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.     

Magnetic resonance imaging of flow-distributed oscillations

The formation of stationary concentration patterns in a packed-bed reactor (PBR), using a manganese-catalyzed Belousov-Zhabotinsky (BZ) reaction in a mixed sulfuric-phosphoric acid medium, was studied using magnetic resonance imaging (MRI). The PBR was composed of a column filled with glass beads, which was fed by a continuous stirred tank reactor (CSTR). As the reactor is optically opaque, investigation of the three-dimensional (3D) structure of these reaction-diffusion-advection waves is not possible using conventional image capture techniques. MRI has been used to probe this system and the formation, 3D structure, and development of these waves has been studied. At reactor startup, traveling waves were observed. After this initial period the waves stabilized and became stationary. Once fixed, they were found to be remarkably stable. There was significant heterogeneity of the reaction fronts, which were not flat, as would be expected from a plug-flow reactor. Instead, the reaction wave fronts were observed to be conical in shape due to the local hydrodynamics of the bed and specifically the higher velocities and therefore lower residence times close to the wall of the reactor.     

Effect of moisture on the extraction efficiency of polycyclic aromatic hydrocarbons from soils under atmospheric pressure by focused microwave-assisted extraction

The performance of a large commercial chromatographic column was investigated using a short pulse of a tracer and an extension of the reverse-flow technique. This technique permits separate determination of the unavoidable irreversible microscopic processes and the reversible effects of flow maldistribution, and allows for the separation of flow maldistribution in the flow distributors from flow maldistribution inside the packed bed. This analysis was performed on a 0.44 m Millipore IsoPak column using Cellufine GC 700, cellulosic-based media with an average particle diameter of 75 microm, for the stationary phase. The column efficiency was quantified by analysis of the effluent curve from a short pulse of a 5% aqueous acetone tracer. The study examined behavior of beds of different lengths (10-24 cm) and beds packed from different slurry concentrations (10-75% v/v). The slurry-packed columns were very uniform, and no significant macroscopic flow maldistribution was observed inside the column. The observed bed plate heights conformed to the predictions of available one-dimensional continuum models. Dispersion in the flow distributors was significant, corresponding to 15-25% of the intracolumn dispersion when the full 24 cm available bed length was used and a proportionally larger increase for shorter bed lengths. Thus, the headers are shown to produce a significant increase in the observed plate height.     

Comparison of the mass transfer in totally porous and superficially porous stationary phases in liquid chromatography

The characterization of mass-transfer processes in a chromatographic column during a separation process is essential, since the influence of the mass-transfer kinetics on the shape of the chromatographic band profiles and on the efficiency of the separation is crucial. Several sources of mass transfer in a chromatographic bed have been identified and studied: the axial dispersion in the stream of mobile phase, the external mass-transfer resistance, intraparticle diffusion, and the kinetics of adsorption–desorption. We measured and compared the characteristics and performance of a new brand of shell particles and those of a conventional brand of totally porous silica particles. The shell stationary phase was made of 2.7-μm superficially porous particles (a 1.7-μm solid core is covered with a 0.5-μm-thick shell of porous silica). The other material consisted of totally porous particles of conventional 3.5-μm commercial silica. We measured the first and second central moments of the peaks of human insulin over a wide range of mobile phase velocities (from 0.02 to 1.3 mL/min) at 20°C. The plate height equations were constructed and the axial dispersion, external mass transfer, as well as the intraparticle diffusion coefficients were calculated for the two stationary phases.     

Fast HPLC with non-porous packing materials in the pharmaceutical industry

Summary This paper demonstrates the possibilities in transfering HPLC methods from porous to non-porous stationary phases. The procedures are transferred from 125 or 250 mm columns packed with porous stationary phases to 33 mm columns packed with non-porous particles. It is demonstrated that fast HPLC using non-porous columns reduces analysis times by a factor four to eight. Precision is comparable to HPLC with porous stationary phases. The costs for HPLC with porous and with nonporous packing materials are similar. The implementation of these fast HPLC columns is easy because standard equipment can be used.     

Surface-to-Volume Ratio of Ganglia Trapped in Small-Pore Systems Determined by Pulsed-Field Gradient Nuclear Magnetic Resonance

Pulsed-field gradient (PFG) nuclear magnetic resonance (NMR) measurements of hydrocarbon diffusion are shown to provide a quantitative measure of the surface-to-volume (s/v) characteristics of slowly dissolving hydrocarbon ganglia, trapped in a water-saturated porous medium, for systems with pore sizes below the limit of spatial resolution of magnetic resonance imaging (MRI). The porous medium is in the form of a packed bed of glass ballotini. The PFG NMR approach is validated in two ways. First, both MRI and PFG analyses are performed on the same system containing ballotini with a diameter of 1 mm. The s/v ratio of the dissolving ganglia determined by the two methods is the same to within the accuracy of the experimental data. Second, below the spatial resolution limit of MRI, PFG NMR alone is used to characterize the s/v ratios of ganglia entrapped in two packings of ballotini with diameters 0.1 and 0.5 mm, respectively. The s/v data are then included into a one-dimensional advection-dispersion model of the ganglia dissolution process. The resultant mass transfer coefficients obtained are in agreement with those obtained, under the same conditions of aqueous superficial flow rate, following MRI analysis of hydrocarbon dissolution in larger pore structures. Copyright 2001 Academic Press.     

Surface instability in a finite thickness fluid saturated porous layer

The Rayleigh-Taylor (RT) instability at the interface between fluid and fluid saturated sparsely packed porous medium has been investigated making use of boundary layer approximation and Saffmann [8] boundary condition. An analytical solution for dispersion relation is obtained and is numerically evaluated for different values of the parameters. It is shown that RT instability can be controlled by a suitable choice of the thickness of porous layer, ratio of viscosities and the slip parameter.     

Theoretical investigation of diffusion along columns packed with fully and superficially porous particles

Chromatographic columns packed with shell particles are now nearly twice more efficient than columns packed with conventional, fully porous particles. Shell particles are made of a solid core surrounded by a porous shell of constant thickness. Diffusion through the bed of packed columns is complex due to their heterogeneity. It involves diffusion through the external and the internal fluid, and surface diffusion. Six diffusion models are compared that combine these diffusion mechanisms. They involve the external porosity of the bed (?(e)), the ratio of the core to the particle diameters (ρ), and the ratio of the shell diffusivity to the bulk diffusion coefficient (Ω). Four different theoretical approaches were considered. They are based on (1) the additivity of the mass flux densities modulated by the obstruction factors caused by non-porous spherical inclusions; (2) the effective medium theory of Landauer; (3) the effective medium theory of Garnett for spherical inclusions; and (4) the probabilistic theory of Torquato (for binary composite materials only). The two Landauer models fail because they cannot account for the obstruction factor imposed by the presence of non-porous spherical inclusions. The ternary Garnett model (3) provides an excellent approximation of the actual diffusion mechanism but the most physically relevant model seems to be the one derived from a combination of the Garnett model for a binary core-shell particle and of the Torquato model for random dispersion of contacting spheres in a matrix. Accurate measurements of axial dispersion coefficients are needed to validate or reject the semi-empirical parallel diffusion models and to select the most appropriate one. The results of such measurements made with the peak parking method for various compounds are reported in the companion paper.     

High Performance Size Exclusion Chromatography with Microparticulate Porous Silica Spheres. Influence of Flow Rate,Salute Mass Transfer and Polydispersity of Samples

Abstract

The contributions to peak broadening in Size Exclusion Chromatography with microparticles of porous silica spheres having narrow size distributions have been determined by measuring the plate height dependence on flow rate for toluene and for polystyrene standards covering a wide range of molecular weights. From these contributions, the diffusion coefficients of the macromolecules in the pore matrix and the polydispersities of the samples could be evaluated. It is shown that for permeating polymers the band broadening is determined by the eddy diffusion in the mobile phase, by the slow mass transfer of the solute in the stationary phase and by the polydispersity of the standards. In properly packed columns the eddy diffusion term is of minor importance compared to the other effects, whereas the solute mass transfer, which is a velocity dependent process, can be minimized only at extremely low flow rates.     

Comparison of core-shell versus fully porous particle packings for chiral liquid chromatography productivity

A loading and productivity study was done using three racemates on vancomycin and teicoplanin-bonded chiral stationary phases of different particle formats. Two columns were packed with 2.7 μm superficially porous particles and two columns were packed with identically bonded 5 μm fully porous particles. The last two columns were packed with specially synthesized 4.5 μm vancomycin and teicoplanin superficially porous particles. The loading of different chiral compounds showed that the columns filled with 2.7-μm chiral stationary phases were inappropriate for preparative separations due to their very low permeability which precluded high flow rates. However, columns containing 4.5 μm superficially porous (core-shell) particles were as effective for small-scale preparative chiral separations as columns filled with classical 5 μm fully porous particles. Comparing the 4.5 μm superficially porous particles and 5 μm fully porous particles teicoplanin columns, the observed respective productivities of 270 and 265 mg/g chiral phase/h for 5-methyl-5-phenyl hydantoin enantiomers were obtained. Particular attention was given to the peculiar case of the mianserin enantiomeric separation on vancomycin columns that gave observed productivities of 200 and 205 mg/g chiral phase/h on the 4.5 μm superficially porous particles and 5 μm fully porous particles, respectively.     

Recent developments in the field of monolithic stationary phases for capillary electrochromatography

This review summarizes the contributions to the rapidly growing area of monolithic columns based on both silica and synthetic polymers for capillary electrochromatography and chip electrochromatography, with a focus on those published during the year 2004. A wide variety of both modified approaches to the "old" monoliths and new monoliths have been reported despite the very short period of time covered. This demonstrates that monolithic stationary phases have become a well-established format in the field of electrochromatography. The simplicity of their preparation as well as the good control over their porous properties and surface chemistries make the monolithic separation media an attractive alternative to capillary columns packed with particulate materials.     

Columns in analytical-scale supercritical fluid chromatography: From traditional to unconventional chemistries

Within this review, we thoroughly explored supercritical fluid chromatography (SFC) columns used across > 3000 papers published from the first study carried out under SFC conditions in 1962 to the end of 2022. We focused on the open tubular capillary, packed capillary, and packed columns, their chemistries, dimensions, and trends in used stationary phases with correlation to their specific interactions, advantages, drawbacks, used instrumentation, and application field. Since the 1990s, packed columns with liquid chromatography and SFC-dedicated stationary phases for chiral and achiral separation are predominantly used. These stationary phases are based on silica support modified with a wide range of chemical moieties. Moreover, numerous unconventional stationary phases were evaluated, including porous graphitic carbon, titania, zirconia, alumina, liquid crystals, and ionic liquids. The applications of unconventional stationary phases are described in detail as they bring essential findings required for further development of the supercritical fluid chromatography technique.     

Fluorometric detection with a packed flow cell in micro high-performance liquid chromatography

Summary A packed flow cell was used for fluorometric detection in micro high-performance liquid chromatography. The flow cell consisted of fused-silica tubing packed with the same material as the separation column. A focusing effect of the stationary phase on the signal intensity was observed, leading to an improvement of the mass detection limit, as achieved by on-column detection.     

Flow Rate Dependence of Separation and Broadening Effects in GPC

Abstract

The primary objective of this work is an investigation of the separation and zone brodening effect in columns packed with porous and nonporous materials, and estimation of the accuracy of the broadening parameter, h, obtained by a reverse flow method. In order to study the separation and dispersion phenomenon in the mobile phase, and that caused by a mass-transfer process, columns packed with smooth glass beads and porous silica columns were used.     

Characteristics and Application of Porous Ceramic/Agarose Composite Beads Derived as an Affinity Medium

Porous ceramic/agarose composite beads were derived as a kind of glutathione S-transferase (GST) affinity medium to investigate the characteristics and application in fast protein liquid chromatography. The analysis of back pressure and chromatographic performance in a packed bed indicated that this kind of affinity medium with a rigid structure and a high level of column efficiency would be suitable for protein chromatography under high flow velocity. The good physical stability evidenced under harsh alkaline treatments ensured the application in real chromatography processes. When the porous ceramic/agarose composite beads were used in the purification process of fusion protein GST-ADAM15, the purity of the total GST related protein reached more than 91.6 % and the yield reached 44.6 % even at the flow velocity of 764.3 cm h^?1. The works indicated the characteristics of porous ceramic/agarose composite beads and their potential application in protein purification processes.     

Novel approach for fritless capillary electrochromatography

At present, the main limitation for the further adoption of capillary electrochromatography (CEC) in the (routine) laboratory is caused by the lack of reproducible and stable columns. The main source of column instability is concentrated in the frits needed to retain the packed bed inside the CEC capillary. The sintering process used to prepare the frits can be rather problematic and irreproducible, particularly for small stationary phase particles and wide column diameters. Since the (surface) composition of the frits is different from the bulk stationary phase packing, different electroosmotic flow (EOF) velocities are generated. This effect is assumed to be primarily responsible for rapid column destruction. In this contribution, a novel approach for the preparation of fritless CEC capillaries is presented and evaluated. Using 5 microm Hypersil ODS particles, separation efficiencies in the range of 130,000-200,000 plates/m were obtained. In a 100 microm inner diameter packed column, electrical currents up to 50 microA could be tolerated without negative effects such as bubble formation. The prepared CEC columns were found to be stable and could easily be operated continuously for several days without column damage. An additional advantage of the proposed tapering approach is that application of pressure on the in- and outlet vial during separation was not required to prevent bubble formation.     

Preparation and evaluation of monodispersed,submicron, non‐porous silica particles functionalized with β‐CD derivatives for chiral‐pressurized capillary electrochromatography

Submicron, non‐porous, chiral silica stationary phase has been prepared by the immobilization of functionalized β‐CD derivatives to isocyanate‐modified silica via chemical reaction and applied to the pressurized capillary electrochromatography (pCEC) enantio‐separation of various chiral compounds. The submicron, non‐porous, cyclodextrin‐based chiral stationary phases (sub_μm‐CSP2) exhibited excellent chiral recognition of a wide range of analytes including clenbuterol hydrochloride, mexiletine hydrochloride, chlorpheniramine maleate, esmolol hydrochloride, and metoprolol tartrate. The synthesized submicron particles were regularly spherical and uniformly non‐porous with an average diameter of around 800 nm and a mean pore size of less than 2 nm. The synthesized chiral stationary phase was packed into 10 cm × 100 μm id capillary columns. The sub_μm‐CSP2 column used in the pCEC system showed better separation of the racemates and at a higher rate compared to those used in the capillary liquid chromatography mode (cLC) system. The sub_μm‐CSP2 possessed high mechanical strength, high stereoselectivity, and long lifespan, demonstrating rapid enantio‐separation and good resolution of samples. The column provided an efficiency of up to 170 000 plates/m for n‐propylbenzene.     

Chromatographic performance of monolithic and particulate stationary phases. Hydrodynamics and adsorption capacity

Monolithic chromatographic support structures offer, as compared to the conventional particulate materials, a unique combination of high bed permeability, optimized solute transport to and from the active surface sites and a high loading capacity by the introduction of hierarchical order in the interconnected pore network and the possibility to independently manipulate the contributing sets of pores. While basic principles governing flow resistance, axial dispersion and adsorption capacity are remaining identical, and a similarity to particulate systems can be well recognized on that basis, a direct comparison of sphere geometry with monolithic structures is less obvious due, not least, to the complex shape of theskeleton domain. We present here a simple, widely applicable, phenomenological approach for treating single-phase incompressible flow through structures having a continuous, rigid solid phase. It relies on the determination of equivalent particle (sphere) dimensions which characterize the corresponding behaviour in a particulate, i.e. discontinuous bed. Equivalence is then obtained by dimensionless scaling of macroscopic fluid dynamical behaviour, hydraulic permeability and hydrodynamic dispersion in both types of materials, without needing a direct geometrical translation of their constituent units. Differences in adsorption capacity between particulate and monolithic stationary phases show that the silica-based monoliths with a bimodal pore size distribution provide, due to the high total porosity of the material of more than 90%, comparable maximum loading capacities with respect to random-close packings of completely porous spheres.     

Fluid dynamics in capillary and chip electrochromatography

This review is concerned with the phenomenological fluid dynamics in capillary and chip electrochromatography (EC) using high-surface-area random porous media as stationary phases. Specifically, the pore space morphology of packed beds and monoliths is analyzed with respect to the nonuniformity of local and macroscopic EOF, as well as the achievable separation efficiency. It is first pointed out that the pore-level velocity profile of EOF through packed beds and monoliths is generally nonuniform. This contrasts with the plug-like EOF profile in a single homogeneous channel and is caused by a nonuniform distribution of the local electrical field strength in porous media due to the continuously converging and diverging pores. Wall effects of geometrical and electrokinetic nature form another origin for EOF nonuniformities in packed beds which are caused by packing hard particles against a hard wall with different zeta potential. The influence of the resulting, systematic porosity fluctuations close to the confining wall over a distance of a few particle diameters becomes aggravated at low column-to-particle diameter ratio. Due to the hierarchical structure of the pore space in packed beds and silica-based monoliths which are characterized by discrete intraparticle (intraskeleton) mesoporous and interparticle (interskeleton) macroporous spatial domains, charge-selective transport prevails within the porous particles and the monolith skeleton under most general conditions. It forms the basis for electrical field-induced concentration polarization (CP). Simultaneously, a finite and -- depending on morphology -- often significant perfusive EOF is realized in these hierarchically structured materials. The data collected in this review show that the existence of CP and its relative intensity compared to perfusive EOF form fundamental ingredients which tune the fluid dynamics in EC employing monoliths and packed beds as stationary phases. This addresses the (electro)hydrodynamics, associated hydrodynamic dispersion, as well as the migration and retention of charged analytes.