Combined effect of coulombic and van der Waals interactions in the chromatography of proteins.

We have recently proposed a theoretical framework for the effect of the eluting salt ionic strength of the eluent on the retention factor of proteins in ion-exchange chromatography of proteins. It is based on the solution of the linearized Poisson-Boltzmann equation for two oppositely charged planar surfaces in contact with a salt solution and describes the coulombic interaction between the protein and the oppositely charged stationary-phase surface. At sufficiently high salt concentrations in the mobile phase van der Waals interactions between the protein and the stationary phase become important. In this work we consider the effect of salt on the combined coulombic and van der Waals interactions by combining the electrostatic theory with the theory for van der Waals interactions. The combined theory describes the retention of proteins as a function of eluting salt concentration over a wide salt concentration range. The protein molecules are, according to the proposed theory, held in a diffuse layer close to the stationary phase and are not in a distinct layer, which is assumed in the traditional thermodynamic interpretation of the capacity factor. For this reason, we also examine the thermodynamic interpretation of the capacity factor when it is due to distant dependent interactions.     

A study of the enthalpy and entropy contributions of the stationary phase in reversed-phase liquid chromatography

The goal of this study was to elucidate the roles played by the stationary and mobile phases in retention in reversed-phase liquid chromatography (RPLC) in terms of their individual enthalpic and entropic contribution to the Gibbs free energy of retention. The experimental approach involved measuring standard enthalpies of transfer of alkylbenzenes from typical mobile phases used in RPLC (methanol/water and acetonitrile/water mixtures), as well as from n-hexadecane (a simple analogue of the stationary phase) to the gas phase, using high-precision headspace gas chromatography. By combining the measured enthalpies with independently measured free energies of transfer, the entropies of transfer were obtained. This allowed us to examine more fully the contribution that each phase makes to the overall retention. It was found that the standard enthalpy of retention in RPLC (i.e., solute transfer from the mobile phase to the stationary phase) is favorable, due to the large and favorable stationary-phase contribution, which actually overcomes an unfavorable mobile-phase contribution to the enthalpy of retention. Further, the net free energy of retention is favorable due to the favorable enthalpic contribution to retention, which arises from the net interactions in the stationary phase. Entropic contributions to retention are not controlling. Therefore, to a great extent, retention is due to enthalpically dominated lipophilic interaction of nonpolar solutes with the stationary phase and not from solvophobic processes in the mobile phase. Further, our enthalpy data support a "partition-like" mechanism of retention rather than an "adsorption-like" mechanism. These results indicate that the stationary phase plays a very significant role in the overall retention process. Our conclusions are in direct contrast to the solvophobic model that has been used extensively to interpret retention in RPLC.     

Nanoelectrospray ionization of protein mixtures: solution pH and protein pI

Solutions consisting of single proteins and mixtures of proteins at different pH values have been subjected to both positive ion and negative ion nanoelectrospray ionization to study the influence of solvent pH and protein pI on the ionization responses of proteins. As has been noted previously, it is possible to form protein ions of one polarity despite the fact that the proteins are present as the opposite polarity in solution. However, total response under this condition tends to be at least an order of magnitude less than the condition in which the nanoelectrospray ionization polarity is the same as the net charge of the proteins in solution. Furthermore, maximum signals in positive ion mode were noted when the pH value of the solution was 4-5 units lower than the protein pI. In the negative ion mode, maximum protein anion signals were observed when the pH was roughly 5 units higher than the protein pI. While only small changes in the abundance-weighted average charge were noted as a function of solution conditions, the extent of sodium ion incorporation was seen to depend strongly on the relationship between net protein charge in solution and gas-phase ion polarity. Sodium ion incorporation was minimized under conditions of maximum signal (i.e., low pH positive ion mode and high pH negative ion mode). Sodium ion incorporation was highest when the protein ion polarities in solution and the gas phase were opposite. These observations are consistent with the charged residue model for electrospray ionization and suggest that a degree of selectivity for electrospray ionization applied to protein mixtures can be realized via judicious selection of solution pH and ionization polarity. Furthermore, the relative extent of sodium ion incorporation under a given set of conditions appears to correlate, at least qualitatively, with protein pI.     

Extended thermodynamic approach to ion interaction chromatography

The chromatographic behavior of charged analytes in ion interaction chromatography (IIC) is theoretically investigated. The chemical modifications of the stationary and mobile phases in the presence of ion interaction reagent (IIR) are theoretically shown to change the partition coefficient for charged molecules. The most reliable literature experimental results concerning retention behavior of charged molecules in IIC were used to test the new theory. Retention equations are compared with those that can be obtained from the most important retention models in IIC. The present exhaustive retention model, which is well-founded in physical chemistry, goes further than the previous ones whose retention equations can be viewed as limiting cases of the present theory. The present extended thermodynamic approach reduces to stoichiometric or electrostatic retention models if the surface potential or pairing equilibria are respectively neglected. Moreover, it is able to quantitatively explain experimental evidences that cannot be rationalized by the existing retention models.     

Electrostatic repulsion hydrophilic interaction chromatography for isocratic separation of charged solutes and selective isolation of phosphopeptides

If an ion-exchange column is eluted with a predominantly organic mobile phase, then solutes can be retained through hydrophilic interaction even if they have the same charge as the stationary phase. This combination is termed electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). With mixtures of solutes that differ greatly in charge, repulsion effects can be exploited to selectively antagonize the retention of the solutes that normally would be the best retained. This permits the isocratic resolution of mixtures that normally require gradients, including peptides, amino acids, and nucleotides. ERLIC affords convenient separations of highly charged peptides that cannot readily be resolved by other means. In addition, phosphopeptides can be isolated selectively from a tryptic digest.     

Effect of net surface charge on particle sizing and material recognition by using phase Doppler anemometry

By taking net surface charge into consideration, the scattering field of particles illuminated by dual laser beams of phase Doppler anemometry (PDA) is computed based on Mie's theory, and the effect of net surface charge on the phase-diameter relationship and the phase ratio is studied. It is found that the phase-diameter relationship and the relationship between the phase ratio and the refractive index of charged particles could be significantly different from those of uncharged particles, which would lead to errors in particle sizing and the measurement of refractive indices. A method of recognizing charged particles and determining the value of their surface conductivity, which is related to net surface charge, is proposed by utilizing the effect of net surface charge on the measurement of refractive indices using PDA.     

Determination of effective protein charge by capillary electrophoresis: effects of charge regulation in the analysis of charge ladders

Protein charge ladders are an effective tool for measuring protein charge and studying electrostatic interactions. However, previous analyses have neglected the effects of charge regulation, the alteration in the extent of amino acid ionization associated with differences between the pH at the protein surface and in the bulk solution. Experimental data were obtained with charge ladders constructed from bovine carbonic anhydrase. The protein charge for each element in the ladder was calculated from the protein electrophoretic mobility as measured by capillary electrophoresis using the hindrance factor for a hard sphere with equivalent hydrodynamic radius. The protein charge was also evaluated theoretically from the amino acid sequence by assuming a Boltzmann distribution in the hydrogen ion concentration. The calculations were in excellent agreement with the data, demonstrating the importance of charge regulation on the net protein charge. These results have important implications for the use of charge ladders to evaluate effective protein charge in solution.     

Vancomycin dimerization and chiral recognition studied by high-performance liquid chromatography

The retention and separation of D,L-dansylvaline enantiomers (used as test solutes) were investigated using silica gel as stationary phase and vancomycin as chiral mobile-phase additive. A retention model was developed to describe the mechanistic aspects of the interaction between solute and vancomycin in the chromatographic system. It considered the formation of vancomycin dimers both "free" in the mobile phase and adsorbed on silica. By fitting the model equation to experimental data, it appeared clearly that the approach taking into account the vancomycin dimerization described accurately the retention behavior of the compounds. The examination of the model equation parameters showed that the glycopeptide dimerization increased the enantioselectivity by a factor of approximately 3.7. This study demonstrated the preponderant role of the vancomycin dimerization on the chiral recognition process of D,L-dansylvaline. Also, an additional analysis on a vancomycin chiral stationary phase indicated that the addition of vancomycin in the mobile phase promoted a greater enantioselectivity mediated by the formation of dimers in the stationary phase.     

Ice chromatography. Characterization of water-ice as a chromatographic stationary phase

Water-ice has been characterized as a stationary phase for liquid chromatography. Solutes having two or more polar groups are retained on this stationary phase with THF/hexane as the mobile phase, suggesting that multipoint interactions are required for measurable solute retention. Chromatographic separation of phenols or crown ethers on water-ice is possible. The ice surface is expected to provide two different adsorption sites coming from the OH and O dangling bonds. Although the solute partition into the quasiliquid layer is also considered, the dependence of the retention times on the THF concentration implies that the interaction of solutes with the water-ice surface rather than the partition into the quasiliquid layer is responsible for solute retention. A retention model suggests that the number of adsorption sites for a crown ether depends on its ring size, whereas two sites are involved for the retention of phenols having two hydroxyl groups. Although hydroxyl groups can act as both a hydrogen bond donor and an acceptor, the interaction with the ice OH sites, which are exposed to the surroundings in comparison with the ice O sites, is more important. However, when an acyclic polyether is added to the mobile phase, its adsorption onto the water ice surface allows the creation of the O sites that phenols can approach without steric hindrance. In the presence of the polyethers adsorbed on the ice surface, the retention of phenols is enhanced, whereas crown ethers become less retained due to the competitive adsorption of the polyethers.     

Key to analyte migration and retention in electrochromatography

This work identifies electrical field-induced concentration polarization (CP) as a key physical mechanism influencing the retention behavior of charged analytes in electrochromatography with fixed beds of porous adsorbent particles. Due to an insufficient screening of intraparticle surface charge, under most general conditions the porous (permeable) particles become charge-selective. CP is caused by coupled mass and charge transport normal to the charge-selective external surface of the permeable particles, which leads to concentration gradients of ionic species in the adjoining interparticle electrolyte solution. Cation-exchange (cation-selective) particles were employed to investigate the influence of applied voltage on the retention factor of counterionic, i.e., positively charged, analytes. It is demonstrated by macroscopic retention data and microscopic studies resolving the CP phenomenon on a particle scale that the dependence of CP on electrical field and mobile-phase ionic strengths is directly reflected in concomitant changes of analyte retention. The CP zones that develop at the interface between interparticle and intraparticle pore space are recognized by charged, but not electroneutral analytes while entering or leaving the particles. The intensity of these convective-diffusion boundary layers (CP zones) depends on the applied field strength and charge selectivity of a particle. Thus, it is the charge-selective transport between the interparticle and intraparticle pore space in packed beds that prevails under typical experimental conditions in electrochromatography and that forms the physical basis for a general electrical field dependence of the retention factor of charged analytes.     

Stabilization of gas-phase noncovalent macromolecular complexes in electrospray mass spectrometry using aqueous triethylammonium bicarbonate buffer

The use of triethylammonium bicarbonate (TEAB) solution in electrospray mass spectrometry proved to be a very efficient way for studying proteins or noncovalent protein complexes under "nondenaturing" conditions. The low charge states observed in the mass spectra improve the separation of ions arising from macromolecular species of close masses. Moreover, the multiply charged ions generated in a TEAB solution are significantly more stable than those formed under more conventional conditions (for example, with ammonium bicarbonate or acetate solution). The analytical interest of TEAB for the analysis of macromolecular species that can easily dissociate in the gas phase, such as hemoglobin or other macromolecular noncovalent complexes, is demonstrated.     

Estimates of protein surface areas in solution by electrospray ionization mass spectrometry

The extent of multiple charging of protein ions in electrospray ionization (ESI) mass spectra depends on the solvent-exposed surface area, but it may also be influenced by a variety of other extrinsic and intrinsic factors. Gas-phase ion chemistry (charge-transfer and charge-partitioning reactions) appears to be the major extrinsic factor influencing the extent of protonation as detected by ESI MS. In this work, we demonstrate that under carefully controlled conditions, which limit the occurrence of the charge-transfer reactions in the gas phase, charge-state distributions of protein ions can be used to assess the solvent-exposed surface area in solution. A set of proteins ranging from 5-kDa insulin to 500-kDa ferritin shows a clear correlation between the average charge in ESI mass spectra acquired under native conditions and their surface areas calculated based on the available crystal structures. An increase of the extent of charge-transfer reactions in the ESI interface results in a noticeable decrease of the average charge of protein ions across the entire range of tested proteins, while the charge-surface correlation is maintained. On the other hand, the intrinsic factors (e.g., a limited number of basic residues) do not appear to play a significant role in determining the protein ion charge. Based on these results, it is now possible to obtain estimates of the surface areas of proteins and protein complexes, for which crystal structures are not available. We also demonstrate how the ESI MS measurements can be used to characterize protein-protein interaction in solution by providing quantitative information on the subunit interfaces formed in protein associations.     

Self‐Assembly Toolbox of Tailored Supramolecular Architectures Based on an Amphiphilic Protein Library

The molecular structuring of complex architectures and the enclosure of space are essential requirements for technical and living systems. Self‐assembly of supramolecular structures with desired shape, size, and stability remains challenging since it requires precise regulation of physicochemical and conformational properties of the components. Here a general platform for controlled self‐assembly of tailored amphiphilic elastin‐like proteins into desired supramolecular protein assemblies ranging from spherical coacervates over molecularly defined twisted fibers to stable unilamellar vesicles is introduced. The described assembly protocols efficiently yield protein membrane–based compartments (PMBC) with adjustable size, stability, and net surface charge. PMBCs demonstrate membrane fusion and phase separation behavior and are able to encapsulate structurally and chemically diverse cargo molecules ranging from small molecules to naturally folded proteins. The ability to engineer tailored supramolecular architectures with defined fusion behavior, tunable properties, and encapsulated cargo paves the road for novel drug delivery systems, the design of artificial cells, and confined catalytic nanofactories.     

Disentangling protein-silica interactions

We present the results of modelling studies aimed at the understanding of the interaction of a 7 nm sized water droplet containing a negatively charged globular protein with flat silica surfaces. We show how the droplet interaction with the surface depends on the electrostatic surface charge, and that adhesion of the droplet occurs when the surface is negatively charged as well. The key role of water and of the charge-balancing counter ions in mediating the surface-protein adhesion is highlighted. The relevance of the present results with respect to the production of bioinorganic hybrids via encapsulation of proteins inside mesoporous silica materials is discussed.     

Electrostatic Interactions between Ru(bpy)(3)(2+) and Chromatographic Surfaces

In HPLC, the zones of organic bases tail when silica-based stationary phases are used because the analytes and the surface are oppositely charged. In developing new stationary phases to achieve lower surface charge, a measure of surface charge is needed. The choice of a suitable analyte to quantitate electrostatic interactions is complicated by the acid-base equilibrium of the analyte itself and by nonelectrostatic interactions between the analyte and the surface, which alter the charge-induced tailing. This paper describes the study of the pH dependence of adsorption to isolate electrostatic interaction and the use of a cationic probe, tris(2,2'-bipyridine)ruthenium chloride (Ru(bpy)(3)(2+)), to sense surface charge without the complication of the probe's acid-base equilibria. The paper further describes the application of Gouy-Chapman theory to reveal the surface charge density. The results confirm that type A silica is considerably more acidic than type B silica and that horizontal polymerization makes type A silica perform as well as type B silica.     

Aqueous chromatography utilizing pH-/temperature-responsive polymer stationary phases to separate ionic bioactive compounds

Cross-linked poly(N-isopropylacrylamide-co-acrylic acid) (poly(IPAAm-co-AAc))-grafted silica bead surfaces were prepared and applied as new column matrix materials that exploit temperature-responsive anionic chromatography to separate basic bioactive compounds, specifically catecholamine derivatives, in aqueous mobile phases. Since poly(IPAAm-co-AAc) has a well-known temperature-responsive phase transition and apparent pKa shift, polymer-grafted silica bead surfaces are expected to exhibit simultaneous hydrophilic/hydrophobic and charge density alterations under thermal stimuli. Elution behavior of catecholamine derivatives from a copolymer-modified bead packed column was monitored using aqueous mobile-phase HPLC under varying temperature and pH. Catecholamine derivatives had higher retention times on poly(IPAAm-co-AAc) columns at higher pH in comparison with those on noncharged PIPAAm reference columns, suggesting an electrostatic interaction as a separation mode. Temperature also affected the retention behavior of catecholamine derivatives. Optimal separation of four catecholamine derivatives was achieved at elevated temperature, 50 degrees C, and at pH 7.0. This is due to the increased hydrophobicity of the stationary phase as evidenced by the elution of a nonionic hydrophobic steroid. From these results, mutual influences of both electrostatic and hydrophobic interactions between basic catecholamine derivatives and pH-/temperature-responsive surfaces are noted. Consequently, elution of weakly charged bioactive compounds is readily regulated through the modulation of stationary-phase thermoresponsive hydrophilic/hydrophobic and charge density changes.     

Temperature-Responsive Chromatography Using Poly(N-isopropylacrylamide)-Modified Silica

A new concept in chromatography is proposed that utilizes a temperature-responsive surface with a constant aqueous mobile phase. The surface of the silica stationary phase in high-performance liquid chromatography (HPLC) has been modified with temperature-responsive polymers to exhibit temperature-controlled hydrophilic/hydrophobic changes. Poly(N-isopropylacrylamide) (PIPAAm) was grafted onto (aminopropyl)silica using an activated ester-amine coupling method. These grafted silica surfaces show hydrophilic properties at lower temperatures which, as temperature increases, transform to hydrophobic surface properties. The elution profile of five mixed steroids on an HPLC column packed with this material depends largely on the temperature of the aqueous mobile phase. Retention times increase with increasing temperature without any change in the eluent. Changes in the retention times of hydrophobic steroids were larger than those for hydrophilic steroids. The temperature-responsive interaction between PIPAAm-modified silica and these steroids is proposed to result from changes in the surface properties of the HPLC stationary phase by the transition of hydrophilic/hydrophobic surface-grafted IPAAm polymers. We demonstrate a novel and useful new chromatography system in which surface properties and the resulting function of the HPLC stationary phase are controlled by external temperature changes. This method should be effective in biological and biomedical separations of peptides and proteins using only aqueous mobile phases.     

Fundamentals of capillary electrochromatography: migration behavior of ionized sample components

The mechanism of separating charged species by capillary electrochromatography (CEC) was modeled with the conditions of ideal/linear chromatography by using a simple random walk. The most novel aspect of the work rests with the assumption that in sufficiently high electric field ionized sample components can also migrate in the adsorbed state on the ionized surface of the stationary phase. This feature of CEC leads to the introduction of three dimensionless parameters: alpha, reduced mobility of a sample component with the electrosmotic mobility as the reference; beta, the CEC retention factor; and gamma, the ratio of the electrophoretic migration velocity and the velocity of surface electrodiffusion. Since the interplay of retentive and electrophoretic forces determines the overall migration velocity, the separation mechanism in CEC is governed by the relative importance of the above parameters. The model predicts conditions under which the features of the CEC system engender migration behavior that manifests itself in a relatively narrow elution window and in a gradient like elution pattern in the separation of peptides and proteins by using pro forma isocratic CEC. It is believed that such elution patterns, which resemble those obtained by the use of external gradient of the eluent, are brought about by the formation of an internal gradient in the CEC system that gave rise to concomitant peak compression. The peculiarities of CEC are discussed in the three operational modalities of the technique: co-current, countercurrent, and co-counter CEC. The results suggest that CEC, which is often called "liquid chromatography on electrophoretic platform" is an analytical tool with great potential in the separation of peptides and proteins.     

Electrophoretic protein transport in gold nanotube membranes

Gold nanotube membranes are ideal model systems for exploring how pore size affects the rate and selectivity of protein transport in synthetic membranes. This is because these membranes have cylindrical, monodisperse pores (the nanotubes) with diameters that can be varied at will from tens of nanometers down to less than 1 nm. We report here on the effects of nanotube inside diameter, solution pH, and applied transmembrane potential on the rate and selectivity of protein transport in PEG-thiol-treated gold nanotube membranes. The transport properties of four proteins of differing sizes and pI values--lysozyme, bovine serum albumin, carbonic anhydrase, and bovine hemoglobulin--were investigated. In general, membranes containing larger diameter nanotubes showed higher fluxes and lower selectivities than membranes with smaller diameter nanotubes. Transmembrane electrophoresis can be used to augment the diffusive transport selectivity. For example, for proteins that are oppositely charged, a combination of a large transmembrane potential and a large nanotube diameter can be used to optimize both selectivity and flux. In addition to transmembrane potential and nanotube diameter, solution pH value plays an important role in determining the transport selectivity. This is because pH determines the net charge on the protein molecule and this, in turn, determines the importance of the electrophoretic transport term.     

Theory and use of the pseudophase model in gas-liquid chromatographic enantiomeric separations

The theory and use of the "three-phase" model in enantioselective gas-liquid chromatography utilizing a methylated cyclodextrin/polysiloxane stationary phase is presented for the first time. Equations are derived that account for all three partition equilibria in the system, including partitioning between the gas mobile phase and both stationary-phase components and the analyte equilibrium between the polysiloxane and cyclodextrin pseudophase. The separation of the retention contributions from the achiral and chiral parts of the stationary phase can be easily accomplished. Also, it allows the direct examination of the two contributions to enantioselctivity, i.e., that which occurs completely in the liquid stationary phase versus the direct transfer of the chiral analyte in the gas phase to the dissolved chiral selector. Six compounds were studied to verify the model: 1-phenylethanol, alpha-ionone, 3-methyl-1-indanone, o-(chloromethyl)phenyl sulfoxide, o-(bromomethyl)phenyl sulfoxide, and ethyl p-tolylsulfonate. Generally, the cyclodextrin component of the stationary phase contributes to retention more than the bulk liquid polysiloxane. This may be an important requirement for effective GC chiral stationary phases. In addition, the roles of enthalpy and entropy toward enantiorecognition by this stationary phase were examined. While enantiomeric differences in both enthalpy and entropy provide chiral discrimination, the contribution of entropy appears to be more significant in this regard. The three-phase model may be applied to any gas-liquid chromatography stationary phase involving a pseudophase.