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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 TechnologyCapillary 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:
Putting CE to Work--Applying the Technology to Real World ApplicationsAlthough 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 ions when rapid results are desired, and has become the predominant technique for the analysis of both basic and chiral pharmaceuticals. This technology is making its mark in biotechnology, replacing traditional electrophoresis for the characterization and analysis of macromolecules such as proteins and carbohydrates, and promises to be a valuable tool in tackling the characterization challenges posed by proteome-wide analysis. CE technology has also served to accelerate the accumulation of genome-level knowledge by automating DNA sequencing and genotyping. The "in solution" approach, which is a key feature of this technique, is also ideal for creating environments in which molecular interactions may be detected and studied. Valuable applications of CE include:
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 CEQ 8000 and 8800 are fully automated genetic analysis systems that employ 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 and genotyping. 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 platform with photo-diode array detection 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 platform configured with a UV detector has proven ideal for this work. 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.
The ProteomeLab PA 800 Protein Characterization platform 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 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 four major analytical platforms which have a foundation of CE technology:
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