Determination of the electrophoretic mobility of bacteria and their separation by capillary zone electrophoresis

Conditions for the determination of electrophoretic mobilities of bacteria by capillary electrophoresis (CE) were explored. Most precise values are obtained using fused silica capillaries of 1–3 m length (0.25 mm inner diameter), a background buffer with an ionic strength of 0.0015 mol/L and a pH value of 7–10 at a field strength of 120 V/cm. Capillary electrophoretic separation of three different bacteria populations on the basis of their mobility differences could be realized. Electrophoretic band widths of all bacteria populations investigated are relatively large compared to molecule bands. It finds its explanation in the different distribution of surface charge density to cross-sectional area of each single cell of a population.     

Determination of the electrophoretic mobility of bacteria and their separation by capillary zone electrophoresis

Conditions for the determination of electrophoretic mobilities of bacteria by capillary electrophoresis (CE) were explored. Most precise values are obtained using fused silica capillaries of 1–3 m length (0.25 mm inner diameter), a background buffer with an ionic strength of 0.0015 mol/L and a pH value of 7–10 at a field strength of 120 V/cm. Capillary electrophoretic separation of three different bacteria populations on the basis of their mobility differences could be realized. Electrophoretic band widths of all bacteria populations investigated are relatively large compared to molecule bands. It finds its explanation in the different distribution of surface charge density to cross-sectional area of each single cell of a population. Received: 30 January 1997 / Revised: 15 May 1997 / Accepted: 22 May 1997     

Electrophoretic mobility, zeta potential, and fixed charge density of bovine knee chondrocytes, methyl methacrylate-sulfopropyl methacrylate, polybutylcyanoacrylate, and solid lipid nanoparticles

The electrophoretic mobility and zeta potential of bovine knee chondrocytes (BKCs), methyl methacrylate-sulfopropyl methacrylate (MMA-SPM) nanoparticles (NPs), polybutylcyanoacrylate (PBCA) NPs, and solid lipid nanoparticles (SLNs) were investigated under the influences of Na+, K+, and Ca2+ with various ionic strengths. The fixed charge density in the surface layers of the four biocolloidal particles was estimated from the experimental mobility of capillary electrophoresis with a theory of soft charged colloids. The results revealed that, for a specific cationic species, the absolute values of the electrophoretic mobility, the zeta potential, and the fixed charge density decreased with an increase in ionic strength. For a constant ionic strength, the effect of ionic species on the reduction in the absolute values of the electrophoretic mobility, the zeta potential, and the fixed charge density followed the order Na+>K+>Ca2+ for the negatively charged BKCs, MMA-SPM NPs, and SLNs. The reverse order is true for the positively charged PBCA NPs.     

Preparation and characterization of carboxylic-containing poly(methyl methacrylate) nanolatexes

Poly(methyl methacrylate) (PMMA)-based latex particles bearing carboxylic groups at the surface were prepared via emulsion polymerization. The polymerization recipe and process were optimized in order to target monodisperse particles with diameters around 100 nm. The polymerizations were performed using 4,4-azobis(4-cyanopentanoic) acid (ACPA) as initiator and sodium dodecyl sulphate (SDS) as surfactant. The polymerization conversion was determined by both gas chromatography and gravimetry. The final latexes were characterized with respect to particle size, size distribution, surface charge density, electrokinetic properties (i.e. electrophoretic mobility vs pH and ionic strength) and colloidal stability (i.e. coagulation rate constants vs pH and stability factor vs ionic strength).     

Reliable electrophoretic mobilities free from Joule heating effects using CE

Ionic electrophoretic mobilities determined by means of CE experiments are sometimes different when compared to generally accepted values based on limiting ionic conductance measurements. While the effect of ionic strength on electrophoretic mobility has been long understood, the increase in the mobility that results from Joule heating (the resistive heating that occurs when a current passes through an electrolyte) has been largely overlooked. In this work, a simple method for obtaining reliable and reproducible values of electrophoretic mobility is described. The electrophoretic mobility is measured over a range of driving powers and the extrapolation to zero power dissipation is employed to eliminate the effect of Joule heating. These extrapolated values of electrophoretic mobility can then be used to calculate limiting ionic mobilities by making a correction for ionic strength; this somewhat complicated calculation is conveniently performed by using the freeware program PeakMaster 5. These straightforward procedures improve the agreement between experimentally determined and literature values of limiting ionic mobility by at least one order of magnitude. Using Tris-chromate BGE with a value of conductivity 0.34 S/m and ionic strength 59 mM at a modest dissipated power per unit length of 2.0 W/m, values of mobility for inorganic anions were increased by an average of 12.6% relative to their values free from the effects of Joule heating. These increases were accompanied by a reduction in mobilities due to the ionic strength effect, which was 11% for univalent and 28% for divalent inorganic ions compared to their limiting ionic mobilities. Additionally, it was possible to determine the limiting ionic mobility for a number of aromatic anions by using PeakMaster 5 to perform an ionic strength correction. A major significance of this work is in being able to use CE to obtain reliable and accurate values of electrophoretic mobilities with all its benefits, including understanding and interpretation of physicochemical phenomena and the ability to model and simulate such phenomena accurately.     

Electrokinetic behavior and colloidal stability of polystyrene latex coated with ionic surfactants

This work is focused on analyzing the electrokinetic behavior and colloidal stability of latex dispersions having different amounts of adsorbed ionic surfactants. The effects of the surface charge sign and value, and the type of ionic surfactant were examined. The analysis of the electrophoretic mobility (mu(e)) versus the electrolyte concentration up to really high amounts of salt, much higher than in usual studies, supports the colloidal stability results. In addition, useful information to understand the adsorption isotherms was obtained by studying mu(e) versus the amount of the adsorbed surfactant. Aggregation studies were carried out using a low-angle light scattering technique. The critical coagulation concentrations (ccc) of the particles were obtained for different surfactant coverage. For latex particles covered by ionic surfactants, the electrostatic repulsion was, in general, the main contribution to the colloidal stability of the system; however, steric effects played an important role in some cases. For latices with not very high colloidal stability, the adsorption of ionic surfactants always improved the colloidal stability of the dispersion above certain coverage, independently of the sign of both, latex and surfactant charge. This was in agreement with higher mobility values. Several theoretical models have been applied to the electrophoretic mobility data in order to obtain different interfacial properties of the complexes (i.e., zeta potential and density charge of the surface charged layer).     

The effect of acrylic acid amount on the colloidal properties of polystyrene latex

Poly(styrene-co-acrylic acid) (St/AA) latices were prepared by using a batch soap-free emulsion copolymerisation in non-buffered medium. Polymerisation kinetics, followed by gravimetric method, revealed that increasing AA comonomer concentration was directly proportional to the copolymerisation rate, while adding AA comonomer caused a strong decrease of particle size of final St/AA latex particle without affecting the size distribution. Transmission electron microscopy indicated that the particles were monodispersed and spherical in shape irrespective of AA amount used in the investigated range. The colloidal stability of the latices was increased upon increasing the AA concentration; owing to the electrosteric stabilisation originated from AA-rich layer on the particle surface. In addition, electrophoretic mobility of formed particles versus polymerisation conversion exhibited the constancy of the surface charge density during the polymerisation process and was inferred for discussion of the polymerisation mechanism of this system.     

Counterion mobility and effective charge of polyelectrolytes in solution

Polyelectrolytes are macromolecules containing dissociable or charged groups. The charge, that is effectively accessible, is determined by counterion condensation, which is strongly influenced by the ionic strength of the solution under study. In general a rapid exchange between free and condensed counterions is expected. In the present study diffusion and electrophoretic mobility of poly(diallyldimethylammoniumchloride) and perfluorinated succinic acid have been monitored simultaneously. Condensation of the perfluorinated succinic acid to the macroion shows in the electrophoretic mobility of succinic acid monitored by pulsed field gradient NMR. In the concentration dependence of the averaged diffusion coefficient and electrophoretic mobility an exchange fast on the time scale of the NMR experiment is manifested.     

Change in electrophoretic mobility of HL-60RG cells by apoptosis

We measured the electrophoretic mobilities of HL-60RG cells and their apoptotic cells triggered by Actinomycin D as a function of the ionic strength of the suspending medium at pH 7.4. Both types of cells showed negative mobilities. The apoptotic HL-60RG cells exhibited larger mobility values in magnitude than intact HL-60RG cells in the whole range of the electrolyte concentration measured. The obtained data were analyzed via a mobility expression for soft particles, that is, colloidal particles with ionpenetrable surface layers. The observed mobility difference between the intact and apoptotic HL-60RG cells was found to be due mainly to the difference in friction exerted by the cell surface layers on the liquid flow around the cells between these two types of cells rather than the difference in charge density in their surface layers. A possible explanation for this mobility change by apoptosis is given.     

Dielectric friction as a mechanism for selectivity alteration in capillary electrophoresis using acetonitrile-water media

The mobilities of a series of aromatic carboxylates and sulfonates, ranging in charge from -1 to -4, were investigated as a function of acetonitrile concentration in the electrophoretic buffer. Absolute mobilities were determined by extrapolation of the effective mobilities to zero ionic strength according to the Pitts' equation. In general, anions of higher charge were more strongly influenced by ionic strength, with similarly charged anions experiencing ionic strength effects that were not significantly different at the 95% confidence level. Furthermore, the relative magnitudes of the Onsager slopes varied with acetonitrile content according to the z/(etaepsilon(1/2)) dependence in the electrophoretic effect of the Pitt's equation. Addition of acetonitrile to the electrophoretic media resulted in changes in the absolute mobilities of the anions. These acetonitrile-induced selectivity alterations were attributed to dielectric friction. As predicted by the Hubbard-Onsager model of dielectric friction, changes in sulfonate mobility were shown to correlate to changes in solvent viscosity (eta), dielectric constant (epsilon), and relaxation time (tau). The combined effects of ionic strength and dielectric friction caused analytes with higher charge-to-size ratios to be slowed to a greater extent upon addition of acetonitrile compared to those with lower charge-to-size. For example, at 75% acetonitrile and 20 mM ionic strength, a migration order reversal occurred between the triply and singly charged sulfonates.     

Modelling of migration behaviour of inorganic anions in ion-exchange capillary electrochromatography

A theoretical model to explain the observed mobility of inorganic anions in capillary electrochromatography (CEC) using ion-exchange (IE) stationary phases has been derived. The model divides contributions to the observed mobility of an analyte ion into capillary electrophoretic (CE) and IE components. The CE component includes the influence of varying the ionic strength of the background electrolyte on the electrophoretic mobility of the analyte, while the IE component accounts for the variation in retention of the analyte ion caused by changing the composition of the background electrolyte. The model was verified using a mixture of UV-absorbing inorganic ions in electrolytes of differing eluotropic strength in both packed and open-tubular CEC systems, with excellent agreement (r2 > 0.98) for both systems. Values of constants in the model equation determined by nonlinear regression were used to estimate the relative strengths of the interactions of different analytes with the stationary phase and these were found to agree well with elution orders observed in conventional IE chromatography.     

Electrophoretic characterization of insulin growth factor (IGF-1) functionalized magnetic nanoparticles

The synthesis of composite nanoparticles consisting of a magnetite core coated with a layer of the hormone insulin growth factor 1 (IGF-1) is described. The adsorption of the hormone in the different formulations is first studied by electrophoretic mobility measurements as a function of pH, ionic strength, and time. Because of the permeable character expected for both citrate and IGF-1 coatings surrounding the magnetite cores, an appropriate analysis of their electrophoretic mobility must be addressed. Recent developments of electrokinetic theories for particles covered by soft surface layers have rendered possible the evaluation of the softness degree from raw electrophoretic mobility data. In the present contribution, the data are quantitatively analyzed based on the theoretical model of the electrokinetics of soft particles. As a result, information is obtained on both the thickness and the charge density of the surrounding layer. It is shown that IGF-1 adsorbs onto the surface of citrate-coated magnetite nanoparticles, and adsorption is confirmed by dot-blot analysis. In addition, it is also demonstrated that the external layer of IGF-1 exerts a shielding effect on the surface charge of citrate-magnetite particles, as suggested by the mobility reduction upon contacting the particles with the hormone. Aging effects are demonstrated, providing an electrokinetic fingerprint of changes in adsorbed protein configuration with time.     

Influence of methanol as a buffer additive on the mobilities of organic cations in capillary electrophoresis

The mobilities of a series of aromatic ammonium ions, ranging in charge from +1 to + 3, were investigated by capillary electrophoresis using buffers consisting of 0-75% v/v methanol. This is an extension of our previous studies involving anion mobility in methanol-water media [1]. Absolute mobilities were determined by extrapolation of the effective mobilities to zero ionic strength according to the Pitts' equation. For all of the buffer compositions studied, the ionic strength effect increased with increasing cation charge, and varied as a function of solvent 1/eta epsilon (1/2) as predicted by the electrophoretic term within the Pitts' equation. In the presence of methanol, the ionic strength effects became more dramatic. The absolute mobilities of the cations were altered by the addition of methanol to the electrophoretic media. For example, at 75% MeOH, a migration order reversal was observed between the + 2 and + 3 ammonium ions. These solvent-induced selectivity changes are attributed to dielectric friction. As predicted by the Hubbard-Onsager dielectric friction model, dielectric friction increased with increasing methanol content and with increasing analyte charge. Further, the changes in cation mobility correlated to the changes in solvent relaxation time (tau), epsilon and eta. Although not predicted by the Hubbard-Onsager theory, the + 3 ammonium ion experienced more dielectric friction than the - 3 sulfonate and - 3 carboxylate investigated previously [1]. This apparent failure of the Hubbard-Onsager model results from its continuum nature, whereby ion-solvent interactions are not taken into account.     

Capillary electrophoresis in aqueous-organic media. Ionic strength effects and limitations of the Hubbard-Onsager dielectric friction model

The mobilities of three aromatic sulfonates, ranging in charge from -1 to -3, were investigated by capillary electrophoresis using buffers containing 0 to 75% ethanol or 2-propanol. Absolute mobilities were determined by extrapolation of the effective mobilities to zero ionic strength according to the Pitts' equation. For all buffers studied, ions of higher charge experienced larger ionic strength effects. The resulting ionic strength-induced selectivity alterations were more dramatic when organic solvents were present in the media. Furthermore, for different organic modifier types and contents, the magnitude of the ionic strength effect was governed to a large extent by the 1/(eta epsilon 1/2) dependence in the electrophoretic effect of the Pitts' equation. Addition of ethanol or 2-propanol to the electrophoretic media resulted in changes in the absolute mobilities of the ions. These solvent-induced mobility changes are attributed to dielectric friction. As predicted by the Hubbard-Onsager model, dielectric friction increased with increasing organic content and with increasing analyte charge. As a result, dramatic changes in the relative absolute mobilities were observed, such as a reversal in migration order between sulfonates of -1 and -3 charge in 75% 2-propanol. Within the alcohols, the Hubbard-Onsager model was successful at predicting the relative mobility trends upon changing solvent. However, the relative trends observed between acetonitrile-water and alcohol-water media were not consistent with the model. This may be explained by the continuum nature of the model, whereby the different ion-solvent interactions characteristic to each solvent class are not taken into account.     

Influence of detergent additives on mobility of native and subviral rhinovirus particles in capillary electrophoresis

The electrophoretic properties of two human rhinovirus (HRV) serotypes, HRV2 and HRV14, their subviral particles, and their capsid proteins were investigated by CE using borate buffer, pH 8.3, as BGE and three different detergents as additives. In addition, the influence of modification of the capsid with an amine reactive fluorescent dye, Cy3.5, on migration in the electric field was assessed. We found that the reproducibility of the electrophoretic results was decisively dependent on the presence of the detergents above their respective CMC. As compared to the strong ionic detergent SDS, the nonionic, mild detergent dodecylpoly(ethyleneglycol ether) (D-PEG) efficiently and reproducibly resolved both, native viruses as well as subviral particles. Most of the analytes behaved as expected except native HRV2; this serotype showed a dramatically higher anionic mobility in SDS than in D-PEG. Additionally, its mobility decreased when each positive charge contributed from a lysine at the capsid surface was substituted by four negative charges upon derivatization with Cy3.5. We discuss the possibility that this effect is caused by differences in number and in arrangement of exposed lysines in the two serotypes leading to differences in the amount of bound SDS micelles.     

Relating chromatographic retention and electrophoretic mobility to the ion distribution within electrosprayed droplets

Ions that are observed in a mass spectrum obtained with electrospray mass spectrometry can be assumed to originate preferentially from ions that have a high distribution to the surface of the charged droplets. In this study, a relation between chromatographic retention and electrophoretic mobility to the ion distribution (derived from measured signal intensities in mass spectra and electrospray current) within electrosprayed droplets for a series of tetraalkylammonium ions, ranging from tetramethyl to tetrapentyl, is presented. Chromatographic retention in a reversed-phase system was taken as a measure of the analyte’s surface activity, which was found to have a large influence on the ion distribution within electrosprayed droplets. In addition, different transport mechanisms such as electrophoretic migration and diffusion can influence the surface partitioning coefficient. The viscosity of the solvent system is affected by the methanol content and will influence both diffusion and ion mobility. However, as diffusion and ion mobility are proportional to each other, we have, in this study, chosen to focus on the ion mobility parameter. It was found that the influence of ion mobility relative to surface activity on the droplet surface partitioning of analyte ions decreases with increasing methanol content. This effect is most probably coupled to the decrease in droplet size caused by the decreased surface tension at increasing methanol content. The same observation was made upon increasing the ionic strength of the solvent system, which is also known to give rise to a decreased initial droplet size. The observed effect of ionic strength on the droplet surface partitioning of analyte ions could also be explained by the fact that at higher ionic strength, a larger number of ions are initially closer to the droplet surface and, thus, the contribution of ionic transport from the bulk liquid to the liquid/air surface interface (jet and droplet surface), attributable to migration or diffusion will decrease.     

Significance of cell electrokinetic properties determined by soft-particle analysis in bacterial adhesion onto a solid surface

The influence of extracellular polymeric substances (EPSs) on bacterial cell electrokinetic properties and on cell adhesion onto glass beads in connection with bacterial cell electrokinetic properties was investigated using 12 heterotrophic bacterial strains. Bacterial cell surface properties such as the softness 1/lambda and charge density ZN were determined by Ohshima's soft-particle analysis using the measured electrophoretic mobility as a function of ionic strength. In 10 of 12 strains, when EPSs covering the cell surface were removed, the softness of the cell decreased, indicating that EPS adsorption enhanced the ease of liquid fluid in the ion-penetrable layer on the cell surface. On the other hand, the negative charge density of the cell surface increased for 9 of 12 strains, suggesting that EPSs covering the cell surface decreased the negative charge density of the cell surface layer. In addition, the characteristics of bacterial cell adhesion onto glass beads were evaluated by the packed-bed method and the data were interpreted to indicate cell adhesiveness. As a result, the efficiency of cell adhesion onto glass beads increased as negative cell surface potential psi0 decreased, whereas there seemed to be no correlation between zeta potential and cell adhesiveness. Cell surface potential psi0, which was derived by taking the bacterial polymer layer with EPSs into consideration, provided a more detailed understanding of the electrokinetic properties of bacterial cells.     

Hindered diffusion of synthetic polyelectrolytes in charged microporous membranes

To examine electrostatic effects on the diffusion of macromolecules in membranes, diffusivities of narrow molecular-size fractions of the polyelectrolytes ficoll sulfate and dextran sulfate were measured in polycarbonate track-etch membranes. Radius, number density, and surface charge density of membrane pores were determined from a combination of hydraulic permeability, glucose diffusion, and streaming potential measurements. Molecular charge and Stokes—Einstein radius for each macromolecule fraction were determined from free-solution electrophoretic mobility and diffusivity in a large pore radius membrane, D∞, respectively. As ionic strength was increased from 0.005 to 0.1 M, D∞ for ficoll sulfate remained constant while D∞ for dextran sulfate increased slightly (15-18%). Macromolecule diffusivities in small pore membranes, D, were much more sensitive to ionic strength. For membranes where the ratio of Stokes—Einstein radius to pore radius ranged from 0.08 to 0.29, D/D∞ for ficoll sulfate and dextran sulfate increased by factors ranging from 2.5 to 14 for the same increase in ionic strength. Recent theoretical results for electrostatic double layer interactions in hindered diffusion are in good quantitative agreement with these findings.     

A study of oil-in-water emulsions stabilized by whey proteins

Like many other emulsifiers, whey protein concentrates stabilize oil-in-water emulsions. However, the emulsifying capacity of whey proteins is affected by several factors, e. g., type of homogenizer, degree of homogenization, protein concentration, oil volume fraction, pH and ionic strength of the aqueous phase. For the present study, oilin-water emulsions were made by homogenizing known amounts of whey protein concentrate with a vegetable oil (i. e. grapeseed oil) at different pH. The emulsifying properties of whey proteins are expressed as a function of the particle size and size distribution of oil droplets as measured by light scattering, and of the surface charge density derived from the electrophoretic mobility.The whey protein concentrate was shown to have an isoelectric point at pH 4.4. Near this pH value, the oil-in-water emulsions exhibited poor stability as expected from the low surface coverage.     

Electrophoretic properties of BSA-coated quantum dots

Low toxic InP/ZnS quantum dots (QDs), ZnS:Mn²⁺/ZnS nanocrystals and CdSe/ZnS nanoparticles were rendered water-dispersible by different ligand-exchange methods. Eventually, they were coated with bovine serum albumin (BSA) as a model protein. All particles were characterised by isotachophoresis (ITP), laser Doppler velocimetry (LDV) and agarose gel electrophoresis. It was found that the electrophoretic mobility and colloidal stability of ZnS:Mn²⁺/ZnS and CdSe/ZnS nanoparticles, which bore short-chain surface ligands, was primarily governed by charges on the nanoparticles, whereas InP/ZnS nanocrystals were not charged per se. BSA-coated nanoparticles showed lower electrophoretic mobility, which was attributed to their larger size and smaller overall charge. However, these particles were colloidally stable. This stability was probably caused by steric stabilisation of the BSA coating.