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Electrophoresis Separation Technique
Electrophoresis Separation Technique
Electrophoresis Separation Technique
1957
Review
Sergey P. Radko1, 2 Andreas Chrambach2
1
Separation and characterization of sub-m- and m-sized particles by capillary zone electrophoresis
The analytical separation and characterization of particles in the size range of sub-mm and mm diameters by capillary zone electrophoresis (CZE) has been reviewed. The theoretical basis, on which the mobility can be interpreted to provide information regarding characteristics of particle surface, has shortly been presented. Particular emphasis was put on the model dependence of that interpretation and the need in most applications to forego the classical idealized model of spherical particles with smooth surfaces and to apply more realistic models, which take the hairy surface of real particles into account. Some highlights of the literature on the CZE of polystyrene latex microspheres, organic and inorganic colloids, lipoprotein particles, viruses, liposomes, biological membrane vesicles, and biological cells have been discussed. Also summarized are the reports on the particle size dependence of mobility and peak broadening in CZE and on electrophoretic behavior of rodlike particles and particle aggregates. Finally, the effects of neutral polymers in the background electrolyte on particle mobility and peak width are reviewed.
Keywords: Capillary zone electrophoresis / Microparticles / Review EL 4969
Biotechnology Department, Research Center for Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russia 2Section on Marcromolecular Analysis, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
Contents
1 2 3 3.1 3.2 3.3 3.4 3.5 3.6 4 4.1 4.2 4.3 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Physical mechanisms underlying the electrophoretic migration of particles . . . . . . CE of various microparticles . . . . . . . . . . . . . Polystyrene latex size standards . . . . . . . . . . Organic and inorganic colloidal particles . . . Lipoprotein particles . . . . . . . . . . . . . . . . . . . Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liposomes and biological membrane vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological cells . . . . . . . . . . . . . . . . . . . . . . . . Special cases of microparticle electrophoretic behavior . . . . . . . . . . . . . . . . . . . . . . CZE of nonspherical particles: the effect of the electric field-induced orientation . . . . Size-dependent electrophoretic migration of microparticles . . . . . . . . . . . . . . . . . . . . . . Source of peak width in CZE of microparticles . . . . . . . . . . . . . . . . . . . . . . 1957 1958 1960 1960 1961 1962 1962 1963 1964 1966 1966 1966 1968
4.4 4.5 5 6
Spike-like peaks in CZE of microparticles and particle aggregation . . . . . . . . . . . . . . . . CZE of microparticles in solutions of neutral polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 Introduction
During the past two decades, after emerging in the early 80s, capillary electrophoresis (CE) has become a wellestablished analytical technique widely applied to analysis and separation of both small ions and protein or DNA macroions (e.g., [1]). One of the features of CE is that electrophoresis can be performed in free solution, in the absence of a gel, since convection is effectively suppressed in capillaries of inner diameter of less than 0.2 mm. A restriction in the size of analyte, imposed by the dimensions of gel pores, is thus removed while the inner diameter of capillaries generally used allows them to easily accommodate particles of up to a few mm in radius, which is of the order characteristic for biological cells. As a result, the interest in CE as an analytical technique to analyze particulate species of different origin (with sizes well exceeding those of macromolecules) [2], including various types of biological cells was steadily growing over the last decade. We will limit our consideration to the species with sizes lying within the range of 0173-0835/02/13071957 $17.501.50/0
Correspondence: Dr. Andreas Chrambach, Bldg. 10, Rm 9D50, NIH, Bethesda, MD 20892-1580, USA E-mail: acc@cu.nih.gov Fax:1301-402-0263 Abbreviations: DLV, Doppler laser velocimetry; PC, phosphatidylcholine; PEO, polyethylene oxide; PG, phosphatidylglycerol; PSL, polystyrene latex; RBC, red blood cells; TBE, Tris-borateEDTA
CE and CEC
1958
Electrophoresis 2002, 23, 19571972 cells. Distinction will be made between largely artificial particles with smooth surfaces and biological particles with preponderantly hairy surfaces. Some aspects of microparticle electrophoretic behavior such as electrophoresis of non-spherical particles, size-dependent electrophoretic migration, peak broadening, migration of particle conglomerates, and particle electrophoretic migration in solutions of neutral polymers will be shortly discussed.
about 10 nm (which is above the average size of most globular proteins) to about 10 mm in diameter and we will not consider the case of long chain-like macromolecules. For the sake of simplicity, we will refer to those species as particles or microparticles. Since the overwhelming majority of surfaces possess an electric charge in electrolyte solutions, electrophoretic methods have long been among those employed to separate or to analyze microparticles both in science and in industry. Zone electrophoresis and isoelectric focusing in media stabilized by a density gradient, rotation, or agarose gels of low concentration have been employed to separate microparticles with the purpose of profiling, purification, or measuring their pI (e.g., [39]). Free-flow electrophoresis was mostly used for preparative separation of particles on the basis of electrophoretic mobility differences and isolation of the species of interest [10, 11]. Microelectrophoresis [12, 13] and Doppler laser velocimetry (DLV) [13] were applied for measuring electrophoretic mobilities of particles. CE appears to be well suited for both mobility and pI measurements, for profiling microparticle mixtures, and quantification of microparticles. Even micropreparative isolations of microparticles can be achieved, using the standard CE instrumentation [14]. The technique allows for on-line detection producing quantitative information in the form of peak area or height, single sample analysis in a serial fashion, and the possibility to carry out separation in the presence of flow (generated by electroosmotic current). These features, which render CE automated and easy to perform can be thought to make it a method of choice for many applications where electrophoretic characterization or separation of microparticles is needed. A concern may arise as to mobility measurements in capillary zone electrophoresis (CZE) since the relatively large surface-to-volume ratio of thin capillaries, coupled with the relatively large surfaces of microparticles, can result in an interaction between particles and capillary walls that affects particle mobility. Nonetheless, a consistency of mobility values derived by CZE and DLV both for model latex microspheres (at least, those possesing a net negative charge) [2] and for biological cells [15] was found in general. Capillary zone electrophoresis (CZE) is the simplest of CE modes and straightforward to perform. In regard to analytical separation and characterization of microparticles, CZE has been the CE mode most widely employed. The aim of the present paper is to review applications of CZE to separation and characterization of microparticles such as synthetic latex nano- and microspheres, organic and inorganic colloids, lipoprotein particles, viruses, liposomes, biological membrane vesicles, and biological
Electrophoresis 2002, 23, 19571972 where e is the dielectric permittivity of the medium and z the particles zeta potential. The zeta potential is an electrostatic potential at the so-called shear surface an imaginary surface in the proximity of the solid surface, forming a sheath enveloping the particle. Thus, the particle migrates as a kinetic unit, accompanied by a certain quantity of the surrounding liquid. Within the frame of the classical electrokinetic theory (e.g., [17]), z-potential (and therefore particle mobility) monotonically decreases with increasing I due to a progressive screening of the surface charge by counterions. The function f(kR) ranges from 1 at kR , 1 (the Hckel limit) to 1.5 at kR . 1 (the , . Smoluchowski limit). In fact, the increase occurs within the kR-region of approximately 1 to 100 (f = 1.027 and 1.460 at kR = 1 and 100, respectively) [20]. The underlying assumptions in deriving Eq. (1) were that the medium is structureless and characterized by uniform e and Z, and that values of z are smaller than 25 mV (at 257C). One important feature of the ionic atmosphere, neglected in the derivation of Eq. (1) is its distortion (polarization). Once the particle moves, the ionic atmosphere lags behind the particle, imposing an additional drag on particle motion, known as the relaxation effect. While affecting the particle migration negligibly at small z-potentials (z # 25 mV), this effect makes Eq. (1) invalid at moderate or high z. The relaxation effect gives rise to a much stronger dependence of m on kR [21] than that according to Eq. (1). The other consequence of the relaxation effect is that, at kR . 3, the electrophoretic mobility undergoes a maximum as a function of z at a given kR (ibid.). Therefore, the particle mobility can in some cases become independent of, or even start to decrease with, an increasing z-potential. In practice, particles of higher surface charge density can migrate at a particular kR slower than those of lower charge density and the derivation of z-potential from the mobility data may become ambiguous. The z-potential (or the electrokinetic charge) that governs the particles electrokinetics is known to depend on the surface electrostatic potential, cs (or the particle surface charge). The electrostatic potential near the charged surface (and, thus, cs as its part) is basically calculated according to the Gouy-Chapman theory of a diffuse double layer [17]. In the more elaborate theory, the so-called Gouy-Chapman-Stern model, the ion adsorption onto the surface is taken into account based on the Langmuir adsorption isotherm (ibid.). However, the relation of the z-potential to the surface potential is not straightforward: electrokinetically only the hydrodynamically mobile part of the double layer is seen. A significant part of the countercharge is thought to be embedded in a hydrodynamically stagnant layer (a layer between the shear and particle surfaces), resulting in a sometimes substan-
CZE of microparticles
1959
tial discrepancy between the electrokinetic and the surface charge (e.g., [22]). Though the stagnant layer is often assumed to coincide with the Stern layer (a region encompassing ions and molecules adsorbed at the particle surface), not all ions in the stagnant layer are necessarily bound to the surface and thus electrophoretically immobile. Their motility may be taken into account by ion transport processes of one or another sort in the stagnant (or the Stern) layer [17, 23, 24] but such anomalous surface conductance further complicates the interpretation of electrophoretic mobility in terms of particle surface properties. Moreover, the coion adsorption may occur in the Stern layer (e.g., [24] and references therein). Both the anomalous surface conductance and the co-ion adsorption if operative result in the appearance of a maximum on the curve of m vs. I, a deviation from the behavior expected according to the classical electrokinetic theory [17]. The other important implication in the derivation of Eq. (1) is the ideality of the particle surface. One may often neglect irregularities on the particle surface if their characteristic dimensions are much smaller than the particle size and the Stokes drag is the dominant retarding force. Yet, if the electrophoretic retardation is the dominant force, it is necessary to compare the characteristic size of these irregularities with the thickness of the EDL (e.g., k1 = 3 nm in a 10 mM 1:1 electrolyte solution upon complete dissociation [17]). An example of a particle with a rough (or hairy) surface may be that with a layer of neutral polymers adsorbed on, or grafted to, its surface (e.g., [2527]). If the thickness of such hairy layer is comparable with or exceeds k1, the drag on the particle due to the electrophoretic retardation will increase. The medium then is no more characterized by a uniform Z and the bulk viscosity in Eq. (1) must be substituted by some effective viscosity. However, the calculation of this effective viscosity involves a number of parameters characterizing the hairy layer [28], which are often not experimentally accessible. Formally, the roughness of the particle surface may be accounted for by assuming a thicker stagnant layer that results in the outward shift of the shear surface and a consequent reduction of z-potential [29]. In that case, a possibility of anomalous surface conductance [22, 23] should be taken into account. The hairy layer can also be composed of polyelectrolytes. The typical examples are biological cells and membrane vesicles, or colloids with adsorbed, grafted, or naturaly occuring layers of charged polymers on their surfaces. Such particles may be described as having a relatively hard core covered with a polyelectrolyte layer permeable to both solvent (water) molecules and small electrolyte ions and are sometimes called soft particles [30]. Their important feature is that the surface charges
1960
Electrophoresis 2002, 23, 19571972 mass ratio (ibid.). Ballou and co-workers [40, 41], investigating the dependence on CZE operational parameters of selectivity and efficiency of separation for microspheres of known surface charge density (particles ranging from 30 nm to 1.16 mm in diameter, 75 mm ID capillaries) drew the opposite conclusion, viz. that the separations were electrophoretic in nature. However, it is thought that neither a qualitative agreement between the electrophoretic mobility and the titrated surface charge nor the absence of one allows for a firm conclusion regarding the nature of PSL separation in CZE, since first, the electrophoretic migration is governed by the electrokinetic charge and, second, the surface of polystyrene microspheres is not smooth but has a rather complex structure. Later on, the notion of the electrophoretic nature of PSL separation in CZE was supported by observing a reasonable agreement between electrophoretic mobilities of negatively charged polystyrene microspheres, measured by CZE and DLV [42]. It should be noted, however, that, when measured for the same microparticle concentration and in the same electrophoretic buffer, absolute values of mobility, derived by CZE were found to be about 10% higher than those derived by DLV for all PSL (100300 nm in diameter) studied. A similar discrepancy was observed for mobilities of PSL particles (3001100 nm in diameter), measured in the presence of a 100 mM sodium lauryl sulfate [43]. Moreover, when the latex diameter was decreased to 100 nm, the absolute values of mobility, provided by CZE were about twice of those by DLV (ibid.). The cause of such size-dependent discrepancy is unclear but presumably arises from the inherent technical differences between CZE and DLV. The authors [43] nonetheless speculate that the more pronounced polarization of the EDL due to external electric fields, which are much stronger in CZE than in DLV, may give rise to the observed mobility increase. However, such polarization will result in the relaxation effect that is known to impose an additional drag and to consequently reduce particle mobility [18, 21]. The mobilities of positively charged PSL in CZE were also found to differ substantially from those derived by DLV, probably in view of the strong interaction of these microspheres with negatively charged silica (10 mM phosphate buffer, pH 7.4) in bare 50 mm ID capillaries [42]. PSL of up to 1.1 mm in diameter produced well-defined peaks even in 25 mm ID bare capillaries (though in the presence of detergents) [43]. Polystyrene microspheres of up to 10 mm in diameter were demonstrated to electrophoretically migrate in 150 mm ID capillaries coated internally with linear polyacrylamide [44]. The UV detection appears to be well suited to monitor particle migration, presumably due to the scattering of UV light by the particles. Sample concentrations of 0.01 0.1% of solid latex (30 nm to 1.16 mm diameter microspheres) allow for a
cannot be considered as being located at a thin interface but must be viewed rather as being smeared out over a substantial region adjacent to the particle core. The friction imposed by polyelectrolyte chains on the electroosmotic flow in this region also has to be taken into account. A theoretical description of soft particle electrokinetics was pioneered by Jones [31] and Levine et al. [32] They have considered the case of a thin EDL (compared to the particle size) and low z-potential (25 mV) [31, 32]. Snabre et al. [33] have analyzed the case of an arbitrary z (to model electrokinetic behavior of erythrocytes under conditions of low ionic strength). In most reports on the electrokinetics of soft particles, a numerical computerbased modeling of particle electrophoretic behavior is employed (e.g., [3235]). An analytical expression, though of a quite complicated form, for the electrophoretic mobility of an arbitrary soft particle has also been obtained [36]. It should be noted that theoretical descriptions of soft particle electrokinetics do not necessarily employ the concept of a z-potential (e.g., [30, 36]).
3 CE of various microparticles
3.1 Polystyrene latex size standards
Polystyrene latex microspheres are widely used as model systems for colloids. The advantage of polystyrene latices (PSL) is that their sizes and geometrical shape are well defined. Nowadays, the microspheres are commercially available in a broad variety of sizes, each usually within a narrow size distribution. They possess a charge arising from reaction with polymerization initiators or from charged groups introduced upon polymerization (e.g., sulfate and carboxyl groups, respectively, for negatively charged PSL). The surface electrical properties of PSL are, however, still poorly understood. The reason is thought to be a high degree of hydrophobicity of PSL (charged groups cover as a rule only a few percent of particle surface), which strongly influences adsorption onto the particle surface. Moreover, most PSL possess a charged polymer (hairy) layer on their surfaces (e.g., [26, 37, 38]). A mixture of PSL of different sizes was the first particulate system used to demonstrate the utility of CZE for an analytical separation of microparticles. Six latex microspheres ranging in diameter from 39 to 683 nm were electrophoresed in a bare fused-silica capillary of 50 mm ID in buffers of low ionic strength [39]. The conclusion was that the observed separation of PSL is a result of their differential retardation due to particle-capillary wall interactions since the observed mobilities did not correlate well with the surface (titrated) charge or the charge-to-
Electrophoresis 2002, 23, 19571972 detection with confidence at 254 nm in 75 mm ID capillaries [40]. Optical spectra of PSL suspensions were shown to have a maximum in the short UV range for 50- to 200 nm diameter particles and a broad maximum in the long UV range for 400- to 600 nm diameter particles [45]. Thus, the detection sensitivity for a particle of a given size depends on wavelength. This fact was used for on-line identification of peaks corresponding to PSL of different sizes by monitoring separation at two wavelengths [43]. The fast, well-resolved analytical separations by CZE of mixtures of both chemically different latices [40, 46] and those with similar chemical composition but differing in size were demonstrated [39, 43, 45, 47, 48]. It should be noted that PSL of similar chemical composition exhibited in general a size-dependent electrophoretic migration in CZE. This phenomenon as well as a possible underlying mechanism will be discussed in Section 4.2. The feasibility to study physical interactions between different particles by CZE was shown by using acrylic and urethane latices as model particles [46]. The interacting particles were observed in form of a new peak appearing on the electropherogram (ibid.).
CZE of microparticles
1961
and DLV in the identical buffer were compared, the CZE values were found to exceed those derived by DLV by 3050% (depending on the pH of 10 mM phosphate buffer) [53]. The cause of this discrepancy is unclear. One may speculate that particle interactions with the capillary walls (CZE was carried out in a bare capillary) result in some retention of iron oxides and thus in an apparent increase of mobility. However, the peak shape is rather symmetrical, thus not supporting such speculation (ibid.). Perhaps, thermal or concentration effects are responsible for the discrepancy. It is thought that special attention must be paid to CZE operational parameters if a precise value of m is of interest. A linear relation was reported [56] between mobility of organic colloids (28350 nm in diameter) in CZE and their values of z-potential derived by DLV (the Zetamaster apparatus) in the identical buffer. Unfortunately, the equation is not provided in [56], by which the apparatus software calculated the reported values of z. Hence, no conclusion on a degree of consistency of mobilities measured by CZE and DLV may be drawn. Sub-mm-sized polymeric particles of different composition, possessing carboxylate and borate moieties on their surface were subjected to CZE in buffers supplemented with salts of di- and trivalent metals [51]. Their mobitilies were shown to decrease in the presence of the salts, as should be expected assuming strong binding of the multicharged counterions onto the particle surface. CZE was successfully used to study effects of pH, electrolyte concentration, and anion types on electrophoretic mobility of metal oxide particles [5355]. It was shown [54] that, contrary to the classical electrophoretic theory, the magnitude of m generally increased with increasing I for each oxide studied. This finding was interpreted in terms of coions (anion) binding into the Stern layer. The interpretation agrees with a significant effect of anion type (phosphate, carbonate, and borate anions) on the mobility of oxides and, hence, on the selectivity of separation (ibid.). CZE carried out in indifferent electrolyte solutions (such as those of sodium nitrate in these electrolyte systems, there is no specific adsorption of ions to the surface of the oxides) was utilized to measure isoelectric points of metal oxides (ranging from 0.01 to 1 mm in diameter) and to study effects of pH on the surface charge of the oxides [55]. Nearly baseline-resolved separations by CZE under optimized conditions for various two- and three-component mixtures of oxide particles were reported [57]. The choice of the anion type and the proper ionic strength of electrophoretic buffer were decisive (ibid.). The electrophoretic migration in a size-dependent manner was revealed in the CZE of colloidal gold nanoparticles [52] and silica sols [58]. In the latter, the separation of fine silica particles of different sizes was demonstrated. This particular kind of separation will be discussed in Section 4.2.
1962
Electrophoresis 2002, 23, 19571972 Soane [67] have used the tobacco mosaic virus as a model particle to investigate the effect of orientation on the electrophoretic mobility in CZE of rod-shaped polyions (this effect will be discussed in Section 4.1). However, until recently, no attempts to utilize CZE for characterizing viruses have been made. Over the last few years, a series of papers dealing with CZE of human rhinovirus (HRV) has been published by Okun et al. [6870]. HRV, the main causative agent of common cold infections, is an icosahedral particle of approximately 30 nm in diameter. It consists of a RNA genome and a shell composed of capsid proteins (e.g., [71]). When a HRV preparation was subjected to CZE in 50 mm ID capillaries, the electropherograms showed one major peak upon detection at both 205 and 254 nm. This peak was unambiguously identified as originating from the native virus (i) by heat denaturation of native virus samples, (ii) by enzymatic treatment of native and heat-denaturated virus, (iii) by depletion of virus in the sample by a specific monoclonal antibody [68]. The successful separation and identification of native virus and subviral Bparticles were also reported [69]. The latter are the end product of structural rearrangements that HRVs undergo during infection. In vitro, these rearrangements can be induced by exposure to elevated temperature or to low pH (ibid.). The electrophoretic buffer (100 mM borate buffer, pH 8.3) was supplemented with a ternary mixture of detergents at low concentrations tolerated by both viruses and subviral particles. The detergents were found to be important for preventing virus particle aggregation and adsorption to the capillary wall [69]. The utility of CZE to differentiate between some HRV serotypes even in crude preparations, based on differences in their electrophoretic mobility in the borate buffer supplemented with 10 mM SDS, was demonstrated in [70]. In that study, an infectivity assay was carried out on fractions collected at the capillary outlet, thus allowing for the biospecific identification of CZE peaks. A specific CZE-based approach, affinity capillary electrophoresis, was applied by the same group [7274] to study binding of monoclonal antibodies and lipoprotein receptor fragments to HRVs. The behavior typical for a ligand binding was observed, viz., shifts in the virus mobility in response to ligand (antibody or receptor fragment) concentrations, accompanied by a peak broadening at low ratios of antibody (or receptor fragment) to HRV. The quantitative analysis of this behavior has allowed one to estimate the binding stoichiometry [7274] and the dissociation constant [74] of the complexes. It appears that CZE may be a usefull method both for a quality control and quantification of virus production and for studying ligand binding to virus particles.
3.4 Viruses
Electrophoresis of a tobacco mosaic virus in a thin capillary (100 mm ID, 20 mM Tris-HCl buffer, pH 7.5, on-line detection at 260 nm), demonstrated by Hjertn et al. in 1987 [66], was the first example of the application of CZE to viruses. The migrating sample zone produced a relatively narrow, well-defined peak. Later on, Grossman and
CZE of microparticles
1963
dye exhitited a strong binding to silica when subjected to CZE in 75 mm ID bare capillaries, using 9.5 mM phosphate buffer (pH 7.4) [81]. The mean diameter of the liposomes, controlled by extrusion, was 350 nm. A capillary conditioning by means of three preliminary injections of the liposomes was found necessary to obtain reproducible peaks. This is the only report where such pronounced liposome binding was seen. The liposomes had a negative electrophoretic mobility, likely due to uneven incorporation of the acid and the dye: a slight precipitation of the dye was observed during liposome preparation (ibid.). It is not clear why liposomes with a net negative charge would interact so strongly with negatively charged silica. Interestingly, dimyristoyl-PC, which was the major constituent (about 70%) of these liposomes, has a gel-to-fluid transition temperature of 23.97C (e.g., [83]). This temperature may be close to that in the capillary during electrophoresis. In multicomponent bilayers, a lateral phase separation is known to occur at the transition temperature [83]. One may speculate that the strong adsorption of the liposomes, reported in [81] may be due to a nonuniform redistribution of charged constituents upon a phase separation. Separation by CZE of liposomes similar in lipid composition but differing in size was demonstrated [84] (details in Section 4.2). The utility of CZE to analyze SUV with compositional heterogeneity (liposomes prepared by dispersing mixed lipid powders in aqueous solutions rather than from lipids mixed in an organic solvent and dried prior to the dispersing and, therefore, presumably homogeneous in composition) was also shown [85]. CZE appears to present a robust method for evaluating liposome preparations in respect to heterogeneity in both composition and size [8486]. Interestingly, extruded liposomes prepared from PC, phosphatidylglycerol (PG), and CH, mixed in an organic phase were found to exhibit a sharp peak either adjacent to, or superimposed on, the broad original liposome peak when subjected to CZE in a 25 mM (or below this concentration) Tris-HCl buffer (pH 8.0) [86]. No such peak was observed for the same preparations in 50 mM buffer. The height and area of the sharp peak were reported to depend on average liposome size and surface charge density at a given buffer concentration and the peak was shown to disappear if the concentration of the sample buffer was elevated in relation to the background electrolyte (ibid.). The cause of this phenomenon is questionable but an original liposome compositional heterogeneity seems unlikely (unless one is hypothetically induced by the electric field in the capillary). Thus, in spite of the obvious utility of CZE for studying liposome heterogeneity, caution in the interpretation of experimental data should be exercised and CZE conditions must be well examined.
1964
Electrophoresis 2002, 23, 19571972 gation [94]. A mitochondrion-selective dye, 10-nonyl acridine orange, was used for fluorescent labeling (ibid.). Use of a continuous electrokinetic injection (50 mm ID capillaries, with an internal polymer coating to suppress EOF) and a specific fluorescent dye, MitoTracker Green, which accumulates in the mitochondrial inner membrane and covalently binds to proteins, were shown to allow for counting the organelles and for measuring protein abundance in individual mitochondria [95]. CZE, using a commercial instrumentation, was also employed in studying microsomesmembrane vesicles formed from intracellular membranes upon mechanical disruption of the cell in aqueous solutions [96]. Microsomes (100250 nm in size) formed by membranes of the rough endoplasmic reticulum were isolated from a homogenate of rat liver and subjected to CZE in Tris-borate buffer (pH 8.3). While original microsomes exhibited one major peak (detection at 280 nm), those incubated under conditions promoting the delayed vesicle fusion were found to exhibit a quite complicated pattern (ibid.). Several approaches to on-line detection of liposomes were employed in CZE: the direct UV absorbance [85, 88, 89], the absorbance in the visual range when the appropriate dye was incorporated into the membranes [81], the laser-induced fluorescence with a fluorescent dye either incorporated into the membrane [84], or entrapped in the internal volume of the liposomes [84, 86, 93], and the postcolumn chemiluminescence [80, 82, 97]. In the latter case, eosin-containing liposomes were allowed to migrate by EOF and mixed at the capillary tip with appropriate reagents to disrupt the vesicles and to induce the chemiluminescent signal. It should be noted that, if species encapsulated into the liposome interior are detectable online by some of the detection techniques, two peaks are typically recorded: one is due to liposomes and another due to unencapsulated (free) species. The change in the ratio of peak areas with time may easily and rapidly provide useful information as to the permeability of liposome membranes to these species as suggested in [97].
CZE was employed in investigating the binding of amphiphilic polymers to cationic and anionic liposomes [87] and to evaluate relative variations in surface charge of liposomes of different lipid compositions in various buffer systems [88]. It was also suggested that CZE might be a convenient method to assess the rigidity of a lipid bilayer [89], assuming a reduced frictional hindrance experienced by the elongated liposomes compared to that by the spherical ones. Liposomes are known to undergo the elongation and the subsequent orientation in electric fields as shown by electric field-induced transient birefringence (e.g., [90] and references therein), though the extent of the deviation from the spherical shape has not been estimated. In [89], a jump in liposome mobility was indeed found at the temperature of the gelto-fluid (rigid-to-soft bilayer) transition. The softening of membranes by incorporating CH or by using lipids with shorter acryl tails did give rise to an increase of absolute mobility (ibid.). However, the interpretation of such changes in mobility is not straightforward. The increase (about 50100%) in values of absolute mobility of MLV upon the bilayer phase transition, measured by microelectrophoresis [91] was previously accounted for by a change in surface charge density since a lipid head occupies different areas in gelled or fluid states of the bilayer [76, 77, 91]. The extruded liposomes are also assumed to possess a substantial degree of elongation as a result of the extrusion procedure ([92] and references therein). Thus, one may not rule out that the observed differences in mobility of the softened liposomes [89] are caused by a shape variance of liposomes of different composition due to extrusion (see also Section 4.2). An original approach to measuring electrophoretic mobilities of individual liposomes, using a home-made CE setup has recently been published [93]. A diluted suspension of MLV (0.31.8 mm in size) was subjected to CE in 10 mM HEPES buffer (pH 7.5) supplemented with 250 mM sucrose, in 50 mm ID capillaries, internally coated with a neutral polymer to suppress the electroosmotic flow (EOF). Individual liposomes labeled with fluorescein encapsulated upon preparation were detected online by means of a postcolumn laser-induced fluorescence (LIF) detector. This approach allows one to similtaneously determine mobility and apparent size (entrapped volume, based on a signal intensity) of an individual liposome. Interestingly, the MLV prepared by simply dispersing lipid films (formed by drying of lipid mixtures in an organic phase) in an aqueous phase exhibited apparently a substantial compositional heterogeneity as revealed by the mobility distribution of MLV in a similar size range [93]. Aside from liposomes, this CE-LIF based approach was applied to study mobility distributions of mitochondria isolated from different cells by density gradient centrifu-
Electrophoresis 2002, 23, 19571972 CZE as a new electrophoretic technique for analytical cell separations and analyses has been continually increasing. Several years after the work of Hjertn et al. [66], showing the feasibility of zone electrophoresis of bacterial cells in thin fused-silica capillaries, Ebersole and McCormick [100] have reported a CZE separation of a ternary mixture of bacteria. CZE was performed in 100 mm ID bare capillaries in 4.5 mM TBE (Tris-borate-EDTA, pH 8.3) buffer, employing UV (190 or 200 nm) detection. Four discrete electrophoretic peaks were observed. Two of those peaks were related to individual coccal species of a particular bacterium and their chain assemblages, respectively. The purity of fractions corresponding to near-baseline separated peaks (the electroeluted fractions were manually collected at the tip of capillary) was found to be at least 98% [100]. Most bacteria were shown to survive the electrophoretic process and could be recovered in a viable form (ibid.). Later on, the separation of three bacterial populations by CZE, using 250 mm ID, bare capillaries of 1 to 3 m length and UV detection at 208 nm has been demonstrated [101]. The peaks were broad compared to those for most molecular species. This was attributed to the inherent electrophoretic heterogeneity of bacterial populations. As a result, a baseline-separation of different bacterial populations can be realized only if there is a substantial difference in their mean mobilities. As a compromise between reproducibility, cell stability, and peak width and height, the most appropriate conditions for CZE of bacteria were a low ionic strength of electrophoretic buffer (I = 0.0010.002), basic pH (710), and a moderate field strength (slightly above 100 V/cm) [101]. A good agreement between bacterial electrophoretic mobilities measured by CZE and microelectrophoresis was demonstrated [15]. The lower detection limit was found to be 108 cells/mL (or 2500 cells for the injected volume of 25 nL) in 75 mm ID capillaries with detection at 214 nm. Interestingly, when electrophoresed in bare capillaries in a 10 mM MOPS buffer (pH 7.0), two out of three bacterial populations studied exhibited two peaks. No bimodal mobility distributions were revealed by DLV for the same cell populations [15]. The authors proposed that the cause of the observed discrepancy was the higher resolving power of the capillary electrophoresis (ibid.). The bimodal mobility distribution was interpreted in terms of interpopulational differences in the charge density of the bacterial surface [102]. These differences were postulated to be also responsible for the chromatographically observed decrease in the affinity of the bacteria for glass beads with distance traveled through a column (ibid.). The electrophoretic mobilities of a number of bacterial cells, derived by CZE [103] were also used to evaluate bacterial surface properties, based on the
CZE of microparticles
1965
concept of soft particles [30, 36, 104]. Mobilities were measured using 50 mm ID bare capillaries and a 10 mM phosphate buffer (pH 7.0 or 7.8), the ionic strength of which was adjusted with NaCl to vary from 0.02 to 0.23. The charge density per volume and a frictional characteristic of the glycocalyx region were estimated by the bestfitting of theoretical dependencies of m on I to the experimental points. It should be noted that the mobilities were reported to agree well with those measured by DLV for the same set of bacteria [103, 104]. It appears that CZE is well suited for measuring electrophoretic mobilities of various bacteria. However, due to a rather complex structure of their surfaces [71, 98], the interpretation of the mobility in terms of surface electrostatics is model-dependent. The mobility data for bacterial cells, derived either by CZE or other techniques, may be converted to z-potential values [71], using the Helmholtz-Smoluchowski equation (Eq. 1 with f(kR) = 3/2) since bacterial size is relatively large and kR . 1 for all . practical cases. It was realized early on that such conversion is merely formal and, consequently in most cases of electrophoresis of biological cells, results are expressed in terms of electrophoretic mobilities, the measured values [98, 99]. The more elaborate theories consider different dynamic processes, which may affect electrophoretic mobility and, hence, the derivation of z-values from mobility data (e. g., the ion transfer within the stagnant layer, which is assumed to coincide with bacterial walls, treated as a surface conductance [105]). Other approaches do not employ the concept of z-potential at all [103, 104]. The improved separation of a mixture of two bacterial strains by CZE in a polymer solution (1% dextran, 10 kDa molecular mass), compared to the separation in buffer alone (10 mM phosphate buffer, pH 7.0) was demonstrated [106]. When cells under the peaks were collected at the tip of the capillary, the purity of the cell population for each peak was found to be .98% and about 90% for CZE in the buffered dextran solution and in buffer alone, respectively. It was also shown that peak areas were well correlated with the injected amount of cells, thus, allowing for an easy quantification of mixed bacterial cultures by CZE (ibid.). A series of reports on CZE of bacterial and yeast cells, carried out in the presence of low concentrations (0.01250.025%) of a neutral polymer, polyethylene oxide (PEO, molecular mass of 100 or 600 kDa), has recently been published by Armstrong and co-authors [107111]. The striking feature of the reported separations was an extremely high efficiency (of the order of 106 theoretical plates/m), apparently due to the presence of a small amount of PEO. The separations were performed in
1966
Electrophoresis 2002, 23, 19571972 ionic strength of electrolyte solutions employed in CZE often lies within 0.0010.01 (k1 ranges approximately from 10 to 3 nm, respectively), the condition of kb. .k1 is not necessarily fulfilled for nonspherical microparticles, and their mobilities, in principle, may be shape- and orientation-dependent. The effect of orientation of the rod-like particle on its electrophoretic mobility was studied by the CZE of tobacco mosaic virus (TMV) as a model particle [67]. TMV may be considered as a rigid rod of 15 nm in diameter and approximately 340 nm in length. When subjected to CZE in 50 mm ID capillaries, using 2 mM borate (pH 8.4) buffer as an electrolyte solution, TMV was shown to exhibit an about 8% increase in absolute mobility upon increase of electric field strength (E) from 100 to 400 Vcm1. No appreciable change in mobility was observed for a latex microsphere (364 nm in diameter) under identical conditions. The monotonic mobility increase with E was accounted for by a progressive aligning of TMV particles in the direction of the electric field. It is noteworthy that the calculation of the degree of orientation was carried out assuming the Stokes drag as the only retarding force [67]. Yet, k1 in 2 mM borate used is of the order of 10 nm. Comparing that to the characteristic dimensions of TMV, it is clear that the electrophoretic retardation force cannot be ignored for TMV particles under the given conditions. Irrespective of the real interplay of retarding forces, the effect of orientation of even such elongated particle as that of TMV on its electrophoretic mobility appears to be rather slight, at least in the range of E commonly used in CZE. It is not as yet known at the present time whether the selectivity of separation in CZE of nonspherical rigid particles can be regulated or improved to any practical degree by varying the electric field strength.
100 mm ID uncoated capillaries, mostly in TBE buffer (pH 8.3) diluted to a concentration of 0.5 mM. Either UV [107110] or LIF [110, 111] detection was employed. When electrophoresed bacteria were detected by absorbance at 214 nm, the peak area was found to correlate with cell concentration in the sample, thus allowing for on-line quantitation of bacteria in the sample [110]. The feasibility of rapid identification and quantitation by CZE of pathogenic bacteria in human urine or bacterial ingredients in consumer products was demonstrated [108, 110]. It was shown that, using specific fluorescent dyes, cell viability in single or mixed bacterial populations can be determined on-line during a CZE run [110, 111]. The CZE in the presence of PEO was also applied to separate microbial aggregates [109] (Section 4.4). Surprisingly, there were only two attempts to utilize CZE for analysis of cells other than microbes. In an early attempt, red blood cells (RBCs) were electrophoresed using fluorinated ethylene-propylene copolymer (FEP) tubing as a capillary [112]. A number of various electrolyte solutions supplemented with glucose to maintain isotonicity and with hydroxypropylmethylcellulose (0.1%) to suppress EOF was tested as electrophoresis buffers. Sample concentration was of the order of 105 cells/mL and a well-defined peak was observed upon UV detection at 206 nm when 0.45 mm ID FEP tubing was used. Yet, no peak was observed in tubing of ID # 0.3 mm under the same conditions [112]. Recently, human RBCs have been separated by CZE at the single cell level, employing a direct microscopic detection [113]. A single injection of a very diluted blood was shown to produce tens of peaks, all of them of almost equal height. Each peak was attributed to a single RBC. This appoach allows one to obtain a distribution of electrophoretic mobility in the RBC population.
Electrophoresis 2002, 23, 19571972 85]) were subjected to CZE, they also exhibited a sizedependent migration and particles in a mixture were in most cases separated according to their size. Except for the gold nanoparticles, all microparticles studied in CZE exhibited a similar pattern: absolute mobility either did not change or increased with increase in particle size. The relaxation effect as the likely physical mechanism underlying this phenonenon was suggested early on [58]. Recently, two studies aimed at pinning down the operating mechanism have been published [48, 84]. A set of four latex microspheres of 138 to 381 nm in diameter was electrophoresed in a 100 mm ID capillary coated with linear polyacrylamide to suppress EOF, using a TBE buffer (pH 8.3) of various dilutions (I ranged from 0.0003 to 0.005) [48]. A size-dependent migration was indeed found to be an explicit function of kR. The selectivity of particle separation exhibited a maximum as a function of ionic strength. The relative increase in particle mobility was about 1.5 over the kR-range of 7 to 87, compared to that of 1.2, expected from Eq. (1) (ibid.). In a recent publication of Vanifatova et al. [45], the increase in absolute mobility of PSL particles was reported to be 1.7-fold over the kR-range of 12100, while a 1.16-fold increase should be expected according to the Henry formula within this kR-range (Eq. 1 and [17]). Estimating the lower limit for the z-potential as about 100 mV (based on mobility data of [45, 48]) one should anticipate the relaxation effect to appreciably contribute to the electrophoretic migration of the particles. However, one complication with PSL is that they appear to possess a hairy layer on the surface that can to a large extent affect their electrokinetic behavior as well. It is thought that the expansion of this layer upon decreasing ionic strength of the electrophoretic buffer has been responsible for the decrease in absolute mobility of the PSL microspheres with decreasing I [48]. Later on, extruded liposomes with defined mean size were employed as model particles to elucidate the mechanism of size-dependent migration [84]. The advantages of liposomes for that purpose are that their surface is smooth and that, changing the ratio of zwitterionic to charged lipids, one may easily modify the surface charge density for liposomes of similar size. Liposome preparations ranging in mean diameter from 120 to 500 nm and in PG/PC ratio from 1.6 to 0.14, were studied by CZE, using 100 mm ID capillaries coated with linear polyacrylamide and Tris-HCl buffers (pH 8.0) of various dilution (I = 0.001 to 0.027). The electrophoretic behavior was found to be qualitatively consistent with that expected if the relaxation effect was the dominant operating mechanism giving rise to a strong size-dependent migration of liposomes (e.g., the absolute mobility of highly charged liposomes increased twice over a kR-range of approximately 725) [84]. The separations by CZE of mixtures of liposome pre-
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parations identical in composition but differing in mean size were also demonstrated (ibid.). It should be pointed out that liposomes might undergo an elongation in the electric fields employed in CZE [90], and that the extruded liposomes are assumed to be intrinsically ellipsoidal [92]. To what extent, if at all, the deviation of liposomes from sphericity can contribute to their electrophoretic behavior including the size-dependent separation is unclear. Gold nanoparticles of 5 to 15 nm in diameter, subjected to CZE in electrolyte solutions of low ionic strength (I = 0.00030.006, kR ranges from 0.3 to 3.7) demonstrated a quite complicated pattern of size-dependent migration [52]. At higher ionic strengths used (0.003 and 0.006), the smaller particles migrated faster. Yet, the migration order changes to the opposite at I = 0.0003 and 0.0006. For the particle of a given size, mobility was found to decrease with decreasing ionic strength. This is a clear indication of ion binding onto the particle surface. The authors [52] have interpreted their findings by viewing the nanospheres as conducting, though at the same time realizing that the gold particles with adsorbed ions are widely considered as nonconducting particles. They also assumed that the particles possess an equal z-potential. However, if a uniform surface charge density is assumed (which is reasonable since the binding constant would hardly depend on particle size) rather than a uniform z-potential, the observed electrokinetic behavior may be accounted for without invoking any particle conductance. In the case of a small z-potential (# 25 mV), that is easily demonstrated as follows: The surface density of the electrokinetic charge, se, and z-potential are known to relate as (e.g., Eq. 2.3.37 of [17]): z = Rse/e (11kR) Equation (1) may be rewritten: m = (2se/3Z) [f(kR)R/(11kR) ] (3) (2)
Note that for large kR values, z-potential (and therefore particle mobility for species possessing a similar or identical surface charge density) becomes independent of particle size. This is not true for small kR values where mobility will be increasing with particle size. For instance, upon changing kR from 0.3 to 0.8 (I = 0.0003), the expected increase in absolute mobility is 1.9-fold, thus completely accounting for the 1.6-fold increase, which was experimentally observed [52]. When a higher particle charge and consequently z-potential is built up by the ion binding at elevated ionic strengths, the relaxation effect may come into play, overpowering the increases of m with particle size. Note that within the particular kR-range under study (1.3 to 3.7 at I = 0.006 [52]), the relaxation of the ionic atmosphere is shown to decrease particle mobility in absolute values with increasing kR [18, 21].
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Electrophoresis 2002, 23, 19571972 bacterium subjected to CZE in 1 mM, 2 mM, and 6 mM TBE buffer, the peak width was found to increase (about 3-fold) with increasing buffer concentration [101]. This peak broadening was assumed to manifest a progressive destruction of cells upon increase of the ionic strength (ibid.).
Electrophoresis 2002, 23, 19571972 peak [103, 106]. The longer the duration of sonication of cell suspensions prior to CZE analysis was, the fewer spike-like peaks appeared [106]. When microbial cells in the form of pre-existing multiple aggregates were subjected to CZE in 0.5 mM TBE buffer supplemented with 0.012% PEO (600 kDa molecular mass), several narrow spike-like peaks were observed [109]. It should be noted that under these conditions, even single cells migrate as a narrow zone, giving rise to a spike-like peak [107111]. In a specially designed experiment, the injections of pre-existing aggregates of yeast cells, with a counted number of cells in the aggregate, were carried out under microscopic control [109]. A reproducible relationship was found between the mobility and the aggregation number (the cluster size) under conditions of the study: the absolute mobility linearly increased with lnN, where N is the number of cells in the aggregate (ibid.). It is not clear why large aggregates would exhibit mobility higher in absolute value than that of its constituent particles. In the case of cells, these are large at the scale of kR, even in electrolyte solutions of low concentrations used in CZE. Besides, the binary aggregates are known to migrate with a velocity intermediate between the velocities of particles they are composed of ([116] and references therein). Except for the specific case of CZE of cell aggregates in a dilute PEO solution, the migration time of large aggregates was irreproducible. Roberts et al. [81] suggested that the aggregates are formed during a CZE run. Nonetheless, this still would not explain the higher mobilities. Perhaps, larger aggregates are more susceptible to interactions with capillary walls than small ones. If so, each aggregate can randomly be retarded in the capillary which, in the capillaries with EOF, would give rise to apparent mobilities higher in absolute values than those of single particles and small aggregates.
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ence in mobility of RBCs from Alzheimer patients and from normal individuals was found in a buffered dextran solution but not in the buffer alone [121, 122]. In CZE, solutions of water-soluble neutral polymers were employed in an attempt to bring about or to enhance the particle separation in a size-dependent manner, analogous to that in gel electrophoresis. Using polystyrene size standards as model particles, the particle retardation (expressed by the retardation coefficient, KR, the slope of a linear dependence of logm vs. polymer concentration) in solutions of linear polyacrylamide (PA) was found to be a biphasic function of particle size range [96, 123]. Up to approximately 30 nm in diameter, KR increased linearly with particle diameter and was field strength-independent. Above that size, the retardation became field strength-dependent and its dependence on size of sulfated PSL became complex [123]. Moreover, the relative decrease in mobility was considerably less than that expected based on the solution bulk viscosity and inverse relation between m and Z (Eq. 1) [124, 125]. This behavior was hypothetically interpreted in terms of (i) a layer with a depleted polymer concentration (the depletion layer), progressively forming at the particle surface-polymer solution interface and (ii) a shear-dependent deformation of the polymer network by the migrating particle [123125]. For carboxyl-modified latex (CML) microspheres, a steady decrease of KR was observed with increasing diameter within the range of 100500 nm (ibid.). Interestingly, CML particles possess an extensive hairy layer, according to the manufacturer (Interfacial Dynamics, Portland, OR, USA; Product Guide 7, 1994, p. 8). The interrelation of the hairy and depletion layers and their mutual effect on particle electrophoretic mobility are challenging theoretical problems ([120] and references therein). In a practical respect, it was shown that the selectivity of separation for CML microspheres (100450 nm in diameter) might be increased by the presence of PA in the buffer [126]. For 110- and 280-nm diameter microspheres which comigrate in the absence of polymer, a baseline separation was demonstrated in 0.7% solution of PA (7001000 kDa molecular mass). However, peak broadening was generally observed with increasing polymer concentration [124, 126]. Consequently, a maximum in resolution (when such exists) would correspond to a particular polymer concentration for a given pair of particles (ibid.). The remarkable effect of PEO on electrophoretic behavior of microbial cells in CZE was demonstrated by Armstrong and co-authors [107111] and further exploited for highly efficient separations. In the presence of 0.01250.025% PEO in the electrophoretic buffer, cells were found to migrate within narrow zones giving rise to spike-like peaks. As a representative case, a complete separation
1970
Electrophoresis 2002, 23, 19571972 ing the surface, relative size, and geometry of the particle. A particular difficulty in this respect arises from the fact that real particle surfaces in most cases are not smooth as assumed in the more simplistic models but rather hairy and heterogeneous to unknown degrees and in unknown ways.
Received March 1, 2002
by CZE of the mixture of four microorganisms was presented [107]. The resolution was rather insignificantly affected by the relative cell retardation induced by the polymer (change in separation selectivity), compared to that by peak sharpening. The mechanism of this phenomenon is unclear. PEO is known to induce aggregation of various biological species but not at such low concentrations. Nevertheless, one may speculate that cells of different type would partition upon aggregation induced by PEO (and electric field?), forming specific cell typedependent clusters migrating at some intermediate velocity and thus reducing the intrapopulational diversity. The other possibility is that binding of PEO to cells can somehow eliminate the intrapopulational diversity in cell surface properties, making the apparent z-potential uniform. Earlier, about 10-fold reduction in peak width was reported for PSL microspheres electrophoresed in dilute solutions of PA (0.20.25%, 18 000 kDa molecular mass) [124]. The cause of this reduction was also in question. Irrespectively of the mechanisms responsible for the zone sharpening in dilute polymer solutions, one may conclude that the use of polymers for improving particle separations has a potential that is not as yet explored to any substantial degree at the present time.
6 References
[1] Khaledi, M. G. (Ed.), High-Performance Capillary Electrophoresis: Theory, Techniques, and Applications, J. Wiley & Sons, New York 1998. [2] Radko, S. P., Chrambach, A., J. Chromatogr. B 1999, 722, 110. [3] Korant, B. D., Lonberg-Holm, K., Anal. Biochem. 1974, 59, 7582. [4] Jaspers, E., Overmann, J., Appl. Env. Microbiol. 1997, 63, 31763181. [5] Tulp, A., Verwoerd, D., Benham, A., Jalink, K., Sier, C., Neefjes, J., Electrophoresis 1998, 19, 11711178. [6] Hasan, Z., Pieters, J., Electrophoresis 1998, 19, 11791184. [7] Hjertn, S., in: Townshend, A. (Ed.), Encyclopedia of Analytical Science, Academic Press, New York 1995, pp. 1106 1112. [8] Tietz, D., J. Chromatogr. 1987, 418, 305344. [9] Serwer, P., J. Chromatogr. 1987, 418, 345357. [10] Canut, H., Bauer, J., Weber, G., J. Chromatogr. B 1999, 722, 121139. [11] Bauer, J., J. Chromatogr. B 1999, 722, 5569. [12] Lemp, J. F., Jr., Asbury, E. D., Ridenour, E. O., Biotechn. Bioeng. 1971, 13, 1747. [13] Preece, A. W., Brown, K. A., in: Chrambach, A., Dunn, M. J., Radola, B. J. (Eds.), Adv. Electrophoresis, V.3, VCH, Weinheim 1989, pp. 351404. [14] Altria, K. D., Isol. Purif. 1996, 2, 113125. [15] Glynn, J. R., Jr., Belongia, B. M., Arnold, R. G., Ogden, K. L., Baygents, J. C., Appl. Env. Microbiol. 1998, 64, 25722577. [16] Cevc, C., Biochim. Biophys. Acta 1990, 1031, 311382. [17] Hunter, R. J., Zeta Potential in Colloid Science: Principles and Applications, Academic Press, London 1981, pp. 11 58, 98123. [18] Overbeek, J. T. G., Wiersema, P. H., in: Bier, M. (Ed.), Electrophoresis: Theory, Methods, and Applications, Vol. II, Academic Press, New York 1967, pp. 152. [19] Dukhin, S. S., Derjaguin, B. V., in: Matijevic, E. (Ed.), Surface and Colloid Science, Vol. 7, J. Wiley & Sons, New York 1974. [20] Henry, D. C., Proc. R. Soc. London, Ser. A 1931, 133, 106 129. [21] OBrien, R. W., White, L. R., J. Chem. Soc. Faraday Trans. 1978, 77, 16071626. [22] Lyklema, J., Rovillard, S., De Coninck, J., Langmuir 1998, 14, 56595663. [23] Mangelsdorf, C. S., White, L. R., J. Chem. Soc., Faraday Trans. 1990, 86, 28592870. [24] Zukoski, C. F., Saville, D. A., J. Colloid Interface Sci. 1986, 114, 3244; 4553. [25] Churaev, N. V., Nikologorskaja, E. A., Colloids and Surfaces 1991, 59, 7182. [26] Rosen, L. A., Saville, D. A., J. Colloid Interface Sci. 1992, 149, 542552.
5 Concluding remarks
CZE appears to be an effective and experimentally simple way to analytically separate and characterize particles. A great variety of on- and postcolumn approaches to particle detection feasible in CZE is thought to make this technique exceptionally suited for electrophoretic analysis of particles in many applications. CZE has been shown to be applicable to particulate species within a wide size range, up to about 10 mm in diameter. The migration of particles in CZE is electrophoretic in nature though in particular cases a substantial contribution of particle-capillary wall interactions to the apparent mobility cannot be ruled out. Separations of particles are mostly based on differences in their electrophoretic mobilities. The effectiveness of those separations depends on particular conditions of buffer, ionic strength, the presence of detergents or specific polymeric media. In most applications, the optimal conditions at this time must still be found empirically. Except for the case of (quasi)spherical particles with smooth surface, the electrokinetic theories can appear to provide only a very general guidance in predicting and optimizing those conditions. CZE is well suited for measuring particle mobility but special attention must be paid to operational parameters if a precise value of mobility is of interest. As in general, an interpretation of particle mobility in CZE is model-dependent to the degree that assumptions are made regard-
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