Capillary electrophoresis - ACS Publications - American Chemical

Anal. Chem. 1992, 64, 389 R-407 R (P70) Andersen, S. I.Birdi, ; K. S. hog. ColkM Po/ym. Sci. 1990, 82 (Surfactants Macromoi.: Seif-Assem. Interfaces Bulk), 52-61. (P71) Pirkle, W. H.; Readnour, R. S. Anal. Chem. 1991, 63, 16-20. (P72) Rodionov, V. N.; Chernyaev, B. V.; Verpovskii, N. S.; Vodicka, L.; Zh. Anal. Khim. 1990, 45, 1791-7 (Russ); Chem. Abstr. 1991, 774, 41990~. (P73) Veciana, J.; Crespo, M. I.Angew. Chem., Znt. Ed. Engl. 1991, 30, 74-6. (P74) Serra, M. A.; Aviles, F. X.; Giralt, E.; Cuchillo, C. M. J. Chromafogr. 1989, 479, 27-37. (P75) Preston, J. F., 111; Rice, J. D. Carbohydr. Res. 1991, 275, 137-45. (P76) Preston, J. F., 111; Rice, J. D.; Chow, M. C.; Brown, B. J. Carbohydr. Res. 1991, 275, 147-57. (P77) Berger, G.; Girault, G.; Galmiche, J. M. J. Liq. Chromatogr. 1990, 73, 4067-80. (P78) Giorgio, A. D. Comput. Appl. Biosci. 1990, 6, 395-8. (P79) Nakanishi, S.; Kase, H.; Matsuda, Y. Anal. Biochem. 1991, 795, 3 13-1 8. (P80) Tsygankov, A. Yu.; Motorin, Yu. A.; Vol'fson, A. D.; Kirpotin, D. B. J. Biochem. Biophys. Methods 1990, 27, 145-53. (P81) Doadrlo, A. L.; Sotelo, J. An. R. Acad. Farm. 1989, 55, 203-12 (Span); Chem. Abstr. 1990, 172, 1648419. (P82) Doadrio, A. L.; Iribarren, M. An. R . Acad. Farm. 1990, 56, 9-18 (Span); Chem. Abstr. 1991, 774, 234933f.

(P83) Doadrio, A. L.; Iribarren, M. An. R. Acad. Farm. 1990, 56,367-74 (Span); Chem. Abstr. 1991, 774; 1924239. (P84) Feng, M.; Huang, W.; Weng, X.; Zhao, J.; Shi, Y. Sepu 1989, 7, 330-4 (Ch); Chem. Abstr. 1990, 772, 164838m. (P85) Klimes, J.; Zahradnicek, M. Folia Pharm. Univ. Carol. 1988, 17, 41-55 (Ger); Chem. Abstr. 1991, 774, 41861~. (P86) Fortier, G.; Bernier, N.; Bates, D. A. Anal. Left. 1991, 24, 961-70. (P87) Wood, M. J.; Irwin, W. J.; Scott, D. K. J. Clin. Pharm. Ther. 1990, 75, 291-300. (P88) Ulvi, V.; Tammilehto, S. J. Chromatogr. 1990, 507, 151-6. (P89) Brandsteterova, E.; Kiss, F.; Miertus, S.; Garaj. J. Mikrochim. Acta 1990, 3, 11-19. (P90) Manan, F.; Guevara, L. V.; Ryley, J. J. Micronutr. Anal. 1990, 7, 349-55. (P91) Lee, K. C.; DeLuca, P. P. J. Chromatogr. 1991, 555, 73-80. (P92) Pesek, C.A.; Warthesen, J. J.; Taoukis, P. S. J. Agric. Food Chem. 1990, 38, 41-5. (P93) Yang, S. K.; Lu, X. L. J. "n. Sci. 1989, 78, 789-95. (P94) Fellegvari, I.;Visy, J.; Valko, K.; Lang, T.; Simonyi, M. J. Llq. Chromatogr. 1989, 72, 2719-32. (P95) Mannschreck, A.; Kiessi, L. Chromatographh 1989, 28, 263-6. (P96) Rahn, R. 0. Anal. Chim. Acta 1991, 248, 595-602. (P97) Chen, R.; Deng, G.; Jiang, Q.; Mu, R. Wuli Huaxue Xmbao 1989, 5, 626-9 (Ch); Chem. Abstr. 1990, 772, 26250f.

Capillary Electrophoresis Werner G. Kuhr* and Curtis A. Monnig* Department of Chemistry, University of California, Riverside, California 92521-0403

INTRODUCTION This is the second fundamental review of capillary electrophoresis (CE). The focus of this review will center on the work in electrophoresis which has contributed to the development of this technique as an instrumental method. Only documents published in the period from January 1990 through December 1991 will be examined here. While a significant effort was made to be comprehensive, several areas were deliberately omitted. Traditional electrophoretic methods, as are commonly used in most biochemical analyses, will not be discussed. Likewise, the historical development of CE as an analytical method will be omitted. Related advances in other electrophoretictechniques, such as isotachophoresis,isoelectric focusing and electrophoresis in gel-stabilized media will only be included if they directly impact the optimization of capillary electrophoretic methods.

Werner G. Kuhr (left) is Assistant Professor of Chemistry at the University of California, Riverside, CA. He received his B.S. degree in Chemistry from Stevens Institute of Technology in 1980. After working in the pharmaceutical industry for 2 years and completing an M.S. degree at Stevens in 1982, he returned to graduate school to earn a Ph.D. in analytical chemistry at Indiana University in 1986. He was awarded a NATO Postdoctoral Fellowship to study in the Department of Biological Psychiatry, University of Groningen, The Netherlands, in 1986 and 1987, and then an Ames Lab Postdoctoral Fellowship in Analytical Chemistry in 1988. Dr. Kuhr assumed his current position at the University of California in 1988. His current research is focused on the development of microchemical techniques for in vivo, real time measurement of chemical dynamics in the mammalian brain.

BOOKS AND REVIEWS Capillary electrophoresis continues to be a popular topic for review, with almost 80 reviews appearing in the last 2 years. The first fundamental review in this series covered the developments in the field from 1981 to 1989 ( I ) . A number of fairly extensive, general overviews of the field have appeared in the last 2 years. Terabe published an extensive review on basic principles (2),Chen and Zhu summarized the history and recent development of capillary zone electrophoresis (3), while Foret and Bocek provided a brief overview of the present state of the art (4). Goodall et al. published extensive reviews which discussed applications of complexation and inclusion methods, gel-based separations for protein and peptides, and purification of oligonucleotides on a micropreparative scale (5, 61, Wamright reviewed small molecule separations in uncoated and coated capillaries (7). Schombur reviewed polymer coatings for CE and chromatography (87. Gareil had an extensive review of basic principles of CE of small molecules (9, 10). A number of reviews were published in trade journals, discussingvarious aspects of CE and its applications (11-31). Many other short reviews emphasized the power and utility of the technique (32-50), while others reviewed the progress presented at various symposia featuring CE (51-56). Several reviews compared separations in HPLC with those in CE (57, 58). A scientometric review by Braun and Nagydiosi discussed 0003-2700/92/0364-389R.$lO.OO/O

Curtis A. Monnig (right) is Assistant Professor of Chemistry at the University of California, Riverside. He received his B.S. degree in biochemistry and a M.S. degree in analytical chemistry from the University of Missouri in 1982 and 1984, respectively. I n 1989, he earned a Ph.D. in analytical chemistry at Indiana University. After graduating, he continued his studies as a NSF Postdoctoral Fellow at the University of North Carolina, Chapel Hill. I n 1990, he moved to his current position at the University of California. Dr. Monnig's research interests are primarily concerned with developing microscale analytical tools for monitoring biochemical events associated with fertilization and early embryonic development.

the growth of CE in the last decade (59). There were also many reviews which emphasized the separation of large biomolecules. A significant fraction of these reviews were concerned with the analysis of peptides and proteins (26,6046). Aguilar reviewed the theory, practice, and applications of capillary electrophoresis (67). Guzman reviewed the use of multiple detectors, micropreparative operation and automation in the analysis of proteins, peptides, and amino acids (68). Kasicka and Prusik had an extensive 0

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review of capillary isotachophoresis (ITP)in pe tide analysis (69). Novotny et al. discussed protein, pepti&, and amino acid separations (70). Recent advances in suppressing electroosmotic flow and irreversible adsorption of proteins at the capillary wall were reviewed. Detection aspects emphasized the role of laser-induced fluorescence and capillary electrophoreais/mass spectrometry in high-sensitivity measurements. Novotny also considered microcolumn LC and CE methods that allow isolation of biolo ical macromolecules a t subnanogram levels. Included in tiis discussion were examples of amino acid, peptide, and oli onucleotide separations (71). Issaq et al. examined the simikities and differences between HPLC and CE (72). CE was found superior whenever high ak capacity is required (i.e., the analysis of DNA fragments). renz and Hancock reviewed the ap lication of CE to recombinant DNA methods for the profIuction of therapeutic proteins (73). Turner reviewed the separation of DNA seuencing fragments, carbohydratea, and proteins (74). Mazzeo iscussed coated columns for protein se arations (75) while Landers (76) and Strickland (77) provi&d more general reviews. Several reviews presented the basic concepts of micellar electrokinetic chromatography (78-81). Final1 , a number of authors summarized the progress of various Jetection methods for CE. Yeung reviewed new approaches for absorption detection in capillary columns (82), while Yeung and Kuhr reviewed the general principles of indirect detection in CE (83). Jandik and Jones summarized the parameters relevant to the optimization of detection sensitivity in capillary electrophoresis, emphasizing indirect photometry of anions (84). Lmasaka reviewed the use of lasers for detection (85). Curry et al. discussed electrochemical detection for CE (86),while Pentoney reviewed radioisotope detection (87). Deyl and Struzinsky provided an overview of CE detection strategies, emphasizing CE-mass spectrometry and the cou ling of electrophoretic and chromatographic procedures &2). Huang et al. reviewed some important fundamentals of atmospheric pressure ionization mass spectrometr , including coupling technology for interfacing LC, SFC, C I, and ion chromatography with mass spectrometry (42,88). Several other authors provided general overviews of CE-MS (89, 90).

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FUNDAMENTALS The past 2 years have been characterized by an improvement in our understanding of the fundamental processes which influence electrophoretic separation. Beckers et al. measured ion mobilities in two different electrol systems at different pH values. These data allowed the a solute ionic mobilities and pK, values for the analyte to be calculated (91). If an ion with a high effective mobility is present in the sample at a very high concentration, other sample components can migrate in an isotachophoretic mode. Beckers and Everaerts described under what conditions this effect can be observed (92) and present a model and computer programs to calculate the parameters (e.g., specific zone resistance, response values, etc.) of the different zones (93). Issaq et al. investi ated the effect of the buffer type and concentration on a n i @ mobility, selectivity, and resolution (94,951. Low-conductivity buffers, higher buffer concentrations, and or higher o rating vol e8 were found to improve column e ficiency angesolution. he separation factor was directly related to analysis time, and therefore, selectivity improved with increasing buffer concentration but decreased with increas' applied voltage. Jones and Jandik established a correlatioxetween ionic e uivalent conductance and analyte mobility for CE (96). Itamna et al. investigated the influence of the buffer cation on mobility, resolution, and selectivity of the material being separated (97). Rasmussen and McNair demonstrated that the relative velocity difference of two zones increased with increasing buffer concentration but remained constant for a given concentration regardless of electrical field strength (98).They also noted that increased dispersion due to heating from the increased buffer concentration counteracted an gains derived from the enhanced velocity differences. &ren utilized substituted methylyridines to investigate the theoretical and experimental rektionships between pH and separation in free solution capillar electrophoresis (99). Rickard and co-workers determinei that the mobility for a group of 33 eptides and 10 proteins was proportional to the charge on t1e molecule and

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ANALYTICAL CHEMISTRY, VOL. 64, NO. 12, JUNE 15, 1992

the molecular weight of the species raised to the -2/3 power (100).

Smith et al. (101) examined the reproducibility of retention time and mobility of a seven-component test mixture under ng conditions. The solutions used to rinse the capillary L T t h e frequency of this rinsing were found to have the greatest effect on migration reproducibility. Lee and Yeung (102) developed a migration index and an adjusted migration index to allow the comparison of migration data obtained under changing conditions. The performances of the indexes, migration time, relative migration, and electrz:% mobility were compared, and the relative merits and drawbacks of each were discussed. Modeling. A semiempirical procedure for modeling the electrophoretic mobility of proteins in free solution was presented by Compton et al. This model uses the DebyeHueckleHenry the0 and Henderson-Hasselbalch equation to predict protein mgility (103). Furthermore, determining a protein's mobility at one pH allows calculation of its mobility at other pH values (104). Dose and Guiochon describe computer simulations of CE and isotachophoresis experiments (105). Empirically determined peak parameters match the shapes, areas, and widths predicted with this model. Gas and mworkers derived equations that describe the electrophoretic migration of monovalent ionic substances in solution containing a significant concentration of H+ or OH- (106). Datta employed a model for the electrokinetic dispersion to explore theoretical performance of CE in terms of plate height, plate number, peak resolution, resolving power, and the time of analysis (107). In a collaborative effort with Kotamarthi, Datta considered the merits of superim osing a Poiseuille flow on the natural electroosmotic flow of &e instrument (108). Under some conditions, this was found to lower dispersion. Atamna et al. formulated expressions to predict solute migration time and resolution as a function of applied voltage and buffer concentration (109). H'erten developed equations to describe the observed zone widti as a function of the width of the starting zone and of the zone broadening caused by diffusion, Joule heat, adsorption, and the difference in conductivity between a solute zone and the surrounding buffer (110). The desi of multibuffer systems for sam le stacking capillary were also discussed. tinther and at the head of Soeeberg developed a mathematical model to quantitatively describe the dispersion processes in free solution CE under both stackin and nonstackin conditions (111). Demana et al. considerefithe effect of inaiequate focusing of the analyte at the head of the column, inadequate buffer concentration, and analyte adsorption/desorption kinetics on zone shapes (112). Terabe et al. reversed the electrophoresis voltage several times during a run to cycle a sample zone between two oncolumn detedors. The temporal evolution of the zone allowed reversible and irreversible zone-broadening mechanisms to be distinguished (113). Jones et al. proposed a theoretical and ex erimental approach for isolating time-independent contrigutions to band spread (e.g., injection and detection) from the time-de ndent contributions (molecular diffusion, Joule heating angonideal electroosmotic plu flow) (114). Model systems consisting of amino acids, pepti es, and proteins was used to test the applicability of these methods. Jandik and co-workers developed a definition of optimum conditions for ionic analysis by CE and demonstrate that highly efficient ( N > 500 OOO) separations of complex mixtures of both anions and cations can be obtained using such optimized analysis conditions (115). Kenndler and Schwer demonstrated that the plate height in CE is dependent exclusively on the charge number of the individual analyte, but not on p and D in the absence of electroosmotic flow (116). Kenndler compared the information content (i.e., number of resolvable peaks) of CE with isotachophoresis (117). The information content of CZE is was found to be slightly better than the information content of isotachophoresis. Kenndler and Gassner employed cluster analysis to determine the o timum buffer solution as a solvent for buffering electrolytes f; 18). Methanolic buffer solutions were found to be useful for the separation of many sam les Grushka derived an expression for the late height in cap& electrophoresis for the case when hygostatic flow is present

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(119).

Dose and Guiochon considered the problems associated with electrokinetic sample introduction and developed a method


of internal standardization to correct for these deficiencies (120). The use of two internal standards allows the analyst to uantitatively correct for differences in sampling efficiency an] to account for sample-to-sample injection variations. For isotachophoretic analysis, Ackermans et al. determined that an internal standard helped correct for fluctuations in the electroosmotic flow and irre roducible injections (121). Improved results were obtained gy suppressing the electroosmotic flow with additives such as methylhydroxyethylcellulose. Thermal Effects. The influence of heat dissipation and temperature on electrophoretic separations remains an area of active investigation. Davis developed a numerical algorithm to calculate the radial profiles of temperature and ion mobility in an electrophoretic ca illary from a steady-state equation of heat conduction (1227. A calculation of plate height employing this theory suggests significant zone broadenin only occurs when the difference in temperature between the capillary center and wall exceeds 5 OC. Kurosu et d. investigated the influence of capillary tem erature on migration time and eak area (123). For appliec?voltages less than or equal 15 EV, stabilization of the capillary temperature improved peak area reproducibility but had a lesser effect on migration time. Kobayashi et al. coated a capillary tube with conductive materials (e. ., Al) to stabilize the temperature along the capillary surface (124). This coating allowed separations to be performed in a shorter period of time with improved reproducibility. Guttman and Cooke studied the effect of tem rature on the separation of DNA restriction fragments ( 1 g Under a constant electric field, the migration time and resolution decrease as temperature increased from 20 to 50 OC. Under constant-current conditions, increasing the column tem erature resulted in a maximum migration time for all f r a yents and provided maximum resolution to the lower mo ecular weight fragments (lo). The mass detection limit of fructose was 2 fmol in a 5-pm4.d. capillary. Gross and Yeun (279) used indirect fluorescence for detect' cations separate! by CE. In contrast to previous work w% anions, quinine cations were found to adsorb to the capillary wall. The effect of adsorption on resolution and reproducibility is discussed. Hogan and Yeung (280) used indirect fluorescence detection for the analysis of trace quantities of macromolecular mixtures. Subfemtomolar quantities of tryptic digest mixtures were separated within 3 min. Mass limits of detection were a factor of 180 lower than those of UV absorbance detectors. Grant and Steuer employed axial illumination with indirect fluorescence detection to determine the absorbance within a given length of capillary (281). Although the authors reported a considerable increase in absorbance si al com ared with the usual transcolumn UV absorption teckque, tfeir results were limited b the stability of the li ht source. Amankwa and Kuhr (282fdemonstrated the utigty of indirect fluorescence detection of neutral analytes separated b MECC using quinine sulfate as the visualizing agent. $he detection mechanism involves a combination of displacement of the fluorophorefrom the micelle by the analytea and net reduction of the quantum efficiency of the fluorophore in the sample zone. The selectivity of this detection technique is demonstrated with data showing the separation of several aliphatic alcohols and some phenolic compounds. The effect of quenching by the analyte on the sensitivity of detection was also discussed.

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APPLICATIONS Cyclodextrin-Modified MECC. Terabe et al. (283) roposed the idea of cyclodextrin-modified MECC (CD-ME&) in which CD is added to the micellar solution for the sepal ration of neutral, highly hydrophobic compounds. Waterinsoluble hydro hobic solutes are partitioned between the micelle and the ED cavity. The CD cavities and the micelles have different mi ation velocities and so the migration time through the cap& depends on the relative partitioning between these two pgases. Hi hly hydrophobic and closely related compounds such as chforinated benzene congeners, 3981 ANALYTICAL CHEMISTRY, VOL. 64, NO. 12, JUNE 15, 1992

polychlorinated bi henyl congeners, tetrachlorodibenzo-pdioxin isomers an$ polycyclic aromatic h drocarbons were successfully separated by this method. &ishi et al. (284) performed chiral separations of several drug molecules with CD-MECC. Chiral recognition depended on the type of CD; in particular, y-CD was effective for this purpose. The addition of an or anic solvent or a chiral compound such as sodium D-campior-10-sulfonate or Z-menthox acetic acid to the solution improved the enantioselectivity. biyashita and Terabe (285) used CD-MECC with cyclodextrin to separate racemic dansyl-amino acids. Nine of twelve DL-amino acid derivatives were resolved. Nishi and Matsuo (286) used MECC with cyclodextrins additives to improve the resolution of several lipophilic compounds. Imasaka et al. (287) separated two polycyclic aromatic com unds, 1-aminoanthracene and benzo(a)pyrene, by CD-MEG and detected each species by fluorescence stimulated with a blue semiconductor laser. The concentration detection limit for 1-aminoanthracene was approximately M. Ong et al. (288)separated a mixture of seven water- and two fat-soluble vitamins using MECC and y-cyclodextrin, 8-cyclodextrin, and isopropanol. Ueda et al. (289) separated the DL forms of NDA-derivatized amino acids in the presence of cyclodextrin. The extent and order of separation of the CBI-DL-aminoacids was dependent on the type of cyclodextrin present and the amino acid structure. Fenali separated terbutaline and propranolol by CD-MECC (290). Lee et al. (291) used MECC to separate both normal and modified dansylated nucleotides. The high separation power allows detection of minor components present in less than 1 part per thousand of the major components. Laserexcited fluorescence was used to detect the separated components at attomole levels (nanomolar concentrations). Ong et al. (292) used a photodiode-array UV-visible detector for on-column detection in a MECC system. The separation of phthalate esters and priority pollutants in different concentrations of SDS and at different pH values was investigated. Inorganic Ions. Romano et al. (293) discussed the parameters which influence CE separations of inor anic ions, including choice of electrolyte anion, electrolyte p k , and the addition of an electroosmotic flow modifier. Optimized conditions were established for the separation of inorganic anions, organic acids and alkylsulfonates and the technique was applied to the determination of a variety of anionic solutes in several complex sample matrixes (e.g., Kraft black liquor). Several authors have used complexation to enhance either the separation or the detection of inorganic ions. Foret et al. (294) separated 14 lanthanides by CE in a background electrolyte containing hydroxyisobutyric acid as a complexing counterion and creatinine as a W absorbing mion for indirect detection. Com lete separation of the lanthanides was achieved in less &an 5 min. Fukushi and Hiro studied the effects of crown ethers (295) and a-,8- and y-cyclodextrins (296) on the effective mobilities of various metal ions in capillary ITP. Hirokawa et al. separated 20 metal ions by ITP and the zones were fractionated and analyzed off-line by particle-induced X-ray emission. The recovery, migration order, and separation efficiency were studied as a function the concentration of a complexing egent, a-hydroxyisobutyric acid. The recovery of the metal cations was 100% with both electrolyte systems, exce t for Fe(I1) and Zr"0 (297). Krib to determine Fe(III)-EDTA and vankova and Bocek used I free EDTA in a scrubbing liquid for desulfurization of waste gasea from brown-coal gasification (298). Prosser and Bulman used ITP to demonstrate that Nb(V) can exist in a variet of anionic complexes (299). Tanaka et al. used capillary IT% in similar se arations (300). Swaile and Sepaniak (301) used laser-excite fluorescence to detect metal cations after complexation with t2-hydroxyquinoline-5-sulfonicacid (HQS-). By controlling mobile-phase parameters that affect the complexation reaction, the observed electro horetic mobility of the metal was manipulated. Ca(II), M g h , and Zn(1I) were detected at ppb levels and in complex sample matrices (Le., blood serum). Saitoh et al. (302) separated metal chelates in SDS micellar solution with a,~,y,6-tetrakis(4-carboxyphenyl)porphine as chelating reagent. The method shows great romise for the ultratrace detection of metal chelates (303). fiord et al. used MECC to determine the solute-micelle bindin constants (Kmw)of different compounds (304). Saitoh et af (305)separated neutral complexes of chromium(III), cobalt(III), rho-

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dium(III), and platinum(I1) (or palladium(I1))by MECC. The distribution coefficient of each metal complex was calculated from the capacit factor. The linear log-log relationship between the distriiution and the partition coefficient was used for prediction of both the distribution coefficients and the migration times of other metal complexes, such as alladium(I1) acetylacetonate and chromium(II1) 3-methyracetylacetonate. Organic Acids. Several authors used derivatized amino acids to characterize their systems. Waldron et al. used fluorescence detection of FITC and dimethylaminoazobenzene (DAAB) derivatives of amino acids and demonstrated much higher sensitivi for the fluorescein derivative mol) than for the DAAB erivative (10-l6 mol) (229). Nielen et al. used positional isomers of substituted benzoic acids as model compounds to study the affect of system parameters (i.e., pH, electrolyte, ionic strength, addition of alcohols, counterion, and temperature) on the electrophoretic separation. pH was again found to be the most effective parameter in optimizin resolution. However, thermostating of the capillary at elevatei temperatures increased resolution and decrease analysis time (306). Nishi et al. (307) resolved isomers of 2,3,4,6-tetra-Oacetyl-&D-g~ucopyranosy~ isothiocyanate (GITC) derivatized D L - U ~ ~ Oacids using micellar electrokinetic chromatography ~ O optical resolution with SDS solutions. Of 21 D L - ~ acids, was achieved in 19 with neutral and alkaline conditions; aspartic acid and glutamic acid have an additional carboxyl group. Simultaneous resolution of more than 10 GITC-derivatized DL-amino acids was achieved within 40 min in 0.2 M SDS. Tanaka et al. used cyclodextrin derivatives to separate dansyl-amino acids by CE (308). Karovicova et al. (309) determined formic, acetic, sorbic, and benzoic acids in mustards, jams, and s ps by dissolving in water, adjusting the pH to 10, and p e c i n g ITP analysis. Foret et al. separated triazine herbicides and their solvolytic products by CE in mixed water-ethanol buffers (310). Jokl and Petrzelkova separated propranolol metipranolol(I), and desacetylmetipranolol(I1) with ITP (311). Karovicova et al. determined nitrates in nine vegetables by ITP (312). Kenney determined several organic acids in food samples (313). Kopacek et al. (314)separated complex mixtures of humic substances by ITP after the addition of polyvinyl yrrolidone (PVP) to the leading electrolyte. Interaction of P P with humic substances was found to differentiate humic and fulvic acids. Watarai devel0 ed a variation of MECC which used oil-in-water microemukons as the electrophoretic media for the separation of ionic and nonionic samples containing aromatic compounds (315). On et al. (316) separated 15 dansyl (Dns)-amino acids by ME&. All the amino acids were separated within 26 min in a 40 mM SDS/phosphakborate buffer ( H 7.56). Otauka et al. used sodium N-dodecanoyl-L-valinate&DVal) for chiral separations of PTH derivatives of six DL-amino acids (serine, a-aminobutyric acid, norvaline, valine, tryptophan, and norleucine) (317). Otsuka and Terabe (318) examined the effects of methanol and urea on enantiomeric resolution of PTH-DL-amino acids by MECC with sodium N-dodecanoylL-valinate (SDVal). A mixture of four PTH-mamino acids were separated and each pair of enantiomers was also optically resolved. Otauka and Terabe (319) obtained enantiomeric resolution of PTH-amino acids by MECC using a chiral surfactant (di 'tonin) and an anionic chiral surfactant, sodium N-dodecanoyk-valinate under neutral conditions. Chadwick and Hsieh (320)used CE to separate cis and trans double-bond isomers of ionic species. The separation of fumaric acid and maleic acid in both the dianion and monoanion forms was demonstrated. Because these two isomers carry the same charge under the basic conditions of the running buffer, the separation is attributed to the difference in their hydrodynamic radii, and measurement of the electrophoretic mobilities allowed for quantitation of this difference. All-trans-retinoic acid and 13-cis-retinoic acid were also baseline separated. Karovicova et al. used capillary ITP to determine citric, malic, oxalic, lactic, and ascorbic acids in fruit homogenates (321)and or anic acids and H3P04during lactic fermentation of cabbage 8urin sauerkraut manufacture and durin the maturation of r e f wine (322). Krivankpva-and Bocek 7323) used an ITP method for the determination of pyruvate, acetoacetate, lactate, and 3-hydroxybutyratein 1-10 pL of the untreated heparin plasma of patients with diabetes mellitus.

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Lutonska found that the determination of several organic acids (lactic, acetic, and butyric) on silages by capillary ITP was more accurate than an officially reco nized classical method (324). Vindevogel et al. found that f!E and MECC, where all possible sources of metal ions (silica gel, injector, frits) were excluded, allowed the separation of hop bitter acids in beer production (325). Oefner et al. evaluated the operating conditions for the isotachophoretic separation of organic acids (326). The time of analysis was observed to be a function of the concentration of the leading electrolyte. Hernandez et al. (327) used CE for the se aration and characterization of CAMP,cGMP, and cIMP. fackim and Norwood (328)separated a benzo(a)pyreneGMP adduct from normal nucleotides and detected via UV absorption. Row and Raw examined the effect of isopropanol as a organic modifier in CE in terms of resolution using the four deoxyribonucleotides (329). Carbohydrates. Liu et al. (330) described the use of 3(4-carboxybemyl)-2-quinolinecarboddehyde as a precolumn derivatization agent for amino sugm analysis. This procedure was used to analyze amino sugars in various biological mixtures. Low-attomole (10-ls mol) limits of detection were reported for laser-induced fluorescence detection. This procedure was found to be useful for the analysis of reducing monosaccharides and oligosaccharides after transformation to the amino sugar via reductive amination (331). Under optimized conditions, the minimum detectable quantities for monosaccharide solutes was determined to be at low-attomole levels (0.5 am01 for the CBCA derivative of galactose). Complex oligosaccharides, isolated from bovine fetuin by hydrazinol sis, were successfully mapped using this procedure. Honia et al. derivatized various aldoses to their 3-methyl-lphenyl-2-pyrazolin-5-one derivatives for UV detection and separated as borate complexes (332). This system also provided good separation of derivatives of homolo ous oligoglucans having different interglycosidic linkages. m e r and Yeung (278) used indirect fluorescence detection and CE to detect mixtures of monosaccharides. The mass detection limit of fructose was 2 fmol using a 5-pm-i.d. capillary with an efficiency of >600000 theoretical plates. Deyl et al. (333)used CE to separate the roducta arising from the Maillard reaction of free amino aci& (glycine, alanine, and isoleucine) with aldehydic sugars (glucose or ribose). The products of this reaction were separated without derivatization (W detection at 220 nm), as phenylthiocarbamyl derivatives, and after derivatization with 2,4-dinitrophenylhydrazine.These separations were compared with those obtained by high-performance liquid and thin-layer chromatography. Honda et al. (3%) analyzed 1-phen 1-3-methyl-5pyrazolone (PMP) derivatives of reducing carbo ydrates by CE with W detection using an electrolyte containing alkaline-earth metal salts. The PMP derivatives of isomeric aldopentoses were completely separated from each other by the interaction with these metal ions. The order of mobility for these derivatiyes was different from that observed in borate buffer, suggesting formation of different types of complexes. The extension of this procedure to the analysis of other monosaccharides and several oligosaccharides was also discussed. A1 Hakim and Linhardt used CE for the analysis of non-, mono:, di-, and trisulfated disaccharides derived from chondroitin sulfate, dermatan sulfate, and hyaluronic acid (335). Quantitation of disaccharides derived from chondroitin sulfate using chondroitin ABC lyase and mixtures of unsaturated disaccharide standards required only picogram uantities of sample. Ampofo et al. (336) separated eight &saccharide standards prepared from heparin, heparan sulfate, and derivatized heparins. Two of the standard heparin heparan sulfate disaccharides, having an identical charge o -2, were not fully resolved in the sodium borate/boric acid buffer. The structure and purity of each of the eight disaccharides were confirmed usin fast-atom-bombardment mass spectrometry and high-field NMR spectroscopy. Heparin and heparan sulfate were then depolymerized using heparainase, heparin 1 ase 11, heparinitase, and a combination of all three enzymes. 8E analysis of the products provided the disaccharide composition of each glycosaminoglycan. Other Small Organic Molecules. Kenndler et al. (337) used CE for the determination of impurities in riboflavin 5'-phosphate (I). Tanaka et al. (338) se arated the niacin derivatives of weak bases by capillary I&. Yik et al. used electrochemical detection with CE for a mixture of BBvitamins

8

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on a 50-pm4.d. column (270). A calibration plot was linear over 2 orders of magnitude with a lower limit of detection of approximately 4 fmol. Zweigenbaum se arated anionic components of Triton 770 b CE and ME& (339). Stover reported ap lications of C 6 developed for detergent, food additive, herEicide, animal nutrition, and biotechnology samples (340). Wainright reviewed small-molecule separations in uncoated and coated capillaries (7). Yik et al. investigated the application of CE to the analysis of environmental pollutants (341). The se aration of selected groups of compounds, i n c l u T b p h e n o l s , r l y c y c l i c aromatic hydrocarbons, and p thalate esters were iscussed. The effects of relevant parameters, such as pH of the electro horetic media, surfactant concentration, capillary length, a n i applied voltage on separation efficiency were also considered. Northrup et al. used MECC for separation and detection of organic gunshot and explosive constituents (342). Twenty-six of these constituents were separated in less than 10 min with efficiencies in excess of 200000 plates. The presence of gunshot residues in spent ammunition casings and the composition of six reloading powders and four plastic explosives were determined. Se aniak et al. (343)examined the role of the mobile phase in C# and MECC for determining separation performance. The influences of ionic salt, surfactant, and organic solvent mobile-phase additives on se aration efficiency, retention, and elution ran e were discussetand demonstrated. Ghowsi and Gale (344)%iscussedCE with smaller capillary diameters for use as chemical sensors, electrokinetic field devices based on capillary field effect electroosmosis, and potential ap lication for se aration-based sensors. Takigiku and Schneifer (345) used 8 E for the separation and quantitation of ribonucleoside and deoxyribonucleoside triphosphates. Capillaries were treated to reduce electroosmotic flow and capillary zone electrophoresis was performed with negative voltage. Karovicova and Polonsky analyzed colorants by capillary isotachophoresis (346). Cole et al. (347) optimized binaphthyl enantiomer separation by CE. Bile salts were used in the mobile phases instead of conventional sodium dodecyl sulfate to provide a lower k’ value and an optimal resolution for moderately hydrophobic compounds. Fanali and Bocek (348) separated enantiomers of tryptophan by CE, using a-cyclodextrin as a chiral active component in the background electrolyte. The separation of (-) and (+)-epinephrine was achieved by supplementing the background electrolyte with heptakis(2,6-di-O-methyl-j3cyclodextrin). As a practical a lication of the method, the quantitative analysis of (-) antP+) enantiomers in common pharmaceutical solutions of adrenaline is shown. Leopold and Gouesclou (349) separated chiral amino acids and peptides derivatized with Marfey’s reagent. Vindevogel et al. (350) identified beer bitter acids in a preisomerized hop extract by means of their UV spectra. Preliminary data for the analysis of these compounds by MECC in beer samples was presented. Lu et al. (352) used an ethanol-water micellar solution in MECC to separate several weak polar compounds. Nielen and Mensink (352) separated N-tert-butyl-2-benzothiazolesulfenamide (I) and N-cyclohex 1-2-benzothiazolesulfenamide(11) by MECC. Lahey and Qt.John (353) separated 14 nucleosides and nucleotides by MECC and compared the separation with ion-pair HPLC. Videvogel and Sandra (354)optimized the resolution of teatmterone esters in a buffer with an organic modifier. By using a Plackett-Burman design, the effects of five buffer parameters were investigated: the buffer pH, the buffer concentration, the level of organic modifier, the surfactant concentration, and the use of mixed micelles. Terabe et al. (355)added urea to a micellar solution for the separation of hydrophobic compounds by MECC. The logarithm of the capacity factor (k? decreased linearly with increasing concentration of urea. The use of a high concentration of urea allowed the MECC separation of hydrophobic compounds, which were mostly included in the micelle and could not be resolved by conventional MECC. The addition of urea also expanded the migration-time window and hence enhanced the resolution. Fu et al. used a mixed ethanol-water solvent in MECC to enhance separation of polycyclic aromatic hydrocarbons (356). Ong et al. (357) used MECC for the separation of 11 substituted phenols listed as priorit tants. The retention behavior of the phenols in ME&!%; 398R


related to their physicochemical properties. Rasmussen et al. (358) evaluated the use of sodium decyl sulfate and SDS as micellar phases for the MECC separation of ASTM test mixture LC-79-2. Des ite separation efficiencies of a proximately 3000 theoreticafplatea/cm, benzene and benzaliehyde coeluted with both surfactants. Separation was readily achieved by addition of Bri’ 35 to the micellar hase. Cole et al. (359)used bile salt sudactants in the MECe separation of various hydrophobic compounds. The effects of methanol on CMC was investigated for SDS and the bile salt sodium cholate. The bile salt micelle was much more tolerant of organic solvents than SDS, thereby increasing the scope of applications of MECC to include hydrophobic compounds. Cole et al. (360)then used bile salt surfactants in the MECC separation of polyaromatic hydrocarbons. Pharmaceutical Compounds. Salomon et al. (361) optimized the separation of seven tricyclic antidepressants (protriptyline, desipramine, nortriptyline, nordoxe in, imipramine, amitriptyline, and doxepin). Optimal resorution of this mixture was achieved by the addition of methanol to the buffer to decreased both the electroosmotic flow and the electrophoretic mobilities of the samples. Chmela and Stransky (362) described the isotachophoretic behavior and separation of similar drugs (amitriptyline, dosulepine, chlorpromazine, chlorprothixene,chlorothepin,levopromazine, nortriptyline, oxyprothepine, prochlorperzine, metipranolol, tranylcypromine) and their determination in pharmaceuticals (dosulepin, chlorpromazine, clorotepine). Snopek et al. used as a-, &, y-, and heptakis(2,6-di-O-methyl)-j3-cyclodextrin stereospecific selectors or electrol modifiers, both in capillary zone electrophoresis an isotachophoresis (363). Several model isomeric com unds (including optical isomers of pharmaceutical interestywere resolved. Soluble alkylhydroxyalkylcellulose derivatives were added to the cyclodextrin-modified background electrolytes, and their presence was found to improve enantioselectivity and separation efficiency. Ong et al. used CE with UV detection at 214 nm to separate a group of nine antihistamines (364). Altria and Smith used CE and MECC for the separation of the antidepressant GR50360A from potential manufact impurities (365). Arrowwood and Hoyt developed a m e 3 for the determination of cimetidine (the active ingredient in the ulcer medication Tagamet) in commercial preparations. Analysis of over 60 samples from common available formulations gave relative standard deviations of 1.9-6.4% (366). Kenndler et al. determined arbutin in a crude drug repaUV detection at 214 nm (367). Pietta et a f (368) % i % for% the determination % of flavonol 3-0-glycosides. These results were compared with those obtained by reversed-phase HPLC. Swedberg et al. (369)used nonionic and zwitterionic surfactants to enhance separations of desipramine/nortriptylene and angiotensin III/ValCangiotensin III. The surfactants are only effective at or above the critical micelle Concentration. Nishi and Terabe reviewed the principle, separation characteristics, and a plication of MECC to the analysis of pharmaceuticals (3707 Nishi et al. performed the chiral separation of a bronchodilator (Inolin) and three related compounds (371), diltiazem-HC1, trimeto uinol-HC1, and related compounds (372)by MECC using a ble salt as a chiral surfactant. Chiral recognition was affected b the structure of bile salts, pH, and the structure of the sorutes. Nishi et al. (373)separated corticosteroidsand benzothiazepin analogs by MECC with a bile salts micelle phase. MECC was also applied to the determination of the drug substances in tableta and cream and to wity testing of drug substances and tablets. Weinberger and Eurie (374) compared MECC to HPLC for the determination of illicit drug substances. For a complex mixture consisting of acidic and neutral impurities present in an heroin sample, MECC resolved at least twice as many peaks as HPLC. Altria and Rogan used MECC for determination of impuritiesin drugs,for resolution of closely related impurities, and for analysis of these species in urine (375). Miyashita et al. (376)separated various penicillins, cephalosporins and corticosteroids by MECC. Nishi et al. (377) separated aspoxicillin in human blood plasma by MECC. Plasma proteins, which mi ht interfere with drug analysis in conventional CE, were solu%ilized by the micelles and eluted later than the drugs. This permitted the determination of the drugs in plasma by a direct sample injection. Nishi et al.

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(378,379)examined 12 active ingredients used in cold medicines. The role of five anionic surfactants was investigated by MECC and the results were compared with those obtained by conventional CE. The relative retention order of the 12 ingredients was significantly different among the five surfactants; the different elution orders were ascribed to differences in the hydrophilic groups of the surfactants. Krivankova et al. (380)used a combination of capillary ITP and CE to determine the coccidiocidic drug halofuginone in feed concentrates. The high load ca acity of the isotachophoretic ste and high sensitivity of t i e zone electrophoretic step enable: analysis of up to 25 pL of sample solution conM halofu 'none with excellent reprotainin as little as ducibihy (1%RSD). Meier and hormann (381) uantitated thiopental in human serum and plasma by MECC. h e s e data were compared with e uivalent data obtained by HPLC. Steuer et al. com ared &e utility of HPLC, SFC, and CE for drug analysis. actors considered in this analysis included separation efficienc , performance, sensitivity, optimization parameters, methoBdevelopment time, sample preparation, technical difficulties, orthogonality of the information obtained ible application to various substance groups (382). Swartz iscussed various applications of CE to the Dharmaceutical lab (383). Lloyd et al. (384)developed a CE assa for the antileukemic agent cytosineb-Darabinoside (ara-C). golid- hase extraction and on-ca illary eak concentration are uselto improve the detection L i t . 8uzman et al. used CE for the determination of a recombinant cytokine in a pharmaceutical dosage form (385). Tsikas et al. developed capillary ITP methods for the analysis of 8-lactam antibiotics, i.e. penicillins and cephalosporins, in chemical and pharmaceutical preparations (386). A leading electrolyte with a pH of 7.0 allowed the sensitive detection of several 8-lactam antibiotics independent of the chemical structure of the side chain of the penicillin or cephalosporin nucleus. Yeo et al. reported the separation of six antibiotics by CE with UV photodiode-array detection (387). Clinical Analyses. Clinical applications of CE have own dramatically within the last 2 years. Analytes rangefirom simple metabolic acids to dru s and metabolites in both plasma and urine samples. Widman et al. analyzed oxalate, citrate and several inorganic anions (chloride, sulfate, nitrate, phosphate, and carbonate) as well as oxyanions of arsenic (i.e. arsenite and arsenate) in diluted urine (388). Atamna et al. developed a CE assay for the separation of xanthines and uric acid derivatives normally present in human plasma and urine as metabolites of caffeine. While the uric acid analogs could be se arated with free zone CE, the se aration of the methyl-sugstituted xanthines required ME& (389). Guzman et al. (390)described a quantitative UV detection method for the determination of several urinary metabolites. This method M for creatinine and was only linear between lo-* and between lo-' and 1.0 M urea. Masson et al. compared the CE assay of uric acid in urine analysis with enzymic assays that use urate oxidase (391). Determination of uric acid in reconstituted serum samples also 'elded results in satisfactory agreement with those obtainerby enz ic assays and the value rovided by the supplier. Weingger et al. demonstratel the separation of urinary porphyrins with MECC. They compared the relative sensitivities of absorbance and fluorescence detection, and found that only fluorescence methods had sufficient sensitivity to monitor porphyrins in clinical urine samples (392). Tomita et al. (393)used CE to quantitate gl hosate and its major metabolite, (aminomethyl)phosp!?kic acid (AMPA), in human serum. The two compounds, after derivatization with p-toluenesulfonyl chloride, were separated reproducibly with detection limits of 0.1 pg mL-I in spiked sera. Jellum et al. used CE in a multicomponent analysis system des' ed to diagnose metabolic disorders (394). Comparative a n g e s , using CE, HPLC, and an automated amino acid analyzer, were carried out on urine and blood samples from atienta with various deficiencies. The possible connection ktween deficiency of taurine (2-amino-1-ethanesulfonicacid) in the heart and the develo ment of cardiomyopathy and heart failure insteated the devegpment of a simple CE method for the determination of taurine in submilligram samples of bio ies of the myocardium. Schoota et al. separated organic a c i g accumulated in blood serum of patients with chronic

8

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renal failure with CE (395). Hippuric acid (HA), p hydroxyhippuric acid, and uric acid were identified by their coelution with standards prepared in ultrafiltered normal serum and by comparison with the correspondingW-detected peaks identified in the HPLC analyses. Tanaka and Thormann analyzed S-carboxymethyl-L-cysteineand some of its metabolites in human urine using CE and capillary ITP with on-column detection of underivatized solutes, minimal sample pretreatment, and capillary columns with minimized electroosmosis (396). Results obtained with coated fused-silica capillaries of 25-pm i.d. and Teflon capillaries of 500-pm i.d. were presented. Several papers describe the use of CE to monitor drugs and metabolites in clinical samples. De et al. described a CE method for the determination of 5-fluorouraciland metabolites in buffer solutions (397). Meier and Thormann uantitated thio ental in human serum and plasma with ME8C and the resu ts were compared with reversed-phase HPLC (381). Thormann et al. analyzed barbiturates in human serum (or plasma) and urine by CE-MECC with on-column fast-scanning multiwavelength detection (398). Seven barbiturates were characterized by their retention and absorption spectra between 195 and 320 nm and these compared to results from urine samples after extraction from the sample matrix. Comparison of these com uter-stored data with those of unknown samples allowed t i e identification of barbiturates in samples of patients undergoing pharmacotherapy and in toxicolo ical urine and serum s ecimens. Ling et al. (399) & p J a mixture of several thio[ of pharmacological interest after optimizing buffer pH and concentration, applied voltage, and loading conditions. Human whole blood samples were pretreated, derivatized and in'ected into the optimized CE s stem, which provided a satisiactory method for the specific &termination of glutathione in red blood cells. They also com ared micro-LC and CE for the determination of several thioi derivatized with the fluorogenic reagent SBD-F (400). The performance of the CE method was compared with that of capillary LC and hi h-performance TLC. Gurley et al. developed a CE methofto analyze the proteins in lung fluid (401).Proteins were precipitated from the fluid with acetone and concentrated in 2 mL of water/trifluoroacetic acid. Comparison of the CE fractions detected by UV absorption at 200 nm with similar fractions obtained by HPLC confiied albumin, transferrin, and IgG as three major proteins translocated into the alveolar space. Soini et al. (402)used an eledrochromatographicsolid-phase extraction and preconcentration method for the determination of cimetidine in serum in the concentration ran e 0.2-10 p mL-'. Preconcentrated samples were determine by MECC! Wernly and Thormann (403) separated common drugs of abuse and (or) their metabolites, including opioids, benzoylecgonine,amphetamines, and methaqualone by MECC. After solid-phase extraction of 5 mL of urine, drug concentrations down to about 100 n mL-' could be monitored. Peak assignment was achievef through comparison of retention times and absorption s ectra of eluting peaks with those of computer-stored mo el runs. Miyake et al. (404)separated creatinine and uric acid in human plasma and urine using MECC. The sample was introduced into the capillary by siphoning untreated plasma or urine spiked with an internal standard (antip ine). The results were in agreement with conventional methods. those obtained Catecholamines. Olefirowicz and Ewin used CE with electrochemical detection to separate and dietect attomole levels of neurotransmitter in picoliter volumes of cytoplasm withdrawn from single neurons of the pond snail, Planorbis corerus (405). This work demonstrates, for the f i t time, the direct detection of the cyto lasmic concentration of dopamine in single, intact neurons. $hey further optimized the use of CE with electrochemical detection by decreasing the inner diameter of the separation/sampling capillary to 2 pm (160). Sample volumes as low as 270 fL were in'ected into the electrophoresis ca illary with subattomole detection limits for easily oxidizefspecies. Sam ling of the cytoplasm is accomplished by inserting one encfof the electrophoresis capillary directly into a single nerve cell. The hi h-voltage end of the electrophoresis capillary was etched wit1 hydrofluoric acid to form a microinjector. Chien et al. compared results obtained with voltammetric electrodes and CE for dynamic and static monitoring of dopamine in the somal cytoplasm

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of the giant dopamine neuron of P.comeus (406).The current status of the authors’ microCE technique for the sampling and determination of neurotransmitters/catecholaminesin single nerve cells was reviewed by Olefirowicz and Ewing (407). Kaneta et al. (408)improved the resolution of catecholamines by controlling both electroosmotic and electrophoretic mobilities. The former was controlled by the addition of borate ion and a change in pH, resulting in a separation of 10 catecholamines. Ong et al. (409)investigated the migration behavior of selected catechols and catecholamines in MECC at different concentrations of micellar solutions and at different pH values for the electrophoretic media. The results succeesfully demonstrated the use of MECC for the separation of a mixture of two catechols and six catecholamines. Tanaka et al. (410)also separated various catecholamines using comlexation with boric acid. A short review by Lee and Heo Eoked at the detection of catechols by CE (47). Tanaka et al. performed an isotachophoretic separation of catecholamines based on inclusion complex formation with 0-cyclodextrin. Se arability was improved with increasing concentration of @-EDin the leading electrolyte. Six catecholamines were separated by using complex formation with 0-CD (411). Other Biological Samples. Guzman et al. had a brief symposium report describing the analysis of brain tissue constituents CE with laser-induced fluorescence detection (54). Further work in this area by Hernandez et al. (412)examined the effects of cocaine and two other local anesthetics applied directly into the nucleus accumbens for 20 min by diffusion from a 4-mm microdialysis robe in freely moving rats. Cocaine (7.3 mM) was fountf to increased the extracellular concentration of do amine (DA). Liu and Chan analyzed anglioside micellesy! CE in uncoated fused-silica capillaries $413). The mass sensitivity usin UV absorption (195 nm) was lo-” mol. Ganglioside micejes including GM1, GDlb, and GTlb were resolved into separate peaks by CE. Prolonged incubation caused the ganglioside peaks to merge into a single species. Nguyen (414)monitored domoic acid in mussel hom by CE. UV absorbance detection at 242 nm p r o v i d e r g tection limit in wet tissues of 10 ppm. Nguyen et al. also used CE to quantitate nucleotide degradation in fish tissues and to provide a basis for determination the K value, an indicator of fish freshness (415). The values obtained for the three compounds of interest, inosine monophosphate, inosine, and hypoxanthine, correlated very well with those obtained by enzymic assays. Thibault et al. (416)described a CE method with UV detection for the separation and determination of underivatized toxins associated with aralytic shellfish poisonin Confirmation of the electropIoretic peaks was facil i t a d by mass spectrometric detection using an ion-spray CE-MS interface and by HPLC with fluorescence detection. Mereish et al. (417)used CE and HPLC methods for the anal ais of palytoxin. The detection limit of the HPLC methoiwas 125 ng/injection, while the CE method was 0.5 pglinjection. Yeo et al. proposed the use of a systematic optimization scheme for the CE separation of nine plant owth regulators with a mixed carrier system consisting of t g e e cyclodextrin modifiers (418).The scheme utilizes the overlapping resolution mappin procedure, and interpretive optimization scheme, to precct the optimum cyclodextrin composition for the separation of the plant growth regulators. Tsuda et al. determined free polyamines in rat tissues by CE with fluorescence detection (419).After precipitation of proteins with perchloric acid, the sample solution was derivatized with fluorescamine using a microscale procedure. Ethylenediamine was added to the medium to avoid adsorption of polyamines onto the capillary wall. Matsumoto et al. (420)also described a CE method for polyamine analysis and compared it to HPLC. Huan et al. determined the nucleotide composition of base-hydrofyzed bulk RNA in 5 min by capillary zone electrophoresis with UV absorbance detection (421). Peptides and Proteins. The past 2 years have seen an explosion of papers utilizing CE for the se aration of com lex mixtures of peptides and proteins. Mi ler and co-wor!ers synthesized a 53-peptide and assayed its purity by capillary zone electrophoresis and mass spectrometry (422).Rivier et al. utilized capillary zone electrophoresis to determine the purity of a synthetic peptide mixture (423).CE was used to monitor the identity and purity of human growth hormone

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(hGH) by Nielsen and Rickard (424).The use of internal standards was found to significantly improve the precision of this measurement. These same authors also optimized the mobile-phase composition for the separation of tryptic digest fragments of human growth hormone by CE (425). Chen et al. separated the anticoagulant peptide from its deletion byproducts by free zone electrophoresis (426).Prusik et al. utilized CE and continuous free-flow zone electrophoresis to analyze and prepare pure fractions of synthetic growth hormone releasin peptide (GHRP) (427). Guarino and Phillips used capifary electrophoresis to assay for the urity of peptides (428). Liu et al. derivatized amino aci& and peptides from both standard solutions and biological samples at low with 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde sample concentration to form highly fluorescent isoindole derivatives (429).Minimum detectable quantities with CE and laser-induced fluorescence detection were in the low-attomole (10-l8 mol) range. Hortin and co-workers separated sulfated and nonsulfated forms of pe tides by CE (430). Kruegar et al. utilized CE and reversexphase HPLC to determine the s ecificity and rate of cleav e of ACTH pe tide bonds by enLproteinase Arg C (I) (437. Tran et al. &rivatized samples of amino acids and ptides and analyzed these mixtures by free zone and mice lar CE (432). The use of MECC in combination with L and D Marfey’s reagent offered unequivocal means to confirm the presence ob D-amino acid in an unknown sample. Bullock (433)developed a series of buffers encompassing the pH ran e from 3.5 to 9.0 for free solution capillary electrophoretic (%E)analysis of basic proteins in uncoated fused-silica capillaries. Separations of model roteins possess isoelectric points between 9.1 and 11.0 have efficiencies ranging from 95OOO to 690 OOO gee, achieved theoretical plates. In each case, the modifier 1,3-diaminopropane was incorporated in the operating buffers a t concentrations of 30-60 mM along with moderate levels of alkali-metal salts to suppress protein-capillary wall interactions. The combination of these buffer additives allows the analyses to be performed at pH values below the protein isoelectric points. Florance separated 14 of the 24 peptides that are fragments of the protein motilin. The influence of charge, hydrophobicity, secondary structure, and length of the motilin fragments on mi ation time was investigated (434). Buoen and co-workers &cussed the effect of slow conformational inversion of pro1 lproline residues on the separation of proline-rich peptiles by CE and HPLC (435).The analysis of synthetic branched peptides and multiple-anti en peptides by CE was described by Pessi et al. (436).DifFerences of a single residue between homologous proteins from different species could be observed. Yannoukakos et al. analyzed tryptic digest of proteins to determine sites of phosphorylation (438). Young and Merion investigated s ecies variations in the tryptic maps of cytochrome c by E (439). Ferranti et al. generated tryptic maps for each globin in human Hbs then compared these maps to those for normal samples to identify abnormal globin (440). Bushey and Jor enson employed a two-dimensional reversed-phase HPL8-ca illary electrophoresis instrument to identify differences in &e tryptic digest fingerprints of horse heart c ochrome c and bovine heart cytochrome c (186). Liu an co-workers demonstrated the value of electrokinetic capillary chromatography for separating structurally similar model peptides and tryptic digest mixture (441).These workers also investigated the behawor of model peptides in MECC buffer systems containing dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, sodium dodecyl sulfate, and two cyclodextrins. Hogan and Yeung utilized indirect fluorescence detection to monitor subfemtomolar components in tryptic digests (280). Guzman and co-workers used CE to analyze in vivo release of probabl neuropeptides from the median eminence of the brain (4427. The authors comment on the potential power of CE when combined with localized brain rfusion sampling techniques. Florance suggested that capgary zone electrophoresis could be used to separate deamidated motilin peptides (443).Improved resolution was obtained by modif ‘ng secondary structure and the hydrodynamic profile of t c s e peptides with solvents. Satow and co-workers investigated the effects of the a n le matrix on the separation of peptides by CE (444).Use of pow salts in the sample zone resulted in much better resolution due to refocusing effects. A practical

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guide for high-sensitivity,high-resolution separation of saltcontaining peptide mixtures is proposed. Towns and Regnier reported that se aration of proteins by CE was enhanced dramatically by aerivatizing the silica surface of the separation capillary with octadecylsilane followed by deposition of a layer of nonionic surfactant from an aqueous solution (176). Protein adsorption onto the walls of the ca ill and electroosmotic umping were both dramaticafy r!uced. Christiansen an8mworkers demonstrated the separation of proteins, eptides and DNA fragments in capillaries with walls coatejto minimize electroendosmosis and adsorption of solutes (445). Digests of bovine serum albumin, polymerase chain reaction products, and the deamidation of human growth hormone were all analyzed by this procedure. Tran et al. used free solution CE to separate the glycoforms of recombinant human erythropoietin (446). After a systematic investigation of the factors which influence resolution, regulation of the eledrmmotic flow of the runn' buffer and the reduction of solutewall interactions was f o a to be most important. Mosher used metal ion-containing buffers to enhance the resolution of peptides in capillary zone electrophoresis (447).The dependence of resolution on metal ion concentration was documented for two samples. Castagnola et al. separated approximately 8 pmol of t tic peptides from horse myoglobin in acrylamide-coated c a p g e s (448).

Gurle and co-workers used CE to fractionate histones ?r,e separation of histones by CE was compared with the separation b acid-urea polyacrylamidegel electrophoresis and in reversedIphase HPLC (449). Josic and co-workers se arated h drophobic membrane roteins by CE and caITP &O). The addition of 7 urea to the separation guffers was necessary to achieve reproducible results for CE. In the ITP experiments, splitting of peaks (caused by differently glycosylated and/or phosphorylated proteins) was observed. Nowicka et al. utilized preparative free-flow capillary isotach0 horesis to fractionate apoB-containing lipoproteins from asting and postprandial sera derived from normoli idemic individuals (451). Gebauer and Thormann reporteJon the isotachophoretic determination of proteins in uncoated fused-silica capillaries (452). They reported that small amounts of hydroxypropylmethylcellulose added to the leader provide an efficient method of d amic column conditioning for high-resolution isotachop oretic Separation. Ca illary electrophoresis was used for the analysis of Ca and #n binding proteins by Kajiwara (453). Kajiwara and co-workers investigated the chan e in mobility of calmodulin and parvalbumin caused by the %indingof Ca2+(454). Carbonic anhydrase (a Zn2+binding rotein) also showed a shift in mobilit following metal bin ing. Rush and co-workers changed tge analysis temperature to separate two forms of myoglobin corresponding to different oxidation states of the heme Fe (455). Temperature-related conformational c in a-lactalbumin type I11 were also observed. Chen and u employed a bfh-voltage gradient to separate proteins mixtumj in times less t an 200 s (456).Separation of model proteins demonstrated that the retention times correlate with the isoelectric points of the molecules. The electrophoretic properties of calmodulin were investigated by Chan and Chen (457). These investi ations sug ested that CE separation at near physiological p!-I may dif erentiate the microheterogeneity of calmodulin. Ward et al. evaluated the various strate ies for obtaining internal amino acid sequence data from ekctrophoretically separated proteins (458). CE was used to &98e88 peptide purity before sequence analysis. Cysteine residues were identified in unmodified proteins or peptides by a characteristic phenylthiohydantoin (PTH)-amino acid derivatization during sequence analysis. Tanaka and co-workers presented a sim le and sensitive method for C-terminal sequencin of pe tiles with an anhdrotrypsin affinity column and capiflary egctrohoretic monitoring (459). The C-terminal peptide fragment for Lys-C-endoproteinase-digested horse myoglobin was sequenced by this method. Meyer and co-workers described a procedure for uence analysis of phosphotyrosine-containing peptides (460)?olid-phase sequencing of phosphotyrosineelectrophoresis con-peptides and subsequent cap of PTH-p osphotyrosine permits the unam iguous identification of the phosphoamino acid. Less than 1 pmol of PTH-phosphotyrosine was needed for determination. Berg(449).

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man et al. used repetitive runs and fraction collection to obtain sufficient material for direct sequence analysis of peptides (461).

Camilleri and Okafo (462) demonstrated that CE in D20based buffer solutions provided e n h a n d resolution compared to electrophoresis carried out in water solution of the same acidity. These effects are thought to result from a lowerin of electroosmoticflow due to the hi her viscosity of DzO an to a reduction of the ( potential. t h e y also found that the use of D,O-based buffers lowers Joule heating and that pK, differences can result in enhanced resolution (463). Electrophoresis in the D 0-based solution gave complementary information to that o h i n e d in H,O-based electrolytes of the same acidity (464). For the analysis of the tryptic digest of salmon calcitonin and elcatonin, buffer solutions prepared with D20proved superior to buffers pre ared in HzO (465).From a single separation of a elcatonin igest, three pure cleavage peptides were recovered in sufficient quantity to determine the amino acid sequence. Nashabeh and E1 (466) evaluted CE with fused-silica tubes with a hydrophilic coating for the separation of peptide and glycopeptide fragments from trypsin digestion of al-acid lycoprotein. Submapping of glycosylated and nonglycosy l a d tryptic fragments of the glycoprotein by CE was facilitated by selective isolation of the glycopeptides via solid-phase extraction. In addition, the electrophoretic map and submaps of the whole tryptic digest and its Con A fractions allowed the elucidation of the microheteroaeneitv of the glycoprotein. Kenndler and Schmidt reported that the addition of SDS to protein sam les (conalbumin and ovalbumin), but not the carrier e l e c t r o b (borate, 0.1 mol L-I, pH lo), produced a total lw in resolution in the separation (467). No such effects are found if SDS was added to the carrier electrolyte. They were able to demonstrate that the observed effects were not caused by binding of SDS onto the proteins but are generated by conductivity gradients in the column. Widhalm et al. used a linear, non-cross-linked polyacrylamide gel as a sieving medium to separate proteins according to their masses (214). The four test proteins, covering a molecular mass range of between 17 800 and 77 000, were separated as SDS complexes. Van de Goor et al. separated modified adrenocorticotropic hormone fragments (468). Relative migration in aqueous buffers with and without sodium dodecyl sulfate were considered. Vinther and co-workers separated bovine a rotinin (a ancreatic trypsin inhibitor) from contaminanta p469). HPL8 and mass spectrometry were used to identify the contaminates as aprotinin missing one and two amino acids at the C-terminus. Wenisch and co-workers utilized isoelectric focusing to purified isoforms of human monoclonal antibodies against the gp-41 of AIDS virus and of human recombinant superoxide dismutase (470). CE was used to monitor of the content of each chamber of the electrolyzer. Wu and co-workers demonstrated the separation charge variants of recombinant DNA-derived proteins which arise as a consequence of natural microheterogeneity or due to degradative processes such as deamidation (471). Samples of human growth hormone, a T4 receptor rotein, and tissue plasminogen activator were all e x a m i n d Wiktorowicz and co-workers used CE to analyzed the protein structure of RNase T1 and its site-directed mutanta (472). Yim analyzed the glycoprotein rtPA and d i s d the resulting electropherogram patterns in light of its known structures (473). Nielsen and co-workers demonstrated the separation of antibody-antigen complexes by free-solution CE in less than 10 min (474). Rosenblum analyzed carbonic anhydrase by CE and compared these results to Ag-stained slab gels (475). Cunico et al. was able to characterized PEG-proteins by ap lying a positively charged coating to the fused-silica cap i & y to prevent the adsorption of basic and PEG-proteins (476). The peak widths recorded during these analyses were e, reflecting the broad molecular masa distribution of PEGS an the heterogeneous nature of the PEG conjugates. A procedure to separate proteins in a untreated fused-silica capillary by raising the pH of the running buffer and washing between runs with 1.0 M sodium hydroxide was described by Lee and Heo (477). Capillary electrophoresis-mass spectrometry is an increasingly popular method of characterizing peptide and protein mixtures. Several groups utilized atmospheric pressure

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ionization sources (i.e., electrospray, ion spray, etc.) to detect and characterize these mixtures (257,255). Thibault et al. analyzed native and tryptic peptides and high molecular weight proteins by CE mass spectrometry (478). Noncovalently coated capillaries were found to be useful for minimizing sample adsorption onto the walls of the capillary. Peptidema pin detection of modification sites in proteins was descrged 6y Metzger and Jung (479). A brief report is given on atmospheric pressure ionization mass spectrometry and its usefulness for the analysis of peptides and proteins when cou led with HPLC and CE. Spectra are shown for crude foot an mouth disease VP1 peptide and a lipopeptide vaccine. Moseley et al. employed a coaxial continuous-flowing fastatom-bombardment interface to acquire MS and MS-MS spectra in electrophoretic real time from femtomole levels of the peptides (262). Oligosaccharides. The biolo ical significance of oligosaccharides and their complexes as encouraged efforts to examine these mixtures by CE. Hoffstetter and co-workers investigated the influence of different borate buffers on the electrophoretic mobilities of underivatized mono- and oligosaccharides as well as glycosylated peptides (480).The addition of borate to aqueous solutions of mono- and oligosaccharides resulted in an increase molecular absorbance at 195 nm, allowing the sugars to be detected without derivatization. Furthermore, elevated temperatures (u to 60 "C) enhanced the resolution of the separation. Hon a et al. analyzed the oli osaccharides in ovalbumin (a lycoprotein model) by CZd (481). Honda and co-workers afso analyzed monoterpene lycosides (paeoniflorin and oxypaenoiflorin) and gallic acicfand its derivative from a methanolic extract of P. radix (482). Carney and Osborne developed methods to separate unsulfated, monosulfated, and trisulfated isomers of chondroitin digests of connective tissues (483). The separation of oligomers of hyaluronan was accomplished with similar protocols. Liu and co-workers separated and detected low-attomole quantities of saccharides by laser-induced fluorescence 3-(4carboxybenzoyl)-2-quinolinecarboxaldehyde derivatives (331).This methodology was used to map complex oligosaccharides isolated from bovine fetuin. Nashabeh and El separated pyridylamino derivatives of oligosaccharides (484). The mobility of the oligosaccharide was found to be a linear function of the number of glucose residues in the homologous series. Lee et al. examined the products formed by chitinase acting on N - a c e t y l c h i t-o o i d e - f l u o r e s c e n t conjugates (4851.Oligonucleotides. Macek and co-workers examined the effects of column leneth and amlied electric field on column efficiency and resoluiion of ti6oligonucleotides in capillary gel electrophoresis (486). Best resolution was obtained by using long columns in combination with optimum (and not the highest possible) electric field. Guttman and co-workers separated polydeoxyoligonucleotides on polyacrylamide gel capillary columns (487). Gels with relatively low monomer content were found to be ve efficient. The collection of purified fractions from these coyknnswas shown to be feasible using field-programmin techniques. Baba et al. se arated polynucleotides by capiflary gel electrophoresis a n l H P L C (488). Electropherograms of sin le-stranded homopolynucleotides were compared with HP C separations according to the chain len of the polynucleotides. Paulus and Ohms optimized el- ed capillaries to separate oligonucleotideswith a len h of20-50 bases (489). In addition to homopolymeric samp es, the separation of heteropolymeric oligonucleotides with one nucleotide difference in size was presented. Demorest and Dubrow examined instrumental and sample matrix factors for their effect on the resolution, sensitivity and detectability of oligodeoxynucleotides separations in gel-filled capillaries (490). They determined that substantial errors in sample quantitation could result from if peak areas were not corrected for peak velocity. Dubrow directly compared capillary gel electro horesis with slab gel electrophoresis and reversed-phase HPL8 (491). Paulus and co-workers used polyacrylamide-filled capillaries to se arate oligonucleotide samples (213). Calibration of the g e l d e d capillary rmitted the determination of the mass of isolated molecuc. DNA. Zhang et al. utilized CE with laser-induced fluorescence detection to analyzed labeled amino acids and DNA-se uencing fr ents (492). Chen et al. reported lowzeptomoye detectionymits for DNA sequencing by capillary

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el electrophoresis (230, 493). A green helium-neon laser 7543.5 nm) was F e d to excite tetramethylrhodamine-labeled DNA fragments in a sheath-flow cuvette and the resulting fluorescence detected in a single s al channel. S uenc' data was generated at a rate of a out 70 baseslh. C en an co-workers also demonstrated the feasibility of using gel capillary electrophoresis to perform Southern blotting with online detection to study parameters that affect hybridization of DNA molecules in solution (494). Cohen and co-workers utilized capillary gel electrophoresis with laser-induced fluorescence detection as a tool for sequencing synthetic 01i onucleotides and sin le-stranded pha e DNA (495). Jwerdlow et al. separate%dideoxycytidine c ain-terminated DNA fragments in gel-filled capillaries (496). A postcolumn laser-induced fluorescencedetector provided a mass detection

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nucleotides in the size range of 12-60-mers was achieved in 35 min by electrophoretic methods. Serdlow et al. compared in the capillary format the four spectral-channel sequencing technique of Smith, the two spectral-channel sequencing technique of Prober, and the one spectral-channelsequencing technique of Richardson and Tabor (231). Sequencing rates up to lo00 bases/h were reported at high field strengths (465 V cm-'). At lower field strengths, capillary electrophoresis produced useful data for fr ents greater than 550 nucleotides in length with 2-foldytter resolution than slab gel electrophoresis. In collaboration with Gesteland, Swerdlow employed enzymic dideoxy nucleotide chain termination with fluorescently tagged oligonucleotide primers and laser-based on-column detection to sequence DNA samples (498). Caillary gel separations were shown to be 3 times faster, with etter resolution and higher separation efficiency than a automated slab gel DNA-sequencing instrument. Drossman and co-workers fluorescently labeled DNA fragments by enzymic sequencing reactions, then rapidly separated the mixture by ca illary gel electrophoresis (499). Laser-induced fluorescenceaetedion was used to monitor the labeled s ecies at attomole levels. Heiger and co-workers separatezmixtures of DNA restriction fragments in gel-filled ca illaries (500). Low crodinking of the el ermitted high edciency separations of double-stranded bNw fragments up to 12000 base pairs in length. Reducing the intensity and pulsing the electric field was found to enhance the separation. Demana et al. separated DNA fragments by velocity modulation in gel-filled capillary columns (501). Analyte velocity modulation improved resolution and shortened migration times of the nucleic acid fragments. Luckey et al. decreased the time of DNA sequence analysis by separati the mixture on ultrathin capillary gels (502). The possi%ty of an instrument of this type which can simultaneously analyze many samples was considered. CE separations employin electroosmotic flow in an untreated silica capillary provicfed on1 partial resolution of the 23 fragments in a 1-kbp DNA laddr. By coating the inner walls of a silica capillary with polyacrylamide and filling.these capillaries with buffers contain' methylcellulose as a sievlng medium, Strege and Lagu werexle to separate all f r y e n t s in a 1-kbp DNA ladder (180). Furthermore, this tec nique was able to separation very lar e fragments in a X DNAHindIII digest. The separation o f DNA restriction fragments in solutions of (hydroxyeth 1)cellulose was described by Grcesman and Soane (503,504f An effective mesh size 1order of magnitude smaller than that foun in agarose gels was calculated by using polymer-entanglement theory and confirmed by electrophoretic measurements. Schwartz et al. separated DNA restriction fr ments and polymerase chain reaction (PCR) products by C% on a olysiloxane-coatedcaillar in the presence of polymeric {uffer additives (505). bobifty data and Ferguson plots of the DNA fragments at different polymer concentrations indicated that effective molecular sieving was obtained. This was demonstrated by the analysis of specific coamplified DNA sequences. The addition of ethidium bromide to the buffer resulted in longer mipation times but better peak resolution. Yama ata and Shirasaki separated restriction enzyme-treated DkA in a borate buffer containing low melting point agarose (506). Yamagata separated restriction enzyme-treated 4x174 phage DNA in a borate buffer containing agarose (507).

E

CAP I LLARY ELECTROPHORESIS

Yin et al. measured diffusion coefficients of oligonucleotidea in p l Y a y *h i d e gel capillaries in the absence of an electrical

fluid (508 Com aring this data with data from an electrophoretic system Ed them to conclude that diffusion coefficients of oligonucleotides and also their intermolecular interaction with the polyacrylamide el may be considerably changed by ap lication of electricaffields and thus may influence their erectrophorectic behavior. Song and Maestre employed epifluorescence microsco y to obtain ima es of single DNA molecules inside gel-fi&d capillaries and thus determine their motions and configuration during the separation process (509).The mobilities of three types of DNA were measured as a function of electric field. A particularly significant observation was that at hi h electric fields (>400 V cm-*) aggregates of DNA molecules formed. When the field was reduced, the aggregates dissociated into a cloud of single DNA molecules. Biotechnology-DerivedSamples. Hurni and Miller exploited the speed of CE to monitor individual purification steps in the production of a licensed vaccine product (510). Lookabaugh et al. evaluated CE for the regulatory analysis of commercial dos e forms of insulin and compared these results to the HPL? protocol (511). Stover reported on the analysis of deter ents, food additives, herbicides and biotechnology sam fes by CE (340). Vinther and co-workers utilized biosyntEetic human growth hormone (B-hGH) and closely related analogs as model proteins to evaluate of the separation capability of CE (512). Banke and co-workers utilized CE to analyze an unknown protein from a fermentation broth of Aspergillus oryzae (513). Careful analysis of the reaction mixture allowed this enzyme to be identified as an alkaline rotease of the subtilisin family. The rapid evaluation o f plasmid concentration and stability from fermentations of enetically engineered bacteria was discussed b Hebenbroc! and co-workers (514). Tsuji (515) used SbS-PAGE to separate proteins derived from recombinant DNA. Guzman and co-workers analyzed a solution of highly purified monoclonal antibody (240). Multiple parallel capillaries were employed to increase the detector response and sample load. Harrington et al. evaluated CE as a method for characterizing enzyme-antibody conjugates (516). Methylcellulose (0.5%) in the running buffer was used to help separate the con’ugate and unreacted components of a solution containing alialine phosphatase and IgG. Polymers and Particles. Amankwa and co-workers determined the oligomer distribution of Jeffamine ED-600 and Jeffamine ED-2001 (517). In less than 15 min, 30 oligomers which differ in size by only a methylene unit were separated. Chiari et al. (518)monitored the hydrolysis and degradation kinetics of Immobiline polymers. The decrease in the Immobiline peak and the appearance of ita hydrolytic products (acrylic acid and a diamine in the case of the a lamido bases) could easily be monitored and quantified inyZE. Righetti et al. also anal zed Immobiline polymers for acrylic acid contamination $19). Jones and Ballou reported on the separation of polystyrene latexes containing carboxylate and sulfate groups (520). Rapid and sensitive analysis of polys ene particle size fractions was re rted by VanOrman and $Intire (521). While the charge o g h e particle did not play a major role in the separation, the resolution achievable via CE offers an o portunity to examine complex multiple adsorbate/ art& interactions. Simonova and Eremova considered g e behavior of particles in a strong electric field (522). McCormick separated silica sols (5-500 nm) with CE (523). Baseline resolution of sub-100-nm sols was accomplished in less than 20 min. The ionic strength of the separation buffer had a significant effect on the resolution and mobilities of the sols, with hi her ionic stren hs substantially improving resolution of &e smaller colloi$ while greatly increasing the elution time of the larger sols.

ACKNOWLEDGMENT This work was sup orted by NSF Grant CHE-8957394 (W.G.K.), NSF Grant 8HE-9108530 (C.A.M.), and the Arnold and Mabel Beckman Foundation (C.A.M.). LITERATURE CITED

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Kinetic Determinations and Some Kinetic Aspects of Analytical Chemistry Horacio A. Mottola* Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078-0447

Dolores PBrez-Bendito Department of Analytical Chemistry, University of Cdrdoba, 14071 Cdrdoba, Spain This review retains, basically, the organizational structure of the revious one in this series (I). Topical itemization in the ‘dscellaneous” section is the only minor novelty introduced in this review and the kinetics of separation processes has now been included in that section. The papers reviewed

have been selected from those that appeared since November 1989 and that were available for the authors’ consideration through approximately November 1991. Professor Harry B. Mark, Jr., who since 1972 has been the senior author of these reviews, has decided to hand down this

0003-2700/92/036~407R~10.00/0 0 1992 American Chemlcal Society

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Capillary electrophoresis - ACS Publications - American Chemical

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