Electrophoresis electrophoretic velocities determination

Electrophoresis. Electrophoresis, the movement of charged particles in response to an electric potential, has become very important in biochemistry and colloid chemistry. In the present study an apparatus similar to that described by Burton( M2-M5) was used. A U-tube with an inlet at the bottom and removable electrodes at the two upper ends was half filled with acetone. The a Au-acetone colloidal solution was carefully introduced from the bottom so that a sharp boundary was maintained between the clear acetone and the dark purple colloid solution. Next, platinum electrodes were placed in the top ends of the U-tube, and a DC potential applied. The movement of the boundary toward the positive pole was measured with time. Several Au-acetone colloids were studied, and electrophoretic velocities determined as 0.76-1.40 cm/h averaging 1.08 cm/h. 

FIG. 12.13 Net charge of egg albumin versus pH. The points were determined by electrophoresis, and the solid line by titration the broken line represents 60% of charge from titration. (Data from L. G. Longsworth, Ann. NY Acad. Sci., 41, 267 (1941). (Redrawn with permission from J. Th. G. Overbeek, Quantitative Interpretation of the Electrophoretic Velocity of Colloids. In Advances in Colloid Science, Vol. 3 (H. Mark and E. J. W. Verwey, Eds.), Wiley, New York 1950.)... 

In practical applications of electrophoresis, collections of colloidal particles in bounded systems are usually encountered and the experimentally measured electrophoretic mobility is actually the average value for the entire suspension. It is therefore necessary to determine the average electrophoretic velocity for a suspension of colloidal particles. For dilute dispersions, the first order correction to the mobility of an isolated particle can be determined from the... 

Electrophoresis of nonconducting colloidal particles has been reviewed in this chapter. One important parameter determining the electrophoretic velocity of a particle is the ratio of the double layer thickness to the particle dimension. This leads to Smoluchowski s equation and Huckel s prediction for the particle mobility at the two extrema of the ratio when deformation of the double layer is negligible. Distortion of the ion cloud arising from application of the external electric field becomes significant for high zeta potential. An opposite electric field is therefore induced in the deformed double layer so as to retard the particle s migration. 

The application of laser Doppler velocimetry (LDV) to measure the electrophoretic mobility n of charged colloidal particles is known as laser Doppler electrophoresis (LDE). In a typical LDE experiment, an applied electric field drives the collective motion of charged colloidal particles. The particles pass through an interference pattern created by a dual-beam experimental setup (Section III.A.2). The collective electrophoretic velocity of the particles is then determined via intensity- or spectrum-based analysis of the scattered light, and the electrophoretic mobility n is calculated by dividing the velocity by the applied electric field strength. 

A number of methods for the determination of electrophoretic velocity and electrokinetic potential of particles have been developed. These methods include the moving boundary method (a direct study of motion of the boundary between the disperse system and the free dispersion medium due to the applied potential difference), microelectrophoresis (a direct observation of moving particles using a microscope or ultramicroscope), electrophoresis in gels, paper electrophoresis, etc [ 13]. These methods are broadly used to study disperse systems formed with low molecular weight substances, as well as polymers, especially those of natural origin. Electrophoretic methods allow one to separate and analyze mixtures of proteins, and thus are effectively used in scientific research and medical diagnostic applications. 

Good descriptions of practical experimental techniques in conventional electrophoresis can be found in references 29, 30, and 32. For the most part, these techniques are applied to suspensions and emulsions, rather than foams. In bubble microelectrophoresis, the dispersed bubbles are viewed under a microscope, and their electrophoretic velocity is measured taking the horizontal component of motion, because bubbles rapidly float upwards in the electrophoresis cells (33, 34). A variation on this technique is the spinning cylinder method, in which a bubble is held in a cylindrical cell that is spinning about its long axis. An electric field is applied, and the electrophoretic mobility is determined (2, 35). Other elec-trokinetic techniques, such as the measurement of sedimentation potential (36) have been used as well. 

Electrophoresis The motion of colloidal species caused by an imposed electric field. The term replaces the older term cataphoresis. The species move with an electrophoretic velocity that depends on their electric charge and the electric field gradient. The electrophoretic mobility is the electrophoretic velocity per unit electric field gradient and is used to characterize specific systems. An older synonym, no longer in use, is kataphoresis. The term microelectrophoresis is sometimes used to indicate electrophoretic motion of a collection of particles on a small scale. Previously, microelectrophoresis was used to describe the measurement techniques in which electrophoretic mobilities are determined by observation through a microscope. The recommended term for these latter techniques is now microscopic electrophoresis (see reference 1). 

The principle of electrophoresis, the most straightforward method for electrodeposition is based on the electric field-driven charged particles (silicate, organosilicate, metal oxides, micelles, or polymer composite particles) to an electrode at an electrophoretic velocity, v, which is determined by Stoke s law. 

The determination of electrophoretic velocities may be carried out experimentally by the use of methods suitable for transport number measurements. Moving boundary techniques have proved useful despite the problem of a difficulty in selecting suitable indicator ions. Reliable estimates of electrophoretic velocities make possible the determination of zeta-potentials. Since colloids migrate at characteristic rates under the influence of an electric field, electrophoresis provides an important means of separation. Coatings, such as rubber or graphite, may be deposited on metal electrodes by this means and additives to these may be co-deposited. 

Although it has been pointed out that experiments on electrophoresis are sometimes difficult t o interpret they remain important in several respects Firstly when the electrophoretic velocity is small, v is proportional to K and especially the zeropoint of electrophoretic velocity and thus of the -potentisi can be determined accurately. Secondly when the double layer is thin, s can be evaluated with confidence and finally in the field of biocolloids electrophoretic velocity has been used to characterize different colloids especially proteins) and has been applied as a means of separating them. 

The equivalence of electrophoresis and electro-osmosis has also been repeatedly tested It has been explained in 6b that reliable values of the ( -potential can only be calculated from electroph.oretic measurements if the time-of-relaxation effect can be neglected. If is not very small this is only realised in the case of large particles with a thin double layer. It follows from Henry s considerations (c/ 6a) that just in this case the electrophotelic velocity is equal to the velocity of electro-osmosis, both obeying the equation of Helmholtz Smoluchow ski (4, 26). This equality can be demonstrated very clearly by the ultramicroscopic method for the determination of the electrophoretic velocity. 

In electrophoresis in mixtures of electrolytes it is possible to show rather directly the influence of the time-of-rehxation effect Troelstra and Kruyt determined the electrophoretic velocity of silver iodide sol particles, with addition of Ba(N03)2 and of mixtures of Ba(N03)2 KNO3. In small con ... 

The zeta potential can be measured by electrophoresis, which determines the velocity of particles in an electric field of known strength [144]. This particle velocity, v, can then be related to the electrical field strength, E, as the electrophoretic mobility, fi. This is shown by... 

The parameter normally measured in capillary electrophoresis is migration (retention) time, /. In a given CE system this parameter is inversely proportional to the electrophoretic mobility, pi. The pt (cm /V) is a normalized parameter allowing for comparison of data obtained in different CE systems. If a series of analytes are analyzed under the same conditions then the 1/r and pt are equivalent. There are only a few reports on QSRR analysis of CE data. This may suggest the unsuitability of routinely determined mobility parameters as the LEER descriptors of analyte behaviour. Probably the reproducibility of analyte migration times in CE is poor due mainly to the non-reproducible electroosmotic flow velocity 26. ... 

Determination of the Electrophoretic Mobility, To evaluate the equation for the double-layer interaction (eq 5), the zeta potential, must be known it is calculated from the experimentally measured electrophoretic mobility. For emulsions, the most common technique used is particle electrophoresis, which is shown schematically in Figure 4. In this technique the emulsion droplet is subjected to an electric field. If the droplet possesses interfacial charge, it will migrate with a velocity that is proportional to the magnitude of that charge. The velocity divided by the strength of the electric field is known as the electrophoretic mobility. Mobilities are generally determined as a function of electrolyte concentration or as a function of solution pH. 

Capillary electrophoresis is an exciting, new, high resolution separation technique useful for the determination of drugs and their metabolites in body fluids. The first commercial capillary electrophoresis instruments began to emerge on the market in 1988. Today approximately a dozen companies manufacture electrokinetic capillary instrumentation, with many of these fully automated, that comprise auto samplers with computerized data evaluation.f Capillary electrophoresis involves the electrophoretic separations of minute quantities of molecules in solution according to their different velocities in an applied electrical field. The velocity of these molecules... 

Identification of sample components based solely on migration time in capillary electrophoresis (CE) requires reproducibilities not normally obtained. These are caused, mainly, by two effects temperature effects and electro-osmotic affects. Migration times in CE are determined by the electro-osmotic velocity Ueof and effective electrophoretic migration velocity Ueef the net migration velocity Vt is the vector sum of both velocities ... 

A schematic of a particle electrophoresis apparatus is shown in Fig. 4.17. The suspension is placed in a cell, and a dc voltage V is applied to two electrodes at a fixed distance I apart. The sign of the particle charge is obtained directly since it is opposite to that of the electrode toward which the particle is migrating. The particle velocity is measured by using a microscope, and the velocity per unit field strength (the electrophoretic mobility) is used to determine the -potential and the surface charge. 

The photomicrographic measurements refer directly to polymer motion under the influence of an external force. However, measurements of migration velocity v as a function of applied electrical field E show that some of these electrophoretic measurements were made in a low-field linear regime, in which the electrophoretic mobility jx is independent of E. Linear response theory and the fluctuation-dissipation theorem are then applicable they provide that the modes of motion used by a polymer undergoing electrophoresis in the linear regime, and the modes of motion used by the same polymer as it diffuses, must be the same. This requirement on the equality of drag coefficients for driven and diffusive motion was first seen in Einstein s derivation of the Stokes-Einstein equation(16), namely thermal equilibrium requires that the drag coefficients / that determine the sedimentation rate v = mg/f and the diffusion coefficient D = kBT/f must be the same. 

The physical significance of the zeta potential is discussed in the following section. The suspension could be characterised by particle charge density, which can in principle be determined from the electrophoretic mobility, but which requires certain assumptions regarding the particle size and shape distribution and conductivity effects. The zeta potential is the most commonly used parameter for characterising a suspension, and can be determined from measurements of particle velocity or mobility in an applied field using commercially available electrophoresis cells. In practice electrophoretic mobilities are not easy to measure accurately, and since the Smoluchowski equation is based on a model of doubtful validity, the view sometimes expressed that "zeta potentials are difficult to measure and impossible to interpret" has a ring of truth but is probably unduly pessimistic. The Smoluchowski relation is valid provided that the double... 

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