein characterization processes described above, including molecular weight determination, peptide mapping, isoelectric focusing, carbohydrate profiling and mechanisms to study protein/protein interactions. Based on Beckman Coulter's well-established automation and capillary electrophoresis technologies, the PA 800 plus addresses important issues in the characterization of a given proteome -- managing the analysis of low levels of proteins that range from acidic to basic to membrane-bound.

Beckman Coulter currently supplies three major analytical platforms which have a foundation of CE technology:

• PA 800 plus Pharmaceutical Analysis System
• P/ACE™ MDQ Series Capillary Electrophoresis Systems
• GenomeLab™ GeXP Genetic Analysis System

Capillary electrophoresis (CE) is one of the most powerful separation techniques available today because it utilizes both electric field and chemical equilibrium as driving forces to separate analytes.  Their migration behavior is determined by the differences in their charge-to-size ratio, and their affinity to the additives present in the background electrolyte (BGE).

Electrophoretic Mobility of an Analyte in CE
An analyte molecule migrates in a CE column with an electrophoretic mobility determined by its charge to size ratio. When it forms a complex with an additive molecule (or micelles and nano particles), it acquires a new mobility. The net mobility of this analyte molecule can be anywhere between the free analyte mobility and the complex mobility, depending on the amount of additive molecules present in the BGE. Analyte-additive binding (equilibrium) constant is also an important factor because it determines the portion of the analyte that will be in the complexed form in a certain additive concentration.

If the electric field is taken away from a CE setup, the analyte molecules will no longer migrate by itself, and will have to be pushed through the column by pressure. If the additive is immobilized onto the column, this system becomes a liquid chromatography system. On the other hand, if the additive is taken away, the setup becomes a free zone electrophoresis system, and the separation process is driven only by electric field.  This separation mechanism is essentially the same as other field driven separation techniques such as ultracentrifugation. If the column is filled with a gel, the setup becomes similar to gel electrophoresis apparatus and is capable to perform separations of large DNA and protein molecules.

In summary, CE is a central technique for chemical separation. Once a CE method is developed, it also provides guidance for developing complementary separation methods using either chromatography or electrophoresis techniques.

The following table shows the fundamental equations used in CE and other separation techniques.

Equations for Analyte Migration and Resolution in Various Separation Techniques

	Dynamic Complexation CE	Chromatography	Carrier-Free CE	Ultracentrifugation
Equilibrium			-	-
Equilibrium Constant			-	-
Capacity Factor			-	-
Fraction of Free Analyte			-	-
Field	Electric Field	-	Electric Field	Centrifugal Field
Response to Field	Electrophoretic Mobility	-	Electrophoretic Mobility	Sedimentation Coefficient
Analyte migration with one interacting additive present
Analyte Migration in more complex systems	The electrophoretic mobility of the analyte is equal to the sum of the products of the fraction of each species multiplied its mobility.	The velocity of the analyte is equal to the fraction of the free analyte multiplied by the velocity of the mobile phase.	The velocity of the analyte is determined by the field strength and its charge to size ratio.	The velocity of the analyte is determined by the speed of rotation and its sedimentation coefficient.
Separation Factor
Resolution Equation

Michael T. Bowser, Gwendolyn M. Bebault, Xuejun Peng, David D. Y. Chen: Redefining the separation factor: A potential pathway to a unified separation science. Electrophoresis 18, 2928 - 2934 (1997). Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.