Capillary Electrophoresis - Analytical Chemistry (ACS Publications)

CLINICAL CHEMISTRY

Capillary Electrophoresis Yan Xu Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115 This section of the review will discuss the topic of capillary electrophoresis (CE) in clinical analysis, although selected topics of CE in the areas of biochemical analysis will he also included. Generally, CE is electrophoresis performed in a capillarytube. It is ananalyticaltechnique for theseparation of molecules such as inorganic and organic ions, neutral compounds,aminoacids, peptides, proteins, oligonucleotides, andDNA. Due toitsspeed,columnefficiency,andselectivity, CE has become a owerful complementary technique, sometimes a better sugstitute, to both conventional gel electrophoresis and high-performance liquid chromatography. The continuousdevelopmentofCEwil1certainlyhave great impact on modern clinical analysis. This review covers the Chemical Abstract period from January 1991 to October 1992.

REVIEWS CEisthefastestgrowinganalyticaltechniqueinseparation science. Nearly 90 reviews have been published during 1991 and 1992. Kuhr and Monnig ( M I )wrote an extensive review on fundamentals, instrumentation, column technology, detection, and applications. Chen e t al. ( M 2 ) published an overview on the potential utility of CE for clinical applications. Examples of separating proteins from serum, urine, and cerebral spinal fluid were given, with particular emphasis on the use of an open-tube, untreated fused-silica capillary. McLaughlin et al. ( M 3 )provided the practical guidelines of pharmaceutical drug separations. Wren and Rowe (M4) presented a simple model for separation of chiral molecules. Olefirowicz and Ewing (M5)published a review on analyzing cytoplasm withdrawn from a single nerve cell with a microcapillary electrophoresis technique. Several reviews (M6M I 8 ) discussed the separations of pharmaceuticals, amino acids, peptides, proteins, nucleotides, DNA, inorganic ions, and small organic compounds. A number of reviews discussed the separation modes of CE. They were as follows: (a) capillary zone electrophoresis ( M I S M 2 2 ) ;(b) micellar electrokinetic capillary chromatography(MIS,MB,M24);(c) isotachophoresis (MI9,M21, M22, M25); (d) capillary gel electrophoresis (M19, M20, MZZ);(e) capillary isoelectric focusing (M19, MZZ, M26). Olechno et al. (MZ7) summarized the injection techniques used in various modes of CE. Chien and Burgi (M28) discussed the mechanism and application of on-column sample concentration, also known as sample stacking. There were many reviews on the detection methods of CE, such as (a) UVlvisible absorbance ( M I ,M29), (h) fluorescence ( M I , M30, M31), (c) laser-based detection (M31-M33), (d) mass spectrometry (M34-M36), electrochemical detection (M37). (e) indirect detection (M38),and (0 radioisotope detection (M39). A number of authorsdescribed the columntechnology for CE. Turner (M40) focused on the columns used for the separations of DNA fragments, carbohydrates, and proteins. Schombur (A4411discussed the polymer modified columns, whereas dazzeo and Krull (M42)provided the strategies for the use of coated capillaries and hufferlsample additives in protein separations. Finally, Novotny (M43) illustrated the application of microcolumn liquid chromatography and CE by selected separation examples of amino acids, peptides, and oligonucleotides. Krull and Mazzeo (M44)com ared CEversus liquid chromatogra hy (LC) for the study of giopolymers. Schomburg (M45)&cussed the problems and achievements in the instrumentation and column technology for chromatography and CE. Lauer and Ooms (M46)reviewed the challen es of CE to liquid chromatography and conventional sla gel electrofhoresis: Terahe (M47) and Issaq et al. (M48) examine the similarities and differences among analytical LC, traditional electrophoresis, and CE with regard to theory, characteristics, instrumentation, and fields of application.

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CAPILLARY ZONE ELECTROPHORESIS Background. Capillary zone electrophoresis (CZE), also known as freesolution capillary electrophoresis, is t he simplest

Van Xu is currently an Assistant Professor 1 olChemiS~atCkve!andStaleunivemitv. He received his B.S. degree in chemistry from Znongshan Universny n 1982 Aner graduation. he worked as a researcn chem'slin China lor t h r e e years betore he came 10 me U n b d Slams. He earned a M S degree in analytical chemslv al CaifanaStaleUnlverslry.Fresno. n 1987. and a Pn D. degree n analytical chem slry allheUniverslfyotCncnnatiin 1991. Dr. Xu's currenlreiearchinterestsarefocused on medevelopmentof microscale enzyme immunoassays and lhe use of capillary

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form of CE. A uni ue feature of CZE is its effectiveness in heat dissipation. !Solution heating occurs when electric current passes a solution between two electrodes. The heat roduced,H (J),isdirectly proportional to theapplied voltage etween the electrodes, V (volts), to the electric current, I (A), and to the time, t (9):

E

H = VIt (1) If the heat produced does not dissipate from the system effectively, it can result in temperature and density gradients in the separation medium and subsequent band-broadening of the species being separated. Due to the large surface area to volume ratio, the capillary column provides more efficient heat dissipation than the slab gel. As a result, high applied potentials (up to 30 kV) are commonly used in free solution capillary electrophoresis for fast efficient Separations. The separation in CZE is based on differences in the apparent (or observed) velocities of charged solutes in a free solution capillary tube. The apparent velocity of a charged solute, u . ~ (cmls), is directly proportional to the apparent mobility ofthe charged solute, pWp(cmW"), and the potential gradient across the separation capillary, E (Vlcm): VePP

=b p E

(2)

The apparent migration time of the charged solute. t,p, (9). is related to the capillary length, L (cm), and the apparent velocity of the charged solute: (3) tap, = LIv,, = LIPappE The apparent mobility of the charged solute can he determined by itselectrophoreticmobility, itep(cmVV.s),and electroosmotic mobility, ps0 (cm2lV.s): Papp

= Pep + P..

pep = q

l6w

(4)

(5)

P.. = 4/'/4*7 (6) where q is the net charge of a solute, r is the hydrodynamic radius (or Stokes radius), 7 is the viscosity of the medium, 6 is the dielectric constant of the medium, and f is the zeta potential across the solid-liquid interface. Both pepand Pare vectors. The paPpis a vector sum known as the steadystate mobility. I t is of benefit to us to understand that the buffer concentration, the type of cations and anions, pH, surface charge (or potential). medium dielectric constant, and medium viscosity are the important factors that influence CZE separations. Clinical Analyses. (I)Plasma, Serum, and Blood Samples. Lloyd e t al. (M49) developed an assay for the antileukemic agent, cytosine 8-D-arabinoside, in human plasma. Solid-phaseextraction andon-column concentration were used to improve the detection limit. The separation time was less than 5 min. The limit of detection was 0.5 r M (SIN = 3). Theassayhadbeenvalidatedforthedetermination of cytosine 0-D-arabinoside in human plasma over the concentration range 1-10 rM. Reinhoud et al. (M50) described a rapid method for determination of daunorubicin,

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doxorubicin, and epirubicin in human plasma. A liquidliquid extraction pretreatment procedure was used prior to CZE, which resulted in a sample matrix of low ionic strength in comparison with the electrophoresis buffer. The zone sharpening effect of electrokinetic injection permitted a 2030-fold increase in the injected amount. The limits of detection for daunorubicin, epirubicin, and doxorubicin in plasma were from 125to 250 pg/mL, achieved by laser-induced fluorescence detection. Ling et al. (M51) separated and detected lutathione in human whole blood using CZE with UV and fluorescence detection. They also discussed the sample pretreatment, analyte derivatization, and condition optimization. Jellum et al. (M52) used CZE for the diagnosis and study of human metabolic disorders. The analyses were carried out on blood and urine samples from patients with homocystinuria, cystinuria, lutathione synthetase deficiency, and adenylosuccinase eficiency. The sulfur-containing amino compounds were derivatized with monobromobimane, then separated in CZE, and detected by a fluorescence technique. This method was a quick and simple alternative to classical amino acid analysis. The detection of the characteristic succinylpurines associated with adenylosuccinase defect was well achieved. Owing to the possible connection between acid) in the deficiency of taurine (2-amino-1-ethanesulfonic heart and the development of cardiomyopathy and heart failure, they also developed a sim le CZE method for the determination of taurine in submi ligram levels in myocardium biopsy samples. The homologue, 3-amino-1-propanesulfonic acid, was the internal standard. Both taurine and the homologue were derivatized and determined by CZE with fluorescence detection. Tomita et al. (M53) investigated the separation of two bipyridyllum herbicides, paraquat and diquat. Both herbicides were extracted from fortified sera with disposable ODSsilica cartridges and then separated in a fused-silica ca illary (50-pm i.d., 750 mm). The separation was completefin 10 min at an applied potential of 20 kV. The recovery of both herbicides added to serum was about 97 % a t a concentration range of 0.5-2.0 pg/mL. In a different study, Tomita et al. (M54) resolved glyphosate from its metabolite, (aminomethy1)phosphonic acid (AMPA), in serum using CZE. The two compounds were first derivatized with -toluenesulfonyl chloride and then separated using a 0.1 M toric acid/sodium hydroxide buffer (pH 9.6) containing 10% methanol. The separation was completed within 15 min at an applied potential of 30 kV. Calibration curves for the assay were linear over both the lower (0.5-10 pg/mL) and the higher (10-100 pg/mL) concentration ranges. The detection limit of both derivatives was 0.1 pg/mL in spiked sera, and the recoveriesof glyphosate and AMPA were 87.9-88.8 and 78.486.9 % , respectively. (II) Urine Samples. Wildman et al. (M55) reported the simultaneous analysisof weak acid anions (oxalateand citrate) and inorganic anions (chloride, sulfate, nitrate, phosphate, and carbonate) in diluted urine, and oxyanions of arsenic (arsenite and arsenate) in urine. Guzman et al. (M56) developed two methods for collection of urinary components. The first method utilized multiple capillary bundles which were coupled to a single capillary through a glass connector. The second method used an analyte concentrator containing an antibody bound to a solid support. Both methods allowed greater amounts of sample to be loaded into the capillary, and the continuous collection of purified material in nanogram to microgram quantities. The collected samples were identified by mass spectrometry. In a separate report, Guzman et al. (M57) described a simple, fast, and reproducible CZE method for determination of urinary metabolites, creatinine and urea. The linear range was from 1.0 X 10-4 to 1.0 X le2 M for creatinine and from 0.10 to 1.0 M for urea using UV detection. The detection limit for creatinine was in the picogram (fmol) range, and for urea was in the nanogram analyzed uric acid in urine (pmol) range. Masson et al. (M58) and in reconstituted serum by CZE. They found that ascorbic acid, gentisic acid, caffeine, theophylline, theobromine, glucose, acetylsalic lic acid, and acetaminophen did not interfere with the dretermination of uric acid. The results were in satisfactory a reement with those obtained by enzymatic assays and t i e value provided by the supplier.

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(III) Other Biological Samples. Olefirowicz and Ewing (M59)directly determined attomole levels of neurotransmitter (dopamine) in picoliter volumes of cytoplasm withdrawn from singleneurons of the ondsnail, Planorbis corneus, with CZE. and %u et al. (M61) se arated eight cationic Lai et al. (M60) proteins, histidine-rich peptides ( H R h 1, 2, 3, 4,5, and 6, lysozyme, and histatin 6, in human parotid saliva. Histatins 2 and 4 were identified as the autoproteolytic degradation products of HRP-1 and HRP-3, respectively. Tsuda et al. (M62) reported the separation of free polyamines in rat tissues. After precipitation of proteins with perchloricacid, the sample solution was derivatized with fluorescamine. Ethylenediamine was added to the medium to avoid adsorption of polyamines onto the capillary wall. 6-Amino-n-caproic acid was used as the internal standard. The fluorescence signal was collected through a cylindrical lens. The minimum detectable amount by this method was 200 fmol (or 10 nmol/ mL). Gurley et al. (M63) measured proteins from the lung fluid of rats exposed to erfluoroisobutylene for diagnosing and assessing lung disorlers. The lung fluid was obtained by a bronchoalveolar lavage procedure using 48 mL of physiological saline to wash out the lung fluid of rats. The proteins were precipitated from the fluid with 10 volumes of acetone and then dissolved in 2 mL of 0.2% of trifluoroacetic acid aqueous solution. The protein samples were electrokinetically injected into a 50 cm X 50 pm i.d. coated capillary a t 10 kV for 10 s. Six major proteins were resolved in 35 min in phosphate buffer (0.1 M, pH 2.5) by CZE and detected by UV absorption a t 200 nm. (IV)Microdialysis-CZE. Hernandez et al. (M64) used CZE for calibration of outward diffusion of drugs in an in uiuo bidirectional microdialysis for studying dopaminergic potency of cocaine, procaine, and lidocaine in the nucleus combined the localized brain accumbens. Guzman et al. (M65) perfusion samplin techniques, such as push-pull and microdialysis, with 8ZE to analyze in uiuo release of brain constituents from the median eminence, a discrete hypothalamic area rich in peptider ic-containin neuronal terstuiied the blooi-brain barrier minals. Tellez et al. (M66) permeability of phenobarbital using dual microdialysis couused microdialysis and pled to CZE. O'Shea et al. (M67) CZE with electrochemicaldetection for continuous monitoring of the pharmacokinetics of L-dopa in the rat. The detection limit for L-dopa was 98 am01 or 3.9 ng/mL. The elimination half-life was 10.3 min. Voltammetric characterization was used to verify the identities of L-dopa and its metabolites. The effects of electrochemical pretreatment on the detector performance were also investigated. Enzymes and Enzyme Digests. Wiktorowicz et al. (M68) described the structural analysis of native and site-directed separated mutants of RNase T1 by CZE. Banke et al. (M69) an alkaline protease of the subtilisin family from a crude fermentation broth of Aspergillus oryzae using CZE. Amankwa and Kuhr (M70)published an analytical procedure involving trypsin immobilized on the inner surface of a fused-silica capillarythrough aminoalkylsilane pretreatment and biotin-avidin-biotin coupling for on-line di estion of minute amounts of 8-casein. The tryptic peptites in the collected digest were separated in a 100 cm X 50 pm i.d. capillary for about 25 min at room temperature. Laserinduced fluorescence was used to detect the separated NDAlabeled tryptic peptides in the digest. Nashabeh and El Rassi (M71)immobilized RNase T1 hexokinase and adenosine deaminase on the inner walls of short fused-silica capillaries through glutaraldehyde attachment and then coupled the capillary enzyme reactors in series with CZE. The system allowed on-line digestion and mapping of minute amounts of transfer ribonucleic acids, and the slmultaneous s thesis and separation of nanogram quantities of oligonuc eotides. Kruegar et al. (M72) studied the specificity and rate of cleavage of ACTH peptide bonds by endoproteinase Arg C (I). Acidic cleavage products were readily resolved in uncoated capillaries using low ionic strength electrolytes. The addition of salts and zwitterions to the electrolyte decreased capillarypeptide interactions so that all of the ACTH eptides examined were eluted with hi h efficiency. ACT??v:ptide bond cleavage rates for Are-kpg, Arg17-Arg18, Lys' Lysl6, and L ~ s % 4 r gwere ~ ~ 1.46,0.096,0.057, and 0.029 pmol min-l mgl, respectively. They concluded that CZE exhibited better resolution and required shorter time than Clo HPLC, and

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CZE was an appropriate method for kinetic analysis of roteolytic enzymeaction on peptide substrates. Bushey and or enson (M73) used a two-dimensional reversed-phase HPLC-capillary electrophoresissystem to compare the tryptic digest fingerprints of horse heart cytochrome c and bovine heart cytochromec. The peptide fragments were labeled with fluorescamineand detected with a fluorescencedetector. The system has been proven to be reproducible enough to identify the differences in the fin erprinta of the tryptic digest. Nashabeh and El Rassi (d74) evaluated the separation of peptide and glycopeptide fragments from trypsin digestion of al-acid glycoprotein using a fused-silica capillary with h drophilic coating. Submappin of glycosylated and nong r cosylated tryptic fragments o the glycoprotein was facigtated by selective isolation of the gl co eptides on Con A silica-basedstationary phases prior to t i e erectrophoretic run. In addition, the electro horetic map and submaps of the whole t tic digest and the t o n A fractions allowed the elucidation o%e microhetero eneity of the glycoprotein. They proved that CZE was suita%lefor the mapping of the oligosaccharide chains cleaved from the lyco roteins in endoglycosidase digestion. Rickard et al. ( 75) o served excellentcorrelation between the electrophoretic mobilities and q/MW2I3in CZE, where q was the calculated charge and MW was the molecular weight. Mobilities of a set of 33 diverse peptides from enzymatic digests and 10 intact proteins were measured for separations at pH 2.35, 8.0, and 8.15 with constant ionic strength, temperature, and viscosity. Antibodies. Nielsen et al. (M76) resolved an antigen, a monoclonal antibody specific for the antigen, and the antibody-antigen complex in less than 10min with on-column UV detection. Izumi et al. (M77) used a flat capillary tubing which had a cross-sectional area of about 0.1 mm2 for separation of IgG molecules. Harrington et al. (M78) evaluated the separation of alkaline phosphatase, monoclonal antibody, and monoclonal antibody-alkaline phosphatase conjugate. Due to its speed and resolution, CZE appeared to be a good candidate for in-process evaluations of conjugates for immunoassays. Blood Proteins. Ferranti et al. (M79) used CZE with mass spectrometry for the characterization of genetic variants of human hemoglobin. Globin chains obtained by precipitation of erythrocyte hemolyzate in cold acetone were directly separated by CZE in coated capillaries without any further treatment. The speed and the resolvingpower of CZE allowed fast differentiation of Hbs with similar char es. CZE tryptic mapping was performed for each globin and &ect comparison of the variant tryptic map with the corresponding normal one was done to characterize the variant. They also coupled the electrophoretic data with analysis of enzymatic digests by mass spectrometry via fast atom bombardment mappin to identify amino acid variations. Ong et al. (M80) describe! the se aration of globin chains. Sam les of adult and newborn hemohates from normal individud and children suspected of having thalassemia were analyzed. Separation of globins was accomplished using a 25 mM phosphate buffer at pH 11.8. Distinct peaks of a-,8-, and y-chains were resolved within 8 min. The coefficient of variation (CV) for withinday and between-day runs was 4.1 and 5.7 %, respectively. This method was simple and rapid and could be used for screenin thalassemia and for the clinical study of various hemoglo%inopathies. Gordon et al. (M81) re orted the analysis of proteins in human serum. The pro lem of protein adsorption was overcome by adding eth lene glycol to the protein sample and by choosingdifferent tuffers, pHs, and protein molarities. presented a rapid method for protein analysis Chen (M82) in an untreated fused-silica column. Separation of model proteins showed that the retention times of these proteins correlated well with each of their isoelectric oints. By using e gradient, most proteins coulfbe separated in a200 high-vo% s by C with excellent reproducibility. Similflrl ,human serum proteins could be resolved and detected in reas than 100 s. Binding Proteins. Kajiwara et al. (M83) reported the shifts in mobility caused b the binding of Ca2+to calmodulin and arvalbumin and tge binding of Zn2+ to carbonic anh !rase. Hettiarachchi and Cheung (M84) developed a metiod for Con A and its succinyl derivatives (SCA). The tetramer, trimer, dimer, monomer, and protein fragments of

f

f

dYJ

I !

Con A were separated in less than 50 min. SCA was resolved into more than 10 isomeric succinyl derivatives of Con A. The minimum detectable concentration of Con A in water by measured the binding CZE was 1pg/mL. Chu et al. (M85) constants of li ands to roteins with CZE. The proteins, EC 4.2.1.1, from bovine erythcarbonic anhycfrase B rocytes), glucose-6-phosphate dehydrogenase (EC 1.1.1.49, from Leuconostoc mesenteroides), and calmodulin (from bovine testes) were studied using benzenesulfonamides (substituted in the 4-position with negative groups), NADP+ and NADPH, and calcium ion as ligands. The values of binding constants were calculated from the relationship between the electrophoretic mobilities of these roteins and their ligand concentrations. These values a eetwell with those estimated by conventional assays. H o n g e t al. (M86) determined the association constant of monovalent protein-sugar interaction by CZE. &Galactose-specificlectins and lactobionicacid were used as the model proteins and sugar. The lectin peaks were retarded by addition of lactobionic acid in the carrier. The association constants of monovalent interactions could be obtained from tl (migration time of protein), t z (migration time of complex, obtainable as the mi ration time at the plateau), and the slope of the (t - tl)-k versus [SI-' plots, where t and [SIwere the migration of protein in the presence of lactobionic acid and the concentration of lactobionic acid, respectively. The values for Ricinus communis agglutinin, peanut agglutinin, and soybean agglutinin at pH 6.8 were 3.3 X lO3,g.l X lo2,and 1.1X 102/M,respectively. This method required only small amounts of protein samples and was reproducible. Other Proteins. Tsuji (M87)interfaced a Beckman P/ACE 2050 CE instrument with a Vestec electrospray ionization mass spectrometer for the analysis of recombinant proteins, recombinant bovine and porcine somatotropins (rbSt and rpSt). The standard curve for rpSt was 7-300 fmol by CZE/UV. The theoretical plates were about 410 OOO/m. The relative standard deviation (RSD) for the rpSt peak mi ation time was less than 1%.The multiply charged ion cKsters obtained in the CZE-electrospray ionization mass spectrometer for rpSt were from m / z 1363.2 for the cluster with 16 charges to m / z 1982.5 for the cluster with 11 charges. The average molecular weight of 21 812.6and 21 798.3for a sample of rbSt and rpSt determined in this study were nearly identical to the theoretical values of 21 812.0 and 21 797.9, respectively. The detection limit of the CZE-electrospra ionization mass spectrometer was approximately 1OOfmol. iendaet al. (M88) used CZE with an uncoated fused-silica capillary column to separate the reduced and oxidized forms of plastocyanin, cytochrome c, NAD, NADP, redox proteins, and nucleotides involved in photosynthetic electron transfer. T w o types of ferredoxin, commonly present among the plants, were also clearly separated by this technique. Compton (M89) derived a semiempirical model for describing the electrophoretic mobility of proteins in free solution. Protein mobility was found to be influenced by parameters such as protein valence, size, and shape, solution ionic stren h, pH, viscosity, and temperature. His model predicted t at the molecular weight de endenc of mobility should be a continuous function of M-l! to M-2J, depending on the magnitude of the protein molecular weight and buffer ionic strength. Many aspects of the model were demonstrated by resolving IgG isoelectrotypes, which have previously been resolved only by isoelectric focusing. Peptides. Saria (M90) reported the separation of human and rat a- and &calcitonin gene related peptides in nerve tissue. The separation was achieved at pH between 3.5 and 4.5 and a potential of 20 kV in a fused-silicacapillary. Ouaglia et al. (M91) developed a method for se aration of leucinostatins, the nonapeptides obtained by sutmerged cultures of Paecilomyces marquandii or Paecilomyces lilacinus. Hortin et al. (M92) se arated the sulfated and nonsulfated segments of cholecystofinin, enkephalin, and hirudin in hosphate buffer at pH 6.5. Moseley et al. (M93) analyzefbioactive peptides with CZE-MS through an on-line coaxial continuousflow fast-atom-bombardment interface. High separation efficiencies (up to 410 OOO theoretical plates) were obtained to low femtomole levels of peptides using aminopropylsilylated capillary columns. Sugars. Liu et al. (M94)used 3-(4-carboxybenzoyl)-2quinolinecarboxaldehydeas a precolumn derivatization agent

(ZB,

f

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for various amino sugars. Constituents of various biological mixtures were converted to hi hly fluorescent isoindole derivatives and separated b CZk. Low-attomole levels of amino sugars were detected gy a laser-induced fluorescence developed a method for carbohydetection. Kakehi (M95) drates. The su ars were derivatized by 3-(4-carboxybenzoyl)2- uinolinecargoxaldehyde and separated by CZE withlaserinluced fluorescencedetector. The sensitivity and resolution of this method indicate the possibility for analysis of carbohydrates a t the single-cell level. Al-Hakim and Linhardt (M96) applied CZE for the first time in the analysis of non-, mono-, di-, and trisulfated disaccharides derived from chondroitin sulfate, dermatan sulfate, and hyaluronic acid. These glycosamino lycans were f i i t depolymerized using polysaccharide lyases. $he resulting unsaturated disaccharide products were detected by UV absorbance at 232 nm. An ei ht-component mixture was resolved within 40 min. Nasfiabeh and El Rassi (M9T) examined the electrophoretic behavior of derivatized linear and branched oligosaccharides from various sources using CZE with polyether-coated fused-silica capillaries. Two UVabsorbing (also fluorescent) derivatizin agenta (2-aminopyridine and 6-aminoquinoline) were usef for the detection of neutral oligosaccharides (e.g., N-acetylchitooligosaccharides, high-mannose glycans,and xyloglucan oligosaccharides).The oligosaccharides labeled with 6-aminoquinoline yielded 8 times higher signal than those ged with 2-aminopyridine. Furthermore, the oli osacchari es were better resolved in the presence of tetratutylammonium bromide. Pharmaceutical Compounds. Swartz (Ma) outlined the approach for separation of small pharmaceutical molecules using analgesics as examples and examined various parameters separated seven tricyclic on selectivity. Salomon et al. (M99) antidepressants (protriptyline, desipramine nortriptyline, nordoxepin, imipramine, amitriptyiine, and doxepin). Full resolution was achieved by the addition of methanol to the buffer, which decreased both the electroosmotic flow and the electrophoretic mobilities of the sam les. Yeo et al. (M100) separated six antibiotics with CZE/Uf detection. Kurosu et described the separation of vitamin BBgroup by al. (M101) CZE with an improved fluorescent detector. Johansson et al. (M102) reported the separation of sulfonamides and benzodiazepines in an uncoated fused-silicaca illary. In this work, the capillary was connected to a 1iquid)junction-ion spray interface that was coupled to an atmospheric pressure ionization (API) triple-quadrupole mass spectrometric system. On-line UV detection occurred at 20 cm from the inlet of the capillary and the API mass spectrometric detection occurred a t the outlet of the capillar The major metabolite of flurazepam, N-1-(hydroxyethyl)Jurazepam,was detected in human urine by this system in the presence of other flurazepam metabolites. Kuhn et al. (M103) reported the use of cyclodextrins and chiral crown ether ([18l-crown-6 tetracarboxylic acid) as buffer constituents for separation of chiral drugs and amino acids. Results of separation of racemic a-amino acids using both chiral selectorswere compared with respect to resolution, efficiency, and retention time. For separation of (*)quinagolides, 8-cyclodextrin was used as the chiral agent with buffer concentration and temperature being examined. The optimum conditions were 30 mM 8-cyclodextrin at pH 2.5. resolved cefixime,an oral cephalosporin Honda et al. (MI04) antibiotic, and its metabolites in human urine by various modes of capillary electrophoresis. In CZE mode, the use of a phosphate buffer at pH 6.8 containing 3-[(3-cholamidopropy1)dimethylammoniol -1-propanesulfonate ave complete resolution of cefixime and all five of its metaiolites. Inorganic Ions. Swaile and Sepaniak (M105) developed a novel method for analyses of Ca(I1) and Mg(I1) in blood serum with on-column chelation using 8-hydroxyquinoline5-sulfonic acid. The complexes were separated by CZE and detected by laser fluorescence. The limit of detection was in the ppb range. Koberda et al. (MIO6)used a weak complexing agent, a-hydroxyisobut ic acid, in the carrier electrolyte solution to separate a l k J a n d alkaline-earth cations (sodium, potassium, calcium, and magnesium) in parenteral solutions. The cations were detected indirectly at 214 nm using a strongly absorbing electrolyte co-ion called UV-Cat I. Motomizu et al. (MI07) examined these aration of several divalent metal ions, as well as iron(II1) anzsilver (I), by CZE using chelating

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agents with on-column UV-absorption detection. When 1,2cyclohexanediamine-N,N,N,N’-tetraaceticacid (CyDTA)waa used as the chelating agent in the carrier solution (pH 9), the order of mi ration time was as follows: Ba2+ < Sr2+< Ca2+ < Mg2+ (Bef+ could not be detected), Fe3+ < Mn2+< Co2+ < Ni2+ < Cu2+< Zn2+,Cd2+< Ag+,Hg2+ < Pb2+. The detection limits of common metal ions were 1o-S M.

MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY Background. In CZE, neutral molecules migrate at the speed of electroosmotic flow without separation. The development of micellarelectrokineticcap chromatography (MECC) has extended the capability of C for separation of neutral compounds in the presence of cationic and anionic solutes. Micelles are aggregates of amphiphilic molecules known as surfactants that possess a hydrophilic head group and a hydrophobic region. The hydrophobic region of the molecule is either a straight or branch chain of hydrocarbon or a steroidal structure, whereas the hydrophilic head group can be cationic, anionic,zwitterionic,or nonionic. Normally when surfactant molecules exceed their critical micelle concentration (cmc), micelles are formed in aqueous solution with the hydrophobic regions pointing inward and the hydrophilic heads pointing outward into the aqueous solution, resulting in reduced free energy of the system. In MECC, micelle serves as a pseudostationary hase that resembles the stationary phase in the reversed-piase liquid chromatography. Solutes interact with micelles via various mechanisms including hydrophobic, electrostatic, and hydrogen bonding. The partition process of the solutes between micelles and bulk solution can be controlled by the type of surfactant; surfactant concentration; and additives such as organic solvents, ionic salts, chiral selectors, ion-pairing and complexing agenta; or manipulation of experimental parameters such as pH and temperature. The separation in MECC is the result of differential partitioning of solutes between the bulk aqueous phase and the micellar phase, and differential migration of the solutes in an electric field. Clinical Analyses. Atamna et al. (M108) described the se aration of methyl-substituted uric acids and methylsuistituted xanthines, the metabolites of caffeine present in human plasma and urine. Twelve of 13 compounds were resolved with 0.15 M sodium dodecyl sulfate (SDS) added to 0.05 M phosphate buffer ( H 7.0) a t an ap lied vol kV. Thormann et al. (h109)discusse the an ysis of barbiturates in human serum and urine with on-column multiwavelen h detection. Seven barbiturates were characterized by t eir retention and absorption spectra between 195and 320 nm in a pH 8 buffer containing SDS. Comparison of these computer-stored data with those of unknown samples allowed the identification of barbiturates in urine and serum samples of patients undergoing pharmacotherapy. Meier and investigated the quantitation of thiopental Thormann (M110) in human serum and plasma. Thiopental and an internal standard (carbamazepine) were extracted from serum or plasma using pentane and phosphate buffer (pH 6.4). The separation took place in a phosphate-borate buffer with 50 mM SDS. Results of 66 patients’ samples by MECC method correlated well with an HPLC method. Prunonosa et al. (MI11)developed aprocedure for the determination of S-(+) and R-(-) enantiomers of cicletanine in human plasma. This procedure consisted of an extraction step with diethyl ether and a separation step in a fused-silica capillary using y-cyclodextrins in the run buffer. The linear range was from 10 to 500 ng/mL. The limit of detection was 10 ng/mL for each enantiomer in plasma samples with UV detection. The within-run precision of the method expressed as a relative standard deviation was 10.4 and 9.6 76 at 25 ng/mL for S-(+) and R-(-) cicletanine, and 4.2 and 4.6% a t 500 ng/mL, also developed a simple and respectively. Lloyd (M112) accurate method for quantitation of diastereoisomers of L-buthionine-(R,S)-sulfoximine.This method may be used to detect buthionine sulfoxime in plasma over the expected therapeutic range. Miyake et al. (M113) separated creatinine and uric acid in untreated human plasma and urine. The samples of untreated plasma or urine spiked with internal standard, antipyrine,

%

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CLINICAL CHEMISTRY

were first introduced into the capillary by siphoning. Creatinine, uric acid, and antipyrine were then se arated from each other within 18 min in the resence oPendogenous components. The calibrationplots sIowed good linearity over the concentration range needed for clinicalanalysis. Standard addition tests indicated that the recoveries of creatinine and uric acid from urine samples ranged from 97 to 106%and 97.4 to 108% with a CV of 3.3 % , and those from plasma samples ranged from 100 to 112% and 101 to 107% with a CV of 4.7%. The results were in agreement with those obtained by conventional methods. Small Biomolecules. Miyashita and Terabe (M114) described the separation of dansyl DL-amino acids by MECC with and without the presence of cyclodextrins. Nine of 12 DL-aminO acid derivatives were resolved. Nishi et al. (M115) separated the optical isomers of 2,3,4,6-tetra-O-acetyl-8-Dglucopyranosyl isothiocyanate- (GITC-)derivatized DL-amino acids. Nineteen of 21 DL-amhO acids were resolved under studied neutral and alkaline conditions. Ong et al. (M116) the mi ation behavior of selected catechols and catecholamines r y MECC with SDS in phosphate-borate buffer. A mixture of two catechols and six catecholamines were successfully resolved. Binding Proteins. Kajiwara (M117)applied CE to the analysis of Ca- and Zn-bindingproteins. Ca-bindin proteins (calmodulin, parvalbumin, thermolysin, and roteoyytic eptides of calmodulin),Zn-bindin proteins (cargonicanhy8aae and thermolysin), and intern3 standard proteins (carbonic anhydrase and lacto lobulin) were separated completely by both CZE and ME&. The binding shifts of these roteins were de ended on the type of cations presentelin the electrop oretic buffer. PharmaceuticalCompounds. Altria and Smith (MI18) developed a method for harmaceutical separation of the antidepressant GR503601 from a potential manufacturing reported the determiimpurity. Altria and Rogan (M119) nation of cefuroxime axetil in suspensions and tablets. Yik et al. (MI201described MECC with amperometric detection for a mixture of Be vitamers usin a 50-pm4.d. column. The detection limit was 4 fmol. The [near dynamic range of the calibration lot was from 1to 200p m. Iwagarni et al. (M121) ap lied Ml8CC to the analysis of ggcyrrhizin in glycyrrhizae ra8x and oriental pharmaceutical preparations. A high volta e (-180 V/cm) was ap lied to a 70 cm X 50 pm i.d. fusecfsilicacapillary tube fillexwith borate-phosphate buffer containing 100 mM of SDS at H 9. Hydroxybenzoate was used as the internal standard. he on-column detection was performed at 254 nm. The calibration plots obtained from either peak hei ht ratios or peak area ratios gave good linearities over %e concentration range of 50-1000 pg/mL. The CVs for the detection of glycyrrhizin in six common preparations were 2.7-7.0%. Inorganic Ions. Saitoh et al. (M122) applied MECC to metal complexes of acetylacetone (both stable and labile complexes) using SDS solution containing 100 mM acetylacetone. The migration velocitiesof the complexes of Sc(III), V(IV), Cr(III), Mn(III), Fe(III), Co(III), and Cu(I1) were overned by the partition between the micellar hase and [ulk aqueous phase, whereas those of Ti(IV), Co(fI), Ni(II), and Zn(I1) were governed b the electrophoresisin the aqueous described the phase. In another study, gaitoh et al. (M123) separation of electricall neutral acetylacetonate complexes of Cr(III), Co(III),Rh(I$, andPt(I1) or Pd(II) in SDS solution by MECC. The distribution coefficient of each metal complex between the SDS micelle and the aqueous solution surrounding the micelle was calculated from the capacity factor. A linear log-log relationshipwas found between the distribution coefficient and the partition coefficient of the complex between dodecane and water. The linear relationship was effectively used for prediction of both the distribution coefficientsand the migration timea of other metal complexes, such as Pdn acetylacetonate and Crm 3-methyl acetylacetonate.

K

5

ISOTACHOPHORESIS Background. Isotachophoresis (ITP) stands for “is0

(same) + tacho (velocity) + phoresis (electrophoresis)”. It is also known as displacement electrophoresis, or multizonal electrophoresis. In ITP, the sample is inserted between a

leading electrolyte and a trailing (or terminatin ) electrolyte in a capillary without electroosmotic flow. he leading electrolytehas the highest mobility and the trailing electrolyte has the lowest mobility with respect to the ions of interest in the sample zone. Separation in ITP depends on the different velocities of the analyte ions, Ui, in the sample zone:

%r

ui = pep,iEaz (7) where pe j is the electrophoretic mobility of s ecies i and E, is the efectric field strength of the sam& zone before separation. During the transient separation process, the electric current and tem erature are kept constant. According to the Ohm’s law, the eyectric field strength increases as the mobility of the consecutive analyte ion bands decreases (an ion band is defined as a homogeneous solution separated by moving or stationary boundaries). After the separation process, all the analyte ion bands migrate at the sam le velocity, u; therefore, a steady-state stacking is achieve8

u = pep,iEi = ~

(8) where pe j, E i , and pe,j, Ej are the electrophoretic mobility and eleclric field strength for species i and j’ in analyte ion bands i and j , respectively. The concentrations of analyte ions in their mi atin bands will be adjusted t o the leaiing electrolyte ion according to the concentration of Kohlraugh regulating function: e p p j

tE

ci

pi(pl+

= c,

PJ

+ PJ

(9)

where C i is the concentration of s ecies i in ion band i, CIis the concentration of the leadin e ectrolyte, pi, p1, V d pc are the mobilities of species i, the yeading electrolyte ion 1, and the counterion c in the steady state, respectively. ITP is an enrichment method in those cases where the analyte ion concentration in sample is lower than the concentration of the ion in the steady state. Only one type of ion, either anions or cations, can be determined in a particular run. Detection is based mainly on conductivity, differential conductivity, or direct UV adsorp tion. In the case of direct UV detection, spacers are needed, which are the nonabsorbing solutes with mobility values that fall in between the mobilities of analyte peaks. Plasma Sample. Krivankova and Bocek (M124) developed an isotachophoretic method for simultaneous determination of pyruvate, acetoacetate, lactate, and 3-hydroxybutyrate in 1-10 p L of untreated heparin plasma of patients with diabetes mellitus. An instrument with a column switching system was used. The preseparation capillary was filled with a leading electrolyte of pH 4.2 and the analytical capillary was filled with a leading electrolyte of pH 3.0. The concentrations of individual ketone bodies and lactate found in human plasma varied from 0.01 to 1.4 and 0.8 to 11.8 mM, respectively. Analysis time was about 30 min. Proteins. Thorn andRiebe (M125) described the analysis of 99% pure iron-free human serum transferrin. The best results were obtained with a buffer of 10 mM Tris-HC1 (pH 8.7), as the leading electrolyte, 12.5 mM glycine-barium hydroxide (pH 10.5), as the terminating electrolyte, and 1-methylhistidine and taurine as spacers. Fifteen fractions used ITP of transferrin were collected. Tsikas et al. (M126) to separate cysteinyl leukotrienes (LTs): Cd, D4, and El. The method was based on anionic separation and UV detection (254 nm), The total analysis time was 30 min. The limit of detection was 0.5 nmol of LTEd. Despite the similarity of chemical structures, all three cysteinyl LTe were separated. Caslavska et al. (M127)tested the suitability of using recycling isotachophoresis for the purification of ovalbumin (OVA) and/or lysozyme (LYSO)from a common OVA product containing LYSO and conalbumin (CAL) as the major proteinaceous impurities. Typically 700 mg of the common roduct was processed within 2 h. All three proteins could e! separated and fractionated usi suitable spacers. Deending on the chosen conditions,e%er OVA or LYSO could {e purified at rates of mutiple milli a m s per hour (OVA 300 mg/h; LYSO 10 mg/h). The insta%ity of CAL in solution prevented ita isolation in the investigated configurations. reported the determination Gebauer and Thormann (M128)

P


4281

CLINICAL CHEMISTRY

of proteins in uncoated open-tubular fused-silica capillaries of 50- and 75-pm i.d., with on-column multiwavelength detection. A small amount of hydroxypropylmethylcellulose was added to the leading electrolyte, which provided an efficient a proach for dynamic column conditioning. This method algwed the high-resolution isotachophoretic determination of most proteins in the presence of electroosmotic flow. Different approaches for cationic and anionic analyses were discussed and illustrated with selected examples. Peptides. McDonnell and Pawliszyn (M129) separated several synthetic eptides by ITP with a concentration gradient detector. b h e results were com lementary to those obtained by reversed- hase HPLC. T i e detection of low levels of underivatizef peptides (nmol) was accomplished using a universal, low-volume optical cell. A slider injector was designed and used in this work. Pharmaceutical Compounds. Tsikas et al. (M130) developed qualitative and uantitative methods for 8-lactam antibiotics (penicillins an! cephalosporins) and their precursors (6-aminopenicillanicacid and 7-aminocephalosporanic acid) in chemical and pharmaceutical preparations. Two different electrolytes were emplo ed with pH values of the leading electrolytes of 4 and 7. T i e leading electrolyte with a pH of 7 allowed for the detection of several B-lactam antibiotics that had differences in the side chain of the penicillin or cephalosporin structures. Jokl and Petrzelkova (M131)investigated the separation of propranolol, metiranolol (I), and desacetylmetipranolol (11) in drug tablets y ITP with an acetate leading electrolyte (10 mM, pH 4.75). The linear range of calibration was from 1W to 1 WM. Tanaka separated six niacin derivatives of 8-picoline, et al. (M132) pyridine, 3-(hydroxymethyl)pyridine,6-aminonicotinamide, isonicotinic acid hydrazide, and nicotinamide using 40 mM potassium acetate and acetic acid as the leading electrolyte and 10 mM gl cine and hydrochloric acid as the terminating electrolyte. $he linear calibration ran es were 1-5 nmol. described the use ofalkylhydroxyalkylSnopek et al. (M133) cellulose derivatives in cyclodextrin-modified electrolytes to improve both enantioselectivity and separation efficiency for isomeric compounds. Catecholamines. Tanaka et al. (M134)reported the isotachophoretic separation of six catecholamines based on inclusion complex formation with 8-cyclodextrin. A neutral surfactant was used to suppress the electroosmotic flow in this work. Inorganic Ions. Fukushi and Hiiro (M135) studied the effects of crown ethers on the effective mobilities of various metal ions in ITP. The effective mobilities of thallium, lead, and silver ions decreased when the concentration of crown ethers in an ordinary leadin electrolyte was increased to 50 mM. Use of 18-crown-6 fed to complete separation of ammonium and thallium ions and of thallium and lead ions. Similarly, use of 15-crown-5led to complete separation of potassium and thallium ions and of ammonium and silver ions.

E

CAPILLARY GEL ELECTROPHORESIS Background. Capillary el electro horesis (CGE) is the adaptation of traditional $ab gel el!ktrophoresis to the capillary format. CGE is potentially useful for separation of many large biological molecules, such as polynucleotides and proteins that have similar migration rate in free solution, due to virtually identical char e to mass ratio. CGE is a size-base separation technique contucted in an anticonvectivemedium such as polyacrylamide gel. The gel serves as a molecular sieve because mobility of a molecule in a gel network decreases as the size of molecule increases. The resolving ability of a gel can be optimized for analytes of given molecular weight range using proper gel densit that is determined by the total monomer concentration (% and the degree of cross-linking (% C). The use of gel can also minimize solute diffusion, prevent solute adsorption to the capillary wall, and suppress electroosmoticflow, thus permitting maximum resolution on a short column. Gel Columns. Holloway (MI361prepared a polyacrylamide gel column using a fused-silica tube, a polyacrylamide gel matrix, and a polydimethylsiloxane la er that was covalently attached to both the tube and the germatrix. After

6

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the polymerization, buffer ions were drawn into the column under an electric field of -lo00 V/cm and the reaction products were flushed out at the same time. Durin polymerization, the polyorganosiloxane layer stretchef as the polyacrylamide shrank so the polyorganosiloxane was at least twice as thick as it would have been without the gel matrix attachment. The process yielded a gel matrix that was securely attached to the tube, void-free, and under minimal mechanical stress. Shieh (M137)reported the use of a bifunctional agent for preparation of gel column by adsorbing a positively char ed amine group to the inner surface of the capillary wall ancfcopolymerizingthe remaining functionality of the agent with a polymeric el material. Karger et al. (M138) described a highly stabfe capillary column usin a coating matrix on the inner surface of the capillary. +he coating matrix had an intermediate hydrophobic layer chemically bonded to the inner wall of the capillary and a detergent that covalently bonded to polymerized monomer. The intermediate hydrophobic layer interacted with the detergent forming a hydrophilic outer layer of coating matrix. Novotny et al. (M139)produced a gel column through electrophoreticallydrawing a char ed polymerizationinitiator into a capillary tube that was preilled with monomer, crosslinking agent and catalyst. The travel rate of the initiator was slow enough to maintain a sharp leading edge or front, thereby causing polymerization to occur in a progressive manner from one end of the capillary to the other. The shrinkage inherent in the polymerization reaction was compensated for by fluid movement ahead of the traveling initiator front; thus, the discontinuities in the gel were avoided. Rose (M140) prepared a gel column by filling a capillary with a gel-formingprepolymer, initiating polymerization at one end of the capillary, and causing the polymerization to progress longitudinally, with fresh monomer solution filling vacancies formed from polymerization. Wang et al. (M141) made polyacrylamide gel-filled fusedsilica capillariesthrough hotopolymerizationusin riboflavin as the initiator. The pol$nerization was initiatefby adding riboflavin to the acrylamide-bisacrylamide solution before filling the capillary. After introduction of the solution into a UV-transparent capillary, UV radiation was used to start the polymerization. This method seemed to have fewer complications with regard to bubble formation in the gel in comparison to other methods because of the use of persulfate as the initiator and TEMED as the catalyst. The performance was demonstrated by the separation of oligonucleotides. Schomburg and Lux (M142)developed a ?-irradiation procedure for producing el-filled capillaries. A fused-silica capillary was first filletwith a degassed monomer (e. acrylamide and/or bisacrylamide) in buffer. Then, mixture in the capillary was polymerized through a radical reaction initiated by y-radiation (@To). Baba et al. (M143) reported a method for the production of polyacrylamide gelfilled capillaries. A solution of acrylamide was injected into a capillary without inner surface pretreatment and polymerized in situ by radical initiators. Bubble formation in capillaries was avoided by using well-designed injection equipment. Performance of the gel-filled capillary was examined in terms of stability, reproducibility of migration time, feasibility of method, and resolving power of polynucleotides. Motsch et al. (M144) produced capillaries that filled with agarose gel. The capillaries were used in the separations of medium-size oligonucleotides (DNA restriction fragments) and disaccharides. Baba et al. (M145) reported the use of poly(94nyladenine) as an affinity macroligand that was entrapped in a polyacrylamide el matrix for high-speed and high-resolution separation of o$odeoxynucleotides. Yin et al. (M146)reported the improvement of erformance and stability of polyacrylamide gel-filled capitaries through a surface pretreatment procedure. Nongel Sieving Columns. Zhu et al. (M147)used dissolved, non-cross-linked polymers, such as poly(ethy1ene glycol) or cellulose derivative, which had molecular weight ranges that overlapped with that of the analyte producin an electrophoreticsievink media for the separations of myoglotin, substance P, albumin components, and DNA fragments. Sudor et al. (M148)re orted a rocedure for makin polyacrylamide columnsy! filling inear non-cross-linke polyacrylamide solution from the stock through pressure

tk

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CLINICAL CHEMISTRY

loading. By this way, the in-capillary polymerization was avoided. Oligonucleotide. Dubrow (M149) used gel-filled capillaries to resolve synthetic oli onucleotides from single base unit to beyond 120 bases. Ba%a et al. (M150) demonstrated the high-resolution separation of single-stranded homopolynucleotides. The plate numbers achieved in CGE were 3 X 106-7 X 1Oe plates/m that were about 10-100 times larger than those of HPLC modes of 4 X 1 0 9 X 106 plates/m. DNA. Chen et al. (M151) reported lowzeptomoledetection limits for DNA sequencing by CGE (1zmol = 1X 10-21 mol). A 750-pW green helium-neon laser (X = 543.5 nm) was used to excite tetramethylrhodamine-labeled DNA fragments in a sheath flow cuvette, and a cooled photomultiplier tube was used to detect fluorescence in a single spectral channel. Se uencing data were generated at a rate of about 70 bases1 demonstrated the use of CGE with h. werdlow et al. (M152) laser-induced fluorescence detection for four-, two-, and onespectral-channel sequencing techniques. Sequencing rates up to 1OOO bases/h were obtained at an electric field strength of 465 V/cm. At lower electric field strengths, CGE produced useful data for fragments greater than 550 nucleotides in length, with 2-fold better resolution than slab gel electrophoresis. An on-column detector produced detection limits of 200 zmol for the four-spectral-channel technique. A poatcolumn detector, based on a sheath flowcuvette, produced detection limits of 20 and 2 zmol for the two- and one-spectralchannel techni ues, respectively. Huang et al. (M153) demonstrated a%NA sequencing method that used capillary array electrophoresis, two-color fluorescence detection, and a two-dye labeling protocol. Sanger DNA sequencing fragments were separated in a 25-capillary array and detected on-column using a two-color, laser-excited, confocal-fluorescence scanner. The four sets of DNA s uencing fragments could be separated in a single capillary an then distinguished by using a binary coding scheme, where each fragment set was labeled with a characteristic ratio of two dye-labeled primers. Since only two d e-labeled primers were required, it was possible to select fyes that have identical mobility shifts. The ratio of the si als in the two detection channels provided a reliable identiEation of the sequencing fragment. used gel-filled capillary electrophoresis to Chen et al. (M154) perform Southern blotting with on-linedetection and to study parameters that affected the hybridization of DNA molecules in solution. A fluorescence-tagged oli onucleotide was used as the probe for hybridization in scfution. The reaction mixture was separated in a polyacrylamide gel column and the hybridized species was detected by UV absorption or laserinduced fluorescence detector. Huang et al. (M155) investigated some critical parameters affecting separations of single-stranded DNA fragments with UV absorbance detection. Although not as sensitive as laserinduced fluorescence, UV absorbance detection allowed the accurate and inexpensive calculation of the molarity of each improved separated DNA fragment. Demana et al. (M156) DNA fragments resolution in CGE using velocity modulation. The system was tested with HaeIII fragments of 4x174 RF described DNA in a polyacrylamide gel. Smith et al. (M157) a robotic system for the performance of enzymatic sequencing reactions and an instrument for high-speed DNA sequencing by CE. The performance of the s tem was tested by sequence analysis of the phage vector G13mp19 usin four DNA sequencing reactions with four fluorophores. he speed of analysis was increased more than 14-fold relative to slab gel electrophoresis. Strege and Lagu (M158) investigated severalCE techniques for separation of DNA restriction fragments up to 23 OOO bp. Methods employingelectroosmoticflow in an untreated silica capillary provided only partial resolution of the 23 fragments in a 1-kbp DNA ladder. By coatin the inner walls of the capillary with polyacrylamide and tflling the capillary with buffers containing methylcellulose as a sieving medium, all ents in the 1-kbp DNA ladders were separated. In rd%on, this technique facilitated the separation of very large fragments in a X DNA-Hind111 digest. Optimum resolution was obtained at low separation potentials using buffers containing at least 0.5 5% methylcellulose. Guttman incorporated an affinity li and within a and Cooke (M159) polyacrylamidegel to provide a generalmeanso manipulatin the selectivity of CGE separation. High resolution of DNW

%

41

4

f

restriction fragments was achieved by adding an intercalating agent, ethidium bromide, to the separation medium. The theoretical plates were as high as 107/m. PCR Products. Schwartz et al. (M160)used a polysiloxane-coated capillary and polymeric buffer additives (hydroxypropylmethylcellulose) to separate DNA restriction fragments and PCR products for detection of the AIDS virus (HIV-1)in blood. Ulfelder et al. (M161) described the anal is of human ERBB2 oncogene. The PCR-am lified R Z P sample was loaded into the capillary with a fielxamplification technique and then se arated in a sieving buffer containing ethidium bromide. T i e separation time was less than 30 min. The capillary was reusable and full automation was feasible. The precision of migration time was better than 0.2% RSD with a 12-bp resolution for the DNA fragment sizes of interest. RFLP samples were analyzed for homo- or heterozygosity based on the presence of 500- and/or 520-bp DNA fragments. Yamqata (M162)reported a rapid and simple assay for clinical diagnosis of eathogens. In this assay, Salmonella typhimurium was grown by a standard method. The genomic DNA was isolated and am lified with two synthetic oligonucleotideprimers for 42 cycpes by PCR. The amplified DNA product was separated by CE. Mayer et al. (M163) described the conditions for rapid separation and detection of PCR products from the human immunodeficiency virus type 1gag gene and the HLA-DQ-a gene amplified from the human immunodeficiencyvirus rovirus-containing U1.l cell line. The sensitivity achieve: with CE was roughly equivalent to that of polyacrylamide gel electrophoresis by ethidium bromide staining. Proteins. Tsuji (M164) used fused-silicacapillary columns filled with SDS-polyac lamide gel to separate recombinant biotechnological-derivs proteins. An excellent linear relationship (r > 0.999) existed between the peak migration time and the molecular weights of reference proteins in the ranges of 10 000-100 OOO and 40 000-200 OOO using the capillary columns filled with acrylamide gel at total monomer concentrations of 5 and 3 %. The RSD of the peak migration time was about 1% . Theoretical plates of 5 X 106-1 X 1@/m were routinely obtained. Calibration gra hs of peak area versus molecular wei ht of recombinant [iotechnologicalderived proteins were finear (r > 0.999) and the proteins were quantified with an RSD of 3-7 % . As little as 50 nmol of re orted the protein was detected. Widhalm et al. (M165) separation of proteins according to their molecu ar masses by CE with a buffer containing a linear non-cross-linked polyacrylamide el as a sieving medium. Four test proteins, covering molecdar mass range between 17 800 and 77 OOO, were applied as SDS complexes. The separation was carried out at pH 5.5 (phosphate buffer, 0.5% SDS, 10% linear polyacrylamide gel). The retention times of the proteins correlated well with the logarithm of their molecular masses.

P

CAPILLARY ISOELECTRIC FOCUSING Background. Capillary isoelectric focusing (CIEF) is used for high-resolution separation of roteins based on differences in their isoelectric points (pZ). !he premise of CIEF is that a protein will migrate as long as it is char ed in an electric field. If the protein becomes neutral, it wib cease migratin in the electric field. In CIEF, a series of zwitterions (calle! ampholytes) are used to generate a pH adient inside the focusin capillary. The ampholytes t f a t are positively chargelwill migrate toward the cathode while those that are negatively charged will migrate toward the anode. The pH will therefore increase at the cathode side of the capillary and decrease at the anode side. When each ampholyte reaches ita own pZ and is no longer charged, the migration ceases. As a result, a stable pH gradient is formed. If a rotein has a net positive charge, it w i l l mi ate toward the catfode. During its migration toward the cagode, it eventually encounters a pH at which it has a zero net char e and ceases to migrate. The greater the number of amp%olytes in solution, the smoother the pH gradient will be. Furthermore, the pH of the catholyte must be higher than the pZs of all the basic a m p h o p and the pH of the anolyte must be lower than the Is of the acidic ampholytes, to prevent the migrations of 0th buffers into the capillary. CIEF will be most effective when the electroosmotic flow and other convective forces are eliminated or greatly sup-

E


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CLINICAL CHEMISTRY

pressed. However, it is still possible to perform CIEF in presence of electroosmosis, so long as the electroosmotic velocity of the solution inside the capillary does not exceed the electrophoretic velocities of the roteins. A useful means for reducing electroosmotic flow an protein adsorption onto the capillary wall is to use coating materials such as methylcelluloseand polyacrylamideto su press the ionization of the silanol group on the capillary wa 5 Application. Only a few reports in the past two years have discussedthe application of CIEF. Costello et al. (M166) used CIEF to characterize human anti-Tac monoclonal in antibody in a liquid phase. The human antibody had PIS the range of 8.2-9.0, a region in which recast slab polyacrylamide or agarose gels were not stabE. Because of the simplicity in enerating a variety of pH gradients and other advantays, 81EF may become the method of choice for the routine c aracterization of therapeutic monoclonal antibodies and their stabilit profiles. Dubrow (MI67) described the preparation of hig -viscositypolymer matrixes that contained poly(ethy1ene oxide), hydroxyethyl cellulose, and polyacrylamide for CIEF. The matrixes were used in the separation of proteins, oli onucleotides, and hos ho lated and nonphosphorylate poly(adeny1ic aciz). &oh% et al. (MISS) reported the use of a polycationic coating reagent (MicroCoat) to reverse the surface charge of the untreated fusedsilica capillary. After treatment, the surface of the ca i l l 9 became positively charged at neutral and lower pH, dowmg roteins below their pI to move throu h the capillary without geing attracted to the wall through efectrostatic interaction. This method gives a simple and quick estimation of a protein’s p I without using denaturant. Finally, Chen and Wiktorowicz (MI691 developed a quantitative CIEF method for RNase TI and RNase ba that had PIS at the two extremes of a pH 3-10 gradient. The site-directed mutants of the RNase T1were of RNase T1wildseparated from its wild-type form. The PIS t e, three mutants of RNase T1, and RNase ba were Z r m i n e d for the first time to be 2.9,3.1, 3.1,3.3, and 9.0, respectively. The method exhibited high resolution and linearity over a wide pH range.

B

.

E

d

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