Capillary electrophoresis Barry L. Karger Northeastern University, Boston, Massachusetts, USA The past year has seen major advances in capillary electrophoresis in terms of broadening applicability. A variety of successful approaches to peptide/protein and DNA separation and analysis are now available, and techniques for saccharide analysis are developing rapidly. Capillary electrophoresis-mass spectrometry continues to demonstrate its potential as a tool for high-resolution structure analysis. Current Opinion in Biotechnology 1992, 3:59-64
introduction Capillary electrophoresis (CE), the instrumental approach to slab gel electrophoresis, is a method undergoing rapid development at the present time. Nine commercial systems are available, and several other companies are planning to introduce systems in the near future. At this point, the field is in transition from fundamental development to broad applicability. In this review of the literature of the past year (September 1990 to October 1991), I shall provide a taste of the significant applications and new directions of the technology. As befits electrophoretic methods in general, a major thrust has been towards biological applications, in particular the analysis of proteins and nucleic acids, although there have been promising new results for the study of polysaccharides. The potential of CE coupled on-line to mass spectrometry (MS) is being developed, and it is quite clear that this hybrid method will be of great importance in the future. Also, the high resolution and high speed capabilities of CE are being exploited. The success of these developments leads to the conclusion that CE will play an active role in the future in those areas where high throughput (e.g. clinical/diagnostics) and high speed (e.g. process monitoring) are required. Given the breadth of the field, I have, of necessity, had to be highly subjective in what to include. Nevertheless, it is hoped that the reader attains some sense of the potential of CE in analytical biotechnology.
Peptide/protein separation A central feature of the successful separation of peptide or proteins by open-tube CE is the selection of buffer conditions and/or wall coating to minimize adsorption of protein molecules to the fused silica surface of the capillary. The normally negatively charged surface would obviously adsorb positively charged species; in addition,
hydrophobic adsorption is possible because of the fused silica structure and the aqueous buffer conditions. Even a slight amount of adsorption could dramatically reduce column performance. A good review on this topic of work carried out prior to September 1990 can be found in a paper published in late 1990 [1]. For peptides, two strategies involving buffer conditions have generally been found to be useful. At low pH ( < 3), many (but not all) silanol groups will be protonated, and electrostatic adsorption will thus be minimized. At the same time, all peptides will be positively charged and move in the same direction. Alternatively, basic pH (,-,8-10) can also be used; under these conditions the peptides should have net negative charges and thus be repulsed from the negatively charged wall surface. In addition, small amounts of a base (e.g. morpholine) may be added to reduce adsorption further. An example of these strategies used in peptide mapping can be found in the paper by Wheat eta/. [2"] in which a single residue substitution can be determined using small pH changes in either acidic or basic buffers. Results of CE used in peptide mapping have been sufficiently reproducible [3], and it can thus be concluded that this approach can be used along with liquid chromatography for this application. Indeed, the two methods complement each other in providing analytical information, and both can be coupled to MS (see below). In a significant paper, Neflson and Rickard [4,'], using different buffer pHs, correlated the mobilities of individual peptides with the properties of the molecules. For 33 diverse peptides and 10 intact proteins, they found a good fit between q/M2/3 and mobility, where q is the calculated net charge on the molecule and M is the molecular weight. This relationship was previously found by Offord using paper electrophoresis [5]. Neilson and Rickard [4°°] also determined that the adjacent structural environment of a charged group on the pKA of that group and, ultimately, the pI of the species is important. Clearly, as noted above, small pH modifica-
Abbreviations CE---Capillary electrophoresis; E$! clectrospray ionmation; HPLC---high-performance liquid chromatography; IEF---isoelectric focusing; MS--mass spectrometry. (~) Current Biology Ltd ISSN 0958-1669
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60 Analytical biotechnology tions could thus play a central role in selectivity. Molecular shape is also important in the mobility of a species. While the M-2/3 scale of Rickard e t al. implies that the species have spherical shapes, other shapes are possible for biopolymers and these will influence mobility. Indeed, Compton [6"] recently showed that for complex protein species, mobility more generally foUows M - 1/3 than M -2/3. In another example, Grossman and Soane [7"] demonstrated the presence of an electric field orientation for rod shaped molecules. From these studies, it is clear that protein conformation will significantly affect electric field migration and peak shape. As an example, Rush et a t [8..] presented the electrophoresis of calcium-depleted a-lactalbumin as a function of column temperature. A rapid increase in migration rate was observed with temperature, and in the transition region a broadened electrophoresis band correlating with the simultaneous existence of two conformational states was seen. The importance of conformation in the electrophoretic behavior of calcium- and zincbinding proteins has also been shown [9"]. In addition to pH control, buffer additives can be used to minimize adsorption of proteins to the capillary wall. One approach involves the use of high salt concentrations to reduce electrostatic interactions, often in conjunction with high pH (-~ 10.5) [10,11°]. In order to minimize the Joule heat generated by this technique, narrow bore columns (25 ~tm) are typically employed, rather than the more usual 50-100 gtm tubing. Such methods using high ionic strength buffers are being explored for use in high speed analysis of human serum proteins in clinical chemistry. Zwitterionic salts are another buffer additive [12], used to minimize electrostatic adsorption. For both of these approaches, the bare silica tubing can be washed between runs, with a base for example, to regenerate a new capillary wall each time. Polyamine is another additive that has been used successfully to reverse the charge on the wall surface from negative to positive [13"]. This approach is useful for many basic proteins such as histones (pI> 11). In this case, the polyamine is not added in the buffer but rather in a separate loading step to yield an adsorbed coat on the capillary wall. Covalently attached coatings can also be used, and a good deal of work has been done to find suitable ones. Probably the best known coating to date is that developed by Hjerten [14], which is formed using a bifunctional reagent, followed by linear polymerization with acrylamide. This covalently attached linear hydrophilic polymer reduces electro-osmotic flow to a negligible value. A good example of the use of this coating for peptide separation can be found in the paper by Zhu et al. [15"]. Interestingly, the use of basic conditions and the addition of a zwitterionic surfactant helped reduce adsorption even further. More recently, Novotny and co-workers [ 1'6] have used a similar, albeit somewhat more stable, coating for protein separations. The approach of converting the capillary wall to a cationic surface has been used in a covalently attached coating by Towns and Regnier [17-o]. These workers adsorbed a polyamine to the wall surface, but also followed this by cross-linking with bis-epoxide for stabilization.
Their paper outlines the basic principles of wall coatings and the influence of such coatings on electro-osmotic flow, and the importance of film thickness on the final potential on the wall is discussed in depth. These authors also prepared a capillary in which a non-ionic detergent (Brij 35) was adsorbed to an n-alkyl (C18) modified fused silica surface [18.-]. The detergent was also added to the mobile phase to maintain a stable coating. While no net formal charge existed on the coated surface, the potential was still sufficient to cause electro-osmotic flow, such that both positively and negatively charged proteins could be separated in one run. One nice aspect of this approach is that any chemical degradation of the wall (e.g. oxidation of the Brij) can be eliminated by simply washing out the surfactant and adsorbing new material. As hydrophobic capillary columns are commercially available [19], this approach is generally applicable. Results from another covalent coating in the form of a polyether phase have recently been published [20-]. Excellent separations of proteins were again shown. Another approach for peptide/protein separation by open-tube CE is isoelectric focusing (IEF) [21]. As generally practiced, a non-ionic coated tube is used in which the electro-osmotic flow is reduced to a negligible value. Carrier ampholytes are mixed with the sample which is loaded into the capillary. An electric field is then applied, causing the buffer amphotytes and sample to focus, demonstrated by a decrease in the current to essentially zero. The sample can then be mobilized to pass by the detection window, either by pressure, or by addition of salt to the cathotyte or anolyte. In the latter case, a pH gradient is established in which the appropriate salt ion will exchange with H + or O H - in the running buffer. The resolving power of this approach has been studied with hemoglobin variants [22"]. This paper also discusses a number of practical details conceming CE-IEF. Another recent study has demonstrated the potential use of capillary IEF for the analysis of carbohydrate variants in glycoproteins [23]. Although further development of this method may be necessary, capillary IEF would at present appear to be a useful approach in the resolution of proteins. It can be seen that peptide/protein separations by CE are well advanced with many options available for diverse samples. We can anticipate further developments in the coming year, continuing the advance of rapid, high resolution separations.
DNA separation The most powerful technique for the separation of DNA molecules is slab gel electrophoresis. In free solution, to a good first approximation, all DNA molecules migrate with the same mobility as the addition of a base (or base pair) results in the self compensation of mass and charge. Separation in gels occurs through a type of sieving behavior in which molecules migrate as a function of length. It is therefore obvious to use a sieving medium in a capillary to separate DNA by CE.
Capillary electrophoresisKarger The first attempts to use polymer networks (i.e. gels or viscous polymer solutions) as the sieving medium in CE involved crosslinked polyacrylamide gel for the separation of single-stranded DNA molecules. Remarkable separations were achieved, well in excess of 107 plates per meter [24]. Such columns can be used for the assay of synthesized DNA as well as for DNA sequencing [25",26,27,28.]. In the latter case, by analogy with the automated fluorescently tagged slab gel technique, separation of DNA with single base differences is achievable, with identification of individual bases related to specific fluorescent dyes. In principle, CE is 25 times faster than an individual slab gel lane in a standard sequencer; for example, up to ,-, 300 sequences can be read in 1 hour or less. This greater speed of separation is a consequence of the higher fields used in CE (,-,300V/cm) compared with slab gels ( ,-, 10V/cm). The capillary format reduces the current and thus the power or heat generated. The slab format can, however, be accelerated by using thin gel films of 50-100 I~m (compared with 350 ~ or more in normal operation), with which high fields can be used and multiple lanes incorporated [29]. Although thin slab gel electrophoresis appears to be quite fast, parallel processing, using multiple columns, is also possible for CE. The question of which technique is finally preferred will undoubtedly depend on which format yields sequence data for the greatest number of bases per lane. Issues such as temperature control across the capillary or slab gel will thus be critical in the final decision [30]. Ultimately, CE or slab gel operation may be overtaken by even faster methods. Although the final methodology is not yet clear, it is quite likely that either ultra-thin slabs or capillaries will be used, at least as a first stage, for enhancing the rate of DNA sequencing. Polymer networks can also be used for the separation of double-stranded DNA molecules in which non-denaturing buffer conditions are employed. Linear or lightly cross-linked polyacrylamide can be used for high resolution separation of species up to 1000 bp or higher [31"']. Separations have also been observed by Schwartz e t al. [32o.], Strege and Lagu [33"] and earlier by Zhu e t al. [34] with either hydroxypropylcellulose or methylcellulose. The effects of temperature on separation [35] and especially the addition of an intercalating agent such as ethidium bromide [36"] have also been demonstrated by Guttman and Cooke. The intercalating agent alters the flexibility of the DNA and enhances separation, particularly when high fields are used. The use of non-crosslinked polymer networks for sieving was initiated on slab gels many years ago [37]. Recently, Grossman and Soane [38"'] have explained the separation behavior of this sieving in terms of entangled polymer solutions, whose structures provide mesh spacings necessary for sieving purposes. One advantage of using a polymer network is that only relatively low viscosity solutions are required. Such solutions are then replaceable by simply blowing out the matrix and reloading with fresh material. The high resolution separation of doublestranded DNA with such materials provides a means of rapidly analyzing potyrnerase chain reaction products and restriction-fragment length polymorphisms,
among other applications. Successful separations in the 20 000-30 000 bp range should ultimately improve mapping procedures. DNA analysis is widely applicable, and CE should become a standard tool in carrying it out.
Instrumentation considerations In general, UV detection, usIng miniaturized high-performance liquid chromatography (HPLC) cells, is the most widely applied procedure for detection in CE. Unfortunately, although mass detection with HPLC is quite low, sample concentrations for CE are generally in the area of 10-5 M, several orders of magnitude less than those used in HPLC. Researchers have therefore been investigating preconcentration injection methods including field amplification, in which the conductivity of sample matrix is lower than that of the running buffer [39"], isotachophoresis [40.](displacement electrophoresis) or pH manipulation [41.]. A second way of enhancing detection is to use more sensitive detectors such as laser-based detection systems [42]. Although a variety of approaches have been employed, most systems rely on laser-Induced fluorescence for which sample concentrations of 10-1°-10-11M are typical. Derivatizing agents can be used for ultratrace laser analysis, such as fluorescamine, fluoroisothiocTanate, dansyl, etc. Recently, Novotny and co-workers [43"'] have introduced 3-(4-carboxybenzoyi)-2-quinolinecarboxaldehyde to react with primary amines. This reagent appears to be useful in determining reducing sugars. This approach may prove potentially useful in carbohydrate mapping of glycoproteins. In addition, through the use of concentrated polyacwlamide gels, high resolution of the fluorescently tagged sugars, has been achieved for partially hydrolyzed Dextrin 15 [44..]. As mentioned earlier CE-MS is developing at a rapid pace. This hybrid approach to separation and analysis is quite logical, given the very low flow rates (nL/min) for electro-osmotically driven bulk flow or simple ion transport with no bulk flow. Indeed, make-up fluids are often mixed with the CE-separated components prior to entry into the mass spectrometer. One successful CE-MS technique uses fast atom bombardment in which a coaxial continuous flow interface is employed [45°]. In this study, no degradation in separation performance was observed in the coupling of CE with MS and low femtomole levels of peptides could be structurally determined. This would appear to be a generally useful tool. These examples show that the high resolution of CE in combination with the structural information gained from MS, CE-MS will be a widely applied tool in analytical biotechnology. Recently, there has been a great deal of interest in electrospray ionization (ESI). In this technique, ions are evaporated from microcharged liquid droplets at atmospheric pressure under a high field. Following focusing, the ions are brought into a quadruple mass spectrometer. Besides vaporization at atmospheric pressure, another special feature of ESI is the formation of multiple charge states for
61
62 Analyticalbiotechnology an individual species. Multiple peaks (m/z) are thus observed in the mass spectrum and, as a consequence of large charged states (often > + 20), the spectra for high molecular weight species can be observed on an ordinary 0-3000 m/z quadruple instrument. A good review on this can be found in a recent paper [46].
NIElsONRG, RICKARDEC: Method Optimization in Capillary Zone Electrophoretic Analysis of hGH Tryptic Digest Fragments. J C]yromatogr 1990, 516:99-114. This paper correlates the electrophoretic mobility of peptides (33) and intact proteins (10) with q/M2/3 where q = calculated charge and M ~ molecular weight. The correlation will aid in optimization strategies and in the elucidation of peptide/protein structure.
The coupling of CE to ESI has been accomplished especiaUyweU by Smith and co-workers [47"] and Henion and co-workers [48-]. Because of atmospheric ionization, the coupling is particularly straight forward as there is no pressure drop required across the capillary. Although successful and rapidly growing in use, several points are worth mentioning. First, some preconcentration of the sample by the methods discussed above often proves helpful for detection in the picomole range. Secondly, acidic solutions aid in protonation, and hence workers have generally foundthat using a coating with a positive charge (e.g. aminosilane) is of value [49].
5.
OFFORDRE" Nature 1966, 211:591-593.
6
COMPTONBJ: Electrophoretic Mobility Modeling of Proteins in Free z o n e Capillary Electrophoresis and its Application to Monoclonal Antibody Microheterogeneity Analysis. J Cbromatogr 1991, 559:357-366.
Conclusion In such a fast moving field it is unavoidable that much information has been omitted from this review. There is, for example, a good deal of research activity on the analysis of small molecules such as drugs using micellar electrokinetic chromatography. Chiral separations are also being accomplished by CE. In addition, methods for ion analysis that are competitive with ion chromatography are available. Thus, CE is on a very rapid development curve. We can anticipate that it will take its place beside HPLC in many laboratories in the years ahead.
4. ••
7. .
GROSSMANPD, SOANE DS: Orientation Effects on the Electrophoretic Mobility of Rod-shaped Molecules in Free Solution. Anal ~ 1991, 62:1592-1596. This paper shows that tobacco mosaic virus, a rod-shaped molecule, orients in the electric field in free solution CE. This orientation leads to an increase m electrophoretic mobility with field strength. (Similar orientation is found for DNA molecules in polymer networks, e.g. gels.) 8. •.
Rush RS, COHEN AS, KARGERBI; Influence of Column Ternperature on the Electrophoretic Behavior of Myoglobin and ~x-lactalbumin in High-performance Capillary Electrophoresis. Anal Chem 1991, 63:1346-1350. This paper studies the effect of temperature on the conformational change of proteins in capillary electrophoresis. In addition, the on-column reduction of myoglobin from the FE3+ state to the FE2 + state is demonstrated. KAJ1WARA H: Application of High-performance Capillary Electrophoresis to the Analysis of Conformation and Interaction of Metal-binding Proteins. J Ooromatogr 1991, 559:345-356. A study of the separation of Ca2+ - and Zn2+ -binding proteins by CE was undertaken. Electropherograms shifted depending on the presence or absence of the metal ions in the buffer. 9. •
10.
GREENJS, JORGENSONJ'~: MinimizIng Adsorption of Proteins on Fused Silica in Capillary Zone Electrophoresis by the Addition of Alkali Metal salts to the Buffers. J Chromatogr 1989, 478:63-70.
11.
CHENF-T: Rapid Protein Analysis by Capillary Electrophore-
Thi--s sis. J Chromatogr 1991, 559:445-453.
Acknowledgements The author gratefully acknowledges the Department of Energy and Beckman Instruments for their support of this work. Contribution Number 510 from the Barnett Institute.
paper describes a study that used a buffer system of 150 mM borate at pH 10.5 for the high speed (less than 200 s) separation of proteins on a 25 wn internal diameter column. Rapid serum protein separations are also shown. 12
BUSHEYi i , JORGENSONJ~,V. Capillary Electrophoresis of Proteins in Buffers Containing High Concentrations of Zwitterionic Salts. J Chronuttogr 1989, 480:301-310.
13. .
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •. of outstanding interest 1.
NOVOTNYMV, COBB KA, IAU J: Capillary Electrophoresis of Proteins, Peptides and Amino Acids. Electrophoresis 1990, 11:735-749.
2. •
WHEATTE, YOUNG PM, ASTEPHEN NE: Use of Capillary Electrophoresis for the Detection of Single-residue Substitutions in 'Peptide Mapping. J Liq Ooromatogr 1991, 14:987-996. This paper demonstrates the power of open tube CE in the separation of closely related peptides. 3.
NIELSENRG, RICKARDEC: In Analytical Biotechnology, Cap# lary Electrophoresis and Chromatography edited by Horvath C, Nikelly JG. Washington DC: ACS ACS Symposium Series, 1990, 434:36-49.
WIKTOROWICZJE, COmURN JC: Separation of Cationic Proteins via Charge Reversal in Capillary Electrophoresis. E/ectrophoresis 1990, 11:769-773. This study used polyamines adsorbed onto the fused silica for separation of basic proteins, including histones. 14.
HJERTENS: High-performance Electrophoresis: Elimination of Electroendosmosis and Solute Adsorption. J Ooromatogr 1985, 347:191-198.
15. •
ZHU M, RODRIGUEZ R, HANSEN D, WEHR T: Capillary Electrophoresis of Proteins Under Alkaline Conditions. J O0ro matogr 1990, 516:1123--1131. The use of a coated robe improves peak shape and reproducibility of protein migration in CE. In certain cases, buffer additives enhance performance even more. 16.
17. ••
COBBKA, DOtNIKV, NOVOTNYM: Electrophoretic Separations of Proteins in Capillaries with HydrolyticaUy Stable Surface Structures. Anal Chem 1990, 62:2478-2483.
TowNsJK, REGNIERFE. Polyethyleneimine-bonded Phases in the Separation of Proteins by Capillary Electrophoresis. J ~romatogr 1990, 516:69-78. This paper presents a positively charged coating that was stable over many runs, particularly of basic proteins. The coating could be used
Capillary
electrophoresis
Karger
over a wide pH range without substantial change in bulk electro-osmoUc flow. Rapid, high resolulaon of protons was possible with good recovery from the column.
Linear and lightly cross-linked polyacrylamide were used for the highspeed separation of double-stranded DNA.Analysisof polymerase chain reaction products was demonstrated.
18.
32. ..
TOWNSJK, REGNIERFE: Capillary Electrophoretic separations of Proteins Using Nonionic Surfactant Coatings. Anal Chem 1991, 63:1126-1132. A coating, consisting of a detergent Brij 35 adsorbed on a hydrophobic surface, is shown to work successfully In the resolution of proteins. Litde or no change in electro-osmotic flow occurs in the pH range of 4-11, of species both net positive and net negative charge can be separated in one run. ••
19.
DOUGHERTYAM, WOOH.EY CL, WIlaaXiS DL SWJdtE DF, COtE RO, SEPANIAKMJ: Stable Phase for Capillary Electrophoresis. J Liq t~romatogr 1991, 14:907-921.
20.
NASHABEHW, EL RASSIZ: Capillary Zone Electrophoresis of Proteins with Hydrophilic Fused-silica Capillaries. J Cbrc> matogr 1991, 559:367-383 High resolution separation of proteins is described, as well as the fmgerprimLrtg of crude protein mixtures using a fused silica capillary covalently coated with neutral polyether layer. .
21.
HJERTENS, L~o J'L YAO K: Theoretical and Experimental Study of High-performance Electrophoretic Mobilization of isoelectrically Focused Protein Zones. J C.bromatogr 1987, 387:127-138.
22.
ZHU M, RODRIGUEZR, WEHRT: Optimizing separation Parameters in Capillary Isoeletric Focusing. J Chromatogr 1991, 559:479-488. Improvements in capillary isoelectric focusing for detection of acidic and basic proteins, -/-globulins were focused successfully using a nonionic detergent to reduce precipitation. •
23.
24.
YIM KW: Fractionation of the Human Recombinant Tissue Plasminogen Activator (rtPA) Glycoforms by High-performance Capillary Zone Electrophoresis and Capillary isoelectric Focusing. J Clm'omatogr 1991, 559:401-410. COHENAS, NAJARIANDR, PAULUSA, GUTYMANA, SMITH JA, KARGER BL: Rapid Separation and Purification of Oligonucleotides by High-performance Capillary Gel Electrophoresis. Proc Natl Acad Sci U S A 1988, 85:9660-9663.
25.
LUCKEYJA, DROSSMANH, KOSTICHKAAJ, MEADDA, D'CUNHAJ, NORRISTB, SMITH LM: High Speed DNA Sequencing by Capillary Electrophoresis. Nucleic Acids Res 1990, 18:4417-4421. A prototype instrument was built for the simultaneous analysis of Sanger DNA-sequencing reaction products (DNA polymerase chain extension with dideoxynucleotide stops). Four different dyes were attached to a common primer for the recognition of individual bases •
26.
COHENAS, NAJARIANDR, KARGERBL Separation and Analysis of DNA Sequence Reaction Products by Capillary Gel Electrophoresis. J Chromatogr 1990, 516:49-60.
27.
CHENDY, SWERDLOWHP, HARKEHR, ZHANGJZ, DOVICHI NJ: Low-cost, High-seusitivity Laser-induced Fluorescence Detection for DNA Sequencing by Capillary Gel Electrophoresis. J ~ t o g r 1991, 559:237-246.
28. s
KARGERAE, HARRISJM, GESTELANDRF: Multiwavelength Fluorescence Detection for DNA Sequencing Using Capillary Electrophoresis. Nucleic Acids Res 1991, 19:4955--4962. A charge-coupled device was used with capillary gel electrophoresis to determine fluorescently labelled DNA sequencing products. 29.
SMITHLM: High-speed DNA Sequencing by Capillary Gel Electrophoresis. Nature 1991, 349:812-813.
30.
NISHIKAWA T, KAMBARAH: Analysis of Limiting Factors of DNA BAnd Separation by a DNA Sequencer Using Fluorescence Detection. Electrophoresis 1991, 12:623-631.
31. ..
HEIGERDN, COHEN AS, KARGERBL Separation of DNA Restriction Fragments by High Performance Capillary Electrophoresis with Low and Zero Crosslinked Polyacrylamide Using Continuous and Pulsed Electric Fields. J Chromatogr 1990, 516:33-48.
SCHWARTZHE, ULFELDERK. Analysis of DNA Restriction Fragments and Polymerase Chain Reaction Products Towards Detection of the AIDS (HIV-1) Virus in Blood. J Oyromatogr 1991, 559:267-283. HydroxypropytceUulose was used as a sieving medium for DNA products. Polymerase chain reaction-derived HIV-1 sequences were analyzed by this method. 33. .
STREGEM, LAGUA: Separation of DNA REstriction Fragments by Capillary Electrophoresis Using Coated Fused Silica Capillaries. Anal Cbem 1991, 63:1233-1236. Methylcellulose was used to separate double-stranded-DNA fragments according to base size. 34.
ZHU M, HANSENDL, BURDS, GANNONF: Factors Affecting Free Zone Electrophoresis and Isoelectric Focusing in Capillary Electrophoresis. J Cbromatogr 1989, 480:311-319.
35.
GUTrMANA, COOKE N: Effect of Temperature on the Separation of DNA Restriction Fragments in Capillary Gel Electrophoresis. J 03romatogr 1991, 559:285--294.
36. GUTrMANA, COOKE N: Capillary Gel AfFinity Electrophoresis • of DNA Fragments. Anal ~ 1991, 63:2038-2042. Incorporation of an Intercalating agent for double-stranded DNA prorides enhanced selectivity in CE with polyacrytamide gel columns. 37.
BODEHJ: Partitioning and Electrophoresis in Flexible Polymer Networks. Electrophoresis 1980, 79:39-52.
38. ..
GROSSMAN PD, SOANE US: Capillary Electrophoresis of DNA in Entangled Polymer Solutions. J Cbromatogr 1991, 559:257-266. The separation of DNA restriction fragments is modeled In terms of sieving through entangled polymer solutions. 39. •
CHIENR-L, BURGI US: Field Amplified Sample injection in High-performance Capillary Electrophoresis. J Cbromatogr 1991, 559:141-152. A simple on-column concentration technique is presented in which a water plug is first introduced in the capillary. The sample is then dectrokinetically injected in a low conductivity solution. As the current is constant, the electric field drops mainly over the sample to provide focused injection. 40. •
FORETF, SUSTACEKV, BOCEK P: On-line Isotachophoretic Sample Preconcentration for Enhancement of zone Detectability in Capillary Zone Electrophoresis. J Microcolumn Sep 1990, 2:229-233. Isotachophoresis was coupled on-line to CE to yield a 200-fold enhancement in concentration. The direct on-line analysis of thiamine in human blood is demonstrated. 41. .
AEBERSOLDR, MORRISONHn: Analysis of Dilute Peptide Sampies by Capillary Zone Electrophoresis. J Chromatogr 1990, 516:79--88. Injection of a basic sample solulaon into an acidic running buffer provides an on-column focusing step leading to a detectable concentratton lowered by a factor of 5. The method is simple to implement and does not require extra equipment. 42.
CHENGY-F, DOVlCHI NJ: Subattomole Amino Acid Analysis by Capillary Zone Electrophoresis and Laser-induced Fluorescence. Science 1988, 24:562-564.
43 ••
LtU J, SHIROTAO, WIESLERD, NOVOTNYM: Ultraseusitive Fluorometric Detection of Carbohydrates as Derivatives In Mixtures separated by Capillary Electrophoresis. Proc Natl Acad Sci U S A 1991, 88:2302-2306. After reductive amination, reducing monosaccharides and oligosaccharides are derivatized with 3-(4-carboxybenzoyt)-2-quinolinecarboxaldehyde and analyzed by CE and laser-induced fluorescence. Complex oligosaccharides, isolated from bovine fetuIn by hydrazinolysis, were successfully mapped. 44. .•
LIU J, SHIROTA O, NOVOTNY M: separation of Fluorescent Oligosaccharide Derivatives by Microcolum Techniques
63
64
Analytical biotechnology Based on Electrophoresis and Liquid Chromatography. J Oyromatogr 1991, 559:223-235. Separation and analysis of fluorescently tagged oligosaccharides using CE and laser induced fluorescence. Polyacrylamide gel capillary columns with high monomer concentrations achieved very high resolutions on the basis of molecular weight. 45. •
MOSELEY MA, DETERDIN6 LJ, TOMER KB, JORGENSON JW: Determination of Bioactive Peptides using Capillary Zone Electrophoresis/Mass Spectrometry. Anal C,hem 1991, 63:109-114. Mixtures of bioactive peptides were analyzed by CE-MS using an online coaxial continuous flow, fast atom bombardment interface. High separation efficiencies (up to 400000 plates) were obtained from low femtomole levels of sample. 46.
FENNJB, MANN M, MENG CK, WONG se, WH1TEHOUSE CM: Electrospray Ionization for Mass Spectrometry of Large Biomolecules. Science 1989, 246:64-71.
47. •
SMITHRD, UDSETHHR, BARINAGACJ, EDMONDSCG: In$trumentation for High-performance Capillary Electrophoresis-mass Spectrometry. J Chromatogr 1991, 559:197-208.
Coupling of an electrospray interface with a commercial CE instrument has been accomplished. Capabilities of CE-MS for peptide and protein mixtures are demonstrated. 48 •
JOHANSSON M, HUANG EC, HENION JD: Capillary Electrophoresis-atmospheric Pressure Ionization Mass Spectrometry for the Characterization of Peptides: Instrumental Considerations for Mass Spectrometric Detection. J Chro matogr 1991, 554:311-327. CE-MS using an ion spray approach is demonstrated for peptides and a tryptac digest of human hemoglobin. Also examples of structural elucidation by MS are presented. 49.
THIBAULTP, PAPASC, PLEASg~CES: Analysis of Peptides and Proteins by Capillary Electrophoresis/mass Spectrometry Using Acidic Buffers and Coated Capillaries. Rapt~ Comm in Mass Spec 1991, 5:484-490.
BL Karger, Barnett Institute, Northeastern University, Boston, Massachusetts 02115, USA.
introduction Capillary electrophoresis (CE), the instrumental approach to slab gel electrophoresis, is a method undergoing rapid development at the present time. Nine commercial systems are available, and several other companies are planning to introduce systems in the near future. At this point, the field is in transition from fundamental development to broad applicability. In this review of the literature of the past year (September 1990 to October 1991), I shall provide a taste of the significant applications and new directions of the technology. As befits electrophoretic methods in general, a major thrust has been towards biological applications, in particular the analysis of proteins and nucleic acids, although there have been promising new results for the study of polysaccharides. The potential of CE coupled on-line to mass spectrometry (MS) is being developed, and it is quite clear that this hybrid method will be of great importance in the future. Also, the high resolution and high speed capabilities of CE are being exploited. The success of these developments leads to the conclusion that CE will play an active role in the future in those areas where high throughput (e.g. clinical/diagnostics) and high speed (e.g. process monitoring) are required. Given the breadth of the field, I have, of necessity, had to be highly subjective in what to include. Nevertheless, it is hoped that the reader attains some sense of the potential of CE in analytical biotechnology.
Peptide/protein separation A central feature of the successful separation of peptide or proteins by open-tube CE is the selection of buffer conditions and/or wall coating to minimize adsorption of protein molecules to the fused silica surface of the capillary. The normally negatively charged surface would obviously adsorb positively charged species; in addition,
hydrophobic adsorption is possible because of the fused silica structure and the aqueous buffer conditions. Even a slight amount of adsorption could dramatically reduce column performance. A good review on this topic of work carried out prior to September 1990 can be found in a paper published in late 1990 [1]. For peptides, two strategies involving buffer conditions have generally been found to be useful. At low pH ( < 3), many (but not all) silanol groups will be protonated, and electrostatic adsorption will thus be minimized. At the same time, all peptides will be positively charged and move in the same direction. Alternatively, basic pH (,-,8-10) can also be used; under these conditions the peptides should have net negative charges and thus be repulsed from the negatively charged wall surface. In addition, small amounts of a base (e.g. morpholine) may be added to reduce adsorption further. An example of these strategies used in peptide mapping can be found in the paper by Wheat eta/. [2"] in which a single residue substitution can be determined using small pH changes in either acidic or basic buffers. Results of CE used in peptide mapping have been sufficiently reproducible [3], and it can thus be concluded that this approach can be used along with liquid chromatography for this application. Indeed, the two methods complement each other in providing analytical information, and both can be coupled to MS (see below). In a significant paper, Neflson and Rickard [4,'], using different buffer pHs, correlated the mobilities of individual peptides with the properties of the molecules. For 33 diverse peptides and 10 intact proteins, they found a good fit between q/M2/3 and mobility, where q is the calculated net charge on the molecule and M is the molecular weight. This relationship was previously found by Offord using paper electrophoresis [5]. Neilson and Rickard [4°°] also determined that the adjacent structural environment of a charged group on the pKA of that group and, ultimately, the pI of the species is important. Clearly, as noted above, small pH modifica-
Abbreviations CE---Capillary electrophoresis; E$! clectrospray ionmation; HPLC---high-performance liquid chromatography; IEF---isoelectric focusing; MS--mass spectrometry. (~) Current Biology Ltd ISSN 0958-1669
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60 Analytical biotechnology tions could thus play a central role in selectivity. Molecular shape is also important in the mobility of a species. While the M-2/3 scale of Rickard e t al. implies that the species have spherical shapes, other shapes are possible for biopolymers and these will influence mobility. Indeed, Compton [6"] recently showed that for complex protein species, mobility more generally foUows M - 1/3 than M -2/3. In another example, Grossman and Soane [7"] demonstrated the presence of an electric field orientation for rod shaped molecules. From these studies, it is clear that protein conformation will significantly affect electric field migration and peak shape. As an example, Rush et a t [8..] presented the electrophoresis of calcium-depleted a-lactalbumin as a function of column temperature. A rapid increase in migration rate was observed with temperature, and in the transition region a broadened electrophoresis band correlating with the simultaneous existence of two conformational states was seen. The importance of conformation in the electrophoretic behavior of calcium- and zincbinding proteins has also been shown [9"]. In addition to pH control, buffer additives can be used to minimize adsorption of proteins to the capillary wall. One approach involves the use of high salt concentrations to reduce electrostatic interactions, often in conjunction with high pH (-~ 10.5) [10,11°]. In order to minimize the Joule heat generated by this technique, narrow bore columns (25 ~tm) are typically employed, rather than the more usual 50-100 gtm tubing. Such methods using high ionic strength buffers are being explored for use in high speed analysis of human serum proteins in clinical chemistry. Zwitterionic salts are another buffer additive [12], used to minimize electrostatic adsorption. For both of these approaches, the bare silica tubing can be washed between runs, with a base for example, to regenerate a new capillary wall each time. Polyamine is another additive that has been used successfully to reverse the charge on the wall surface from negative to positive [13"]. This approach is useful for many basic proteins such as histones (pI> 11). In this case, the polyamine is not added in the buffer but rather in a separate loading step to yield an adsorbed coat on the capillary wall. Covalently attached coatings can also be used, and a good deal of work has been done to find suitable ones. Probably the best known coating to date is that developed by Hjerten [14], which is formed using a bifunctional reagent, followed by linear polymerization with acrylamide. This covalently attached linear hydrophilic polymer reduces electro-osmotic flow to a negligible value. A good example of the use of this coating for peptide separation can be found in the paper by Zhu et al. [15"]. Interestingly, the use of basic conditions and the addition of a zwitterionic surfactant helped reduce adsorption even further. More recently, Novotny and co-workers [ 1'6] have used a similar, albeit somewhat more stable, coating for protein separations. The approach of converting the capillary wall to a cationic surface has been used in a covalently attached coating by Towns and Regnier [17-o]. These workers adsorbed a polyamine to the wall surface, but also followed this by cross-linking with bis-epoxide for stabilization.
Their paper outlines the basic principles of wall coatings and the influence of such coatings on electro-osmotic flow, and the importance of film thickness on the final potential on the wall is discussed in depth. These authors also prepared a capillary in which a non-ionic detergent (Brij 35) was adsorbed to an n-alkyl (C18) modified fused silica surface [18.-]. The detergent was also added to the mobile phase to maintain a stable coating. While no net formal charge existed on the coated surface, the potential was still sufficient to cause electro-osmotic flow, such that both positively and negatively charged proteins could be separated in one run. One nice aspect of this approach is that any chemical degradation of the wall (e.g. oxidation of the Brij) can be eliminated by simply washing out the surfactant and adsorbing new material. As hydrophobic capillary columns are commercially available [19], this approach is generally applicable. Results from another covalent coating in the form of a polyether phase have recently been published [20-]. Excellent separations of proteins were again shown. Another approach for peptide/protein separation by open-tube CE is isoelectric focusing (IEF) [21]. As generally practiced, a non-ionic coated tube is used in which the electro-osmotic flow is reduced to a negligible value. Carrier ampholytes are mixed with the sample which is loaded into the capillary. An electric field is then applied, causing the buffer amphotytes and sample to focus, demonstrated by a decrease in the current to essentially zero. The sample can then be mobilized to pass by the detection window, either by pressure, or by addition of salt to the cathotyte or anolyte. In the latter case, a pH gradient is established in which the appropriate salt ion will exchange with H + or O H - in the running buffer. The resolving power of this approach has been studied with hemoglobin variants [22"]. This paper also discusses a number of practical details conceming CE-IEF. Another recent study has demonstrated the potential use of capillary IEF for the analysis of carbohydrate variants in glycoproteins [23]. Although further development of this method may be necessary, capillary IEF would at present appear to be a useful approach in the resolution of proteins. It can be seen that peptide/protein separations by CE are well advanced with many options available for diverse samples. We can anticipate further developments in the coming year, continuing the advance of rapid, high resolution separations.
DNA separation The most powerful technique for the separation of DNA molecules is slab gel electrophoresis. In free solution, to a good first approximation, all DNA molecules migrate with the same mobility as the addition of a base (or base pair) results in the self compensation of mass and charge. Separation in gels occurs through a type of sieving behavior in which molecules migrate as a function of length. It is therefore obvious to use a sieving medium in a capillary to separate DNA by CE.
Capillary electrophoresisKarger The first attempts to use polymer networks (i.e. gels or viscous polymer solutions) as the sieving medium in CE involved crosslinked polyacrylamide gel for the separation of single-stranded DNA molecules. Remarkable separations were achieved, well in excess of 107 plates per meter [24]. Such columns can be used for the assay of synthesized DNA as well as for DNA sequencing [25",26,27,28.]. In the latter case, by analogy with the automated fluorescently tagged slab gel technique, separation of DNA with single base differences is achievable, with identification of individual bases related to specific fluorescent dyes. In principle, CE is 25 times faster than an individual slab gel lane in a standard sequencer; for example, up to ,-, 300 sequences can be read in 1 hour or less. This greater speed of separation is a consequence of the higher fields used in CE (,-,300V/cm) compared with slab gels ( ,-, 10V/cm). The capillary format reduces the current and thus the power or heat generated. The slab format can, however, be accelerated by using thin gel films of 50-100 I~m (compared with 350 ~ or more in normal operation), with which high fields can be used and multiple lanes incorporated [29]. Although thin slab gel electrophoresis appears to be quite fast, parallel processing, using multiple columns, is also possible for CE. The question of which technique is finally preferred will undoubtedly depend on which format yields sequence data for the greatest number of bases per lane. Issues such as temperature control across the capillary or slab gel will thus be critical in the final decision [30]. Ultimately, CE or slab gel operation may be overtaken by even faster methods. Although the final methodology is not yet clear, it is quite likely that either ultra-thin slabs or capillaries will be used, at least as a first stage, for enhancing the rate of DNA sequencing. Polymer networks can also be used for the separation of double-stranded DNA molecules in which non-denaturing buffer conditions are employed. Linear or lightly cross-linked polyacrylamide can be used for high resolution separation of species up to 1000 bp or higher [31"']. Separations have also been observed by Schwartz e t al. [32o.], Strege and Lagu [33"] and earlier by Zhu e t al. [34] with either hydroxypropylcellulose or methylcellulose. The effects of temperature on separation [35] and especially the addition of an intercalating agent such as ethidium bromide [36"] have also been demonstrated by Guttman and Cooke. The intercalating agent alters the flexibility of the DNA and enhances separation, particularly when high fields are used. The use of non-crosslinked polymer networks for sieving was initiated on slab gels many years ago [37]. Recently, Grossman and Soane [38"'] have explained the separation behavior of this sieving in terms of entangled polymer solutions, whose structures provide mesh spacings necessary for sieving purposes. One advantage of using a polymer network is that only relatively low viscosity solutions are required. Such solutions are then replaceable by simply blowing out the matrix and reloading with fresh material. The high resolution separation of doublestranded DNA with such materials provides a means of rapidly analyzing potyrnerase chain reaction products and restriction-fragment length polymorphisms,
among other applications. Successful separations in the 20 000-30 000 bp range should ultimately improve mapping procedures. DNA analysis is widely applicable, and CE should become a standard tool in carrying it out.
Instrumentation considerations In general, UV detection, usIng miniaturized high-performance liquid chromatography (HPLC) cells, is the most widely applied procedure for detection in CE. Unfortunately, although mass detection with HPLC is quite low, sample concentrations for CE are generally in the area of 10-5 M, several orders of magnitude less than those used in HPLC. Researchers have therefore been investigating preconcentration injection methods including field amplification, in which the conductivity of sample matrix is lower than that of the running buffer [39"], isotachophoresis [40.](displacement electrophoresis) or pH manipulation [41.]. A second way of enhancing detection is to use more sensitive detectors such as laser-based detection systems [42]. Although a variety of approaches have been employed, most systems rely on laser-Induced fluorescence for which sample concentrations of 10-1°-10-11M are typical. Derivatizing agents can be used for ultratrace laser analysis, such as fluorescamine, fluoroisothiocTanate, dansyl, etc. Recently, Novotny and co-workers [43"'] have introduced 3-(4-carboxybenzoyi)-2-quinolinecarboxaldehyde to react with primary amines. This reagent appears to be useful in determining reducing sugars. This approach may prove potentially useful in carbohydrate mapping of glycoproteins. In addition, through the use of concentrated polyacwlamide gels, high resolution of the fluorescently tagged sugars, has been achieved for partially hydrolyzed Dextrin 15 [44..]. As mentioned earlier CE-MS is developing at a rapid pace. This hybrid approach to separation and analysis is quite logical, given the very low flow rates (nL/min) for electro-osmotically driven bulk flow or simple ion transport with no bulk flow. Indeed, make-up fluids are often mixed with the CE-separated components prior to entry into the mass spectrometer. One successful CE-MS technique uses fast atom bombardment in which a coaxial continuous flow interface is employed [45°]. In this study, no degradation in separation performance was observed in the coupling of CE with MS and low femtomole levels of peptides could be structurally determined. This would appear to be a generally useful tool. These examples show that the high resolution of CE in combination with the structural information gained from MS, CE-MS will be a widely applied tool in analytical biotechnology. Recently, there has been a great deal of interest in electrospray ionization (ESI). In this technique, ions are evaporated from microcharged liquid droplets at atmospheric pressure under a high field. Following focusing, the ions are brought into a quadruple mass spectrometer. Besides vaporization at atmospheric pressure, another special feature of ESI is the formation of multiple charge states for
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62 Analyticalbiotechnology an individual species. Multiple peaks (m/z) are thus observed in the mass spectrum and, as a consequence of large charged states (often > + 20), the spectra for high molecular weight species can be observed on an ordinary 0-3000 m/z quadruple instrument. A good review on this can be found in a recent paper [46].
NIElsONRG, RICKARDEC: Method Optimization in Capillary Zone Electrophoretic Analysis of hGH Tryptic Digest Fragments. J C]yromatogr 1990, 516:99-114. This paper correlates the electrophoretic mobility of peptides (33) and intact proteins (10) with q/M2/3 where q = calculated charge and M ~ molecular weight. The correlation will aid in optimization strategies and in the elucidation of peptide/protein structure.
The coupling of CE to ESI has been accomplished especiaUyweU by Smith and co-workers [47"] and Henion and co-workers [48-]. Because of atmospheric ionization, the coupling is particularly straight forward as there is no pressure drop required across the capillary. Although successful and rapidly growing in use, several points are worth mentioning. First, some preconcentration of the sample by the methods discussed above often proves helpful for detection in the picomole range. Secondly, acidic solutions aid in protonation, and hence workers have generally foundthat using a coating with a positive charge (e.g. aminosilane) is of value [49].
5.
OFFORDRE" Nature 1966, 211:591-593.
6
COMPTONBJ: Electrophoretic Mobility Modeling of Proteins in Free z o n e Capillary Electrophoresis and its Application to Monoclonal Antibody Microheterogeneity Analysis. J Cbromatogr 1991, 559:357-366.
Conclusion In such a fast moving field it is unavoidable that much information has been omitted from this review. There is, for example, a good deal of research activity on the analysis of small molecules such as drugs using micellar electrokinetic chromatography. Chiral separations are also being accomplished by CE. In addition, methods for ion analysis that are competitive with ion chromatography are available. Thus, CE is on a very rapid development curve. We can anticipate that it will take its place beside HPLC in many laboratories in the years ahead.
4. ••
7. .
GROSSMANPD, SOANE DS: Orientation Effects on the Electrophoretic Mobility of Rod-shaped Molecules in Free Solution. Anal ~ 1991, 62:1592-1596. This paper shows that tobacco mosaic virus, a rod-shaped molecule, orients in the electric field in free solution CE. This orientation leads to an increase m electrophoretic mobility with field strength. (Similar orientation is found for DNA molecules in polymer networks, e.g. gels.) 8. •.
Rush RS, COHEN AS, KARGERBI; Influence of Column Ternperature on the Electrophoretic Behavior of Myoglobin and ~x-lactalbumin in High-performance Capillary Electrophoresis. Anal Chem 1991, 63:1346-1350. This paper studies the effect of temperature on the conformational change of proteins in capillary electrophoresis. In addition, the on-column reduction of myoglobin from the FE3+ state to the FE2 + state is demonstrated. KAJ1WARA H: Application of High-performance Capillary Electrophoresis to the Analysis of Conformation and Interaction of Metal-binding Proteins. J Ooromatogr 1991, 559:345-356. A study of the separation of Ca2+ - and Zn2+ -binding proteins by CE was undertaken. Electropherograms shifted depending on the presence or absence of the metal ions in the buffer. 9. •
10.
GREENJS, JORGENSONJ'~: MinimizIng Adsorption of Proteins on Fused Silica in Capillary Zone Electrophoresis by the Addition of Alkali Metal salts to the Buffers. J Chromatogr 1989, 478:63-70.
11.
CHENF-T: Rapid Protein Analysis by Capillary Electrophore-
Thi--s sis. J Chromatogr 1991, 559:445-453.
Acknowledgements The author gratefully acknowledges the Department of Energy and Beckman Instruments for their support of this work. Contribution Number 510 from the Barnett Institute.
paper describes a study that used a buffer system of 150 mM borate at pH 10.5 for the high speed (less than 200 s) separation of proteins on a 25 wn internal diameter column. Rapid serum protein separations are also shown. 12
BUSHEYi i , JORGENSONJ~,V. Capillary Electrophoresis of Proteins in Buffers Containing High Concentrations of Zwitterionic Salts. J Chronuttogr 1989, 480:301-310.
13. .
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •. of outstanding interest 1.
NOVOTNYMV, COBB KA, IAU J: Capillary Electrophoresis of Proteins, Peptides and Amino Acids. Electrophoresis 1990, 11:735-749.
2. •
WHEATTE, YOUNG PM, ASTEPHEN NE: Use of Capillary Electrophoresis for the Detection of Single-residue Substitutions in 'Peptide Mapping. J Liq Ooromatogr 1991, 14:987-996. This paper demonstrates the power of open tube CE in the separation of closely related peptides. 3.
NIELSENRG, RICKARDEC: In Analytical Biotechnology, Cap# lary Electrophoresis and Chromatography edited by Horvath C, Nikelly JG. Washington DC: ACS ACS Symposium Series, 1990, 434:36-49.
WIKTOROWICZJE, COmURN JC: Separation of Cationic Proteins via Charge Reversal in Capillary Electrophoresis. E/ectrophoresis 1990, 11:769-773. This study used polyamines adsorbed onto the fused silica for separation of basic proteins, including histones. 14.
HJERTENS: High-performance Electrophoresis: Elimination of Electroendosmosis and Solute Adsorption. J Ooromatogr 1985, 347:191-198.
15. •
ZHU M, RODRIGUEZ R, HANSEN D, WEHR T: Capillary Electrophoresis of Proteins Under Alkaline Conditions. J O0ro matogr 1990, 516:1123--1131. The use of a coated robe improves peak shape and reproducibility of protein migration in CE. In certain cases, buffer additives enhance performance even more. 16.
17. ••
COBBKA, DOtNIKV, NOVOTNYM: Electrophoretic Separations of Proteins in Capillaries with HydrolyticaUy Stable Surface Structures. Anal Chem 1990, 62:2478-2483.
TowNsJK, REGNIERFE. Polyethyleneimine-bonded Phases in the Separation of Proteins by Capillary Electrophoresis. J ~romatogr 1990, 516:69-78. This paper presents a positively charged coating that was stable over many runs, particularly of basic proteins. The coating could be used
Capillary
electrophoresis
Karger
over a wide pH range without substantial change in bulk electro-osmoUc flow. Rapid, high resolulaon of protons was possible with good recovery from the column.
Linear and lightly cross-linked polyacrylamide were used for the highspeed separation of double-stranded DNA.Analysisof polymerase chain reaction products was demonstrated.
18.
32. ..
TOWNSJK, REGNIERFE: Capillary Electrophoretic separations of Proteins Using Nonionic Surfactant Coatings. Anal Chem 1991, 63:1126-1132. A coating, consisting of a detergent Brij 35 adsorbed on a hydrophobic surface, is shown to work successfully In the resolution of proteins. Litde or no change in electro-osmotic flow occurs in the pH range of 4-11, of species both net positive and net negative charge can be separated in one run. ••
19.
DOUGHERTYAM, WOOH.EY CL, WIlaaXiS DL SWJdtE DF, COtE RO, SEPANIAKMJ: Stable Phase for Capillary Electrophoresis. J Liq t~romatogr 1991, 14:907-921.
20.
NASHABEHW, EL RASSIZ: Capillary Zone Electrophoresis of Proteins with Hydrophilic Fused-silica Capillaries. J Cbrc> matogr 1991, 559:367-383 High resolution separation of proteins is described, as well as the fmgerprimLrtg of crude protein mixtures using a fused silica capillary covalently coated with neutral polyether layer. .
21.
HJERTENS, L~o J'L YAO K: Theoretical and Experimental Study of High-performance Electrophoretic Mobilization of isoelectrically Focused Protein Zones. J C.bromatogr 1987, 387:127-138.
22.
ZHU M, RODRIGUEZR, WEHRT: Optimizing separation Parameters in Capillary Isoeletric Focusing. J Chromatogr 1991, 559:479-488. Improvements in capillary isoelectric focusing for detection of acidic and basic proteins, -/-globulins were focused successfully using a nonionic detergent to reduce precipitation. •
23.
24.
YIM KW: Fractionation of the Human Recombinant Tissue Plasminogen Activator (rtPA) Glycoforms by High-performance Capillary Zone Electrophoresis and Capillary isoelectric Focusing. J Clm'omatogr 1991, 559:401-410. COHENAS, NAJARIANDR, PAULUSA, GUTYMANA, SMITH JA, KARGER BL: Rapid Separation and Purification of Oligonucleotides by High-performance Capillary Gel Electrophoresis. Proc Natl Acad Sci U S A 1988, 85:9660-9663.
25.
LUCKEYJA, DROSSMANH, KOSTICHKAAJ, MEADDA, D'CUNHAJ, NORRISTB, SMITH LM: High Speed DNA Sequencing by Capillary Electrophoresis. Nucleic Acids Res 1990, 18:4417-4421. A prototype instrument was built for the simultaneous analysis of Sanger DNA-sequencing reaction products (DNA polymerase chain extension with dideoxynucleotide stops). Four different dyes were attached to a common primer for the recognition of individual bases •
26.
COHENAS, NAJARIANDR, KARGERBL Separation and Analysis of DNA Sequence Reaction Products by Capillary Gel Electrophoresis. J Chromatogr 1990, 516:49-60.
27.
CHENDY, SWERDLOWHP, HARKEHR, ZHANGJZ, DOVICHI NJ: Low-cost, High-seusitivity Laser-induced Fluorescence Detection for DNA Sequencing by Capillary Gel Electrophoresis. J ~ t o g r 1991, 559:237-246.
28. s
KARGERAE, HARRISJM, GESTELANDRF: Multiwavelength Fluorescence Detection for DNA Sequencing Using Capillary Electrophoresis. Nucleic Acids Res 1991, 19:4955--4962. A charge-coupled device was used with capillary gel electrophoresis to determine fluorescently labelled DNA sequencing products. 29.
SMITHLM: High-speed DNA Sequencing by Capillary Gel Electrophoresis. Nature 1991, 349:812-813.
30.
NISHIKAWA T, KAMBARAH: Analysis of Limiting Factors of DNA BAnd Separation by a DNA Sequencer Using Fluorescence Detection. Electrophoresis 1991, 12:623-631.
31. ..
HEIGERDN, COHEN AS, KARGERBL Separation of DNA Restriction Fragments by High Performance Capillary Electrophoresis with Low and Zero Crosslinked Polyacrylamide Using Continuous and Pulsed Electric Fields. J Chromatogr 1990, 516:33-48.
SCHWARTZHE, ULFELDERK. Analysis of DNA Restriction Fragments and Polymerase Chain Reaction Products Towards Detection of the AIDS (HIV-1) Virus in Blood. J Oyromatogr 1991, 559:267-283. HydroxypropytceUulose was used as a sieving medium for DNA products. Polymerase chain reaction-derived HIV-1 sequences were analyzed by this method. 33. .
STREGEM, LAGUA: Separation of DNA REstriction Fragments by Capillary Electrophoresis Using Coated Fused Silica Capillaries. Anal Cbem 1991, 63:1233-1236. Methylcellulose was used to separate double-stranded-DNA fragments according to base size. 34.
ZHU M, HANSENDL, BURDS, GANNONF: Factors Affecting Free Zone Electrophoresis and Isoelectric Focusing in Capillary Electrophoresis. J Cbromatogr 1989, 480:311-319.
35.
GUTrMANA, COOKE N: Effect of Temperature on the Separation of DNA Restriction Fragments in Capillary Gel Electrophoresis. J 03romatogr 1991, 559:285--294.
36. GUTrMANA, COOKE N: Capillary Gel AfFinity Electrophoresis • of DNA Fragments. Anal ~ 1991, 63:2038-2042. Incorporation of an Intercalating agent for double-stranded DNA prorides enhanced selectivity in CE with polyacrytamide gel columns. 37.
BODEHJ: Partitioning and Electrophoresis in Flexible Polymer Networks. Electrophoresis 1980, 79:39-52.
38. ..
GROSSMAN PD, SOANE US: Capillary Electrophoresis of DNA in Entangled Polymer Solutions. J Cbromatogr 1991, 559:257-266. The separation of DNA restriction fragments is modeled In terms of sieving through entangled polymer solutions. 39. •
CHIENR-L, BURGI US: Field Amplified Sample injection in High-performance Capillary Electrophoresis. J Cbromatogr 1991, 559:141-152. A simple on-column concentration technique is presented in which a water plug is first introduced in the capillary. The sample is then dectrokinetically injected in a low conductivity solution. As the current is constant, the electric field drops mainly over the sample to provide focused injection. 40. •
FORETF, SUSTACEKV, BOCEK P: On-line Isotachophoretic Sample Preconcentration for Enhancement of zone Detectability in Capillary Zone Electrophoresis. J Microcolumn Sep 1990, 2:229-233. Isotachophoresis was coupled on-line to CE to yield a 200-fold enhancement in concentration. The direct on-line analysis of thiamine in human blood is demonstrated. 41. .
AEBERSOLDR, MORRISONHn: Analysis of Dilute Peptide Sampies by Capillary Zone Electrophoresis. J Chromatogr 1990, 516:79--88. Injection of a basic sample solulaon into an acidic running buffer provides an on-column focusing step leading to a detectable concentratton lowered by a factor of 5. The method is simple to implement and does not require extra equipment. 42.
CHENGY-F, DOVlCHI NJ: Subattomole Amino Acid Analysis by Capillary Zone Electrophoresis and Laser-induced Fluorescence. Science 1988, 24:562-564.
43 ••
LtU J, SHIROTAO, WIESLERD, NOVOTNYM: Ultraseusitive Fluorometric Detection of Carbohydrates as Derivatives In Mixtures separated by Capillary Electrophoresis. Proc Natl Acad Sci U S A 1991, 88:2302-2306. After reductive amination, reducing monosaccharides and oligosaccharides are derivatized with 3-(4-carboxybenzoyt)-2-quinolinecarboxaldehyde and analyzed by CE and laser-induced fluorescence. Complex oligosaccharides, isolated from bovine fetuIn by hydrazinolysis, were successfully mapped. 44. .•
LIU J, SHIROTA O, NOVOTNY M: separation of Fluorescent Oligosaccharide Derivatives by Microcolum Techniques
63
64
Analytical biotechnology Based on Electrophoresis and Liquid Chromatography. J Oyromatogr 1991, 559:223-235. Separation and analysis of fluorescently tagged oligosaccharides using CE and laser induced fluorescence. Polyacrylamide gel capillary columns with high monomer concentrations achieved very high resolutions on the basis of molecular weight. 45. •
MOSELEY MA, DETERDIN6 LJ, TOMER KB, JORGENSON JW: Determination of Bioactive Peptides using Capillary Zone Electrophoresis/Mass Spectrometry. Anal C,hem 1991, 63:109-114. Mixtures of bioactive peptides were analyzed by CE-MS using an online coaxial continuous flow, fast atom bombardment interface. High separation efficiencies (up to 400000 plates) were obtained from low femtomole levels of sample. 46.
FENNJB, MANN M, MENG CK, WONG se, WH1TEHOUSE CM: Electrospray Ionization for Mass Spectrometry of Large Biomolecules. Science 1989, 246:64-71.
47. •
SMITHRD, UDSETHHR, BARINAGACJ, EDMONDSCG: In$trumentation for High-performance Capillary Electrophoresis-mass Spectrometry. J Chromatogr 1991, 559:197-208.
Coupling of an electrospray interface with a commercial CE instrument has been accomplished. Capabilities of CE-MS for peptide and protein mixtures are demonstrated. 48 •
JOHANSSON M, HUANG EC, HENION JD: Capillary Electrophoresis-atmospheric Pressure Ionization Mass Spectrometry for the Characterization of Peptides: Instrumental Considerations for Mass Spectrometric Detection. J Chro matogr 1991, 554:311-327. CE-MS using an ion spray approach is demonstrated for peptides and a tryptac digest of human hemoglobin. Also examples of structural elucidation by MS are presented. 49.
THIBAULTP, PAPASC, PLEASg~CES: Analysis of Peptides and Proteins by Capillary Electrophoresis/mass Spectrometry Using Acidic Buffers and Coated Capillaries. Rapt~ Comm in Mass Spec 1991, 5:484-490.
BL Karger, Barnett Institute, Northeastern University, Boston, Massachusetts 02115, USA.
