Rapid Separation of Proteins with Monodisperse Porous Polystyrene-Divinylbenzene(PS/DVB) Chromatographic Packings

It has been recognized for half a century that stagnant mobile phase mass transfer is a dominant limitation in liquid chromatography. The resolution is seriously affected at high mobile phase velocity. One approach to solve this problem is to eliminate the pores. Through the use of 1~2 μm non-porous particles it has been possible to carry out protein separations an order of magnitude faster,albeit at the expense of diminished loading capacity and high operating pressure. A second alternative is to cause liquid to flow through the particles. Because convective transports is much more rapid than that achieved by diffusion, these materials can also be used at an order of magnitude higher velocity for mobile phase in protein separations.     

Pore network modelling: determination of the dynamic profiles of the pore diffusivity and its effect on column performance as the loading of the solute in the adsorbed phase varies with time

A three-dimensional pore network model for diffusion in porous adsorbent particles was employed in a dynamic adsorption model that simulates the adsorption of a solute in porous particles packed in a chromatographic column. The solution of the combined model yielded the dynamic profiles of the pore diffusion coefficient of beta-galactosidase along the radius of porous ion-exchange particles and along the length of the column as the loading of the adsorbate molecules on the surface of the pores occurred, and, the dynamic adsorptive capacity of the chromatographic column as a function of the design and operational parameters of the chromatographic system. The pore size distribution of the porous adsorbent particles and the chemistry of the adsorption sites were unchanged in the simulations. It was found that for a given column length the dynamic profiles of the pore diffusion coefficient were influenced by: (i) the superficial fluid velocity in the column, (ii) the diameter of the adsorbent particles and (iii) the pore connectivity of the porous structure of the adsorbent particles. The effect of the magnitude of the pore connectivity on the dynamic profiles of the pore diffusion coefficient increased as the diameter of the adsorbent particles and the superficial fluid velocity in the column increased. The dynamic adsorptive capacity of the column increased as: (a) the particle diameter and the superficial fluid velocity in the column decreased, and (b) the column length and the pore connectivity increased. In preparative chromatography, it is desirable to obtain high throughputs within acceptable pressure gradients, and this may require the employment of larger diameter adsorbent particles. In such a case, longer column lengths satisfying acceptable pressure gradients with adsorbent particles having higher pore connectivity values could provide high dynamic adsorptive capacities. An alternative chromatographic system could be comprised of a long column packed with large particles which have fractal pores (fractal particles) that have high pore connectivities and which allow high intraparticle diffusional and convective flow mass transfer rates providing high throughputs and high dynamic adsorptive capacities. If large scale monoliths could be made to be reproducible and operationally stable, they could also offer an alternative mode of operation that could provide high throughputs and high dynamic adsorptive capacities.     

A novel stationary phase derivatized from hydrophilic gigaporous polystyrene-based microspheres for high-speed protein chromatography

Using agarose coated gigaporous polystyrene microspheres as a base support, a novel anion exchanger (DEAE-AP) has been developed after functionalization with diethylaminoethyl chloride. The gigaporous structure, static adsorption behavior, and chromatographic properties of DEAE-AP medium were characterized and compared with those of commercially available resin DEAE Sepharose Fast Flow (DEAE-FF). The results implied that there existed some through pores in DEAE-AP microspheres, which effectively reduced resistance to stagnant mobile phase mass transfer by inducing convective flow of mobile phase in the gigapores of medium. As a consequence, the column packed with DEAE-AP exhibited low column backpressure, high column efficiency, high dynamic binding capacity and high protein resolution at high flow velocity up to 2600 cm/h. In conclusion, all the results suggested that the gigaporous absorbent is promising for high-speed protein chromatography.     

Capillary electrochromatography with macroporous particles.

The performance of macro-porous particles in capillary electrochromatography is studied. Three reversed-phase stationary phases with pore diameters between 500 A and 4000 A have been tested for separation efficiency and mobile phase velocity. With these stationary phases, a large portion of the total flow appears to be through the pores of particles, thereby increasing the separation efficiency through a further decrease of the flow inhomogeneity and through enhancement of the mass transfer kinetics. The effects of pore size and mobile phase composition on the plate height and mobile phase velocity have been studied. With increasing buffer concentrations and larger pore diameters, higher mobile phase velocities and higher separation efficiencies have been obtained. Columns packed with 7 microns particles containing pores with a diameter of 4000 A generated up to 430,000 theoretical plates/m for retained compounds. Reduced plate heights as low as 0.34 have been observed, clearly demonstrating that a significant portion of the flow is through the pores. For the particles containing 4000 A pores no minimum was observed in the H-u plot up to linear velocities of 3.3 mm/s, suggesting that the separation efficiency is dominated by axial diffusion. On relatively long (72 cm) columns, efficiencies of up to 230,000 theoretical plates/column have been obtained under non-optimal running conditions. On short (8.3 cm) columns fast separations could be performed with approximately 15,000 theoretical plates generated in less than 30 s.     

Theoretical and experimental comparison of mobile phase consumption between ultra-high-performance liquid chromatography and high performance liquid chromatography

Ultra performance liquid chromatography (UPLC) using small particles and very high pressure has demonstrated higher resolution and speed compared with conventional HPLC. An additional benefit of UPLC is the significantly reduced consumption of mobile phase. This report discusses how column length, particle size, inner column diameter, extra column void volume, and capacity factor contribute to the reduction of mobile phase consumption in UPLC compared with HPLC. In addition, theoretical and experimental comparison of mobile phase consumption was made between isocratic HPLC and UPLC as well as between gradient HPLC and UPLC. Both theoretical and experimental results indicate that UPLC typically saves at least 80% of mobile phase in isocratic and gradient conditions when compared with HPLC.     

Characteristics of superficially-porous silica particles for fast HPLC: some performance comparisons with sub-2-microm particles

Columns of 2.7-microm fused-core (superficially porous) Type B silica particles allow very fast separations of small molecules at pressures available in most high-performance liquid chromatography instruments. These highly-purified particles with 1.7-microm solid silica cores and 0.5-microm-thick shells of 9 nm pores exhibit efficiencies that rival those of totally porous sub-2-microm particles but at one-half to one-third of the column back pressure. This presentation describes other operating features of fused-core particle columns, including sample loading characteristics and packed bed stability. The superior mass transfer (kinetic) properties of the fused-core particles result in much-improved separation efficiency at higher mobile phase velocities, especially for > 600 molecular weight solutes.     

Determination of the intraparticle electroosmotic volumetric flow-rate, velocity and Peclet number in capillary electrochromatography from pore network theory

The results obtained from the pore network model employed in this work, clearly show that the magnitudes of the intraparticle electroosmotic volumetric flow-rate, Qintrap, and velocity, (v(intrap,x)), in the pores of the charged porous silica particles considered in this study are greater than zero. The intraparticle Peclet number, Pe(intra, of a solute in these charged porous silica particles would be greater than zero, and, in fact, the magnitude of the intraparticle Peclet number, Pe(intrap), of lysozyme is greater than unity for all the values of the pore connectivity, nT, of the intraparticle pores and of the applied electric potential difference per unit length, Ex, along the axis of the capillary column considered in this work. Furthermore, the values of the intraparticle electroosmotic volumetric flow-rate, Qintrap, and velocity, (v(intrap,x)), as well as the magnitude of the pore diffusion coefficient, Dp, of the solute increase as the value of the pore connectivity, nT, of the intraparticle pores increases. The intraparticle electroosmotic flow can contribute significantly, if the appropriate chemistry is employed in the mobile liquid phase and in the charged porous particles, in (i) decreasing the intraparticle mass transfer resistance, (ii) decreasing the dispersive mass transfer effects, and (iii) increasing the intraparticle mass transfer rates so that high column efficiency and resolution can be obtained.     

Properties and performance of novel high-resolution/high-permeability ion-exchange media for protein chromatography

There is continued interest in the development of stationary phases for protein chromatography that can provide high resolution at elevated flow rates of the mobile phase. When using porous particles, resolution and dynamic binding capacity decline rapidly as the flow rate is increased. Monolithic columns have been developed to overcome these limitations. However, there are difficulties in manufacturing homogeneous larger scale monoliths. In this paper we investigate the morphology and performance characteristics of columns based on new ion exchangers obtained by mechanically disrupting continuous beds of acrylamido-based polymeric media. Near colloidal suspensions of loose particles obtained with this procedure can be flow-packed in ordinary chromatography columns resulting in beds of unexpectedly high hydraulic permeability. Columns up to 2.2 cm in diameter were studied with both Q and S functionalized media. The hydraulic permeability and interparticle porosity of these columns were rather high. The permeabilities of the S and Q media were 1.5 x 10(-13) and 2.4 x 10(-13) m2, respectively, while the corresponding porosities were 60 and 70%. These porosity values are similar to those of monoliths, suggesting that these particles assemble under flow to give high-porosity bridged structures. The structure of these packed beds was further characterized by embedding small packed columns in resins and obtaining sections for microscopic observation. The sections reveal the presence of small aggregates of non-porous 1-3 microm particles, surrounded by flow channels several micrometers in size. The height equivalent to a theoretical plate under isocratic and gradient elution conditions and the dynamic binding capacity were determined for several proteins and were found to be virtually independent of flow.     

Two-fluid model for the simultaneous flow of colloids and fluids in porous media

To describe the velocities of particles such as ions, protein molecules and colloids dispersed or dissolved in a fluid, it is important to also describe the forces acting on the fluid, including pressure gradients and friction of the fluid with the particles and with the porous media through which the fluid flows. To account for this problem, the use of a two-fluid model is described, familiar in the field of fluid mechanics, extended to include osmotic effects. We show how familiar relationships follow in various situations and give examples of combined fluid/particle transport in neutral and charged membranes driven by a combination of electrostatic, diffusional and pressure forces. The analysis shows how the same modeling framework can be generally used both for multidimensional electrokinetic flow through macroscopic channels and around macroscopic objects, as well as for mean-field modeling of transport through porous media such as gels and membranes.     

A study of the effects of column porosity on gradient separations of proteins

The type of the stationary phase for reversed-phase liquid chromatography significantly affects the sample elution. Hydrodynamic properties, efficiency and gradient elution of proteins were investigated on five commercial C18 columns with wide-pore totally porous particles, with superficially porous layer particles, non-porous particles and a silica-based monolithic bed. The efficiency in the terms of reduced plate height is higher for low-molecular ethylbenzene than for proteins, but depends on the character of the pores in the individual columns tested. The superficially porous Poroshell and the non-porous Micra columns provide the best efficiency for proteins at high mobile phase flow rates, probably because of similar pore architecture in the stationary phase. The Zorbax column with similar pore architecture as the Poroshell active layer, i.e. narrow pore distribution of wider pores shows better efficiency than the packed column with narrow pores and broad pore distribution. The monolithic column shows lower efficiency for proteins at high flow rates, but it performs better than the broad-pore distribution totally porous particulate columns. Different pore architecture affects also the retention and selectivity for proteins on the individual columns. The retention times on all columns can be predicted using the model for reversed-phase gradient elution developed originally for low-molecular compounds. Consideration of the limited pore volume accessible to the biopolymers has negligible effect on the prediction of retention on the columns packed with non-porous or superficially porous particles, but improves the accuracy of the predicted data for the totally porous columns with broad pore distribution.     

Rapid Chiral Separation by Flow-Through Chromatography with a Biporous Stationary Phase

Enantiomer separation is an area of increasing importance in chemistry and in the pharmaceutical industry. Although liquid chromatography is one of the most practical processes for chiral resolution, the high mass-transfer resistance of most commercially available packings makes chiral resolution time-consuming. In this work, a novel chiral stationary phase (CSP) with wide pores was prepared by coupling bovine serum albumin to a biporous resin with triazine as a linker. The rigid biporous medium was fabricated by radical suspension copolymerization with solid granules and solvents as porogenic agents. Studies by scanning electron microscopy and mercury intrusion porosimetry revealed the matrix contained two types of pore—micropores smaller than 180 nm and macropores of 500–7300 nm. Because the macropores provide convective flow channels for the mobile phase, the chromatographic process can be operated at high flow rate with low back-pressure. The biporous CSP was applied to the resolution of d, l-tryptophan (Trp). When the flow velocity was as high as 1800 cm h^–1, d, l-Trp could still be separated. By comparison of the resolution of d, l-Trp on this CSP at high flow velocities with that predicted theoretically for conventional supports we concluded that the increased flow velocity had little effect on the resolution of enantiomers with biporous packings. The results indicate that the protein biporous CSP is promising for high-speed resolution of enantiomers.Revised: 8 October 2004 and 3 January 2005     

光色谱分离方法研究

采用几何光学模型对光色谱分离中激光辐射压力与粒子半径之间函数关系进行了描述,对光色谱分离的动态范围进行了讨论。     

Emulsion templated open porous membranes for protein purification

Approximately 25 cm×25 cm large sheets of crosslinked highly porous poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate-co-ethylhexyl methacrylate) membranes with an average thicknesses between 285 and 565 μm were prepared by casting a high internal phase emulsion (HIPE) containing monomers onto glass substrates and subsequent polymerisation. Open cellular porous polyHIPE type membranes were obtained with large pores (cavity) sizes between 3 and 10 μm while interconnecting pores were between 1 and 3 μm. The percentage of ethylhexyl acrylate and ethyleneglycol dimethacrylate influenced the flexibility and morphology of the resulting membranes. Porous membranes were chemically modified with diethylamine to yield functionalised supports for ion exchange chromatography. Cylindrical housings were used for positioning of the membranes and allowing flow of the mobile phase. Pulse experiments were used to study the flow characteristics and a homogeneous flow through the entire area of the membrane was found. Bovine serum albumin was purified by a 8 ml column containing functional membrane in modular shape; dynamic binding capacity was measured to be as high as 45 mg/ml.     

Partitioning and adsorption in gas-liquid-solid chromatography: Models of temporal moments for retention and variance

Summary The resolution of gaseous chemical species in gasliquid-solid chromatography is influenced by absorption (partitioning) in the liquid and adsorption at the liquid-solid interface. We consider fundamental mass transfer models with adsorption and partitioning effects for solid chromatographic supports covered with thin films of stationary liquid. The dynamic models, based on mass-balance partial-differential equations, include the significant phenomena: convection, axial dispersion, gas-liquid mass transfer, intraparticle diffusion, and liquid-solid adsorption. Expressions for retention time and band variance (first and second temporal moments) are presented and evaluated for four distinct models: (1) capillary tube with inner surface covered with a uniform-thickness liquid film, (2) column of nonporous spheres covered with a uniform-thickness liquid film, (3) porous spherical particles with intraparticle pores covered with a uniform-thickness liquid film, (4) porous spherical particles with intraparticle pores completely filled with liquid.     

Effects of adsorbed polyelectrolytes on convective flow and diffusion in porous membranes

Poly(acrylic acid) and poly(styrene sulfonate) were adsorbed from aqueous solutions to track-etched mica membranes with pores of radius 290 to 1400 Å. Effects of the adsorbed polymers on momentum and mass transport within the pores were studied by measuring the decrease in both the hydraulic and diffusional permeabilities caused by the presence of the polymers. The diffusional permeability was determined by measuring the flux of a small, uncharged solute molecule (thiourea). Polymer-free, aqueous solutions of potassium chloride (KCl) were used in all transport experiments. The reduction in hydraulic permeability increased with a decrease in KCl concentration in the range 10^-2 to 10^-1 molarity (M), but was independent of electrolyte concentration below 10^-2 M, presumably because the small pores constrained expansion of the adsorbed polymer chains. Shear thickening effects, that is, a decrease in hydraulic permeability with increasing solvent velocity through the pores, was observed with both polymers at 10^-1 M KCl in pores of 600 Å or smaller. Effects of the polymer on diffusional permeability of thiourea, on the other hand, were relatively insensitive to electrolyte concentration. Perhaps the most significant result is that the reduction in diffusional permeability was substantially less than the reduction in hydraulic permeability at each pore size and electrolyte concentration, indicating that the blockage of momentum transport by these adsorbed polymers is greater than the blockage of diffusion of a small solute. Measurements of thiourea transport by simultaneous diffusion and convection over a range in Peclet numbers from -2 to +2 showed that thiourea was filtered by the polymer. This filtration was probably not due to steric limitations on the thiourea, rather it is likely that thiourea was excluded from the water which solvated densely packed regions of the polymer chains.     

Assessment of Mobile Phase Flow Resistance of Fused Microplatelet Alumina-Based Stationary Phases for Reversed Phase High Performance Liquid Chromatography

Abstract

The mobile phase flow resistances (φ's) of several stationary phases consisting of surface-modified Unisphere^TM fused microplatelet alumina particles were determined and compared with corresponding values obtained from stationary phases based upon more conventional spherical and angular silica and alumina particles. Although the φ value obtained for a fused-microplatelet alumina-based phase was slightly lower than the value obtained on a spherical alumina-based phase with similar particle dimensions and surface modification, factors other than particle shape were found to have a substantial effect on mobile phase flow resistance. Larger φ values were obtained with phases with smaller average particle diameters. These were attributed to the presence of greater numbers of microparticles with diameters less than 5 microns in such phases. Larger φ values obtained for wide pore (21 nm) and octyl-bonded alumina-based phases over narrow pore (11 nm) and octadecyl-bonded phases were attributed to the former's ablities to entrap larger volumes of stagnant mobile phase within their pores.     

Experimental Van Deemter plots of shear-driven liquid chromatographic separations in disposable microchannels

We present a new stationary phase coating method, yielding a monolayer of densely arrayed porous HPLC beads (d(p)=4 microm) for use in a disposable shear-driven flow LC system. The system is inherently suited for whole-column detection through the small voids between the individual particles of the layer. The chromatographic performance of the system has been characterized by performing a series of coumarin dye separation experiments (reversed-phase mode) and by measuring the theoretical plate height as a function of the mobile phase velocity. The resulting Van Deemter curve, yielding a value of about 90,000 plates/m near the u=u(opt) velocity, shows good agreement with the theoretical expectations, and hence constitutes the first full validation of the theory of shear-driven chromatography.     

An Introduction to Monolithic Disks as Stationary Phases for High Performance Biochromatography

Monolithic stationary phases have revolutionized protein chromatography because they combine speed, capacity, and resolution in a unique manner. Since such stationary phases contain no particles but only flow‐through pores, the usual mass transfer restrictions to the chromatography of large molecules are not observed and extremely fast separations become possible. Recently the area of application of monolith chromatography has been extended to the separation and analysis of small molecules and plasmid DNA. This review summarizes the state of art in high performance monolith and especially high performance monolithic disk chromatography (HPMDC). The current understanding of the theory of protein HPMDC is summarized, while an introduction to the evolving field of small molecule HPMDC is attempted. The basic differences between the monolithic disks and columns packed with conventional stationary phases (including perfusion and micropellicular particles) but also monolithic columns (porous rods) are outlined. Finally, the potential of HPMDC to analytical and preparative biochromatography is demonstrated by a discussion of recent applications of chromatographic disks for protein isolation and bioprocess analysis.     

Insights from theory and experiments on slip flow in chromatography

Slip flow has become a topic of interest in reversed‐phase liquid chromatography because it gives a flow enhancement that facilitates the use of submicrometer particles, providing a large improvement in separation efficiency. Moreover, slip flow provides an additional improvement in efficiency by reducing the velocity distribution in the mobile phase. The phenomenon of slip flow in open tubes is described in chromatographically relative terms. A recent paper in this journal is discussed, as it provides the first theoretical study of slip flow in packed beds, in this case for face‐centered cubic geometry. The theory paper reveals that the presence of the packed bed introduces a heterogeneity in fluid velocities that is absent in open tubes, reducing the additional improvement in efficiency from slip flow. The recent paper also suggests that there is yet another factor improving efficiency, which is size‐exclusion of proteins from regions of stagnant flow. The latter is supported by recently published data on restricted protein diffusion in face‐centered cubic silica colloidal crystals. Extremely low plate heights are enabled by use of submicrometer particles, and further improvement appears to be possible when the analyte size is on the order of 1% of the particle diameter or larger.