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Capillary electrophoresis

Capillary Electrophoresis (CE) is a separation technique that uses narrow bore fused-silica capillaries to separate a complex array of large and small molecules. High electrical 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. CE technology can be further segmented into several separation techniques.

Capillary Zone Electrophoresis (CZE), also know as free-solution CE (FSCE), is the simplest form of CE. The separation mechanism is based on difference 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 solution relies principally on the pH controlled dissociation of acidic group 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 ratio to be resolved by size. This technique is commonly employed in SDS-Gel molecular weight analytes 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 electroph-oresis 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 (pl), migration stops and the sample is focused into a tight zone. In CIEF, once a solute has focused at its pl, the zone is mobilised past the detector by either pressure or chemical means. Micellar Electrokinetic Capillary Chromatography (MECC OR MEKC) is a mode of electro kinetic 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 micelle. 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.

Non-Aqueous Capillary Electropho-resis (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 electro osmotic flow. The use of non-aqueous medium allows additional selectivity options in methods developm-ent and is also valuable for the separation of water-insoluble compounds.

Although CE technology may be applied to many different types of research, it has gained its reputation from the study of molecules that have traditionally been difficult to separate. In general, CE should be considered first when dealing with highly polar, charged analytes. CE excels in the analysis of ion when rapid results are desired, and has become the predominant technique for the analysis of both basic and chiral pharmaceuticals. This technology is making mark in biotechnology, replacing traditional electrophoresis for the charac-terisation and analysis of macromolecules such as proteins and carbohydrates, and promises to be valuable tool in tackling the characterisation challenges posed by proteome-wide analysis.

Valuable applications—

Pharmaceutical analysis

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 non-specific ionic interactions that occur with reverse-phase chromatography. With CE, these highly functional amines are favoured and may be exploited to provide extraordinary resolution.

Enantiomer analysis

Pharmaceuticals with asymmetric carbons that exist as enantiomers provide a significant challenge. As these stereo-isomers are physically and chemically identical, one must construct chiral environments to facilitate separation. One of the attribute 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 introduced by the simple application of pressure.

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 electro osmotic flow. The primary mode of detection for this work is indirect UV absorption, in which a UV-absorbing ion is added as a background electrolyte.

Gentic analysis

CE technology is now in routine use for the purity analysis of oligonucleotides and siRNAs. If not pure, synthesised oligonucleotides can cause problems in hydridisation reactions, so good quality assurance can save significant time and money. This rigorous characterisation is particularly essential in the development of nucleic acid-based therapeutics.

Protein characterisation

CE technology has been valuable for the comprehensive characterisation of macromolecules, used both as biologics and in proteomic study.

• SDS-Gel molecular weight analysis: CE has become an effective replacement for manual slab gel electrophoresis due to its automation, quantification, 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)
• Isoelectric focusing analysis: Isoelectric focusing (IEF) has been widely used for the separation of proteins based on differences in their isoelectric points. The IEF method is accomplished by electrophoresis of proteins or peptides through a stable pH gradient until they reach the pH equal to their isoelectric point (pl), at which time the net charge and mobility are zero. The pl of an unknown isoelectric protein can be interpolated from a pl 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 (pl 3-10) screening and pl identification of proteins and peptides.
• Carbohydrate analysis: Carbohydrate analysis is essential to fully characterise 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 ionisable 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 oligosaccha-rides. APTS has been used successfully for the derivatisation 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 reproducib-ility 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.

(Contributed by Beckman Coulter)