pH-gradient ion-exchange chromatography: an analytical tool for design and optimization of protein separations

This work demonstrates that a highly linear, controllable and wide-ranged pH-gradient can be generated through an ion-exchange chromatography (IEC) column. Such a pH-gradient anion-exchange chromatography was evaluated with 17 model proteins and found that acidic (pI<6) and basic (pI>8) proteins elute roughly at their pI, whereas neutral proteins (pI 6-8) elute at pH 8-9 regardless their pI values. Because of the flat nature of protein titration curves from pH approximately 6 to approximately 9, neutral proteins indeed exhibit nearly zero net charge at pH approximately 9. The elution-pH in pH-gradient IEC or the titration curve, but not the pI, was identified as the key parameter for pH optimization of preparative IEC in a fast and rational way. The pH-gradient IEC was also applied and found to be an excellent analytical tool for the fractionation of crude protein mixtures.     

High entrapment of insulin and bovine serum albumin into neutral and positively-charged liposomes by the remote loading method

Remote loading of insulin and bovine serum albumin (BSA) into neutral or positively-charged liposomes by incubation under a transmembrane pH gradient or non-pH gradient was investigated. Trapping efficiencies in several incubation conditions were compared with those of the conventional reverse-phase evaporation vesicle method (c-REV method). For neutral liposomes, insulin could not be effectively loaded into the liposomes by incubation, regardless of the incubation conditions. The trapping efficiency of insulin into positively-charged liposomes was higher than that of neutral liposomes, especially by the pH-gradient method. Insulin could be loaded into positively-charged liposomes about twofold more efficiently than by the pH-gradient method, compared with the c-REV method. Insulin distributed more on the surface of liposomes by the pH-gradient method than by the c-REV method. BSA showed significantly higher affinity to positively-charged liposomes than to neutral ones by various methods. However, the transmembrane pH-gradient method did not increase BSA loading into liposomes, compared with the c-REV and the non-pH-gradient methods. Our results suggest that the pH-gradient method, combining electrostatic interactions, may be useful for preparation of liposomal insulin and that the high hydrophobicity of BSA may not increase the remote loading of BSA into liposomes by the pH-gradient method.     

The Behaviour of Reduced, Alkylated and Native Proteins in a pH-Gradient LC System

Two dimensional (2D) liquid chromatography (LC) separations of proteins can be obtained faster and more automated than traditional 2D gel electrophoresis. Previously we have described a 2D LC method for separation of native proteins with separation according to pI by pH-gradient strong anion exchange (SAX) chromatography in the first dimension, and according to hydrophobicity by reversed phase chromatography in the second dimension. Since there are few literature reports on the combination of reduced/alkylated proteins and modern LC, a basic study of the chromatographic properties of a few reduced /alkylated proteins was undertaken with a pH-gradient SAX chromatographic system. Proteins where the disulfide groups were reduced, but not alkylated, were also included. The conditions that separated native proteins according to pI could not be used for neither reduced nor reduced/alkylated proteins. High concentrations of urea (4–8 M) were needed in the mobile phase in order to obtain good peak shapes. Addition of urea had an undesired impact on both the retention of the proteins and the pH gradient profile, with the effect that little correlation between reported pI values and elution pH was found. The conclusion was that proteins should be separated in the native state if good pI–pH correlations are important, and in the alkylated state with urea if other considerations are more important.     

A diffusion model for reversible comsumption in emulsion liquid membranes

This work extends previous diffusion models for emulsion globules in which a solute reacts with an internal reagent. This model allows for reversible consumption of the solute by the internal reagent. Local concentration of the internal reagent is nonzero and satisfies reaction and phase equilibria within the reacted zone. Predicted solute absorption rates are lower for the reversible consumption model than for irreversible models.     

Use of GAMESS/COSMO program in support of COSMO-SAC model applications in phase equilibrium prediction calculations

Thermodynamic models based on conductor-like screening models (COSMO) offer viable alternatives to existing group-contribution methods for the prediction of phase equilibria. Normally a COSMO-based model requires input of the distribution of screening charges on the molecular surface, aka. the sigma profile, determined from a specific quantum chemistry program and settings. For example, the COSMO-SAC model requires input of DMol³ generated sigma profiles. In this paper, we investigate the proper settings for an open-source quantum chemistry package GAMESS in order to generate sigma profiles to be used directly in the COSMO-SAC model. The phase behaviors (VLE and VLLE) of 45 binary mixtures from 10 commonly used solvents and the solubilities of 4 complex drug compounds in these solvents calculated from DMol³ and GAMESS generated sigma profiles are compared. While noticeable fine structure differences are observed in the individual sigma profiles for the same chemical compound generated from the two packages, it is found that the accuracy in the VLE/VLLE and solubility predictions from the two packages are comparable. Based on the systems we studied here, the open-source GAMESS/COSMO program with proper program settings could be used as an alternative sigma profile generation source in support of COSMO-SAC model applications in phase equilibrium prediction calculations.     

Isoelectric point separation of proteins by capillary pH-gradient ion-exchange chromatography

In the present work, isoelectric point (pl) separation of proteins by pH-gradient ion-exchange chromatography (IEC) on packed capillary columns is demonstrated. The development of a miniaturized flow-through pH probe for reliable pH monitoring of the column effluent, which was an important technical challenge for adapting this technique to capillary dimensions, was solved by designing a low microliter per minute flow rate housing to a commercially available micro pH probe. Highly linear outlet pH-gradients within the pH range 8.5-4.0 were obtained when applying simple inexpensive buffers consisting solely of piperazine, N-methylpiperazine and imidazole on 10 cm x 0.32 mm i.d. fused silica capillaries packed with anion-exchange poly(styrene divinylbenzene)-based macroporous materials, i.e. 10 microm Mono P from Amersham Biosciences and 10 microm PL-SAX from PolymerLabs. Furthermore, when using a pH-gradient from 6.8 to 4.3, both columns were able to baseline separate the A and B genetic variants of beta-lactoglobulin, which differ with two amino acid residues only, but the PL-SAX column provided almost a two-fold decrease in peak widths compared to the Mono P column. The influence of varying the buffer concentration, injection volume and column temperature on the peak widths and resolution of the beta-lactoglobulins was investigated, e.g. a 100 microl sample of dilute beta-lactoglobulins was injected directly on the column with practically no increase in peak width as compared to what obtained with conventional injection volumes. Finally, a pH-gradient from 6.8 to 4.3 was used to separate proteins in skimmed bovine milk on the PL-SAX column. The milk was simply diluted 1:10 (v/v) with water and filtrated before injection.     

A New Numerical Method for Accurate Simulation of Fast Cyclic Adsorption Processes

In the simulation of fast cyclic adsorption processes, to apply the Fickian diffusion model it is necessary to include an increasing number of numerical discretization points as the cycle time is reduced in comparison to the characteristic diffusional time constant. We propose a new numerical method based on the definition of two distinct regions within an adsorbent particle: an outer layer where the concentration varies significantly with large internal gradients leading to enhanced mass fluxes, and an internal region where the concentration profile is virtually flat. The proposed method leads to the automated generation of a numerical grid that has a constant number of elements independent of the process cycle time. The procedure is demonstrated on a model for the simulation of a heatless dryer pressure swing adsorption process.     

Vapor-liquid and liquid-liquid equilibria of water, 2-methoxyethanol and cyclohexanone: Experiment and correlation

Vapor-liquid equilibria and liquid-liquid equilibria of a ternary mixture consisting of water, 2-methoxyethanol and cyclohexanone and in addition of all binary subsystems were studied experimentally at several temperatures. A ternary corrective term in the expression for the Gibbs free energy based on the NRTL model improves simultaneous representation of binary and ternary phase equilibria.     

Retention in field-flow fractionation with secondary chemical equilibria

Summary The application range of field-flow fractionation (FFF) can be extended to low molecular weight solutes, as demonstrated a few years ago by Berthod et al., by taking profit of secondary chemical equilibria (SCE) occurring between the bulk carrier and a retained carrier component. The theory of solute retention in this SCE-FFF method is developed for any value of the solute distribution coefficient and of the retention ratio of the retained carrier component, provided that the Brownian mode of retention applies for this component and that the flow velocity profile is parabolic. This removes some of the limitations of the model previously developed by Berthod and Armstrong and sheds light on the potentialities of the SCE-FFF method for physico-chemical studies about secondary chemical equilibria in colloidal systems. Remaining assumptions in the model are discussed.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

Effect of lipophylic salts on ise detection limit: application to calixarene-based highly efficient potassium-selective electrodes

A new interpretative model for interpolation of potentiometric data from liquid membrane ion selective electrodes has been formulated; the model allows one to achieve information about partitioning equilibria establishing at the membrane-analysed solution interface of hydrophilic cations present in the membrane as counterions of exchanger lipophylic anions. The fundamental importance of such equilibria is related to their contribution to the detection limit of the electrodes. The soundness of the proposed model was positively verified by employing it in the characterization of new K(+)-selective electrodes based on a calixarenic ionophore. Although the validity of interpolation based on our model does not significantly differ from that obtained with the simpler model providing the inclusion of a constant parameter in the logarithmic argument of Nicolski-Eisenman's equation, the information obtained resulted to be analytically suitable for optimising the membrane composition and, as a consequence, the performance of the electrodes.     

Adsorption equilibria of butyl- and amylbenzene on monolithic silica-based columns

The adsorption isotherms of butyl- and amylbenzene on silica monolithic columns were measured by frontal analysis. The external, internal and total porosities of these columns were determined by inverse size-exclusion chromatography. The adsorption isotherms are concave upward in the entire concentration range investigated. They were fitted to the anti-Langmuir model, an unusual model in liquid-solid and liquid-liquid phase equilibria. Band profiles under overloaded conditions were recorded. They were in good agreement with the profiles calculated using th,e lumped pore diffusion model of chromatography and these adsorption isotherms.     

Assigning vibrational polyads using relative equilibria: application to ozone

We demonstrate how relative equilibria of a vibrating molecule, which are families of principal periodic orbits otherwise known as nonlinear normal modes, can be used to describe the global polyad structure of vibrational energy levels. The classical action integral n(E) computed along these orbits at different energies E corresponds to the polyad quantum number n so that the energy En of different relative equilibria describes the splitting of n-polyads. Further information on the internal polyad structure can be driven from the stability analysis of relative equilibria. We use the ozone molecule as a concrete example where n-polyads or "hyperpolyads" should be distinguished from the well-known polyads of the 1:1 stretching mode resonance; the stretching polyads are structural elements of hyperpolyads. We give dynamical interpretation of the relation between relative equilibria and n-polyads based on the normal form reduction in the limit of small vibrations near the equilibrium.     

Thermal denaturation of some proteins and its effect on their electrospray mass spectrat

The effects of beat on the electrospray mass spectra of eight globular proteins in solution were studied. These ranged from hardly noticeable to a dramatic shift in the mass spectrometric profile and a concomitant increase in ion abundance. This change is believed to be the result of thermal denaturation of the protein species in solution resulting in a transition from a more compact to a less compact conformation. We accounted for this transition by means of a recently proposed model based on aqueous solution acid/base equilibria. For cytochrome c, profiles calculated by means of this model agree well with experimental data. The ΔH of the denaturation reaction of cytochrome c in aqueous solution containing 0.2% acetic acid was calculated from experimental data to be 103.8 ± 9.2 kJ mol^?1, in good agreement with previous measurements.     

Modeling of (vapor + liquid) equilibrium and enthalpy of solution of carbon dioxide (CO2) in aqueous methyldiethanolamine (MDEA) solutions

A thermodynamic model was used to estimate enthalpy of solution of carbon dioxide (CO₂) in methyldiethanolamine (MDEA) aqueous solutions. The model was based on a set of equations for chemical equilibria, phase equilibria, charge, and mass balances. Non-ideality in the liquid phase was taken into account by interaction parameters fitted to (vapor + liquid) equilibrium data.The enthalpies of solution of CO₂ were derived from the model using classical thermodynamic relations and were compared to experimental values obtained in previous works.     

Complexation and Oxygenation in Cobalt(II)–Diamine–Oxygen Systems

The general pattern of equilibria involved in the complexation and oxygenation in cobalt(II)–diamine (phenanthroline or bipyridine)–oxygen systems was suggested. The set of equilibria included the following reactions: dissociation of protonated diamine cations; the formation of cobalt(II) mono-, bis-, and tris-diamine complexes, a hydroxo aqua complex, a hydroxo dimer, and a dihydroxo complex (monomer); various pathways to the dihydroxo dimer; and oxygenation of the hydroxo dimer. A mathematical model of the process was derived based on the material balances for cobalt and diamine and on the electroneutrality equation. The data of pH-metric studies of the systems in air and in an inert atmosphere and manometric data were used to calculate the individual equilibrium constants for each step of the process.     

Chemical Selectivities Disguised by Mass Diffusion. VIII. Influence of pH-gradients on the product distribution in mixing-disguised azo coupling reactions. 9th Communication on the selectivity of chemical processes

In mixing-disguised azo coupling reactions the protons which are released during the electrophilic substitution steps cause a pH-gradient in the reaction zone. Since, owing to the acid-base pre-equilibria, these pH-gradients also determine the local concentrations of the reactants in the reaction zone, they also have an influence on the measured product distribution of such fast reactions. In the present work, the product distribution of the azo coupling of 1-naphthol with 4-sulfophenyldiazonium ion was experimentally measured and the results were compared with the distributions predicted from our mixing-reaction model developed previously [1]. Furthermore, some experimental evidence for the existence of isoselectivity points are presented, at which a change of the initial pH-value has no influence on the product distribution.     

Solid phase extraction of copper(II) by fixed bed procedure on cation exchange complexing resins

The efficiency of the metal ion recovery by solid phase extraction (SPE) in complexing resins columns is predicted by a simple model based on two parameters reflecting the sorption equilibria and kinetics of the metal ion on the considered resin. The parameter related to the adsorption equilibria was evaluated by the Gibbs–Donnan model, and that related to the kinetics by assuming that the ion exchange is the adsorption rate determining step. The predicted parameters make it possible to evaluate the breakthrough volume of the considered metal ion, Cu(II), from different kinds of complexing resins, and at different conditions, such as acidity and ionic composition.