Agilent's CE-MS System
CE refers to a family of related analytical techniques that use very strong electric fields to separate molecules within narrow-bore capillaries (typically 20-100 µm internal diameter). The CE family includes techniques exclusive to the capillary format as well as capillary analogues of other methods such as slab gel electrophoresis, isoelectric focusing, and liquid chromatography.1-3 Separations exploiting differences in analyte size, charge, hydrophobicity, ligand-binding properties, and even chirality can be implemented. CE techniques are employed in seemingly limitless applications in both industry and academia. Gel- and polymer network-based CE has revolutionized studies of nucleic acids; applications include DNA sequencing, nucleotide quantification, oligonucleotide purity evaluation, and mutation/polymorphism analysis. "CE has had an enormous impact on genomics, and more CE instruments are probably sold for DNA sequencing than for any other single use," comments Fred Regnier, professor in the department of chemistry at Purdue University. Other applications include forensic, clinical, and environmental analyses; quality control procedures; drug discovery; binding studies; and characterizations of proteins, glycoproteins, and carbohydrates.
Tool TimeDuring the past few years, a number of automated CE systems have become commercially available. Some of these are general CE systems for a wide variety of CE applications. Others are specifically designed to perform certain applications and analyze the resultant data. For example, PE Biosystems of Foster City, Calif., offers two CE-based sequencers, the ABI Prism® 3700 DNA Analyzer and the ABI Prism 310 Analyzer. "The ABI Prism DNA analyzers enable high-sensitivity, four-color fluorescence detection and are designed specifically for DNA applications such as sequencing and fragment analysis," says Joe Olechno, senior director of marketing services and planning at PE Biosystems. "The ABI Prism 3700 System is the only production-scale DNA analysis system with walk-away automation enabling 24-hour unattended operation." Beckman Coulter offers several P/ACETM CE systems, each optimized for a certain set of applications. Systems that separate, quantify, and characterize nucleic acids, DNA/protein interactions, glycoproteins, carbohydrates, or chiral enantiomers are available. The Capillary Ion Analysis (CIA®) system from Waters of Milford, Mass., is optimized for studying inorganic molecules. "The CIA system may be used for environmental and public health-related applications, such as analyses of drinking water, wastewater, and food. It is also employed for a number of applications in the chemicals industries," explains Eric Fotheringham, product marketing manager at Waters. Kits for optimizing specific applications are also on the market. The different commercially available capillary types sport a mind-boggling array of coatings. Coatings vary with regard to intrinsic characteristics such as charge and hydrophobicity, and some are optimized for specific applications.
The most common means of detection is through UV light absorbance, and this remains popular, especially for measuring relatively large quantities of proteins and peptides. Instruments with fixed- or variable-wavelength detectors are available, as are those that include scanning UV detectors or photodiode arrays. Species that don't absorb UV light well can be detected indirectly using UV-absorbing buffers or conductivity detectors. Increasing numbers of applications require more sensitive methods, such as laser-induced fluorescence (LIF) or mass spectrometry (MS). LIF offers highly sensitive fluorescent-species detection. It is useful for nucleic acid detection, DNA sequencing, carbohydrate analysis, enzyme assays, and affinity techniques such as CE-based immunoassays. CE-LIF can be used with other techniques for very specific applications. For example, a recent report describes the localization of ligand-binding domains of G protein-coupled receptors using photoaffinity labeling with fluorescent probes, receptor proteolysis, and CE-LIF.4 Most commercial LIF instruments use argon-ion lasers that bombard samples with light at 488 nm. Devices with other laser types, including helium-neon lasers (594 nm) and diode lasers with higher-wavelength outputs, are also commercially available. Instruments employing two different lasers ("dual-wavelength LIF") provide extra flexibility and are available from Bio-Rad Laboratories of Hercules, Calif., and Beckman Coulter.
Beckman Coulter's 32 Karat™ Software for compound
CE PrimerA basic CE system consists of a fused-silica capillary that connects two buffer reservoirs, two electrodes, a controllable high-voltage power supply, and a detector. Early CE studies utilized relatively simple "homemade" devices with this configuration. Today's commercially available instruments offer a number of advantages, including computerized control, temperature regulators, and interfaces for sophisticated detection devices.
In CE, the sample is injected, high voltage is applied, and sample components move under the influence of the electrical field. In the simplest scenario, resolved sample components are detected as they pass sequentially through a beam of light near the end of the capillary. The graphical representation of CE data is known as an electropherogram, and the amount of material in each peak can be calculated. An industry expert, Harry Whatley, has created a Web site with a wealth of relevant introductory information, including an animated illustration of the basic CE concept (www.neptune.net/~whatley/capelec.htm).
One of the advantages of CE is that it requires small amounts of sample. "Often nanoliter sample volumes comparable to a fog droplet are actually loaded onto the capillary in CE runs. However, most instruments require at least 1 µl of sample due to problems with manipulating such small volumes," explains Olechno. Even those who aren't particularly concerned with sample conservation might find it difficult to ignore the time efficiency and high-resolution capabilities of CE. Joule heating, the heat generated through the passage of electrical current, imposes upper limits on the voltages applicable for different electrophoretic techniques. The large surface-to-volume ratios of capillaries enable rapid heat dispersion. Thus, capillaries can tolerate voltages far higher than those used for conventional electrophoresis systems. This translates into significant savings in time and increased separation efficiencies for researchers.
Several factors give CE its high- resolution capabilities. Separation protocols of different selectivities can be optimized to resolve a wide range of analytes. Modifying selectivity is the easiest way to optimize CE separations. A number of factors enhance resolving power by increasing separation efficiency or "peak sharpness." These include higher voltages, fast run times, and electroosmotic "pluglike" flow instead of pressure-driven laminar flow.
SGE's UV transparent electrophoresis capillaries
On the MoveCE is an electrophoretic technique, and many modes of CE also employ electroosmotic flow (EOF). In electrophoresis, positively charged (cationic) species are propelled toward the negatively charged electrode (cathode). Negatively charged (anionic) analytes move to the positively charged electrode (anode), and uncharged species do not migrate. In contrast, EOF pumps cationic, anionic, and uncharged species toward the same electrode (usually the cathode).
Capillary zone electrophoresis (CZE) is the simplest form of CE. A subset of CZE uses EOF as a driving force. This enables simultaneous separation of positively charged, negatively charged, and neutral species. EOF results when an electrical field passes through a solution in a capillary that has fixed charges on its surfaces. The internal surface charge is its "zeta potential." These immobilized, charged moieties are under the influence of the electrical field, but are unable to migrate. The fixed, charged groups lining the capillary wall cannot exist alone. They become neutralized by interaction with charged species in the surrounding milieu. As described below, the result of these interactions is movement of fluid toward the electrode with the same charge as the capillary wall. The most commonly used capillaries are composed of fused silica. In CE methods employing noncoated ("bare") fused silica capillaries when the walls are negatively charged, this pumping action is toward the cathode. Fused silica capillaries modified with coatings of certain types (for example, cationic surfactants) can be used in applications that call for EOF toward the anode. Methods employing anode-directed electroosmosis are often referred to as "EOF-reversal" techniques.
In a conventional tube, EOF is not a significant effect; the small size and cylindrical geometry of a capillary create an environment in which EOF can provide a significant pumping action.6 How does this pumping action work? Fused silica capillaries have high zeta potentials because they bear ionizable, acidic silanol groups. The degree of silanol ionization is primarily controlled by the pH of the buffer. At alkaline pH, silanol groups are fully ionized, and EOF is robust. The fixed negative charges attract cations from the buffer, creating a positively charged, electrical "double layer." When voltage is applied across the tube, cations in the upper portion of this double layer migrate in the direction of the cathode, mobilizing the buffer fluid. At neutral to basic pH, EOF is the dominant driving process when employing negatively charged capillaries.
Under these conditions, all species are propelled toward the negative electrode, regardless of their charges. Positively charged species migrate fastest, followed by neutral and then negatively charged species. Positively charged analytes zip toward the cathode fastest because they are ushered in this direction by both EOF and their electrophoretic mobilities. Their velocity is greater than the EOF's. If two different analytes carry an identical positive charge, the component with the lowest mass will migrate fastest. Uncharged species are the middlemen. Unaffected by electrophoresis, they travel at the same velocity as EOF. If multiple neutral species are contained in a sample, these components will migrate together, with no resolution from each other. Negatively charged species are the laggards because they are held back by the opposing electrophoretic process. Their velocity is slower than the EOF's. If two analytes have an identical negative charge, the species with the greatest mass will be detected first because it is less resistant to EOF.
Buffer Power Different buffer conditions, used in conjunction with capillaries bearing different types of coatings, can be used to effect separations based on a wide range of molecular properties. Buffer additives that modify separation conditions include organic solvents, metallic salts, ion-pairing reagents, surfactants, or chiral selectors. Some modifiers act directly on the analytes under study. Charges on analytes are pH dependent, and separations can be dramatically affected by buffer pH. Borate salts can facilitate glycoprotein separations. Borate ions form complexes with the carbohydrate moieties of glycoproteins and can enable separation of glycovariants based on differential charge. Ligand-induced conformational changes can also alter the mobility times of proteins; this is exploited in affinity capillary electrophoresis (ACE). In nonaqueous capillary electrophoresis (NACE), separations occur in organic solvents, which affect both analyte mobility and EOF level.
In the Zone CZE is useful for separating a wide variety of molecules. The conditions required for resolution depend on the specific nature of a given application. CZE-based separations of small charged molecules and ions are often performed at neutral to high pH conditions in which EOF is significant. CZE users sometimes encounter difficulties due to the electrostatic binding of positively charged species to negatively charged capillary walls. When working with proteins, this can be especially prevalent. This problem can be overcome by operating at least two pH units above the pI of the protein. However, this more basic pH is not always optimal for separation or for the condition of the protein itself. Certain protein separations can be performed at acidic pH. Under these conditions, the capillary wall is uncharged. Proteins will be positively charged, and will not electrostatically interact with the wall. (But hydrophobic interaction may still occur.) EOF will be reduced or eliminated, and electrophoretic mobility will primarily drive the separation. An alternative is to use an appropriate coated capillary. Be aware, though, that different proteins can bind to the walls of certain coated capillaries as well, depending on the properties of both the given protein and a particular coating. Another approach is to utilize gel-, polymer network- or chromatography support-based CE techniques. "CE techniques are very useful in protein applications. However, establishing conditions for the successful analysis of an arbitrary range of proteins is sometimes technically difficult and time consuming," cautions George Whitesides, professor in the department of chemistry and chemical biology at Harvard University.
MECC & Friends Micellar electrokinetic capillary chromatography (MECC or MEKC) offers separations that resemble reverse-phase liquid chromatography with the benefits of CE. Techniques similar in concept to MECC can be used for unique applications, such as separation of chiral forms of compounds. Like one subset of CZE, MECC puts EOF to use. However, MECC differs in that micelle-forming surfactants are added to the running buffer. This misleadingly simple modification makes MECC a fundamentally different type of CE mode with "chromatography-like" aspects. Unlike its micelle-free cousin, MECC can not only separate charged species, but also resolve neutral analytes based on hydrophobic interactions.
Surfactants have long hydrophobic tails and hydrophilic head groups. Above a certain concentration known as the critical micelle concentration (CMC), surfactants spontaneously aggregate to form roughly spherical (or ellipsoidal) micelles. Hydrophobic tails generally make up the interiors of micelles that form in aqueous solution, and the hydrophilic heads point outward. Micelles can bind analytes through electrostatic interactions mediated by the exposed head groups. These aggregates can also bind uncharged analytes by hydrophobic core-mediated interactions; this provides the basis for separating neutral sample components by MECC.
Sodium dodecyl sulfate (SDS), an anionic detergent, is the most widely used surfactant in MECC. EOF pulls SDS micelles toward the cathode, but electrophoresis tugs these negatively charged aggregates in the opposite direction. The overall result is that SDS micelles move toward the cathode, but at a reduced velocity compared to the bulk flow of the buffer. Analytes can partition between the slower-moving micelles and the faster-moving, surrounding buffer. The stronger the interaction, the longer a given analyte interacts with the micelle, and the longer its migration time. SDS is by far the most commonly used micelle-forming surfactant, but alternative anionic surfactants, cationic surfactants, nonionic surfactants, and bile salts are also used. The selectivity of MECC can be controlled not only by the choice of surfactant, but also by adding modifiers (for example, organic solvents) to the buffer.
MECC got its name from its use of micelles. However, certain other types of molecules that do not form micelles can be used in separations that involve the same basic principles governing MECC. For example, CD-MECC uses cyclodextrins in lieu of micelles. Cyclodextrins are barrel-shaped macrocyclic oligosaccharides with highly hydrophobic, optically active cores. CD-MECC is primarily used to separate chiral enantiomers. Various cyclodextrins and cyclodextrin derivatives are used for different separation selectivities.
ACE in the Hole In recent years, many papers have reported that CE techniques are helpful when studying noncovalent interactions of macromolecules with ligands. Known as affinity capillary electrophoresis (ACE), these techniques are based on mobility changes that occur when macromolecules bind their ligands.7,8 Examples include antigen/antibody, lectin/sugar, drug/protein, and enzyme/substrate complexes. The equilibrium constant of complex formation can be determined by measuring ligand/macromolecule migration time as a function of ligand concentration. Unlike some of the more traditional affinity- determination methods, ACE does not require radiolabeling or secondary reagents for quantification. ACE can also help determine binding stoichiometries.9 Immunoassays coupling CE's rapid separation of antigen/antibody complexes from other assay components with LIF's high-sensitivity detection are becoming popular. CE and laser-induced fluorescence polarization (LIFP) have recently been combined for the online detection of affinity interactions.10 Comparing ligand binding to differently charged derivatives of proteins ("protein charge ladders") allows assessment of electrostatic contributions to ligand/protein interactions.11
Focusing on CIEF and ITP For many researchers, the mention of isoelectric focusing conjures images of slab gels. However, isoelectric focusing can also be performed in capillaries. Capillary isoelectric focusing (CIEF) is generally used for high-resolution separations of proteins and polypeptides, as well as for pI determinations. In CIEF, the sample and carrier ampholytes are premixed, then injected into a capillary. Basic and acidic buffers occupy the reservoirs at the cathode and anode, respectively. An electric field is applied across the capillary, and the ampholytes establish a pH gradient. Each analyte migrates until it encounters a region in which the pH is equivalent to its pI. At this point, the substance's net charge is zero, and its trek is over. The analytes are "focused"; they remain in a very narrow zone. These tight zones are established because diffusion of a species to a zone of different pH causes it to adopt a charge; the electric field promptly sends any such errant, "freshly charged" analyte back to its proper zone. EOF must be suppressed for effective CIEF. This is usually accomplished using hydrophilic coatings such as polyacrylamide. When focusing is complete in gel-based systems, one need only stain the gel to observe the resolved bands. However, before bands separated in capillaries can be monitored, they must first migrate past the detector. Thus, CIEF employs a final mobilization step. Mobilization is effected either by applying pressure at the inlet while maintaining the electric field, or by making a buffer change to alter the pH.
Like CIEF, isotachophoresis (ITP) requires the suppression of EOF and employs a heterogeneous buffer system. Analytes are sandwiched between leading and trailing electrolytes. The leading electrolyte has a higher mobility than do any of the sample components. In contrast, the mobility of the terminal electrolyte is lower than that of any of the sample components. Separation occurs in the gap between the leading and terminating electrolytes based on the individual mobilities of the analytes. Both anions and cations can be determined using this method, but not in the same run. ITP is unique in that it is characterized by the following two features: All bands move at the same velocity, and the bands are focused. ITP is very useful for concentrating solutions and is sometimes incorporated as a preliminary concentrating stage in CE separations.
"Filling" StationConventional slab gel electrophoresis is conducted in polyacrylamide or agarose, which also serve as molecular sieves to perform separations based on size. Capillary gel electrophoresis (CGE) and a conceptually similar technique that incorporates polymer networks are of wide utility. These techniques are primarily used for size-based separations of macromolecules including proteins, oligonucleotides, and DNA restriction fragments. Polyacrylamide, a chemically crosslinked gel, is relatively viscous and is polymerized within the capillaries. Relatively low-viscosity, noncrosslinked polymer networks are "replaceable"; they can be pumped in and out of capillaries at the end of each round of separation. This feature makes polymer networks very useful for large-scale applications. "The Human Genome Project initiative is now using automated CE-based DNA sequencing systems to sequence the human genome. These systems employ 96 capillaries together in an array, and replaceable polymer solutions are automatically blown out and replaced after each run, allowing unattended operation," explains Barry Karger, professor and director of the Barnett Institute at Northeastern University. Polymer network-based CE with LIF detection has also been used in the capillary equivalent of the electrophoretic mobility shift assay (EMSA) for DNA-protein binding.12 EOF is suppressed in these techniques; either directly by using high viscosity-polymer formulations, or by using coated capillaries in conjunction with low-viscosity matrices. Separation is based on the same principles involved in slab gel electrophoresis: electrophoretic mobility and molecular sieving. As is the case with slab-based methods, gel- and polymer network-based CE can be performed in the presence of denaturants such as SDS and urea.
Capillary electrochromatography (CEC) is a hybrid of CE and liquid chromatography (LC). CEC techniques are primarily used to resolve smaller molecules, and certain media can be used for chiral separations. CEC capillaries are packed with any of a number of different applicable stationery particles similar to those used in LC. When an electric field is applied, EOF carries the mobile phase through the packed column. Separation is achieved by both electrophoretic mobility and partitioning between the stationary and mobile phases. Thus, CEC can yield separation profiles different from either CE or HPLC. An informative Web site maintained by Glaxo Wellcome's Kevin Altria is a good online source of information about CEC and CE (www.ceandcec.com). "There are different schools of thought with regard to the future of CEC. Some think that technical difficulties associated with this method, such as capillary fragility, will cause it to fall into disuse. Others, especially in academia, believe that CEC will be used more and more as future innovations are implemented," comments Altria.
CE'ing Ahead The impact of CE-based DNA sequencing cannot be overstated. "CE has had a tremendous impact on genomic sequencing and single nucleotide polymorphism (SNP) discovery. It allows genome laboratories to rapidly and easily generate high-quality DNA sequence data in a highly automated, hands-off operation. The technology has helped accelerate genome sequencing and has played a key role in enabling the Human Genome Project to meet its accelerated goals," explains Lauren Linton, associate director of the Whitehead Institute Center for Genome Research in Cambridge, Mass. CE is also being employed in a wide range of other applications in both academia and industry. CE can be used as a complementary method to HPLC to analyze small molecules. In recent years, CE has been increasingly used to analyze carbohydrates and proteins. The pharmaceutical sector is using CE techniques for the quality control of antibody- and peptide-based products, chiral separations, and impurity determinations. CE is also being used to measure inorganic anions and metal ions in a wide variety of industries. Much recent effort has been directed toward developing microfluidic circuit technology, which enables faster, less expensive analyses. This lab-on-a-chip methodology is still in the early stages of commercialization, but will undoubtedly become widely used as more technological innovations are made. S
Deborah Wilkinson can be contacted at email@example.com.
1. K.D. Altria, "Overview of capillary electrophoresis and capillary electrochromatography," Journal of Chromatography A, 856:443-63, Sept. 24, 1999.
2. R. Weinberger, Practical Capillary Electrophoresis, 2d ed., San Diego, Academic Press, 2000.
3. M.G. Khaldedi (ed.), High Performance Capillary Electrophoresis: Theory, Techniques and Applications, New York, John Wiley & Sons, 1998.
4. M. Dong et al., "Structurally related peptide agonist, partial agonist, and antagonist occupy a similar binding pocket within the cholecystokinin receptor: Rapid analysis using fluorescent photoaffinity labeling probes and capillary electrophoresis," Journal of Biological Chemistry, 274:4778-85, Feb. 19, 1999.
5. B. Sinclair, "Small wonder," The Scientist 14:25, March 6, 2000.
6. H. Whatley, "Harry's CE Page," www. neptune.net/~whatley/capelec.htm, 2000.
7. N.H. Heegaard et al., "Affinity capillary electrophoresis: important application areas and some recent developments," Journal of Chromatography B, Biomedical Sciences and Applications, 715:29-54, 1998.
8. M.V. Novotny, "Capillary electrophoresis," Current Opinion in Biotechnology, 7:29-34, 1996.
9. Y.H. Chu et al., "Using capillary electrophoresis to determine binding stoichiometries of protein-ligand interactions," Biochemistry, 33: 10616-21, 1994.
10. Q.H. Wan, X.C. Le, "Fluorescence polarization studies of affinity interactions in capillary electrophoresis," Analytical Chemistry, 71:4183-9, Oct. 29, 1999.
11. I.J. Colton et al., "Affinity capillary electrophoresis: a physical-organic tool for studying interactions in biomolecular recognition," Electrophoresis, 19:367-82, 1998.
12. J. Xian et al., "DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method," Proceedings of the National Academy of Sciences, 93:86-90, 1996.