Electrophoresis 1990,11, 661-664
Frantiiek Foret Petr BoEek
CZF - present state of art
66 I
Capillary electrophoresis: Present state of art
Institute of Analytical Chemistry, Czechoslovak Academy of Sciences, Brno
1 Introduction This survey is aimed at giving the readers a brief overview of the present state of art of capillary zone electrophoresis (CZE); therefore, only selected literature is cited here. For more detailed information on capillary electrophoresis, specialized review articles published recently should be consulted [ 1-31.
2 Theory In analogy with chromatography, the theory in CZE is aimed at a mathematical description of the migration dynamics of sample zones, their mutual separation, and the separation power available for a given operational condition. The mathematical description of the separation dynamics, i.e. the development of the concentration profiles of the migration zones with time along the migration path, is well advanced. The transport equations describing electromigration and diffusion have been solved by numerical calculations and extensive work has been done to provide computer simulations ofthe migration ofzones 14-81. As the separationrecord in capillary zone electrophoresis closely resembles the record obtained in elution chromatography, the formal concepts of separation efficiency, number of theoretical plates (N), and resolution ( R ) as defined in chromatography [91 have been widely accepted. Several theoretical and experimental studies have been aimed at the description ofthe number oftheoretical plates generated for a given operational condition. Briefly, the dependence N =f(capillary, background electrolyte, sample, voltage and temperature) has been studied. Speaking in terms of phenomena contributing to the dispersion of zones, the following dependencies have been studied N = f (electromigration, diffusion, length of initial sample pulse, electromigration dispersion, electroosmosis, Joule heat and adsorption). A complex description of all the dispersive effects in one run is not yet available; however, approaches involving some of these effects have already provided valuable conclusions. No matter what their historical hierarchy, we believe it reasonable to mention them here as follows. The injection of the sample brings about the initial variance of the sample zone which limits the maximum accessible separation efficiency. When the starting sample zone, injected as a rectangular pulse, occupies fraction I of the total length L of the capillary the maximim separation efficiency is limited by the value
N = 12 L2/12
(1)
Correspondence: Dr. P. BoEek, Institute of Analytical Chemistry, Czechoslovak Academy o Sciences, Kounicova 82, CS-61142 Brno, Czechoslovakia Abbreviations: BGE, t ickground electrolyte; CZE, capillary zone electrophoresis; HPLC, hil 1 performance liquid chromatography 0VCH Verlagsgesellschaft .nbH, D-6940 Weinheim, 1990
Thus, e.g., when the initial sample zone length occupies 0.5 % of the capillary length, the maximum separation efficiency cannot be higher than 220 000 plates, provided that no stacking of the sample (according to Kohlrausch’s regulation function) during the starting period of the analysis occurs. For the value of 1 to be 0.1 %, the injection limit of the separation efficiency is higher than 5 000 000 theoretical plates. For highly efficient separations the injection procedure is of great importance. The model covering electromigration and diffusion is also important and has attracted the attention of a broad group of experts, initiating the boom of CZE.This model is described by the simple formula [ 101.
N
= uU/2D
where u, D and U are the electrophoretic mobility of the separand, its diffusion coefficient, and the voltage applied across the ends ofthe separation capillary, respectively. Equation (2) was derived at by assuming that diffusion is the only dispersive process during the separation and that the injection volume is negligible. Hundreds of thousands of theoretical plates can easily be generated in agreement with the predictions according to Eq. (2); however, in practice, substantial deviations from this theory are observed. Recently, several papers were published which not only concern the diffusion of the spearnds but also the role of other dispersion effects. It is well known now that the contribution of injection, Joule heating, and electroosmosis towards total dispersion during the analysis can be estimated from known operational parameters such as conductivity of the background electrolyte (BGE), separation voltage, length, and internal diameter of the separation capillary,etc. [3,8,11-171.It stemsfromthesestudiesthatfor a given experimental set-up the dependence of the separation efficiency (expressed by the number of theoretical plates) versus applied voltage is not linear as predicted by Eq. (2) but shows a maximum at a certain voltage. At higher voltages the efficiency decreases again. For the experimental conditions used at present the maximum on the efficiency curve should count millions of theoretical plates, a value only seldom obtained in practice. The reason can be attributed to the sample volume injected into the capillary and, especially in protein analyses, to the dispersion due to the adsorption ofthe sample onto the capillary wall. The adsorption of separands onto the capillary wall in many cases results not only in the broad and tailing zones of the migrating species but could also prevent the detection of these zones since all the material may be bound to the capillary wall. Adsorption represents the most important source of peak broadening and many attempts were made to eliminate it. Besides the operation at extremely high and/or low pHs ofthe BGE, where the protein-fused silica interaction is low, mainly chemical coatings of the inner wall of the capillary are still being tested [ 18, 191. Also examined recently was the use of BGE with high ionic strength where the sorption equilibria are shifted by the high concentration of indifferent sodium chloride or zwitterionic salts in the BGE [20,21I. The role of 0173-0S35/90/0909-0661 $3.50+.25/0
662
F. Foret and P. BoEek
other effects, such as nonequilibrium phenomena and micellar polydispersity in micellar electrokinetic capillary chromatography(MECC),wasonlytreatedinapreliminary study [22].
3 Instrumentation The basic arrangement of the equipment for C Z E is now well established (in fact, several commercial products can now be found on the market [23-291). Principally, it consists of the fused silica separation capillary located between two electrolyte chambers equipped with electrodes for the connection of the high voltage power supply. A common sample introduction procedure is either electromigration or the controlled flow (by siphoning or overpressure') of the sample solution into the capillary during a preset time interval. Separation voltages up to 30 kV are used, with separation capillaries 20- 100 cm long, and an internal diameter as low as 9 pm [30l. The most important part of the instrumentation is the detector since it determines the sensitivity of the analysis. At present, UV absorbance detectors are mainly being used [3 1,321. Recent developments in the design of the capillary on-column flow cells decreased the noise of the detector to A. U. While in microcolumn liquid chromatography a substantial increase of sensitivity was demonstrated with the capillary Z-shaped cell with an optical path of 10 tnm in a 7 5 Fm i. d. capillary [331, this approach is not easy to adopt for high performance C Z E where the detected zone length may be well below the length ofthe detection cell [341. Fluorescencedetectors, especially with laser-induced excitation, exhibit much higher sensitivity and selectivity than absorbance detectors [35, 361. Several high performance liquid chromatography (HPLC) spectrofluorimeters suitable for modification to C Z E are on the market now and laser-induced fluorescence systems are expected to appear soon. The lack of strong native fluorescence ofmany compounds ofinterest can be overcome by suitable tagging with a highly fluorescent agent, and new arrangements for post-column fluorescence derivatization are now available 137,381. Electrochemical detection is useful for selective detection of many electroactive substances. Its sensitivity is comparable to fluorescence detection and several interesting papers recently described its use with extremely narrow capillaries [30, 391. For substances without significant optical or electrochemical activity the use of conductivity [401 andlor the indirect modes of electrochemical, UV absorption and fluorescence detection were suggested [30,41-441. Great potential is expected from the combination of capillary electrophoresis and mass spectrometry. Mass spectra of femtomole amounts of the sample separated by C Z E were reported [45-501 and a CZEIMS interface is already on the market 1.511. New detector schemes for C Z E such as the use of a radioisotope 1.52. 531, fluorescence-detected circular dichroism [541, refractometric detector with analyte velocity modulation [551 and the use ofthermooptical absorbance [56,571 and/or ion mobility spectrometric detectors I581 were reported. A promising approach to improve the detection limit in C Z E is the on-line coupling of isotachophoresis and capillary electrophoresis. This combination brings the concentrating capability of isotachophoresis as well as easy interpretation of the separation record into the zone electrophoretic step. Improvements of detection sensitivityofmany ordersofmagnitudecanbeexpected [59-6 1 I.
Electrophoresis 1990, 11, 661-664
4 Options in achieving resolution The resolution R in electrophoresis is given by the separation efficiency N and by the selectivity (the relative difference in mobilities u ) of separated substances. R = 114 \1TA u l i i
(3)
From Eq. ( 2 )it is clear that the most effective way to increase resolution is to increase selectivity. Several ways of accomplishing this are of particular interest, e.g., acid-base equilibria [62-64], ion pairing [65,661, the use of organic and polymer modifiers in the BGE 167, 681, micellar solubilization [ 69-7 11, and host-guest interactions with nonionic substances such as cyclodextrins [72, 731. Promising modes of electrophoretic separation to achieve high resolution are: the use of gel-filled capillaries [741, capillary isoelectricfocusing 175,761 and gradient elutions 177-801. In gel electrophoresis the baseline resolution of oligonucleotides counting up to 160 bases and differing by only one base was already demonstrated [741 and theoretical plate counts of the order of lo7were shown. The use of capillary isoelectric focusing is still in its infancy, but its separation power, based on differences in isoelectric points of the separands rather than on electrophoretic mobilities, was clearly demonstrated for protein mixtures [75, 761. The separation in capillary isoelectric focusing proceeds in two steps: The sample components are focused in a gradient formed by carrier ampholytes according to their isoelectric points. After the steady state is reached, the separated zones are mobilized (unstacked) by changing the composition of the electrolyte in one of the electrode chambers, and, thus, by shifting the focused species out of their isoelectric conditions. In gradient elution, a regulated flow of organic solvent or ions [ 77-801 is furnished to control the migration of separands in the capillary. Thus, for example, the controlled flow of H+ions can form the pH gradient. The use of gradient elution is expected to be especially useful in protein separations.
5 Optimization of CZE Optimization of a practical analysis by C Z E calls for a compromise between the required sensitivity, speed and resolution. The sensitivity ofthe detector dictates the minimum concentration of the separands in their zones. Generally, it is recommended to keep this concentration two orders of magnitude below the concentration of the BGE to avoid electromigration dispersion [ 8 11. By increasing the concentration of the BGE, the concentration in zones can also be higher and therefore the dynamic range of detection is increased. However, two facts must be kept in mind when increasing the BGE concentration. First, the detector response has limited linearity when using a circular detection cell (the on-column detection) and second, at high BGE conductivity, adverse effects of the Joule heat are to be expected [821. For these reasons, when detector sensitivity is insufficient, the composition of the BGE should be such that the mobility of the background-forming co-ion is close to the mobility of the separated ions [ 4l].When that is the case, the electromigration dispersion can be kept low even when the concentration in zones is close to the concentration of the BGE-forming co-ion.
hlectrophoresry 1990. 11, 661-664
6 Applications Capillary electrophoresis is well suited for both physicochemical measurements and analytical purposes. The primer may be evidenced by the measurement of physicochemical data such as ionic mobilities and/or diffusion coefficients [831 as well as by the measurements for the characterization and control of the electroosmotic flow 184-871. The analytical potential of C Z E may be demonstrated by the fact that capillary electrophoresis can easily compete with HPLC in many application areas, such as rapid and efficient separations of inorganic ions 188-901, pharmaceuticals [9 1, 921, polyamines 1931 and of optical isomers [941.The potential of CZE for the high resolution separation of polar and high molecular weight fuel-related materials was also demonstrated recently 1951. However, the main area of C Z E application is expected to be in the analysis of high molecular weight compounds such as peptides, proteins and polynucleotides, where, in principle, electrophoresis should be superior to other separation methods [96-1041. Here, the low sample volume for the analysis makes capillary electrophoresis an excellent analytical step, e.g., for peptide mapping after enzymatic digestion of a minute amount of sample protein [loll. The use of C Z E in oligonucleotide separations was already mentioned; great activity is expected in this field in connection with the human genome mapping project. Received March 30, 1990
7 References 1 I I Karger, B. C., Cohen, A. S. and Guttman, A,, J . Chromatogr. 1989, 492,585-614. 121 Knox, J. H. and McCormack, K. A,, J . Liquid Chromalogr. 1989, 12,2435-2470. 131 Foret, F. and BoEek, P., Adv. Electrophoresis 1989,3, 273-342. 141 Bier, M., Palusinski, 0..Mosher, R. A. and Saville, D. A,, Science 1983,219, 1281-1287. I51 Thormann, W., Mosher, R. A., Bier, M.,J. Chromatogr. 1986,351. 17-29. 161 Mosher, R. A,, Thormann, W., Kuhn, R. and Wagner, H.,J. Chromatogr. 1989,478, 39-49. 171 Mosher, R. A., Dewey. D., Thormann, W., Saville, D. A. and Bier, M., Anal. Chem. 1989,61,362-366. 181 Roberts, G. O., Rhodes, P. H. and Snyder, R. S., J . Chromafogr. 1989,480,35-68. 191 Giddings, J. C., Sep. Sci. 1969,4, 181-189. 1101 Jorgenson, J. W. and Lukacs, K. D., Anal. Chem. 1981, 53, 1298-1302. [ 111 Knox, J. H., Chroniatogruphia 1988,26, 329-336. 1121 Otsuka, K. and Terabe, S., J . Chromatogr. 1989,480,91-94. 1 131 Huang, X., Coleman, W. F. and Zare, R. N., J . Chromatogr. 1989, 480,95-110. 11'41 Nelson, R. J., Paulus, A.? Cohen, A. S., Guttman, A. and Karger, B. C.,J. Chromatogr. 1989,480, 111-128. 1151 Grushka, E. and McCormick, J., J . Chromatogr. 1989, 471, 421-428. 1161 Janson, M., Emmer, A. and Roeraade, J., J. High Resolul. Chromatogr. Commun. 1989,12, 797-801. 1171 Schwartz, H. E., Melera, M. and Brownlee, R. G., J . Chromatogr. 1989,480. 129-140. 1181 Bruin, G. J . M., Huisden, R., Kraak, J. C. and Poppe, H., J . Chro matogr. 1989,480,339-350. I191 Bruin, G. J. M., Chang, J. P., Kuhlman, R. H., Zegers, K., Kraak, J. C. and Poppe, H.. J . Chrornatogr. 1989.471,429-436. 1201 Green,J.S. andJorgenson,J.W.,J. Chromatogr. 1989,478,63-70.
CZE
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1211 Bushey, M. M. and Jorgenson, J. W., J . Chromazogr. 1989.480, 30 1-3 10. I221 Davis. J. M.. Anal. Chem. 1989,6l. 2455&2461. [23I Applied Biosystems, Foster City, CA, IJSA. 1241 Bio-Rad. Richmond, CA, USA. [251 Beckman, Palo Alto, CA, USA. 1261 Dionex, Sunnyvale, CA, USA. 1271 ISCO, Lincoln, NE, USA. I281 Spectro Vision, Chelmsford, MA, USA. 1291 Spectra Physics, Darmstadt, W. Germany. 1301 Olefirowicz, T. M. and Ewing, A. G., J . Chromatogr. 1990, 4Y9, 7 13-7 19. I3 11 Green, J. S. and Jorgenson. J. W.,J. Liquid Chromatogr. 1989.12, 2527-256 1. 1321 Kobayashi, S., Ueda, T. and Kikumoto, M., 1.Chromarogr. 1989, 480, 179- 184. I33 I Chervet, J. P., Ursem, M., Salzmann, J. P. and Vannoort, R. W., J. High Resol. Chromatogr. 1989, 12, 278-281. \ 341 Grant,I.H. andSteuer, W.,J.Microcolumn Sep. l990.2,74-79. 1351 Wu, S. and Dovichi, N. J.,J. Chromatogr. 1989,450, 141-156. I361 Hernandez, L., Marguina, R., Escalona, J. and Guzman, N. A., J . Chromatogr. 1990,502,247-255. 1371 Nickerson, B. and Jorgenson, J. W.. J . Chromatogr. 1989. 480, 157-1 68. I381 Cheng, Y. F., Wu, S., Chen, D. Y. and Dovichi, N. J.. Anal. C h e m 1990,62,496-503. (391 Wallingford, R. A.. Curry, P. D., Jr. and Ewing, A. G., J . Microcolumn Sep. 1989,l. 23-27. 1401 Huang, X., Gordon, M. J. and Zare, R. N., J . Chromatogr. 1989, 480,285-288. 1411 Foret, F., Fanali, S., Ossicini, L. and BoEek, P.. J . Chromatogr. 1989,470.299-308. 1421 Gross, L. and Yeung, E. S., J . Chromatogr. 1989,480, 169- 178. 143 I Gross, L. and Yeung, E. S., Anal. Chem. 1990,62,427--431. [441 Hogan, B. L. and Yeung, E. S.,J. Chromatogr. Sci. 1990,28,15- 18. 145 I Moseley, A. M., Deterding, C. J.,Tomer, K. B. and Jorgenson,J. W.. Rapid Commun. Mass Spectrom. 1989, 3, 87--93. I461 Edmonds, C . G., Loo, J . A., Barinaga, C. J., Udseth, H. R. and Smith, R. D.,J. Chromafogr. 1989.476, 21-37. 1471 Smith, R. D., Udseth, H. R., Loo, J. A,. Wright, B. W. and Ross, G. A,, Talarttci 1989.36, 161-169. 1481 Moseley, M. A,. Deterding, L. J., Toner, K. B. and Jorgenson. J . W., J . Chromatogr. 1989.480, 197-210. 1491 Smith. R. D., Loo, J. A,, Barinaga, C. J., Edmonds, C. G. and Udseth, H. R..J. Chromutogr. 1989,480, 21 1-232. 1501 Caprioli, R. M., Moore,T. W., Martin, M., DaGue, B. B., Wilson, K . and Moring, S., J. Chromatogr. 1989,480, 247-259. 1511 Sciex, Thornhill, Ontario, Canada. [52] Pentoney, S. L., Jr., Zare, R. N. and Quint, J . F., J . Chromutogr. 1989,480,259-270. 1531 Pentoney,S. L., Jr., Anal. Chem. 1989,6/, 1642-1647. 1541 Christensen, P. L. and Yeung, E. S., Anal. Chem. 1989, 61, 1344- 1347. i55l Chen, C., Demana,'r., Huang, S. and Morris, M. D., Anal. Chem. 1989,61, 1590-1593. [561 Yu, M. and Dovichi, N. J., Microchim. Acta 1988,l I I , 27-40. [571 Earle, C. W. and Dovichi, N. J., J. Liquid Chrornatogr. 1989, 12, 2575-2585. 1581 Hallen, R. W., Shumate. L. B., Siems, W. F., Tsuda, T. and Hill, H. H., Jr., J . Chromatogr. 1989,480. 233-246. 1591 Kaniansky,D. andMarak,J.,J. Chrornarogr. 1990,498,191-204. [601 Dolnik, V., Cobb, K. A. and Novotny. M., J. Microcolumn Sep. 1990, in press. [611 Foret, F., SustaEek, V. and BoEek, P.,J. Microcolumn. Sep. 1990, in press. 1621 Grossman, P. D., Wilson, K. J., Petrie, G . and Lauer. H . H., Anal. Biochem. 1988,173,265-270. 1631 Rasmussen, H. T. and McNair, H. M.,J. High Resol. Chromafogr. 1989.12, 635--636. 1641 Dolnik, V., Liu, J., Banks, F. J., Jr., Novotny. M. and BoCek, P.. J . Chromatogr. 1989,480,321-330.
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1651 Nishi, H. and Tsumagari, N., Anal. Chem. 1989,61, 2434-2439. 1661 Terabe, S. and Isemura, T., Anal. Chem. 1990.62, 650-652. [671 Zhu, M., Hansen, D. L., Burd, S. and Gannon, F., J . Chromatogr. 1989,480,3 11-320. 1681 Liu. J., Cobb, K. A. and Novotny, M., J . Chromatogr. 1988,468, 55-65. 1691 Terabe, S., Shibata, M. and Miyashita, Y., .I. Chromatogr. 1989. 480,403-4 12. I701 Dobashi, A., Ono, T., Hara, S. and Yamaguchi, J., J . Chromatogr. 1989,480,4 13-420. [ 7 1I Swedberg, S. A., J. Chromatogr. 1990,503,449-452. I721 Fanali, S., J . Chromatogr. 1989,474,441-446. [731 Fanali, S., Ossicini,L., Foret,F. andBoEek. P..,J. Microcolumn. Sep. 1989,1, 190-194. I741 Guttman, A., Cohen, A. S., Heiger, D. N. and Karger, B. L., Anal. Chem. 1990,62, 137-141. [751 Kilar, F. and Hjerten, S.,J. Chromatogr. 1989,480, 351-358. 1761 Kilar. F. and Hjerten, S., Electrophoresis 1989,IO, 23-29. 1771 BoEek, P., Deml, M., Pospichal, J. and Sudor, J., J. Chromatogr. 1989,470,309-3 12. [781 Sudor, J., Stransky, Z., Pospichal, J., Deml, M. and BoEek, P., Electrophoresis 1989,10,802-805. 1791 SustaEek, V., Foret, F. and BoEek, P., J . Chromatogr. 1989, 480, 271-277. I801 Sepaniak, M. J., Swaile, D. F. and Powell, A. C., J . Chromatogr. 1989,480, 185-196. 1811 Mikkers, F. E. P., Everaerts, F. M. and Verheggen, T. P. E. M.. J. Chromatogr. 1979, 169, 11-20. 1821 Foret, F., Deml, M. and BoEek, P., J. Chromatogr. 1988, 452, 601-613. 1831 Walbroehl, Y. and Jorgenson, J. W.,J. Microcolumn Sep. 1989, I , 41-45. I841 Tsuda, T., J . Liquid Chromatogr. 1989, 12, ;!501-25 14. I851 Altria,K.D.andSimpson,C.F.,Anal.Proc. 1986,23,453-454. 1861 Van de Goor, A. A. A., Wanders, B. J. and Everaerts, F. M., J . Chromatogr. 1989.470, 95- 104.
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1871 Verheggen,T. P. E.M., Schoots,A. C. andEveraerts,F. M..J. Chromatogr. 1990,503, 245-255. I881 Guzman, N. A., Hernandez, L. and Advis, J. P., J. Liquid Chromatogr. 1989,12,2563-2574. [ 8 9 l Honda, S., Suzuki, K. and Kakehi, K., Anal. Biochem. 1989, 177. 62-66. 1901 Aguilar, M.. Huang, X. andZare, R. N.,J. Chromatogr. 1989,480, 427-432. 1911 Nishi, H., Tsumagari, N., Kakimoto, T. and Terabe, S . , J . Chromatogr. 1989,474,259-210. I921 Nishi, H., Fukuyama, T., Matsuo, M. and Terabe, S., J. Chromatogr. 1990,498,313-323, [931 Tsuda, T., Kobayashi, Y., Hori, A., Matsumoto, T. and Suzuki. O., J. Microcolumn. Sep. 1990,2, 21-25. 1941 Dobashi, A,, Ono, T., Hara, S. and Yamaguchi, J., Anal. Chem. 1989,62, 1984-1986. 195 I Wright, B. W.,Ross,G. A. and Smith,R. D., J. Energy Fuels 1989,3. 428-430. [961 Miick,W.M. andHenion,J.D.,J. Chromatogr. 1989,495,41-59. 1971 RohliEek, V. and Deyl, Z., J. Chromatogr. 1989,496, 87-99. [981 Hjerttn, S., Valtcheva, L., Elenbring, K. and Eaker, D., J. Liquid Chromatogr. 1989,12,2471-2499. 1991 Grossman, P. D., Colburn, J. C., Lauer, H. H., Nielsen, R. G.,
Riggin, R. M., Sittampalam, G. S. and Rickard, E. C., Anal. Chem. 1989,61, 1186-1194. I1001 Deyl. Z., RohliEek, V. and Struiinsky, R., J. Liquid Chromatogr. 1989,12,2515-1526. Il o l l Cobb,K.A.andNovotny,M.,Anal. Chem. 1989,6/,2226-223 1. I1021 Deyl, Z., RohliEek, V. and Adam, M., J . Chromatogr. 1989,480. 371-378. I1031 Frenz, J., Wu, S. L. and Hancock, W. S.,J. Chromatogr. 1989,480, 379-392. 11041 Nielsen, R. G., Riggin, R. M. and Rick&d, E. C., J . Chromatogr. 1989,480,393-402.
Frantiiek Foret Petr BoEek
CZF - present state of art
66 I
Capillary electrophoresis: Present state of art
Institute of Analytical Chemistry, Czechoslovak Academy of Sciences, Brno
1 Introduction This survey is aimed at giving the readers a brief overview of the present state of art of capillary zone electrophoresis (CZE); therefore, only selected literature is cited here. For more detailed information on capillary electrophoresis, specialized review articles published recently should be consulted [ 1-31.
2 Theory In analogy with chromatography, the theory in CZE is aimed at a mathematical description of the migration dynamics of sample zones, their mutual separation, and the separation power available for a given operational condition. The mathematical description of the separation dynamics, i.e. the development of the concentration profiles of the migration zones with time along the migration path, is well advanced. The transport equations describing electromigration and diffusion have been solved by numerical calculations and extensive work has been done to provide computer simulations ofthe migration ofzones 14-81. As the separationrecord in capillary zone electrophoresis closely resembles the record obtained in elution chromatography, the formal concepts of separation efficiency, number of theoretical plates (N), and resolution ( R ) as defined in chromatography [91 have been widely accepted. Several theoretical and experimental studies have been aimed at the description ofthe number oftheoretical plates generated for a given operational condition. Briefly, the dependence N =f(capillary, background electrolyte, sample, voltage and temperature) has been studied. Speaking in terms of phenomena contributing to the dispersion of zones, the following dependencies have been studied N = f (electromigration, diffusion, length of initial sample pulse, electromigration dispersion, electroosmosis, Joule heat and adsorption). A complex description of all the dispersive effects in one run is not yet available; however, approaches involving some of these effects have already provided valuable conclusions. No matter what their historical hierarchy, we believe it reasonable to mention them here as follows. The injection of the sample brings about the initial variance of the sample zone which limits the maximum accessible separation efficiency. When the starting sample zone, injected as a rectangular pulse, occupies fraction I of the total length L of the capillary the maximim separation efficiency is limited by the value
N = 12 L2/12
(1)
Correspondence: Dr. P. BoEek, Institute of Analytical Chemistry, Czechoslovak Academy o Sciences, Kounicova 82, CS-61142 Brno, Czechoslovakia Abbreviations: BGE, t ickground electrolyte; CZE, capillary zone electrophoresis; HPLC, hil 1 performance liquid chromatography 0VCH Verlagsgesellschaft .nbH, D-6940 Weinheim, 1990
Thus, e.g., when the initial sample zone length occupies 0.5 % of the capillary length, the maximum separation efficiency cannot be higher than 220 000 plates, provided that no stacking of the sample (according to Kohlrausch’s regulation function) during the starting period of the analysis occurs. For the value of 1 to be 0.1 %, the injection limit of the separation efficiency is higher than 5 000 000 theoretical plates. For highly efficient separations the injection procedure is of great importance. The model covering electromigration and diffusion is also important and has attracted the attention of a broad group of experts, initiating the boom of CZE.This model is described by the simple formula [ 101.
N
= uU/2D
where u, D and U are the electrophoretic mobility of the separand, its diffusion coefficient, and the voltage applied across the ends ofthe separation capillary, respectively. Equation (2) was derived at by assuming that diffusion is the only dispersive process during the separation and that the injection volume is negligible. Hundreds of thousands of theoretical plates can easily be generated in agreement with the predictions according to Eq. (2); however, in practice, substantial deviations from this theory are observed. Recently, several papers were published which not only concern the diffusion of the spearnds but also the role of other dispersion effects. It is well known now that the contribution of injection, Joule heating, and electroosmosis towards total dispersion during the analysis can be estimated from known operational parameters such as conductivity of the background electrolyte (BGE), separation voltage, length, and internal diameter of the separation capillary,etc. [3,8,11-171.It stemsfromthesestudiesthatfor a given experimental set-up the dependence of the separation efficiency (expressed by the number of theoretical plates) versus applied voltage is not linear as predicted by Eq. (2) but shows a maximum at a certain voltage. At higher voltages the efficiency decreases again. For the experimental conditions used at present the maximum on the efficiency curve should count millions of theoretical plates, a value only seldom obtained in practice. The reason can be attributed to the sample volume injected into the capillary and, especially in protein analyses, to the dispersion due to the adsorption ofthe sample onto the capillary wall. The adsorption of separands onto the capillary wall in many cases results not only in the broad and tailing zones of the migrating species but could also prevent the detection of these zones since all the material may be bound to the capillary wall. Adsorption represents the most important source of peak broadening and many attempts were made to eliminate it. Besides the operation at extremely high and/or low pHs ofthe BGE, where the protein-fused silica interaction is low, mainly chemical coatings of the inner wall of the capillary are still being tested [ 18, 191. Also examined recently was the use of BGE with high ionic strength where the sorption equilibria are shifted by the high concentration of indifferent sodium chloride or zwitterionic salts in the BGE [20,21I. The role of 0173-0S35/90/0909-0661 $3.50+.25/0
662
F. Foret and P. BoEek
other effects, such as nonequilibrium phenomena and micellar polydispersity in micellar electrokinetic capillary chromatography(MECC),wasonlytreatedinapreliminary study [22].
3 Instrumentation The basic arrangement of the equipment for C Z E is now well established (in fact, several commercial products can now be found on the market [23-291). Principally, it consists of the fused silica separation capillary located between two electrolyte chambers equipped with electrodes for the connection of the high voltage power supply. A common sample introduction procedure is either electromigration or the controlled flow (by siphoning or overpressure') of the sample solution into the capillary during a preset time interval. Separation voltages up to 30 kV are used, with separation capillaries 20- 100 cm long, and an internal diameter as low as 9 pm [30l. The most important part of the instrumentation is the detector since it determines the sensitivity of the analysis. At present, UV absorbance detectors are mainly being used [3 1,321. Recent developments in the design of the capillary on-column flow cells decreased the noise of the detector to A. U. While in microcolumn liquid chromatography a substantial increase of sensitivity was demonstrated with the capillary Z-shaped cell with an optical path of 10 tnm in a 7 5 Fm i. d. capillary [331, this approach is not easy to adopt for high performance C Z E where the detected zone length may be well below the length ofthe detection cell [341. Fluorescencedetectors, especially with laser-induced excitation, exhibit much higher sensitivity and selectivity than absorbance detectors [35, 361. Several high performance liquid chromatography (HPLC) spectrofluorimeters suitable for modification to C Z E are on the market now and laser-induced fluorescence systems are expected to appear soon. The lack of strong native fluorescence ofmany compounds ofinterest can be overcome by suitable tagging with a highly fluorescent agent, and new arrangements for post-column fluorescence derivatization are now available 137,381. Electrochemical detection is useful for selective detection of many electroactive substances. Its sensitivity is comparable to fluorescence detection and several interesting papers recently described its use with extremely narrow capillaries [30, 391. For substances without significant optical or electrochemical activity the use of conductivity [401 andlor the indirect modes of electrochemical, UV absorption and fluorescence detection were suggested [30,41-441. Great potential is expected from the combination of capillary electrophoresis and mass spectrometry. Mass spectra of femtomole amounts of the sample separated by C Z E were reported [45-501 and a CZEIMS interface is already on the market 1.511. New detector schemes for C Z E such as the use of a radioisotope 1.52. 531, fluorescence-detected circular dichroism [541, refractometric detector with analyte velocity modulation [551 and the use ofthermooptical absorbance [56,571 and/or ion mobility spectrometric detectors I581 were reported. A promising approach to improve the detection limit in C Z E is the on-line coupling of isotachophoresis and capillary electrophoresis. This combination brings the concentrating capability of isotachophoresis as well as easy interpretation of the separation record into the zone electrophoretic step. Improvements of detection sensitivityofmany ordersofmagnitudecanbeexpected [59-6 1 I.
Electrophoresis 1990, 11, 661-664
4 Options in achieving resolution The resolution R in electrophoresis is given by the separation efficiency N and by the selectivity (the relative difference in mobilities u ) of separated substances. R = 114 \1TA u l i i
(3)
From Eq. ( 2 )it is clear that the most effective way to increase resolution is to increase selectivity. Several ways of accomplishing this are of particular interest, e.g., acid-base equilibria [62-64], ion pairing [65,661, the use of organic and polymer modifiers in the BGE 167, 681, micellar solubilization [ 69-7 11, and host-guest interactions with nonionic substances such as cyclodextrins [72, 731. Promising modes of electrophoretic separation to achieve high resolution are: the use of gel-filled capillaries [741, capillary isoelectricfocusing 175,761 and gradient elutions 177-801. In gel electrophoresis the baseline resolution of oligonucleotides counting up to 160 bases and differing by only one base was already demonstrated [741 and theoretical plate counts of the order of lo7were shown. The use of capillary isoelectric focusing is still in its infancy, but its separation power, based on differences in isoelectric points of the separands rather than on electrophoretic mobilities, was clearly demonstrated for protein mixtures [75, 761. The separation in capillary isoelectric focusing proceeds in two steps: The sample components are focused in a gradient formed by carrier ampholytes according to their isoelectric points. After the steady state is reached, the separated zones are mobilized (unstacked) by changing the composition of the electrolyte in one of the electrode chambers, and, thus, by shifting the focused species out of their isoelectric conditions. In gradient elution, a regulated flow of organic solvent or ions [ 77-801 is furnished to control the migration of separands in the capillary. Thus, for example, the controlled flow of H+ions can form the pH gradient. The use of gradient elution is expected to be especially useful in protein separations.
5 Optimization of CZE Optimization of a practical analysis by C Z E calls for a compromise between the required sensitivity, speed and resolution. The sensitivity ofthe detector dictates the minimum concentration of the separands in their zones. Generally, it is recommended to keep this concentration two orders of magnitude below the concentration of the BGE to avoid electromigration dispersion [ 8 11. By increasing the concentration of the BGE, the concentration in zones can also be higher and therefore the dynamic range of detection is increased. However, two facts must be kept in mind when increasing the BGE concentration. First, the detector response has limited linearity when using a circular detection cell (the on-column detection) and second, at high BGE conductivity, adverse effects of the Joule heat are to be expected [821. For these reasons, when detector sensitivity is insufficient, the composition of the BGE should be such that the mobility of the background-forming co-ion is close to the mobility of the separated ions [ 4l].When that is the case, the electromigration dispersion can be kept low even when the concentration in zones is close to the concentration of the BGE-forming co-ion.
hlectrophoresry 1990. 11, 661-664
6 Applications Capillary electrophoresis is well suited for both physicochemical measurements and analytical purposes. The primer may be evidenced by the measurement of physicochemical data such as ionic mobilities and/or diffusion coefficients [831 as well as by the measurements for the characterization and control of the electroosmotic flow 184-871. The analytical potential of C Z E may be demonstrated by the fact that capillary electrophoresis can easily compete with HPLC in many application areas, such as rapid and efficient separations of inorganic ions 188-901, pharmaceuticals [9 1, 921, polyamines 1931 and of optical isomers [941.The potential of CZE for the high resolution separation of polar and high molecular weight fuel-related materials was also demonstrated recently 1951. However, the main area of C Z E application is expected to be in the analysis of high molecular weight compounds such as peptides, proteins and polynucleotides, where, in principle, electrophoresis should be superior to other separation methods [96-1041. Here, the low sample volume for the analysis makes capillary electrophoresis an excellent analytical step, e.g., for peptide mapping after enzymatic digestion of a minute amount of sample protein [loll. The use of C Z E in oligonucleotide separations was already mentioned; great activity is expected in this field in connection with the human genome mapping project. Received March 30, 1990
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