An apparatus for continuous separation of macromolecules by combined electrophoresis and gel filtration

Summary An apparatus for continuous separation of soluble and particulate macromolecules, and of other electrically charged particles by combined electrophoresis and gel filtration, is described. The two principles, electrophoresis and gel filtration, which operate simultaneously at right angles, enable separation between the various macromelecules, depending upon differences in electric charge and molecular wight. The sample to be fractionated is fed continuously into the apparatus while the separated macromolecules reaching the distal end of the instrument are collected at the outlet ports. Data for a continuous fractionation of human plasma proteins using the instrument are given.     

Comparison of gel permeation chromatography, density gradient ultracentrifugation and precipitation methods for quantitation of very-low-, low- and high-density lipoprotein cholesterol.

Human VLDL, LDL and HDL (very-low-, low-, and high-density lipoproteins) were isolated from plasma by gel permeation chromatography with one pre-ultracentrifugation step. The column effluent was monitored at 280 nm. The cholesterol content of the fractions correlated well with fractions from sequential ultracentrifugation (VLDL, r = 0.839; LDL, r = 0.924; HDL, r = 0.766) or precipitation (LDL, r = 0.975; HDL, r = 0.972) methods. The average triglyceride, phospholipid and protein compositions of the separated lipoprotein fractions were close to those of the ultracentrifugally isolated fractions reported previously. Apolipoproteins A1 and B were determined from fractions to confirm the right distribution between different lipoproteins.     

Gel permeation chromatography of fullerene-centered macromolecules

Gel permeation chromatography (GPC) behavior of fullerene-centered macromolecules is investigated. Because of the globular shape of the macromolecules, their GPC results are different from those of linear polystyrene standards. These macromolecules may serve as alternative molecular weight standards for polymers or dendrimers of similar globular structures. The results also show that the GPC analysis is capable of discerning relatively minor structural differences in the fullerene-centered macromolecules.     

Modification of the universal calibration of gel permeation chromatography to include macromolecules presenting a draining effect

A new universal calibration for gel permeation chromatography is proposed in which the hydrodynamic volume of the macromolecular chains is expressed by the quantity [η]M/Φ instead of the commonly used quantity [η]M (where [η] is the intrinsic viscosity, M is the molecular mass, and Φ is Flory's parameter). Introducing Φ into the hydrodynamic volume is necessary because its value changes from one polymer to another when the polymers present a certain draining effect. The proposed procedure also allows the determination of Φ of any wormlike polymer. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 707–710, 2003     

Effect of temperature and polymer type on gel permeation chromatography

A series of polyisobutene and polystyrene fractions was subjected to gel-permeation chromatography at 150°C. The two types resulted in distinctly different calibration curves in a plot of projected, extended chain length versus elution volume. The average end-to-end distances of the samples were determined by intrinsic viscosity measurements. It was found that these data plotted versus elution volume could be represented by a common curve for both polymer types. The elution volumes of the polyisobutene series were determined at three additional temperatures, 35, 70, and 110°C. It could be shown that elution volume is again determined by polymer coil size at the temperature of measurement.     

An exactly solvable Ogston model of gel electrophoresis. VI. Towards a theory for macromolecules

In this article, we present a generalized version of our lattice model of low-field gel electrophoresis that allows us to treat the case of macromolecules such as short linear or circular oligomers and semi-flexible rods. We show that free-solution electrophoresis problems can be seen as random walks in the conformational space of the analyte. For sufficiently small molecules, our mathematical approach provides exact mobilities. In a quenched gel-like environment, however, both conformational and positional degrees of freedom must be used, but exact solutions can also be obtained. As an example, we then investigate several two-dimensional model gels, as well as a simple channel system where we see evidence of entropic effects that cannot be captured by the traditional Ogston concept of free volume.     

Instrumental broadening in gel permeation chromatography

Summary Instrumental broadening corrections to gel permeation chromatograms have been demonstrated to be of minimal concern when using the newly available high efficiency packing materials. This was demonstrated using -Styragel as packing material on polymer standards with both narrow and broad molecular weight distributions.     

Capillary gel electrophoresis analysis of G-quartet forming oligonucleotides used in DNA-protein interaction studies

DNA-protein binding is among the most frequently studied biomolecular interactions with high importance in modern systems biology research. One interesting aspect of this rapidly developing field is the affinity capture of proteins by G-quartet forming oligonucleotides also referred to as aptamers. G-quartets are structural motifs formed by guanine-rich sequences commonly occurring in the human genome. In this paper, we describe a capillary gel electrophoresis based method to validate G-quartet formation of in-house designed oligonucleotides and discuss the effect of monovalent cation concentration on the development of this structure. The relevant aptamer was then bound to magnetic beads to form an affinity capture surface for target proteins, which were then analyzed by matrix-assisted laser desorption/ionization mass spectrometry.     

New way of presenting the universal calibration of gel permeation chromatography to include macromolecules with very high segment density

The universal calibration of gel permeation chromatography presents deviations when star‐shaped polymers are used, especially when they present high segment density. Polymers presenting high segment density also have higher values of Flory's parameter Φ than the corresponding flexible, linear polymers. This deviation disappears if we express the hydrodynamic volume of the polymers with the quantity [η]M/Φ (where [η] is the intrinsic viscosity and M is the molecular mass) instead of the commonly used quantity [η]M. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 2388–2391, 2005     

Role of diffusion in gel permeation chromatography

A theory is developed that describes the diffusion of solute into the gel particles during a gel permeation chromatographic experiment. The particles are treated as homogeneous spheres of radius a, into which diffusion takes place with diffusion coefficient D_s. The concentration in the mobile phase at any level at any time is supposed to be uniform throughout the cross-section of the column. It is shown that in the usual columns the effect of diffusion in the mobile phase is unimportant. A determinative quantity in the process is the parameter a²/D_st, where t is the time. For large values of a²/D_st an explicit expression for concentration versus time in the mobile phase at the end of the column is derived [eq. (26) and Fig. 1]. It shows a relatively long tail at large efflux volumes V, where the concentration varies at V^?3/2. For arbitrary values of a²/D_st the first three moments of the concentration versus time curve are calculated [eqs. (33)–(37)]. Pronounced skewness of the curve is found unless a²/D_st is small.     

Silver staining of macromolecules separated by electrophoresis

The use of silver, either in the free ionic form or as a diamine complex, has become important for staining proteins, nucleic acids and lipopolyaccharides separated by acrylamide gel electrophoresis. A number of applications and methodologies have been developed for this staining procedure over the last four years.     

Flow-rate dependence of gel permeation chromatography

Analysis of polystyrene standards by gel permeation chromatography over a wide range of flow rates revealed two sources of error in volume measurement. These errors arise from solvent evaporation in the siphon chamber and from solvent continuing to flow into the siphon during discharge. Appropriate corrections are discussed, and a vapor feedback loop to eliminate the solvent evaporation error is described. The flow rate dependence of the GPC calibration curve, expressed in the corrected elution volumes, appears different from that reported in the literature. The corrected flow rate dependence of peak elution volumes is in agreement with the expectation of diffusion and exclusion theories.     

Hydrodynamic interaction between macromolecules in sedimentation

The sedimentation constant of hard spheres decreases with concentration, due to the effect of backflow and distortion of the hydrodynamic field. For macromolecules at the θ-temperature, this effect of concentration is much smaller than that of equivalent spheres. Pyun and Fixman suggest that intertwinement of macromolecules gives rise to increase of the sedimentation velocity. However, their detailed model of intertwined molecules is unrealistic. An alternative model is proposed by considering a pair of intertwined homologous molecules with N_i and N_j segments respectively, as a single molecule with (N_i + N_j) segments. This model leads to a simple expression for the concentration coefficient in the sedimentation velocity, containing an adaptable parameter, χ_ij, characterising the probability of intertwining. The probability of intertwining is put equal to the probability of intermolecular approach to within a distance of R_int and χ_ij is defined by χ_ij = 2R_int/(R₁ + R_j), with R_i and R_j the friction equivalent molecular radii. From experiments on a number of sharp polystyrene fractions in cyclohexane at T = θ, a value χ_ji = 2.2 is deduced, decreasing with increase of molecular weight. Qualitative agreement is found between experimental results and predictions from the model. Quantitative agreement is not expected in view of uncertainties in the model. In ternary systems, containing molecules of different velocity, probably additional effects operate besides backflow, field distortion and intertwinement.     

Multiple peak analysis in gel permeation chromatography

Summary This paper describes the resolution of multimodal GPC chromatograms into their individual components through the use of two optimization techniques, applied sequentially. The principle of the method can be applied to any form of curve but only the mathematics for Gaussian curves is given. The method has been successfully tested against both model and real chromatograms. The model chromatograms consisted of overlapping, perfectly Gaussian, curves whilst the real chromatograms were obtained from oligomeric polyesters submitted for routine GPC analysis and contained from three to five components. These chromatograms, which would have been too complex to resolve by “pencil and ruler” techniques, were resolved by this method to a precision within experimental error.     

Characterization of biological macromolecules by electrophoresis and neutron activation

As analytical techniques have become more sensitive, the role of trace elements associated with biological macromolecules has become the subject of many studies in the past twenty years. Many biologically significant macromolecules, such as nucleic acids and proteins, have trace elements essential to their function and structure. Instrumental neutron activation analysis (INAA) contributes useful information in such studies. A procedure combining polyacrylamide gel electrophoresis (PAGE) with INAA and autoradiography has been developed to study biological macromolecules and their associated trace elements. Results from the application of this method to several metalloproteins are presented.