Oscillatory electroosmosis-enhanced intra/inter-particle liquid transport and its primary applications in the preparative electrochromatography of proteins.

The concept of generating an oscillatory electroosmotic flux inside the porous particle to enhance the intra-particle mass transport was presented and a new kind of electrochromatography carried out in a five-compartment electrolyzer were developed. The adsorbent was packed in the central compartment, while the neighboring compartments were used as the elution compartments and the electrode compartments, respectively. Chromatographic separations of human serum albumin on Blue Sepharose Fast Flow, bovine serum albumin (BSA) on DEAE-Sepharose Fast Flow, and BSA on hydroxyapatite were carried out, respectively. The adsorption isotherms were shown to be independent of electric field, while the increase in the electric field strength resulted in a linear increase in the magnitude of electroosmotic flux and the improvement of the breakthrough behavior in all cases. The experiment results have demonstrated the effectiveness of the oscillatory electroosmosis in enhancing intra- and inter-particle mass transport and its high potential to large-scale chromatography.     

Modeling of retention behavior in capillary electrochromatography from chromatographic and electrophoretic data

A phenomenological approach was presented to describe the retention behaviors of solutes in capillary electrochromatography (CEC). Equations for calculation of the retention time and actual chromatographic retention factor for ionic solutes, weak monoprotic acid and weak monoprotic base were derived, which can be described by two general expressions regardless the charge status of the solute. The general expressions enable calculation of the retention time and retention factor in CEC from chromatographic and electrophoretic data, which were experimentally verified with a variety of compounds and a variety of CEC modes. Based on this approach, the chromatographic retention and the electrophoretic migration in the CEC systems studied were found to be two independent processes. The validity of this approach was also discussed.     

Some theoretical considerations on preparative separation with orthogonal pressurised planar electrochromatography

In this work, separation of multicomponent mixtures containing components with the same and different electrophoretic mobility by using orthogonal pressurised planar electrochromatography is studied theoretically. Additionally, a simple way for determination of a maximum amount of mixture causing volume overload of flat/planar columns used in this technique is presented. In the next stage, effects of change in different parameters on process performance by simulation case studies are investigated. A comparative study of separation productivity of orthogonal pressurised planar electrochromatography with the continuous and periodic modes of mixture delivery and column chromatography is carried out.     

Comparison of the chromatographic behavior of monolithic capillary columns in capillary electrochromatography and nano-high-performance liquid chromatography

Porous monoliths based on N,N-dimethylacrylamide (DMAA) or methacrylamide (MAA) were prepared inside fused silica capillaries as stationary phases for nano-chromatography. The columns were characterized in terms of flow rate and backpressure and showed, e.g. differences as a function of the salt concentration added to the polymerization mixture. When the columns were investigated for the separation of uncharged (polar hydroxylated aromatic compounds) and charged (amino acids) analytes under pressure driven conditions (pLC), differences to the previously observed behavior under voltage driven conditions (CEC) were observed. Whereas the non-charged analytes showed similar behavior in both cases--thus, corroborating the previous assumption of a mainly chromatographic separation mode driven by hydrophilic interactions in CEC--the charged amino acids did not. Assuming that the separation was governed by chromatographic phenomena in the pLC mode and by both chromatographic and electrophoretic effects in the CEC mode, the experiments allowed deconvoluting the two contributions. In particular, the charged amino acids appeared to interact with the stationary phases mainly by electrostatic interactions modified by some hydrophilic effects.     

Interplay of chromatographic and electrophoretic processes in capillary electrochromatography

The separation mechanism in capillary electrochromatography (CEC) is a hybrid differential migration process, which entails the features of both high-performance liquid chromatography and capillary zone electrophoresis, i.e., chromatographic retention and electrophoretic migration. The adsorption of the different sample components on the stationary phase can be modified by the presence of the electric field across the column. Here, we use our previously published approach to decouple chromatographic retention from electrophoretic migration that allows us to investigate the "modification" of the retention process in CEC. This paper presents a methodology for characterization of changes in the retention of neutral and charged sample components, under identical conditions of stationary and mobile phase.     

Continuous full filling capillary electrochromatography: chromatographic performance and reproducibility

Continuous full filling capillary electrochromatography with nanoparticles as pseudostationary phase interfaced with electrospray ionisation mass spectrometric detection was used for reversed phase separations with very high separation efficiency. Several batches of nanoparticles were synthesised and their electrochromatographic performance were evaluated. Different parameters, such as repeatability, reproducibility, limit of detection, and peak asymmetry, were investigated yielding excellent results. The stability of the system over wide pH ranges and over time was found to be excellent. Very high separation efficiencies with over 1.1 million theoretical plates per metre were obtained. The limit of detection for the investigated dialkyl phthalates was approximately 1.0 micromol L(-1), corresponding to 3-5 fmol injected. After preparation, nanoparticle suspensions could be used without further treatment for at least an entire working day with maintained chromatographic qualities.     

Evaluation of the interaction between hydroxyapatite and bisphosphonate by nonlinear capillary electrochromatography

Bisphosphonates are a class of chemical compounds used to treat diseases caused by increased bone resorption. Zoledronate is a third‐generation bisphosphonate drug. Hydroxyapatite is main mineral constituent of bones, which can be bound by bisphosphonates in vivo. In this work, we report a method of nonlinear capillary electrochromatography for study on the interaction between hydroxyapatite and bisphosphonate. Hydroxyapatite was modified on the inner wall of capillary by a biomimetic‐mineralization method. Then nonlinear chromatography was used to fit and analyze the interaction between zoledronate and hydroxyapatite. The association rate constants of zoledronate in hydroxyapatite‐modified capillary and bare capillary are 642.3 and 195/M/min, respectively. This indicates that there is strong binding interactions and affinity between zoledronate and hydroxyapatite. Besides, the interaction between zoledronate and hydroxyapatite was confirmed further by ultraviolet spectroscopy. The method of nonlinear capillary electrochromatography provides a fast and effect approach for studying of bone metabolism disease by evaluation of interaction between hydroxyapatite and bisphosphonates.     

Adsorption of plasmid DNA on ceramic hydroxyapatite chromatographic materials

The adsorption of plasmid DNA onto two different types of ceramic hydroxyapatite beads with a particle diameter of 20 μm, namely Ceramic Hydroxyapatite Type II and the Type III, which is not commercially available, were investigated. Type II and the Type III have a pore diameter of 80 and 240 nm, respectively. Equilibrium and dynamic binding capacity for a 4.9 kbp model plasmid on Ceramic Hydroxyapatite Type II and Type III were enhanced by addition of NaCl to the adsorption buffer. This result indicates that the adsorption mechanism cannot be solely explained by electrostatic interaction. The affinities of plasmid DNA for Ceramic Hydroxyapatite Type II (with a K(D) of ≈0.005 mg/mL) and to Hydroxyapatite Type III (with a K(D) of ≈0.045 mg/mL) were not affected by NaCl, whereas the binding capacity was. This observation corroborates the assumption that a change of the shape of the plasmid molecule is affected and could be the reason for increased binding capacity with salt. The maximal binding capacity shows that at least a part of the CHT II bead must be accessible for the plasmid, whereas CHT III can be saturated with the plasmid. In both cases, an extremely hindered transport takes place.     

Comparison of separation behavior of benzodiazepines in packed capillary electrochromatography and open-tubular capillary electrochromatography

Packed column capillary electrochromatography (CEC), open-tubular CEC and microcolum liquid chromatography (LC) using a cholesteryl silica bonded phase have been studied to compare the retention behavior for benzodiazepines. It has been found that packed column CEC gives better resolution, faster analysis time than microcolumn LC for benzodiazepines maintaining similar selectivity except for some solutes which are charged species under the separation conditions. However, open-tubular CEC gave different selectivities to a larger extent for charged benzodiazepines from that which should be produced by the chromatographic properties of the cholesteryl silica phase. Charged species migration times are mainly influenced by electrophoretic mobility rather than the chromatographic interactions.     

Calculation of retention factors in capillary electrochromatography from chromatographic and electrophoretic data

Retention factors in capillary electrochromatography (CEC) were calculated by means of theoretically derived equations and experimentally determined parameters in microcolumn liquid chromatography and capillary zone electrophoresis. It was found that the retention factor of uncharged components in CEC was about 20% higher than was calculated. The derived equations do not take into account alteration of the nature of the stationary phase or distribution constant by the applied electric field. However, the influence of the electric field on the retention in CEC can be estimated. Individual field contributions could not be determined.     

Optimization of preparative chromatographic separation of multiple rare earth elements

This work presents a method to optimize multi-product chromatographic systems with multiple objective functions. The system studied is a neodymium, samarium, europium, gadolinium mixture separated in an ion exchange chromatography step. A homogeneous Langmuir Mobile Phase Modified model is calibrated to fit the experiments, and then used to perform the optimization task. For the optimization a multi-objective Differential Evolution algorithm was used, with weighting based on relative value of the components to find optimal operation points along the Pareto front. The objectives of the Pareto front are weighted productivity and weighted yield with purity as an equality constraint. A prioritizing scheme based on relative values is applied for determining the pooling order. A simple rule of thumb for pooling strategy selection is presented. The multi-objective optimization gives a Pareto front which shows the rule of thumb, as a gap in one of the objective functions.     

Polymerised bicontinuous microemulsions as stationary phases for capillary electrochromatography. Effect of pore size on chromatographic performance

Monolithic columns for capillary electrochromatography (CEC) were prepared by in situ polymerisation of bicontinuous microemulsions containing butyl methacrylate. The resulting monoliths were found to be permeable to mobile phase flow and their behaviour as CEC stationary phases was investigated. It was found that the monoliths were able to separate a simple test mixture of phthalates, but that efficiencies were low. However, several advantages of the monoliths compared to conventional ODS packed columns were found: preparation time is short, many columns can be prepared from the same batch of microemulsion and column conditioning is much faster. The columns show promise as stationary phases for CEC, but more development is required to improve efficiencies.     

Planar electrochromatography Part 1. Planar electrochromatography on non-wetted thin-layers

Summary Planar electrochromatographic separations of test substances were performed on non pre-wetted, commercially available, thin-layer plates. The behavior of different layers and solvents was studied in an applied electric field of up to 2000 V cm^–1. Evident electrokinetic effects, electroosmosis and electrophoresis were observed only on silica gel and polyamide layers developed with polar solvents. The selectivity of separation of nonionic and ionogenic compounds was greatly enhanced. Although experimental conditions were controlled to a certain extent, results obtained with the same solvents were reproducible within 5%.     

Mass transfer effects in preparative chromatography of eremomycin on polymeric sorbents

This work is devoted to the study of the regularities of the sorption of the new antibacterial antibiotic eremomycin on carboxylic sorbents. The main sorption kinetic equilibrium and dynamic parameters for realization of one-act preparative chromatographic process were determined and the difference between gel-like and structurally segregated carboxylic cation exchangers was analyzed. The optimal conditions for sorption and complete desorption of eremomycin were found.     

Immunochemical characteristics and preparative application of agarose-based immunosorbents

The physical and chemical properties of immunosorbents prepared from Sepharose 4B have been studied, with the objective of improving their utility in preparative applications. The most significant factors influencing efficient use of these immobilized reagents are loss of immunochemical reactivity with increasing protein substitution and the restrictions imposed by the porous nature of the support. Similar effects are observable with controlled-pore glass matrices, but they are of less significance when solid, nonporous supports are used. Considerable improvements in operational efficiencies have been obtained by limiting the amount of protein immobilization and restricting reaction to the more accessible surface regions of the porous supports. Improved operating protocols based on these principles have been adapted to automated chromatographic systems, resulting in a fourfold increase in productive capacity. With the addition of the continuous dialysis and concentration system described here, routine daily production of gram quantities of specific antibodies from antisera becomes possible. Similarly, with a knowledge of the basic characteristics of these immunosorbents, it has been possible to increase greatly the efficiency of small-scale separations and purifications of immunological reagents from limited sources. The general approach toward the practical exploitation of immunosorbents for preparative applications is discussed in the context of these findings.     

Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode

The adsorption isotherms of four model proteins (lysozyme, α-lactalbumin, ovalbumin, and BSA) on eight commercial phenyl hydrophobic interaction chromatography media were measured. The isotherms were softer than those usually seen in ion-exchange chromatography of proteins, and the static capacities of the media were lower, ranging from 30 to 110 mg/mL, depending on the ammonium sulfate concentration and the protein and adsorbent types. The protein-accessible surface area appears to be the main factor determining the binding capacity, and little correlation was seen with the protein affinities of the adsorbents. Breakthrough experiments showed that the dynamic capacities of the adsorbents at 10% breakthrough were 20-80% of the static capacities, depending on adsorbent type. Protein diffusivities in the adsorbents were estimated from batch uptake experiments using the pore diffusion and homogeneous diffusion models. Protein transport was affected by the adsorbent pore structures. Apparent diffusivities were higher at lower salt concentrations and column loadings, suggesting that adsorbed proteins may retard intraparticle protein transport. The diffusivities estimated from the batch uptake experiments were used to predict column breakthrough behavior. Analytical solutions developed for ion-exchange systems were able to provide accurate predictions for lysozyme breakthrough but not for ovalbumin. Impurities in the ovalbumin solutions used for the breakthrough experiments may have affected the ovalbumin uptake and led to the discrepancies between the predictions and the experimental results.     

Pretreatment of body fluids by preparative isotachophoresis prior to chromatographic analysis

Summary This study focusses attention on the possibilities of preparative isotachophoresis (ITP) as a sample pretreatment technique prior to liquid chromatographic (HPLC) analysis. The increased demand for accurate and less time consuming analysis necessitates that sample pretreatment procedures, should be develop in parallel with other improvements (e. g. in detection and separation) which can be observed. The preparation isotachophoresis was performed on gel slabs and the zones of interest were subsequently cut out, desorbed and the desorbates analyzed by HPLC. In this study satisfactory recoveries of between 85–90% with a standard deviation of 1–5% were observed for blank experiments. For spiked serum and urine samples the recoveries in general decreased with decreasing spiked drug concentrations. These observations are discussed in this paper.