Particle interactions in electrophoresis: I. Motion of two spheres along their line of centers

The electrophoretic motion of two charged colloidal spheres with very thin electrical double layers in a constant applied electric field along their line of centers is considered. The particles may differ in radius and in zeta potential at the surface. The electrostatic and hydrodynamic governing equations are solved in the quasi-steady situation using bipolar coordinates and the electrophoretic velocities of particles are calculated for various cases. The interaction effect between particles can be very significant when the distance between particle surfaces gets close to zero. The particle with smaller zeta potential is speeded up by the motion of the other, which is retarded at the same time by the motion of the former one, if the two spheres have unequal zeta potentials of the same electrical sign. For two particles of different signs in zeta potential, motions of both are hindered by each other. The influence of the interaction between particles in general is stronger on the smaller one than on the larger one. For the special case of two electrophoretic spheres with identical zeta potentials, there is no particle interaction for all particle sizes and separations.     

Electroosmotic flow changes due to interactions of background electrolyte counter-ions with polyethyleneimine coating in capillary zone electrophoresis of proteins

The properties and behavior of polyethyleneimine (PEI) covalently coated capillaries with respect to different background electrolytes used in capillary zone electrophoresis (CZE) are described. The coating stability and changes of inner surface charge in the capillary were followed by measurement of electroosmotic flow (EOF). Interest was focused mainly on conjugate bases of carboxylic acids as anionic background electrolyte components (acetate, citrate, malate, malonate, tartrate, and succinate). An interesting phenomenon was observed in PEI-coated capillaries: The direction (and the magnitude) of EOF depends on the composition of the background electrolyte and at a certain pH it can undergo reversible change. Ionic complex formation was suggested as a hypothesis to explain this behavior. With this knowledge, the PEI-coated capillary was used for the separation of basic proteins in the above-mentioned background electrolytes. A standard protein mixture of cytochrome c, ribonuclease A, and lysozyme at a concentration of 0.25 mg/mL each was chosen as model sample.     

Particle interactions in flowing suspensions

For a fully destabilized suspension of non-Brownian praticles in laminar tube flow, the extent of orthokinetic flocculation can be calculated by classical Smoluchowski theory, using the average shear rate $\text{G}$ and the average residence time $\text{t}$. It can be shown very simply that the dimensionless quantity $\text{Gt}$ (and hence the degree of flocculation) depends only on the tube dimension and not on the flow rate. However, calculations based on this approach predict far more flocculation than is observed experimentally. There are two major reasons for the discrepancy: 1) the Smoluchowski treatment of orthokinetic flocculation neglects hydrodynamic interaction between particles, which can be introduced by a semi-empirical method due to van de Ven and Mason and this step leads collision efficiencies which are considerably less than unity and depend both on shear rate and on interparticle forces; 2) the shear rate is not uniform in the tube but varies from zero at the tube axis to a maximum value at the wall. Since the major contribution to the flow comes from regions close to the tube axis, where the shear rate is low, the simple averaging procedure considerably overestimates the degree of flocculation.From experimental measurements on the degree of flocculation of dispersions achieved by laminar flow through narrow tubes at different flow rates it is possible to draw semi-quantitative conclusions concerning particle interaction and the strength of flocs.The effect of helical winding of the tube is briefly considered and shown to give more flocculation than in a straight tube. Some experimental results for latex particles destabilized by cationic polymers flowing through straight and coiled tubes are mentioned.     

Particle electrophoresis and dielectrophoresis in curved microchannels

Studies of particle electrophoresis have so far been limited to primarily theoretical or numerical analyses in straight microchannels. Very little work has been done on particle electrophoretic motions in real microchannels that may have one or multiple turns for reducing the devices size or achieving other functions. This article presents an experimental and numerical study of particle electrophoresis in curved microchannels. Polystyrene microparticles are found to migrate across streamlines and flow out of a spiral microchannel in a focused stream near the outer wall. This transverse focusing effect arises from the dielectrophoretic particle motion induced by the nonuniform electric field intrinsic to curved channels. The experimental observations agree quantitatively with the numerical predictions.     

Applications of affinity interactions in capillary electrophoresis

Capillary electrophoresis (CE) has proven useful for the study of reversible molecular interactions. This is because highly efficient and reproducible separations take place in an environment where molecular interactions may contribute to selectivity without being inhibited by adverse buffer conditions. Affinity CE may be used to estimate quantitative binding data (binding constants and in some cases binding stoichiometries and rate constants) for various molecular interactions. Specific binding interactions (e.g., based on antibodies or aptamers) may also be utilized to quantitatively measure specific analytes using CE. Applications within these areas are here reviewed with focus on the last three years and with emphasis on novel concepts as well as innovative methodology and technology. It is concluded that the affinity CE approach is of growing versatility and will continue to play an integral role in discovering, characterizing, and exploiting biomolecular interactions.     

Selector-selectand interactions in chiral capillary electrophoresis.

This review discusses selected aspects of selector-select and interactions in chiral capillary electrophoresis (CE). Studies performed using nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS) and X-ray crystallography for a better understanding of chiral recognition mechanisms in CE are summarized. The theoretical background of chiral CE in general, mathematical models, method development and optimization strategies, etc., are not covered. A general overview on the most recent developments in chiral CE is presented in this volume in the review paper by Bocek [1].     

Chemical shifts due to long range dispersion interactions

Dispersion chemical shifts, σ_w, for a variety of solute-solvent systems have been obtained by three different methods. The value of σ_w for a given system differs with the method employed for evaluating the characteristic frequency.     

Recent applications of affinity interactions in capillary electrophoresis

Systems biology depends on a comprehensive assignment and characterization of the interactions of proteins and polypeptides (functional proteomics) and of other classes of biomolecules in a given organism. High-capacity screening methods are in place for ligand capture and interaction screening, but a detailed dynamic characterization of molecular interactions under physiological conditions in efficiently separated mixtures with minimal sample consumption is presently provided only by electrophoretic interaction analysis in capillaries, affinity CE (ACE). This has been realized in different fields of biology and analytical chemistry, and the resulting advances and uses of ACE during the last 2.5 years are covered in this review. Dealing with anything from small divalent metal ions to large supramolecular assemblies, the applications of ACE span from low-affinity binding of broad specificity being exploited in optimizing selectivity, e.g., in enantiomer analysis to miniaturized affinity technologies, e.g., for fast processing immunoassay. Also, approaches that provide detailed quantitative characterization of analyte-ligand interaction for drug, immunoassay, and aptamer development are increasingly important, but various approaches to ACE are more and more generally applied in biological research. In addition, the present overview emphasizes that distinct challenges regarding sensitivity, parallel processing, information-rich detection, interfacing with MS, analyte recovery, and preparative capabilities remain. This will be addressed by future technological improvements that will ensure continuing new applications of ACE in the years to come.     

Particle electrophoresis for quality assurance and process control

Process control is an increasingly important issue as life science companies world-wide strive for recognition of their manufacturing and product development quality measures according to International Standards Organization (ISO) or good manufacturing practices (GMP) standards. Analytical particle electrophoresis (APE) has the potential for significant contributions, not just to basic research, but also in process development and control in manufacturing environments. An important feature of colloidal (small) particles, which controls their behavior, is their surface charge. Optimization of life science products and process conditions involving small particles (>100 nm) may be approached by a variety of strategies based upon direct measurements of the charge properties of process particles or "reporter" particles. The availability of increasingly powerful instruments and control particle preparations (National Institute of Standards and Technology ((NIST) and others) for validation of instrument operation make the method more attractive than ever. We summarize highly flexible electrophoretic strategies for assessing process consistency both from the perspective of particles being processed as well as the processing environment and describe principles for the use of polymer microspheres both as control particles for validation of instrument operation as well as for probes of the assay medium.     

Particle interactions and rheological effects in kaolinite suspensions

Particle interactions in kaolinite suspensions are modelled by representing the edge face of a kaolinite platelet as cylinder and flat plate, respectively. Computations of total energy of interaction show that at pH 6,7 and 8 and in the presence of 10^?4 ?10^?I M NaCl both face-edge and edge-edge modes of interaction are likely. Rheological parameters for flocculated suspensions (extrapolated shear stress and plastic viscosity) for dilute sodium kaolinite suspensions (volumetric concentration 0.02) are interpreted in terms of the proposed interaction models.     

Studies of DNA-protein interactions by gel electrophoresis

The use of gel electrophoresis in studies of nucleic acid-protein (especially DNA-protein) interactions has yielded much qualitative and quantitative information about a variety of such systems. The reduction in mobility of complexes relative to free DNA allows isolation and characterization of the complexes as well as determination of thermodynamic and kinetic properties of the interactions. This article begins with a review of recent applications of the "gel retardation" assay, by way of introduction to experiments in two areas. In the first, a hypothesis is tested regarding whether a DNA molecule with sizable proteins bound very near to each end migrates through a polyacrylamide gel differently than does the corresponding complex having the proteins in the middle of the DNA fragment. The data show little mobility differences for these types of complexes, implying that both may move in a linear, "snakelike", manner through the gel. The experiments also provide results pertaining to questions of DNA bending caused by the binding of the E. coli catabolite activator protein (CAP) and RNA polymerase to the lactose promoter region. It appears that DNA bending by CAP at its wild type lac binding site is retained in complexes where RNA polymerase is bound simultaneously at the lac UV5 promoter.     

Particle size limitations due to heat transfer in determining pyrolysis kinetics of biomass

A simplified heat transfer model is analyzed in order to determine an upper bound for biomass particle size in conducting experimental pyrolysis kinetics. In determining intrinsic kinetic rates, it is desirable that the entire particle be at reactor temperature for the duration of the chemical reaction. By comparing characteristic times for reaction rates versus heat-up rates, an approximate boundary for particle size can be constructed as a function of temperature; above this boundary, the reaction rate is strongly heat transfer dominated, and below the boundary the reaction rate is kinetically controlled. Using parameters for cellulose pyrolysis, it is estimated that a 200 μm particle will be heat transfer limited due to internal heat transfer at temperatures above 500°C. This boundary applies for conditions where the surface of the particle is brought to reactor temperature instantaneously. Using our specific experimental conditions, it is found that the limitations imposed by external transfer are reached before those predicted by assuming that only internal heat transfer is limiting. In examining wood pyrolysis, where a global reaction rate approximation is insufficient, it is experimentally shown that the decomposition for hemicellulose can be transport limited, while cellulose remains in the kinetic controlled regime.     

Automated detection and removal of capillary electrophoresis artifacts due to spectral overlap

While DNA detection using capillary electrophoresis has enabled improvements in both resolution and throughput, the use of CE – particularly with multiple dye channels – can introduce artifacts that can complicate analyses. Undetected pull‐up artifacts can pose a challenge to investigators, especially in low‐level samples, while partial pull‐up peaks can distort peak height balance within a locus and impact the downstream likelihood ratio. Current methods for addressing pull‐up are typically manually implemented. This study presents an effective alternative: a series of mathematical models, created using symbolic regression achieved through genetic programming. The models estimate the amount of pull‐up expected in a peak from a true allele for a given dye‐dye relationship and instrument type. This leads to the removal of artifactual pull‐up peaks and peak height corrections when pull‐up is present within true alleles. When models are used in conjunction with a dynamic threshold, pull‐up peaks were automatically detected and removed with an accuracy rate of 96.1%. The removal of partial pull‐up from true allele peaks led to a more accurate heterozygote balance for the affected locus. These models have been optimized for use with any analytical threshold and can be implemented by any lab using a 3100 or 3500 instrument series.     

Electrostatic interactions in peptides. Polarisation effects due to an α-helix

A quantum mechanical/molecular mechanical study of a dodecapeptide made of 11 alanine and one asparagine residues in a helical conformation is carried out by means of the local self-consistent field/molecular mechanical and integrated molecular orbital and molecular mechanics computational schemes. The electronic properties of the asparagine side chain are analysed to extract the influence of electrostatic and induction interaction. One finds that induction may play an important role in the energetic and structural features of the systems modelled with mixed methods. The importance of performing quantum computations which explicitly take account of the electrostatic interactions is pointed out.Contribution to the Jacopo Tomasi Honorary Issue     

Changes in electronic couplings of mixed-valence systems due to through-space intramolecular interactions

The strength of the electronic interactions between the two redox moieties in fullerene-substituted mixed-valence bis(ferrocenylethynyl)ethene derivatives is modulated by the through-space intramolecular electronic interactions of C(60) with the bridging conjugated system.     

Discrimination between peak spreading in capillary zone electrophoresis of proteins due to interaction with the capillary wall and due to protein microheterogeneity

Our study attempts to find an approach to distinguishing between the contribution to peak spreading in capillary zone electrophoresis (CZE) due to protein microheterogeneity and that due to interaction with the capillary wall, by analyzing correlations between observed peak spreading and peak asymmetry. The peak asymmetry was measured as ln[(tm-t1)/(t2-tm)] where tm, t1, and t2 are migration times at the mode of the peak and at the intersection of the peak width at half-height with the ascending and descending limbs, respectively. Two isoforms of recombinant green fluorescent protein (GFP-1 and GFP-2, 27 kDa molecular mass), glucose-6-phosphate dehydrogenase (GPD, 104 kDa), and the naturally fluorescent protein R-phycoerythrin (PHYCO, 240 kDa) were subjected to CZE in polyacrylamide-coated fused-silica capillaries of 50 and 100 microns diameters under varying conditions of protein concentration, field strength, and the initial zone length. Under conditions such that contributions to peak spreading from axial diffusion, thermal effects, and electrophoretic dispersion are negligible, the analysis of the interrelations between peak width and peak asymmetry was found to allow a conclusion as to the cause of peak spreading in CZE of protein. It appears that the peak width of GFP-2 originates mostly in protein microheterogeneity while that of GFP-1 is due to protein-capillary wall interactions. For PHYCO, both microheterogeneity and protein-capillary wall interactions contribute to peak spreading. GPD exhibits relatively little microheterogeneity or interaction with capillary walls. Thus, its peak width appears to be mostly affected by an extracolumn source of spreading such as the initial zone length.     

An attempt to evaluate the vibrational intensity changes due to long-range interactions

The effects of intermolecular interactions within the framework of long-range interactions on IR and Raman intensities are investigated.     

Particle interactions in kaolinite suspensions and corresponding aggregate structures

The surface charge densities of the silica face surface and the alumina face surface of kaolinite particles, recently determined from surface force measurements using atomic force microscopy, show a distinct dependence on the pH of the system. The silica face was found to be negatively charged at pH>4, whereas the alumina face surface was found to be positively charged at pH<6, and negatively charged at pH>8. The surface charge densities of the silica face and the alumina face were utilized in this study to determine the interaction energies between different surfaces of kaolinite particles. Results indicate that the silica face-alumina face interaction is dominant for kaolinite particle aggregation at low pH. This face-face association increases the stacking of kaolinite layers, and thereby promotes the edge-face (edge-silica face and edge-alumina face) and face-face (silica face-alumina face) associations with increasing pH, and hence the maximum shear-yield stress at pH 5-5.5. With further increase in pH, the face-face and edge-face association decreases due to increasing surface charge density on the silica face and the edge surfaces, and decreasing surface charge density on the alumina face. At high pH, all kaolinite surfaces become negatively charged, kaolinite particles are dispersed, and the suspension is stabilized. The face-face association at low pH has been confirmed from cryo-SEM images of kaolinite aggregates taken from suspension which show that the particles are mostly organized in a face-face and edge-face manner. At higher pH conditions, the cryo-SEM images of the kaolinite aggregates reveal a lower degree of consolidation and the edge-edge association is evident.     

Particle electrophoresis as a tool to understand the aggregation behavior of red blood cells

Red blood cell (RBC) electrophoresis measurements in polymer solutions have recently been introduced as a promising approach for investigating polymer-cell interactions near the RBC surface. A polymer-poor depletion layer near the RBC has been demonstrated: for depletion layers thicker than the double layer, viscosity within the depletion layer, rather than suspending medium viscosity, affects cell mobility. Using a well-documented model of sepsis in rats, we have induced RBC membrane damage, and then measured the electrophoretic mobility of rat RBC from control and septic animals. Mobility measurements were carried out for cells suspended in polymer-free buffer and in 0.5-2% solutions of dextran 500 (500 kDa molecular mass); RBC aggregation in autologous plasma and in dextran 500 was also studied. Our results indicate: (i) as anticipated from prior studies, the aggregation of RBC from septic animals is markedly enhanced (p<0.001) in plasma and in 0.5-1% dextran; (ii) the mobility of septic RBC in polymer-free buffer was identical to control, whereas cells from septic animals had lower mobilities in 0.5% dextran; (iii) Over the range studied (0.5-2%), the mobility of RBC from septic animals was less sensitive to increases of dextran concentration and hence medium viscosity. These mobility-aggregation findings can be partially interpreted in terms of a depletion model for RBC aggregation; alterations of RBC surface charge and the hydrodynamic friction within the cell's glycocalyx may also be involved. In overview, we believe that these results suggest the merits of microelectrophoresis for exploring protein or polymer behavior near biological particles and the potential value of future studies for understanding cell-cell interactions.     

Molecular interactions of re-released proteins in electrophoresis of human erythrocytes

Recently, we found that hemoglobin (Hb) could be re-released from live erythrocytes during electrophoresis release test (ERT). The re-released Hb displays single-band and multiple-band re-release types, but its exact mechanism is not well understood. In this article, the protein components of the single-band re-released Hb were examined. First, the re-released band of erythrocytes and the corresponding band of hemolysate, which was used as control, were cut out from starch-agarose mixed gel. Next, proteins were recovered from the starch-agarose mixed gel by freeze-thaw method. After condensing in a vacuum freeze drier, the samples were loaded onto a 5-12% SDS-PAGE. After electrophoresis, three protein bands (16, 28.9, and 29.3 kDa) emerged from the erythrocytes re-released Hb single-band (R-R), but only one band (29.3 kDa) emerged from the corresponding hemolysate control band (H-R). Finally, these bands were analyzed by MALDI-TOF MS. The results showed that these proteins were beta-globin (16 kDa), carbonic anhydrase 1 (CA1, 28.9 kDa), and carbonic anhydrase 2 (CA2, 29.3 kDa). Because CA2 exists in both erythrocytes re-released band and hemolysate control band, we conclude that the single-band re-released Hb is mainly composed of HbA and CA1. Studying the possible interaction between HbA and CA1 will help us further understand the in vivo function of Hb.