Ion-exchange displacement chromatography of proteins Dextran-based polyelectrolytes as high affinity displacerss

Dextran-based polyelectrolyte displacers were successfully employed for the displacement purification of proteins in ion-exchange displacement systems. The effect of molecular mass was investigated by examining the efficacy of DEAE-dextran and dextran sulfate displacers of various molecular masses in cation- and anion-exchange systems, respectively. Induced salt gradients produced during these displacement experiments were measured in order to study their effect on the protein separations. The unique characteristics of these displacements were well predicted by simulations obtained from a steric mass action (SMA) ion-exchange model. These displacements differ from the traditional vision of displacement chromatography in several important ways: the isotherm of the displacer does not necessarily lie above the feed component isotherms; the concentration of the displaced proteins can sometimes exceed that of the displacer; higher-molecular-mass displacers are not necesarily more efficacious than lower-molecular-mass compounds; and the salt gradients induced by the adsorption of the displacer produce different salt micro-environments for each displaced protein.     

Investigation of particle-based and monolithic columns for cation exchange protein displacement chromatography using poly(diallyl-dimethylammonium chloride) as displacer

The overall topic of the investigation was the separation of basic proteins by cation exchange displacement chromatography. For this purpose two principal column morphologies were compared for the separation of ribonuclease A and alpha-chymotrypsinogen, two proteins found in the bovine pancreas. These were a column packed with porous particles (Macro-Prep S, 10 microm, 1000 A) and a monolithic column (UNO S1). Both columns are strong cation exchangers, carrying -SO3(-)-groups linked to a hydrophilic polymer support. Poly(diallyl-dimethylammonium chloride) (PDADMAC), a linear cationic polyelectrolyte composed of 100-200 quaternary pyrrolidinium rings, was used as displacer. The steric mass action (SMA) model and, in particular, the operating regime and dynamic affinity plots were used to aid method development. To date the SMA model has been applied primarily to simulate non-linear displacement chromatography of proteins using low molar mass displacers. Here, the model is applied to polyelectrolytes with a molar mass below 20000 g mol(-1), which corresponds to a degree of polymerization below 125 and an average contour length of less than 60 nm. The columns were characterized in terms of the adsorption isotherms (affinity, capacity) of the investigated proteins and the displacer.     

Salt effects in anion exchange displacement chromatography: Comparison of pentosan polysulfate and dextran sulfate displacers

Summary Experimental studies were carried out to investigate the utility of pentosan polysulfate as a low molecular weight polyelectrolyte displacer for the purification of proteins in anion-exchange displacement systems. In addition, the influence of mobile phase salt concentration on displacer efficacy, protein-protein resolution, and displacement development were studied for several anionic displacers. It was found that while large polyelectrolytes (50 kd dextran sulfate) were efficient displacers for a wide range of salt concentrations, relatively small polyelectrolytes (3 kd pentosan polysulfate) were seen to act as an efficient displacer only under conditions of high salt micro-environments. In addition, for proteins exhibiting similar affinities, zone mixing at the protein-protein boundary was found to be quite sensitive to the salt concentration. Finally, displacement chromatography was successfully implemented for the separation of proteins from milk whey.     

Salt effect on surface modification of silica by interpolyelectrolyte complexes

The effect of a low-molecular-mass salt on the properties of interpolyelectrolyte complexes formed as a result of interactions between poly(diallyldimethylammonium chloride) and copolymers of maleic acid with propylene or α-methylstyrene in their salt containing non-stoichiometric mixtures has been studied. Properties of such interpolyelectrolyte complexes were found to be determined by the chemical nature of the polyelectrolytes and by the salt concentration. The effect of salt on the surface modification of silica particles via their interactions with interpolyelectrolyte complexes has been examined. Two different ways of the surface modification of silica particles were used: (i) silica particles were contacted with previously prepared interpolyelectrolyte complexes and (ii) silica particles were contacted with cationic polyelectrolyte at first and then anionic polyelectrolyte was added. The efficiency of the surface modification was shown to be also dependent on the salt concentration and the chemical nature of polyelectrolytes. Turbidimetry, quasi-elastic light scattering, laser microelectrophoresis, and polyelectrolyte titration were used to characterize studied systems.     

Investigation of polyelectrolyte adsorption by means of a novel concept based upon the use of model surfaces and model polyelectrolytes

A concept to study the influence of molecular parameters in the adsorption of polyelectrolytes is presented. Characteristic for this approach is the use of model surfaces and model polyelectrolytes. The properties of both the surfaces and polyelectrolytes can be varied in a well defined way and over a broad range. First experiments show that the method is able to give structural information about the adsorbed polyelectrolytes. The method is very sensitive, adsorption experiments with polyelectrolyte concentrations in the region of micromoles per litre are possible.     

Entropy and enthalpy of polyelectrolyte complexation: Langevin dynamics simulations

We report a systematic study by Langevin dynamics simulation on the energetics of complexation between two oppositely charged polyelectrolytes of same charge density in dilute solutions of a good solvent with counterions and salt ions explicitly included. The enthalpy of polyelectrolyte complexation is quantified by comparisons of the Coulomb energy before and after complexation. The entropy of polyelectrolyte complexation is determined directly from simulations and compared with that from a mean-field lattice model explicitly accounting for counterion adsorption. At weak Coulomb interaction strengths, e.g., in solvents of high dielectric constant or with weakly charged polyelectrolytes, complexation is driven by a negative enthalpy due to electrostatic attraction between two oppositely charged chains, with counterion release entropy playing only a subsidiary role. In the strong interaction regime, complexation is driven by a large counterion release entropy and opposed by a positive enthalpy change. The addition of salt reduces the enthalpy of polyelectrolyte complexation by screening electrostatic interaction at all Coulomb interaction strengths. The counterion release entropy also decreases in the presence of salt, but the reduction only becomes significant at higher Coulomb interaction strengths. More significantly, in the range of Coulomb interaction strengths appropriate for highly charged polymers in aqueous solutions, complexation enthalpy depends weakly on salt concentration and counterion release entropy exhibits a large variation as a function of salt concentration. Our study quantitatively establishes that polyelectrolyte complexation in highly charged Coulomb systems is of entropic origin.     

Conformation of preadsorbed polyelectrolyte layers on silica studied by secondary adsorption of colloidal silica

The conformation of cationic polyelectrolytes preadsorbed on macroscopic silica surfaces was studied before and after addition of colloidal silica (CS) and compared to the fixation capacity of CS. The study included two polyelectrolytes of equal charge density, cationic polyacrylamide and cationic dextran. Adsorbed amounts were determined with stagnation point adsorption reflectometry (SPAR) and quartz crystal microgravimetry (QCM). Unsaturated layers of polyelectrolyte were formed in SPAR by stopping the adsorption at a fractional coverage relative to saturation adsorption. These layers were probed by secondary saturation adsorption of colloidal silica (CS). At low salt concentrations a high fractional coverage of polyelectrolyte was required to attain adsorption of CS, while significant adsorption of CS was found also for low fractional coverages of polyelectrolyte at salt concentrations above 10 mM NaCl. Saturation adsorption of cationic polyacrylamide (CPAM) and cationic dextran (Cdextran) onto the silica surface was found to be similar, while the secondary adsorption of CS was significantly higher onto preadsorbed CPAM compared with Cdextran. The QCM and SPAR data together indicated that the adsorbed layer of Cdextran was thinner than CPAM, and that a loose, expanded layer was formed after adsorption of CS on CPAM but not on Cdextran.     

Electrostatic model for protein adsorption in ion-exchange chromatography and application to monoclonal antibodies,lysozyme and chymotrypsinogen A

A model for the adsorption equilibrium of proteins in ion-exchange chromatography explicitly accounting for the effect of pH and salt concentration in the limit of highly diluted systems was developed. It is based on the use of DLVO theory to estimate the electrostatic interactions between the charged surface of the ion-exchanger and the proteins. The corresponding charge distributions were evaluated as a function of pH and salt concentration using a molecular approach. The model was verified for the adsorption equilibrium of lysozyme, chymotrypsinogen A and four industrial monoclonal antibodies on two strong cation-exchangers. The adsorption equilibrium constants of these proteins were determined experimentally at various pH values and salt concentrations and the model was fitted with a good agreement using three adjustable parameters for each protein in the whole range of experimental conditions. Despite the simplifications of the model regarding the geometry of the protein–ion-exchanger system, the physical meaning of the parameters was retained.     

Human serum albumin self-assembly on weak polyelectrolyte multilayer films structurally modified by pH changes

Adsorption of proteins onto film surfaces built up layer by layer from oppositely charged polyelectrolytes is a complex phenomenon, governed by electrostatic forces, hydrogen bonds, and hydrophobic interactions. The amounts of the interacting charges, however, both in polyelectrolytes and in proteins adsorbed on such films are a function of the pH of the solution. In addition, the number and the accessibility of free charges in proteins depend on the secondary structure of the protein. The subtle interplay of all these factors determines the adsorption of the proteins onto the polyelectrolyte film surfaces. We investigated the effect of these parameters for polyelectrolyte films built up from weak "protein-like" polyelectrolytes (i.e., polypeptides), poly(L-lysine) (PLL), and poly(glutamic acid) (PGA) and for the adsorption of human serum albumin (HSA) onto these films in the pH range 3.0-10.5. It was found that the buildup of the polyelectrolyte films is not a simple function of the pure charges of the individual polyelectrolytes, as estimated from their respective pKa values. The adsorption of HSA onto (PLL/PGA)n films depended strongly on the polyelectrolyte terminating the film. For PLL-terminated polyelectrolyte films, at low pH, repulsion, as expected, is limiting the adsorption of HSA (having net positive charge below pH 4.6) since PLL is also positively charged here. At high pH values, an unexpected HSA uptake was found on the PGA-ending films, even when both PGA and HSA were negatively charged. It is suggested that the higher surface rugosity and the decrease of the alpha-helix content at basic pH values (making accessible certain charged groups of the protein for interactions with the polyelectrolyte film) could explain this behavior.     

Proteins and polyelectrolytes: A charged relationship

We review the interaction of charged polymeric systems with proteins. In solutions of low ionic strength there are many examples of proteins attracted to polyelectrolytes even if both systems carry the same overall charge. This attractive interaction is widespread, having been observed for single polyelectrolyte chains as well as for polyelectrolytes grafted to surfaces (polyelectrolyte brushes) and charged polymeric networks. In all cases, adding salt weakens the interaction considerably. We discuss the suggestion that the attractive force at low salinity originates from the asymmetry of interaction between charged polymer segments and charged patches on the surface of the protein globule. This can be explained if the attractive force is mainly due to a counterion release force, i.e., the polyelectrolyte chains become the multivalent counterions for the patches of opposite charge localized on the surface of the proteins. We review a selection of simple models that lead to semi-quantitative estimates of this force as the function of salt concentration.     

Steric mass-action model for dye-ligand affinity adsorption of protein

The steric mass-action (SMA) model has been reported in the literature for ion-exchange and metal-affinity interaction adsorption equilibrium of proteins. In this work, an SMA model was developed for protein adsorption equilibrium to dye-ligand affinity adsorbent, Cibacron Blue-modified Sepharose CL-6B (CB-Sepharose). Static adsorption experiments with bovine serum albumin as a model protein were carried out to determine the model parameters, that is, equilibrium constant (K), characteristic number of binding sites (n), and steric factor (sigma). It was found that the linear model parameters, K and n decreased with the increase of ionic strength, while the nonlinear parameter, sigma, increased with ionic strength and the dye-ligand concentration. Thus, expressions correlating these parameters with the dye-ligand concentration and/or ionic strength were derived. With these correlations, the SMA model gave promising results in predicting protein adsorption isotherms. Thus, it is considered that the model would be useful in the theoretical analysis of dye-ligand affinity chromatography.     

Adsorption of polyelectrolyte-nanoparticle systems on silica: influence of ionic strength

In this work the effect of ionic strength on the adsorption behavior of cationic polyelectrolyte (acrylamide-acrylamidopropyltrimethylammonium chloride) and negatively charged silica particles has been studied by means of ellipsometry. The adsorption of the polyelectrolyte was observed to increase with increasing salt concentration, a behavior typical for polyelectrolytes with a screening-reduced solvency and a nonelectrostatic affinity for the surface. A similar dependence on the ionic strength was observed when studying the electrolyte effect on the nanoparticle adsorption to the preadsorbed polyelectrolyte film, suggesting that the polyelectrolyte surface conformations largely govern the binding capacity of the particles to the surface.     

Modelling gradient elution of bioactive multicomponent systems in non-linear ion-exchange chromatography

A theoretical framework for the ion-exchange behaviour of bioactive substances in non-linear ion-exchange chromatogaphy is described. The aim of the study was the creation of a model basis to support a process design for production-scale ion-exchange chromatography. The theory can be applied to a whole variety of biological substances, such as amino acids, polysaccharides, peptides and proteins and either isocratic or gradient elution can be carried out. The influence of the eluent concentration on the ion-exchange as well as on the characteristic charge was considered. Experimental measurements showed a strong non-linear ion-exchange equilibrium with a transition from a Langmuir-type to a sigmoidal isotherm at higher eluent concentrations. Hereby, the compound binds to the surface though it is not ionic. Therefore, the model considered the possibility of ion-exchange as well as adsorption. A simplified distribution of the counter-ions based on the Gouy-Chapman theory with a discrete distribution of the counter-ions was used. The theory was extended by a selectivity in the double layer to allow specific adsorption. Calculations of adsorption-elution cycles showed, in agreement with the experimental observations, the development of non-linear elution profiles with a desorption fronting. As a result, the column loading and the eluent concentration were varied. The effect of contaminants, in this case sodium ions, was investigated and included in the model. Finally, the model was extended to multicomponent systems to investigate the effect of side components on the retention behaviour. The development of the characteristic elution profiles and the effect of the column loading on the separation are discussed. Calculated concentration profiles along the column at discrete time steps were used to reveal the influence of side components and the underlying separation mechanism. The simulations provided a new insight into the phenomena involved in biochromatography and make convenient design concepts at least doubtful as the separation is in this case mainly determined by the loading step and not by the choice of the elution gradient.     

Effect of charge regulation on steric mass-action equilibrium for the ion-exchange adsorption of proteins

A thermodynamic formalism is developed for incorporating the effects of charge regulation on the ion-exchange adsorption of proteins under mass-overloaded conditions as described by the steric mass-action (SMA) isotherm. To accomplish this, the pH titration behavior of a protein and the associated adsorption equilibrium of the various charged forms of a protein are incorporated into a model which also accounts for the steric hindrance of salt counterions caused by protein adsorption. For the case where the protein is dilute, the new model reduces to the protein adsorption model described recently by the authors which accounts for charge regulation. Similarly, the new model reduces to the steric mass-action isotherm developed by Brooks and Cramer which applies to mass-overloaded conditions for the case where charge regulation is ignored so that the protein has a fixed charge. Calculations using the new model were found to agree with experimental data for the adsorption of bovine serum albumin (BSA) on an anion-exchange column packing when using reasonable physical properties. The new model was also used to develop an improved theoretical criterion for determining the conditions required for an adsorbed species to displace a protein in displacement chromatography when the pH is near the protein pI.     

A model for the Gibbs energy of aqueous solutions of polyelectrolytes

A new model for the excess Gibbs energy of aqueous solutions of polyelectrolytes is presented and applied for the correlation of the activity of water in aqueous solutions of polyelectrolytes without as well as with an added (single) salt. The model considers the phenomenon of counterion condensation, i.e., the partial dissociation of highly charged polyelectrolytes in water. Three parameters (a binary interaction parameter between polymer segments, the equilibrium constant of the dissociation reaction and a parameter which accounts for the polymer configuration) were fitted to the experimental results. The model allows for a reliable correlation of experimental results for the osmotic coefficient of aqueous solutions of a single polyelectrolyte (without as well as with an added salt).     

Isolation and Characterization of Cellulosic Pulp Fines and Their Interactions with Cationic Polyacrylamides

Four different types of cellulosic fines were isolated from refiner mechanical and kraft pulp samples to characterize their chemical and physical properties. The pulp fines were flocculated using four different types of cationically modified polyacrylamides. The extent of flocculation was observed with multiple light scattering apparatus. The maximum adsorption of the polyelectrolytes on the pulp fines was determined by polyelectrolyte titration. It was concluded that it is the combination of the molar mass and the charge density of a polyelectrolyte, as well as the origins and characteristics of the fines which define the adsorption and flocculation behavior. None of these properties alone could fully describe these phenomena, but the molar mass of the polyelectrolyte was the predominant factor. The most important fines characteristics were the charge properties and the microstructure.     

Protein-polyelectrolyte cluster formation and redissolution: a Monte Carlo study

Aqueous solutions of proteins and oppositely charged polyelectrolytes were studied at different polyelectrolyte chain length, ionic strength, and protein-protein interaction potential as a function of the polyelectrolyte concentration. One of the protein models used represented lysozyme in aqueous environment. The model systems were solved by Monte Carlo simulations, and their properties were analyzed in terms of radial distribution functions, structure factors, and cluster composition probabilities. In the system with the strongest electrostatic protein-polyelectrolyte interaction the largest clusters were formed near or at equivalent amount of net protein charge and polyelectrolyte charge, whereas in excess of polyelectrolyte a redissolution appeared. Shorter polyelectrolyte chains and increased ionic strength lead to weaker cluster formation. An inclusion of nonelectrostatic protein-protein attraction promoted the protein-polyelectrolyte cluster formation.     

Theory of competitive counterion adsorption on flexible polyelectrolytes: divalent salts

The counterion distribution around an isolated flexible polyelectrolyte in the presence of a divalent salt is evaluated using the adsorption model [M. Muthukumar, J. Chem. Phys. 120, 9343 (2004)] that considers the Bjerrum length, salt concentration, and local dielectric heterogeneity as physical variables in the system. Self-consistent calculations of effective charge and size of the polymer show that divalent counterions replace condensed monovalent counterions in competitive adsorption. The theory further predicts that at modest physical conditions for a flexible polyelectrolytes such as sodium polystyrene sulfonate in aqueous solutions polymer charge is compensated and reversed with increasing divalent salt. Consequently, the polyelectrolyte shrinks and reswells. Lower temperatures and higher degrees of dielectric heterogeneity between chain backbone and solvent enhance condensation of all species of ions. Complete diagrams of states for the effective charge calculated as functions of the Coulomb strength and salt concentration suggest that (a) overcharging requires a minimum Coulomb strength and (b) progressively higher presence of salt recharges the polymer due to either electrostatic screening (for low Coulomb strengths) or coion condensation (for high Coulomb strengths). Consideration of ion-bridging by divalent counterions leads to a first-order collapse of polyelectrolytes in modest presence of divalent salts and at higher Coulomb strengths. The authors' theoretical predictions are in agreement with the generic results from experiments and simulations.     

A Monte Carlo study of spherical electrical double layer of macroions-polyelectrolytes systems in salt free solutions

A canonical Monte Carlo simulation is performed to investigate the microstructure and the electrical double layer (EDL) of polyelectrolytes around macroions in the bulk systems based on the primitive model. We explore the influences of particles size, chain length, and charge density of polyelectrolytes on the microscopic behavior of the macroions-polyelectrolytes systems. The simulation results show that the surface charge density and the chain length of the polyelectrolytes are two key factors that affect the microstructure of polyelectrolytes around the macroions and potential of mean force between the macroions as well as the zeta potential of the spherical EDL constructed by polyelectrolytes. The high surface charge density of a polyelectrolyte leads to the polyelectrolyte acting as a bridge for the aggregation of macroions, causing the presence of the attraction between macroions. The polyelectrolytes with a long chain length present a cooperativity effect for the adsorption of the polyelectrolytes on the surface of the macroions. Furthermore, the two key factors both induce the overcharge of the macroions. The longer the chain length and the higher surface charge density of the polyelectrolytes, the stronger is the overcharge.     

The build-up of polyelectrolyte multilayers of microfibrillated cellulose and cationic polyelectrolytes

A new type of nanocellulosic material has been prepared by high-pressure homogenization of carboxymethylated cellulose fibers followed by ultrasonication and centrifugation. This material had a cylindrical cross-section as shown by transmission electron microscopy with a diameter of 5-15 nm and a length of up to 1 microm. Calculations, using the Poisson-Boltzmann equation, showed that the surface potential was between 200 and 250 mV, depending on the pH, the salt concentration, and the size of the fibrils. They also showed that the carboxyl groups on the surface of the nanofibrils are not fully dissociated until the pH has reached pH = approximately 10 in deionized water. Calculations of the interaction between the fibrils using the Derjaguin-Landau-Verwey-Overbeek theory and assuming a cylindrical geometry indicated that there is a large electrostatic repulsion between these fibrils, provided the carboxyl groups are dissociated. If the pH is too low and/or the salt concentration is too high, there will be a large attraction between the fibrils, leading to a rapid aggregation of the fibrils. It is also possible to form polyelectrolyte multilayers (PEMs) by combining different types of polyelectrolytes and microfibrillated cellulose (MFC). In this study, silicon oxide surfaces were first treated with cationic polyelectrolytes before the surfaces were exposed to MFC. The build-up of the layers was monitored with ellipsometry, and they show that it is possible to form very well-defined layers by combinations of MFC and different types of polyelectrolytes and different ionic strengths of the solutions during the adsorption of the polyelectrolyte. A polyelectrolyte with a three-dimensional structure leads to the build-up of thick layers of MFC, whereas the use of a highly charged linear polyelectrolyte leads to the formation of thinner layers of MFC. An increase in the salt concentration during the adsorption of the polyelectrolyte results in the formation of thicker layers of MFC, indicating that the structure of the adsorbed polyelectrolyte has a large influence on the formation of the MFC layer. The films of polyelectrolytes and MFC were so smooth and well-defined that they showed clearly different interference colors, depending on the film thickness. A comparison between the thickness of the films, as measured with ellipsometry, and the thickness estimated from their colors showed good agreement, assuming that the films consisted mainly of solid cellulose with a refractive index of 1.53. Carboxymethylated MFC is thus a new type of nanomaterial that can be combined with oppositely charged polyelectrolytes to form well-defined layers that may be used to form, for example, new types of sensor materials.