Mean Electrophoretic Mobility Research Articles

Validated micellar electrokinetic capillary chromatography (MECC) method for determination of 5-hydroxymethylfurfural in honey and comparison with HPLC

A simple and validated micellar electrokinetic chromatographic method is described for the determination of 5-hydroxymethylfurfural (HMF). The method was applied to fresh and processed (heated at 65 °C, 85 °C and 105 °C for 5 h and 20 h) multifloral and honeydew honeys. Optimum conditions were: 10 mmol L−1 Tris buffer consisting 110 mmol L−1 SDS and 10% (v/v) MeOH at pH 9.10, 20 kV of applied potential, 3 s of injection time at 5 × 103 N m−2, 280 nm of wavelength and 25 °C of fixed temperature. Mean electrophoretic mobility (m2 s−1 V−1) of HMF and methyl paraben (IS) were − 5.28 × 10−5 (RSD of 0.745%) and − 2.52 × 10−4 (RSD of 0.944%), respectively. The calibration curve was linear in the range of 0.68 µg mL−1 (5.40 × 10−6 mol L−1) and 4.05 µg mL−1 (3.21 × 10−5 mol L−1) with R = 0.9990 for inter-day precision. LOD and LOQ values were 0.007 µg mL−1 (5.48 × 10−8 mol L−1) and 0.023 µg mL−1 (1.83 × 10−7 mol L−1), respectively, as inter-day precision. The accuracy was very high with recovery values between 94.07 and 97.16%. The developed method was fully validated according to ICH guidelines. The results were compared with those of HPLC method adapted from AOAC method no: 980.23.

Electrophoresis of concentrated suspension of soft particles with volumetrically charged inner core

The present study deals with the electrophoresis of a concentrated suspension of soft particles. The inner core of the undertaken particles is considered to possess constant volumetric charges and is surrounded by an ion- and fluid-penetrable polyelectrolyte layer (PEL) with uniformly distributed immobile charges. Such types of particles are paradigm of several bio-particles, for example, virus, bacteria, yeasts, and humic substances, to name a few. A unit cell model is employed to take into account the effect of the interaction between neighboring particles. In the present study, we restrict ourselves with the low charge density and weak applied electric field assumptions. The electric potential distribution is obtained from the linearized Poisson-Boltzmann equation. The fluid velocity field inside the PEL and portion of the cell filled with electrolyte solution are determined by solving the Darcy-Brinkman and Stokes equations, respectively. We provide semi-analytical results for the mean electrophoretic mobility of the concentrated suspension of the undertaken soft particles. We have shown extensively that the suspension of the soft particles may switch its propulsion direction depending on the choice of the pertinent parameters. Unlike suspension of bare colloids or full porous particles, the mean electrophoretic mobility of the soft particles may be nonzero even at a net zero charge case.

Electrophoretic mobility and electric conductivity in suspensions of charge-regulating porous particles

The electrophoresis and electric conduction in a homogeneous suspension of charge-regulating porous spheres such as polyelectrolytes with uniformly distributed ionogenic functional groups and hydrodynamic resistance segments in an arbitrary electrolyte solution are analyzed. The charge regulation relationship between the local fixed-charge density and electric potential because of association and dissociation reactions of the functional groups is linearized and a unit cell model allowing the overlap of the electric double layers of adjacent particles is employed to take account of the effect of particle interactions under an applied electric field. The equilibrium and perturbed electric potential distributions are ascertained by solving the linearized Poisson-Boltzmann equation, whereas the external and internal fluid velocity fields are determined by solving the modified Stokes and Brinkman equations, respectively. Explicit formulas for the mean electrophoretic mobility of the particles and the effective electric conductivity of the suspension are obtained. The variation in the concentration of the charge-determining ions in the bulk solution can cause a reversal in the direction of the electrophoretic velocity and in the difference of the electric conductivity from its reference value for a suspension of neutral porous particles. The magnitude of the electrophoretic mobility decreases with increases in the volume fraction and resistance parameter of the porous particles, but the electric conductivity in general is not a monotonic function of the particle volume fraction.

Electrokinetic Behavior of Rat Blood Erythrocytes: Dependence on Cell Volume

The interrelationship between electrophoretic mobility and the volume of rat blood erythrocyte is described by a non-linear curve, where the deviation of cell volume from the physiological normal is accompanied by a decrease in their mean electrophoretic mobility. In some ranges, the deviations of erythrocyte volume do not affect their electrokinetic properties. It can be hypothesized that the simultaneous shifts of these parameters are caused only by some hematological abnormalities that are manifested in pronounced changes in cell homeostasis.

High Resolution Zeta Potential Measurements: Analysis of Multi-component Mixtures

ABSTRACT Microelectrophoresis is an important technique for characterizing the surface charge and stability of colloidal dispersions. Zeta potential has been calculated from electrophoretic mobility measurements using capillary cell electrophoresis for more than 50 years. This particular new method for obtaining electrophoretic mobility and zeta potential results improves the resolution compared with previous methods, provides accurate mean mobility and mobility distributions in a single measurement, avoids making multiple measurements at different locations through the capillary cell and eliminates the requirement for measurement at the stationary layer of a capillary cell altogether. M3 is an acronym for “Mixed Mode Measurement”. This new measurement method is a combination of measurements that improve the resolution of mean electrophoretic mobility measurements and provide mobility distribution results that will be described herein. The combination of measurements includes a fast field reversal measurement to provide the accuracy and stability of the result, and a slow field reversal measurement to improve the resolution of the result by providing accurate mean mobilities and mobility distributions. The combination of these measurements takes less than 1 min to complete. Examples of high-resolution mean electrophoretic mobility latex studies and latex mixtures resulting in multimode zeta potential distributions are illustrated. Zeta potential distribution differences on the order of 10 mV have been obtained on latex mixtures with this new technique.

Influence of pretreatment and experimental conditions on electrophoretic mobility and hydrophobicity of Cryptosporidium parvum oocysts.

Surface properties of Cryptosporidium parvum oocysts were investigated by using electrophoretic mobility and hydrophobicity measurements. Oocysts purified from calf feces by several sucrose flotation steps and deionized water (DI) washes (DIS method) had an electrophoretic mobility (neutral surface charge) near 0.0 m2 V-1 s-1 over a pH range of 2 to 10. The mean electrophoretic mobility of oocysts stored in DI containing a mixture of antibiotics had a lower standard deviation (sigma = 0.36) than that of oocysts stored in DI without antibiotics (sigma = 0.53); their electrophoretic mobility remained unchanged up to 121 days after collection. The electrophoretic mobility of oocysts purified on a cold Percoll-sucrose gradient after the feces was defatted with ethyl acetate (EAPS method) varied linearly with pH from 0.0 m2 V-1 s-1 at pH 2.4 to -3.2 x 10(-8) m2 V-1 s-1 at pH 10 (sigma = 0.52), thus displaying the negative surface charge at neutral pH observed by other researchers. The hydrophobicity of oocysts and two types of polystyrene beads was measured as a function of ionic strength by adhesion to polystyrene. Oocysts were purified by the DIS method. The ionic strength of the suspending solution was varied from 0 to 95 mmol liter-1. Two-week-old oocysts exhibited strong adhesion ( approximately 85%) at ionic strengths of 0 to 10 mmol liter-1 and moderate adhesion ( approximately 20%) at ionic strengths of 20 to 95 mmol liter-1. Two-month-old oocysts exhibited high adhesion ( approximately 60 to 80%) at all ionic strengths. These results show that adhesion properties governed by the electrophoretic mobility of purified C. parvum oocysts can be altered by the method of purification and that hydrophobicity can change as oocysts age.

Automated Particle Electrophoresis: Modeling and Control of Adverse Chamber Surface Properties

Electrophoretic analysis of colloidal particles is adversely affected by a host of surface phenomena, including electroosmosis, phase wall wetting, and sample or air bubble adsorption. Neutral, hydrophilic polymer coatings control such phenomena on a variety of surfaces. Poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) conjugates significantly reduce electroosmosis and positively control adsorption and wetting in the glass sample chambers (5 mm × 3 mm × 1 mm i.d.) employed in a representative commercial electrophoresis apparatus (Coulter DELSA 440). The reduction in electroosmosis (e.g., 80% in 7.5 mM solution at pH 11) was similar to that exhibited by coated 2-mm-i.d. quartz capillaries in a Rank MK I manual apparatus. PEG-PEI coatings significantly reduce electroosmosis over a wide range of pH (2-11) and ionic strength (1-100 mM) and can be stable for weeks under normal laboratory conditions. They greatly enhance ease of operation and accuracy (sample mean electrophoretic mobility ± SD) of the DELSA 440. The latter results from reduced electroosmosis flow profile gradients near the chamber center-axis stationary levels, where particle mobility is typically measured. Such flow profiles may also be affected by chamber wall surface asymmetries. A hydrodynamic description of electroosmotic fluid flow in rectangular chambers was adapted in order to analyze the propagation of errors due to both nonideal focusing and chamber surface asymmetry. The analysis indicated that the accuracy of rectangular chambered devices may be improved by measuring particle mobility at stationary levels different than chamber center-axes. As a result, some rectangular chambers may confer accuracy advantages over cylindrical chambers.

Cell surface analysis of trypanosomes of the subgenus Schizotrypanum isolated from bats.

Two stocks (M5, M29) of trypanosomes of the subgenus Schizotrypanum were isolated from the bat Phyllostomus hastatus and analyzed for cell electrophoretic mobility (EPM) and lectin binding surface sites. Epimastigotes from the M5 and M29 stocks presented a mean EPM of around -0.57 and -0.56 microns. s-1.V-1.cm respectively. Differences in the agglutination profiles were detected between epimastigotes or trypomastigotes from the two parasite populations using lectins with specificity for D-GlcNAc, D-GalNAc, D-Gal and D-Man as probe. Major variation was observed between epimastigote forms. Additionally, the D-GlcNAc binding lectins WGA and BS II strongly interacted with the trypomastigote from both M5 and M29 stocks; this fact is evidence that these trypanosomes are distinct from Trypanosoma (Schizotrypanum) cruzi.

Electrokinetic properties of human cryopreserved platelets.

A method for the cryopreservation of human platelets with glycerol/glucose is described which was a simplified modification of the method of Dayian and Pert (1979). The effect of cryoinjury of the platelet surface membrane was investigated by studying the surface electrokinetic properties of the platelet. A significant increase in platelet electrophoretic mobility was found after cryopreservation. The fresh platelets had a mean electrophoretic mobility of 1.04 +/- 0.05 microns s-1 V-1 cm-1 and cryopreserved platelets 1.18 +/- 0.05 microns s-1 V-1 cm-1, P < 0.05. However, the total platelet sialic acid of fresh platelets was 62.5 +/- 5.6 nmol 10(-9) platelets compared to 47.2 +/- 4.6 nmol 10(-9) platelets after cryopreservation, P < 0.001. Similarly, the neuraminidase-labile sialic acid was 26.4 +/- 4.3 nmol 10(-9) platelets for fresh platelets and 17.6 +/- 4.0 nmol 10(-9) platelets after cryopreservation, P < 0.001. Using polyacrylamide gel electrophoresis with Western blotting, we showed a reduction in the platelet glycoprotein Gp Ib after cryopreservation, this was confirmed by using crossed immunoelectrophoresis. Electron microscopy revealed a significant change in platelet morphology after the cryopreservation procedure with disruption of the platelet membrane and also platelet shape change. These features may explain the changes in platelet electrokinetic properties.

Anionic site behavior in Leishmania and its role in the parasite—macrophage interaction

The behavior of cationized ferritin (CF) binding sites on the surface of Leishmania mexicana amazonensis (amastigotes, infective and non-infective promastigotes) and their participation in the interaction with macrophages were evaluated. Glutaral-dehyde-fixed parasites treated with CF present a uniform labelling over the whole cell surface. However, living parasites displayed CF patches and caps. Capping was usually seen towards the anterior (flagellated) portion of the cells, where shedding phenomena took place. These processes were inhibited by sodium azide but not by low temperature (4 degrees C). CF treatment of non-infective promastigotes led to an increase in their uptake by macrophages, whereas the uptake of amastigotes or infective promastigotes was not significantly altered. The effect of CF on the parasite surface charge was analyzed by whole-cell microelectrophoresis. The mean electrophoretic mobility (EPM) of non-infective promastigotes was decreased by 26%, while once again the other parasite forms were not significantly affected. Transmission electron microscopy of mouse peritoneal macrophage cultures, fixed after interaction with CF-labelled parasites, revealed that both amastigotes and infective promastigotes quickly removed bound CF. Therefore CF was seen neither in parasite-macrophage attachment areas nor in parasitophorous vacuoles. On the contrary, non-infective promastigote-macrophage attachment areas were remarkably large and preferentially comprised CF-labelled membranes. These results strongly suggest an important participation of cell surface anionic sites in the L. mexicana amazonensis-macrophage interaction.

Immunological characterization and clinical significance of low mobility cells appearing in the peripheral blood mononuclear cells of cancer patients

The characteristics of low mobility cells (LMC) in the peripheral blood mononuclear cells (PBMC) of cancer patients were studied using a Parmoquant-L, a fully automated electrophoresis instrument, and a fluorescence-activated cell sorter (FACS). The L/H ratio (%) of LMC ( <0.95 μm/s/V/cm) to high mobility cells (HMC) ( ⩾0.95) was used to analyze the electrophoretic mobility (EPM) histogram of PBMC. In the cancer patient, the L/H ratio increased as a result of the appearance of LMC. As the ratio closely correlated with clinical stage of cancer, recurrence of cancer, and performance status in the patients, the method is a useful parameter for prognosis of cancer, practically, for early detection of recurrence in the follow-up study of cancer patients. The EPM of monocytes and subsets of lymphocytes was determined to characterize LMC. Monocytes were in LMC. T-Cells such as helper/inducer (Leu- 3 + ), suppressor (Leu-2 + Leu- 15 + ) and cytotoxic (Leu- 2 + Leu- 15 − ) cells and natural killer (NK) cells (Leu-7 + or Leu- 11 + ) were in HMC. The percentage of monocytes correlated significantly with the mean EPM of PBMC. In cancer patients, the percentage of monocytes increased significantly. On the other hand, suppressor T-cells did not increase enough to account for LMC. The appearance of suppressor T-cells correlated with that of monocytes. These results suggest that LMC in cancer patients consisted of mainly monocytes rather than suppressor T-cells.

Determination of an immunosuppressive substance in serum by latex particle electrophoresis

The level of immunosuppressive substance (IS), which increases in the serum of patients with cancer, was determined by an assay based on particle electrophoresis. Polystyrene latex particles (PLP) were coated with IS, which was extracted from the ascitic fluid of patients with cancer. The IS was a glycoprotein with a molecular weight of about 52 000, and an isoelectric point in the range pH 2.7–3.3. When the IS on the surface of the PLP reacted with the anti-IS antibody, the mean electrophoretic mobility of the PLP changed from −3.16 to + 0.21 μm·s −1·V −1·cm in the medium of pH 7.2 and ionic strength I = 0.0154. After preincubation of anti-IS antiserum and tested serum, the PLP coated with IS were added to this solution. It was incubated again and then the surface charge of the PLP was measured by an automatic cell-electrophoretic instrument. This method was used to determine the IS concentration in the serum of cancer patients and pregnant women. When compared to healthy controls, the serum IS level was significantly higher in patients with cancer, and lower in pregnant women. The assay based on latex-particle electrophoresis proved to be a sensitive and rapid method for determining the IS level in serum.

Surface charge of resident, elicited, and activated mouse peritoneal macrophages.

The surface charge of resident, thioglycollate-elicited, and Trypanosoma cruzi-activated mouse peritoneal macrophages was analyzed using cell electrophoresis. All macrophages had a net negative surface charge. Activated macrophages had a lower zeta potential and a higher isoelectrophoretic point than resident and elicited macrophages. The populations of resident, elicited, and activated macrophages were heterogeneous in terms of surface charge. The analysis of the effect of the pH of the solution in which the macrophages were suspended on their cellular electrophoretic mobility (EPM) indicated that their surface contained both positively and negatively charged dissociating groups. The contribution of sialic acid residues to the surface charge was determined by analyzing the effect of neuraminidase treatment on the EPM of the cells. Activated macrophages possessed more sialic acid residues exposed on their surface, and sensitive to the neuraminidase from Clostridium perfringens, than resident and elicited macrophages. Treatment of the cells with the neuraminidase from Vibrio cholerae, however, reduced the surface charge of all macrophages in about the same extent. Macrophages had their mean EPM reduced when incubated in the presence of Ca++, suggesting that some cell surface anionogenic sites have Ca++-binding capacity.

Electrical aspects of the adsorption of ionic surfactants on coal measured by the mean electrophoretic mobility as a function of sample dilution

A dilution method is proposed as a means of studying the adsorption of cationic surfactants on coal. Only the electrical part of the adsorption is observed. It is shown that particle charge increases with surfactant concentration, and some irreversibility occurs because the mobilities of states of high dilution depend on initial surfactant concentrations as well. The magnitude of the adsorption is different for different cations. A comparison of measurements on dilution with surfactant solution and distilled water supports the mechanism of particle charge arising from preferential cation adsorption.

Further studies on the surface charge of various strains ofTrichomonas vaginalis andTritrichomonas foetus

The surface charge of three strains of Trichomonas vaginalis and five strains of Tritrichomonas foetus was determined by direct measurement of the mean cellular electrophoretic mobility (EPM) of cells suspended in solutions of different ionic strength and pH. No differences were observed in the mean EPM among the two species, although significant differences among the strains exist. Strains that are more pathogenic to mouse, as measured using the subcutaneous assay, had a surface more negative. Treatment of the parasites with trypsin or neuraminidase reduced significantly their mean EPM and increased their isoelectric point. Tritrichomonas foetus was more sensitive to the enzyme treatment than T. vaginalis. Enzyme-treated cells recovered their normal EPM if, after enzyme treatment, they were incubated in fresh culture medium. The recovery process of trypsin-treated cells was inhibited 10-20% by addition of inhibitors of either protein synthesis (puromycin) or N-glycosylation of proteins (tunicamycin) to the incubation medium, suggesting that a cytoplasmic pool of sialoglycoproteins may exist. The recovering of the EPM of T. foetus and T. vaginalis previously treated with neuraminidase was inhibited by puromycin or tunicamycin about 40-50% and 17-30%, respectively. These observations suggest that sialoglycolipids exist on the surface of both parasite species, and that they contribute more to the surface charge of T. vaginalis than to that of T. foetus.

Sialoglycoproteins and sialoglycolipids contribute to the negative surface charge of epimastigote and trypomastigote forms of Trypanosoma cruzi

Epimastigote and trypomastigote forms of Trypanosoma cruzi have a net negative surface charge, as determined by direct measurement of the mean cellular electrophoretic mobility. Treatment of the parasites with neuraminidase reduces by 17 and 52% the mean electrophoretic mobility of epimastigote and bloodstream trypomastigote forms, respectively. Neuraminidase-treated cells recover their normal electrophoretic mobility if incubated for 2 h in the presence of fresh culture medium. The recovering process of epimastigotes is almost totally blocked by addition of inhibitors of either protein synthesis (puromycin) or N-glycosidically linked glycoprotein synthesis (tunicamycin). The recovering process of trypomastigotes is not totally inhibited by either puromycin or tunicamycin. Treatment of T. cruzi with trypsin reduces by 11 and 40% the mean electrophoretic mobility of epimastigote and bloodstream trypomastigote forms. Trypsin-treated cells recover their normal electrophoretic mobility if incubated for 4 h in fresh culture medium. The recovering process of trypomastigotes is partially inhibited by puromycin. The results obtained indicate that sialoglycoproteins and sialoglycolipids exist on the surface of T. cruzi, the latter being predominant on the surface of trypomastigotes.

Trypanosoma cruzi: Surface charge and freeze-fracture of amastigotes

Amastigotes of Trypanosoma cruzi, within vertebrate cells or isolated from the supernatant of vertebrate cell cultures (L-A9 fibroblast or J774G8 macrophage-like cell lines), possess glycoproteins or glycolipids on the cell surface according to the periodic acid-thiosemi-carbazide-silver proteinate technique used in association with electron microscopy. The cell surface of isolated amastigotes is negatively charged, as evaluated by the binding of cationic particles (colloidal iron hydroxyde at pH 1.8 and cationized ferritin at pH 7.2) as well as by direct measurement of cellular electrophoretic mobility. Amastigotes (Y strain) isolated from the spleen of infected mice and amastigotes (Y and CL strains) from the supernatant of cell cultures previously infected with T. cruzi have the same mean electrophoretic mobility (−0.85 μm sec −1 V −1 cm). It is intermediate between the epimastigote and the trypomastigote forms (determined previously). Sialic acid is the important component responsible for the negative surface charge, as determined by the use of neuraminidase. Thus, it is possible to use the mean electrophoretic mobility as an indicator for identifying amastigotes of T. cruzi.

Changes in surface charge of mouse neuroblastoma cells during growth and morphological differentiation of the cell population

The surface charge of neuroblastoma cells (clone C1300-N18TG2) was studied by microelectrophoresis. The surface charge of these cells was shown to be determined mainly by anionic groups of the membrane, located in a layer about 10 mm thick, with a density of 0.2 e/nm3, covering its outer surface. These groups interact with Ca ions with a binding constant of KCa=10−50 liters/mole and titrate corresponding to pK=3.8. The electrophoretic mobility of the neuroblastoma cells is reduced by trypsin, neuraminidase, and N-bromosuccinimide, which irreversibly neutralizes carboxyl groups, and is increased on treatment of the cells with tosyl chloride — a specific reagent for amino groups. The value of the surface charge also depends on the conditions of culture of the cell population. The process of morphological differentiation of the cells (termination of division, dendrite formation), induced by removal of serum from the medium, leads to an increase of about 30% in their electrophoretic mobility. If cells are cultured in medium containing 10 and 50% of blood serum, enabling them to multiply, variations are observed in the mean electrophoretic mobility, which are opposite in phase to the 24-hourly increase in the number of cells. It is suggested that these effects are determined by partial "self-synchronization" of the cell population. It is concluded that the surface charge of neuroblastoma cells, measured by the microelectrophoresis method, is determined mainly by carboxyl groups of peripheral proteins and gangliosides of the membrane, and that the content of these compounds in the membrane depends on the phase of cell development.

A video image correlation technique for the measurement of electrophoretic mobility

The electrophoretic movement of blood cells has been examined with a video image correlator using the signal from a 625 line monochrome television camera attached to the optical arrangement. The diffraction pattern of a cell produces a characteristic signal which can be detected and registered in any television scan line to form the data of the first image frame. Under the influence of an electric field, cells moved across the television field of view and after a short interval appeared on different scan lines. The detection and registering process was repeated, and a cross-correlation function between the first and second image frame was computed. This function was stored in an output buffer which was continuously updated as fresh functions were integrated with the existing contents. The mean electrophoretic mobility of a suspension of particles can be calculated from the peak position of the integrated correlation function. Using a triangle deconvolution procedure, the relative contributions of subpopulations lymphocytes prepared from peripheral blood samples were examined.

Polystyrene latex particles as indicators of abnormal plasma properties in multiple sclerosis.

Polystyrene latex particles were incubated in plasma from multiple sclerosis (MS) patients or normal subjects. Compared with plasma from healthy controls, MS plasma produced a significantly lower mean electrophoretic mobility of the latex particles after the addition of linoleic acid. The effect was similar to that found when erythrocytes were used as the indicator particle. However, the result with latex particles demonstrates that the phenomenon originates from a plasma component of MS patients rather than from an intrinsic difference in membranes of the red blood cell.