Marcus N. Myers

Electrical Field-Flow Fractionation of Proteins

Protein separation has been achieved by electrical field-flow fractionation, a heretofore unrealized separation technique. Some advantages of this method relative to electrophoresis are the low voltage required, the lack of adverse heating and support effects, and the existence of the method as an elution technique. A comparison of theoretical and experimental retention shows good agreement.

Observations on Anomalous Retention in Steric Field-Flow Fractionation

Abstract We discuss two anomalies that have characterized our recent work in steric field-flow fractionation (steric FFF): (a) retention has been found to vary with flow rate, contrary to simple theory; and (b) low density cells elute earlier than theory predicts. After reviewing the theory, we describe experiments in both gravitational and centrifugal fields applied to two spherical beads (microporous silica and polystyrene latex) of essentially equal sizes but different densities. The velocity dependence of retention is verified for both kinds of particles, but we find that retention depends dramatically on density as well. We speculate that these anomalous dependencies originate in a velocity-dependent lift force acting to pull the particle away from the wall. This hypothesis is supported by the observation that an increase in field strength increases retention. We conclude that a controllable field strength (as in a centrifuge) is an important asset in steric FFF, permitting increased separation speed...

Colloid characterization by sedimentation field-flow fractionation: I. Monodisperse populations

This paper initiates a series of reports intended to develop, refine, and evaluate the methodology of sedimentation field-flow fractionation (sedimentation FFF) applied to the characterization of colloidal materials. Particle parameters subject to characterization include effective mass, actual mass, density, polydispersity, diffusivity, and various particle dimensions. Monodisperse particle populations are the focus of this paper; size distribution and other studies will follow. The general advantages of sedimentation FFF noted for such studies include the theoretically predictable characteristics of the method, experimental flexibility and redundancy, use of nearly any desired suspending medium, small (<1 mg) sample requirements, high resolution, and fraction collection for additional characterization. For monodisperse populations, it is shown that both retention and peak dispersion measurements lead to the various mass and dimensional parameters noted above. The fundamental theory of retention and peak dispersion is summarized; various equations are then derived showing how particle diameter and other particle parameters relate to retention and peak dispersion measurements. Experimental data are reported for five polystyrene latex beads in the nominal diameter range 0.220−0.945 μm measured in two sedimentation FFF systems. Both rigorous and approximate procedures for calculating particle diameter d were divided into three categories: those based on retention measurements (R) only, those based on peak dispersion or plate height (H) only, and those based on a combination (RH) of the two measurements. Analysis of the results suggests that d obtained from rigorous R methods are accurate to about 1–3%. However, it appears that measured peak width may be ∼20% too large, giving d values ∼5% too small by H methods and ∼40–50% too large by RH methods. Various approximations for R, H, and RH methods are evaluated. In another direction, polydispersity values appear accurate to within 1% or better. However, particle density calculated using a combination of retention and peak dispersion data is in error by a factor of ∼3, in contrast to the ∼0.002 g/cm3 accuracy yielded by retention measurements.

Steric Field-Flow Fractionation: A New Method for Separating I to 100 μm Particles

Abstract Steric field-flow fractionation (steric FFF) is described as a high field limit of normal FFF that separates particles according to their diameter or radius. Retention equations are used to describe the phenomenon; these equations lead to the suggestion that steric FFF is applicable to particles from 1 to 100 μm as a minimum range. Conditions that control resolution are discussed, and fundamental similarities and differences between steric FFF on one hand and normal FFF and hydrodynamic chromatography on the other hand are noted. Optimum flow conditions are discussed and the complication of describing the migration of irregular particles is noted. Preliminary experiments with glass beads of 10 to 30 μm diameter demonstrate the existence of fractionation.

Flow field-flow fractionation as a methodology for protein separation and characterization

Flow field-flow fractionation is introduced as a new tool applicable to protein studies. Specific advantages of this method are discussed, including the capability for measuring diffusivities and Stokes radii directly, even for trace components. The theoretical equations of flow FFF are summarized and expanded to include an explicit dependence on the Stokes radius. Several native proteins are retained. The retention is shown to be systematically controllable by changes in cross flow and the results are in quantitative agreement with theory. Fractograms of different rat plasmas are then shown to produce coincident peaks, while human plasma exhibits several systematic peak shifts with respect to the fractogram of the rat plasma. Finally, changes in the Stokes radii of ferritin peaks are shown after various forms of treatment with SDS. Flow FFF in this study demonstrates a capability of working with a mass range of ∼ 105 in a single run.

High-speed polymer separations by thermal field-flow fractionation

Abstract Factors controlling speed in field-flow fractionation (FFF) are noted, and a basic analogy is shown with high-speed chromatography, which is based on similar dynamical requirements. The theory of speed optimization in FFF is developed along lines parallel to the theory of high-speed chromatography. However, allowance is made for the case in which continuous polymer distributions must be analyzed in place of discrete molecular species. We show that analysis time is proportional to the square of teh channel thickness, among other things, thus demonstrating that the reduction of thickness is an important avenue for gaining speed. Accordingly, we described two FFF column systems with channel thicknesses of 0.127 mm and 0.051 mm, two and five times thinner than conventional channels. A comparison of the performance of the two systems with polystyrene polymer fractions is shown under various flow conditions. A comparison is also made between some of the new results and published results for a conventional system. Plate height plots are also shown and discussed. These studies show —more or less in accord with theory— a dramatic improvement in speed with decreasing channel thickness. Using the thinner-channel system, 0.051 mm, we have obtained the separation of three polymer fractions in about 4 min. A partial separation was obtained in 1 min. Speeds considerably greater than this are suggested by theory under conditions of high retention and high flow.

Steric field-flow fractionation as a tool for the size characterization of chromatographic supports

Abstract Steric field-flow fractionation is described andd its application to the determination of the mean size and size distribution of chromatographic supports is suggested. After measuring systematic departures from theoretical retention values, a calibration curve is constructed using microporous silica beads whose mean diameters (4.4, 6.4, 12.1 μm) are accurately determined by electron microscopy. Other spherical chromatographic support materials are shown to cluster around the calibration curve. Fractograms (elution curves) are then shown for 15 spherical supports and six irregular supports. It is shown that the fractograms yield information on mean particle diameters, dispersion, and skewness, the latter possibly reflecting high concentrations of fine and coarse particles. Some limitations and future needs in the application of the method are noted.

Dense-Gas Chromatography of Nonvolatile Substances of High Molecular Weight

Working at pressures of up to 2000 atmospheres, more than ten times higher than in previous gas chromatography, we used the solvent power of dense gases to enable migration of chromatographic substances of molecular weights as high as 400,000. Carotenoids, corticol steroids, sterols, nucleosides, amino acids, carbohydrates, and several polymers have been caused to migrate, separated, and detected in NH3 and CO2 carrier gases at temperatures of 140� and 40�C, just above the respective critical points. Previously such compounds either defied separation by gas chromatography or had to be chromatographed as their more volatile derivatives.

Fractionation and Size Distribution of Water Soluble Polymers by Flow Field-Flow Fractionation

Abstract Flow field-flow fractionation (flow FFF) is introduced as a chromatographic-like method with a potential for separating and characterizing water soluble polymers. The theory of the method is summarized, showing that one gets a size distribution curve based on the Stokes diameter, d. Problems in interpreting the elution profile in both flow FFF and gel permeation chromatography are discussed in the light of complications arising from electrostatic chain expansion in polyelectrolytes. The experimental approach is described using a channel of 2.00 ml volume. Sulfonated polystyrenes of three different molecular weights are separated from one another with and without added salts. The dependence of retention on sample size is shown to be least in the salt solution, indicating that this is most suitable for analytical work. The sodium salts of polyacrylic acid are also investigated. Distinct elution profiles are noted for two of these polydisperse polymers. Size distribution curves for the 2,000,000 MW ...

A Study of Retention in Thermal Field-Flow Fractionation

Abstract A broad theoretical and experimental investigation of retention in thermal field-flow fractionation is reported here. Equations connecting retention parameters with underlying thermal diffusion constants are reviewed, and new equations are developed to account for the distortion of the flow profile caused by a variable viscosity. Parameters investigated experimentally include channel width, solute molecular weight, channel temperature drop, cold-wall temperature, sample size, and solvent effects. All but the last two of these experimental studies showed good conformity with theoretical predictions. No theory exists for the prediction of sample-size or solvent effects. With regard to the latter, experimental results show that a variety of organic solvents are very effective, at roughly equal levels, in providing retention, while aqueous solvents are generally ineffective.