Specific salt effects in hydrophobic interaction chromatography of proteins

Summary It has been noted in the literature that certain salts enter into specific interaction with proteins. As a result of this, they may act as salting-in agents. We have investigated the effect of magnesium chloride which is known to possess such unusual properties on the retention of proteins in hydrophobic-interaction chromatography. First the retention behaviour of amino acids and small peptides having a wide polarity range was studied on reversed-phase columns using eluents containing (NH₄)₂SO₄, MgSO₄ or MgCl₂, in wide the concentration ranges. For less polar eluites plots of the logarithmic retention factors against the salt concentration were found to be linear, whereas the more polar species showed irregular behavior. The retention of a wide range of proteins was measured on a TSK Phenyl-5-PW column using eluents containing (NH₄)₂SO₄, MgSO₄ or MgCl₂ at different concentrations.The salt-mediated retention was regular with (NH₄)₂ SO₄ and MgSO₄ although MgSO₄ showed a lesser effect than that predicted by the surface-tension increment. The effect of MgCl₂ was quite irregular: the retention factors either increased or decreased or remained unchanged depending on the protein. These results corroborate earlier observations regarding the particular effect of MgCl₂ and suggest the modulation of selectivity in hyrophobic-interaction chromatography by the addition of MgCl₂ to the eluent.     

The new experimental data and a new thermodynamic model based on group contribution for correlation liquid–liquid equilibria in aqueous two-phase systems of PEG and (K2HPO4 or Na2SO4)

The new experimental data of liquid–liquid equilibria for aqueous two-phase systems PEG–K₂HPO₄–water and PEG–Na₂SO₄–water are presented. The effects of pH and molecular weight of polyethylene glycol were investigated and the tie lines with binodal curves for both systems are shown. A new thermodynamic model based on group contribution has been proposed for studying the phase behavior of aqueous two-phase polymer–salt systems. The assumptions of NRTL-NRF model and the activity coefficient equation of UNIQUAC-NRF model have been used for the groups. In this new model, UNIFAC-NRF, the nonrandom state of groups were selected as a reference state. The binary interaction parameters were adjusted using the data of binary salt–water systems and the ternary systems were correlated with only six binary adjustable parameters. The Debye–Huckel equation based on Fowller–Guggenheim equation was used to calculate the long range electrostatic interaction of the ions. The UNIFAC-NRF model was applied to correlate the experimental data of aqueous two-phase systems: PEG–K₂HPO₄–water and PEG–Na₂SO₄–water for two different molecular weight of PEG at different pH. The results of the new model showed that it can be used to correlate the LLE in aqueous solution of polymer–salt very well.     

Study on Binding Properties of Poorly Soluble Drug Trimethoprim in Anionic Micellar Solutions

Binding and distribution properties of trimethoprim (TMP) in the presence of various anionic surfactants; sodium octyl sulfate (C₈SO₄Na), sodium decyl sulfate (C₁₀SO₄Na), sodium lauryl sulfate (C₁₂SO₄Na), and sodium tetradecyl sulfate (C₁₄SO₄Na) has been studied by conductivity, spectrophotometry and surface tension measurements. The surface properties of anionic surfactants, that is, maximum surface excess concentration (Γ _max ) and minimum area per surfactant molecule (A _min ) at the air/water interface have been evaluated in the absence and presence of TMP using Gibbs adsorption isotherm. From conductivity data the ionization degree and counterion binding parameter have been obtained. Spectrophotometric experiments were used to determine binding constants of TMP to anionic micelles. With the increasing alkyl chain of surfactants, the interaction becomes stronger, which shows the importance of hydrophobic forces and incorporation of TMP molecules to the pure micelles of anionic surfactants increased. The results obtained from the surface tension and conductometric studies have been correlated with those obtained from the spectroscopic studies and binding tendency of TMP to anionic micelles followed the order as: C₁₄SO₄Na > C₁₂SO₄Na > C₁₀SO₄Na > C₈SO₄Na. From these results, the study of the interaction TMP in different anionic micellar solutions provided information about the characteristics of binding properties of poorly soluble drugs.     

盐酸胍对疏水色谱中蛋白质保留行为的影响

研究了变性剂酸胍对几种蛋白质在高效疏水色谱中保留行为的影响，用液相色谱中溶质的计量置换保留模型和测流动相表面张力及蛋白质紫外吸收光谱方法研究的结果表明：在低浓度范围内，盐酸胍主要以疏水作用影响蛋白质的保留；而在高浓度区域内，盐酸胍的存在则显著影响蛋白质分子的构象，使其保留行为发生变化。     

Thermophysical properties of pure 1-ethyl-3-methylimidazolium methylsulphate and its binary mixtures with alcohols

The density and surface tension of 1-ethyl-3-methylimidazolium methylsulphate, [EMIM][CH₃SO₄] ionic liquid have been measured from (283.15 to 333.15) K. The coefficient of thermal expansion was calculated from the experimental density results using an empirical correlation for T = (283.15-338.15) K. Molecular volume and standard entropies of [EMIM][CH₃SO₄] ionic liquid were obtained from the experimental density values. The surface properties, critical temperature and enthalpy of vaporization were also discussed. Density and surface tension have been measured over the whole composition range for [EMIM][CH₃SO₄] with alcohols (methanol, ethanol, 1-butanol) binary systems at 298.15 K and atmospheric pressure. Excess molar volumes and surface tension deviations for the binary systems have been calculated and were fitted to a Redlich-Kister equation to determine the fitting parameters and the root mean square deviations.     

Extraction of humic acids and their fractions in poly(ethylene glycol)-based aqueous biphasic systems

The possibility of extraction and fractionation of humic acid (HA) in the aqueous biphasic systems (ABS) was shown for the first time. The 10% PEG-10% (NH₄)₂SO₄-H₂O and 5% PEG-7.5% dextran (or dextran sulphate, sodium salt)-H₂O systems were used. HA originated from peat, soddy-podzolic soil and chernozem were studied. The distribution coefficients were measured for HA of different origin, size of the molecules, molecular weights (MW) of the polymers and pH of the system. The PEG-(NH₄)₂SO₄-H₂O system was found to be better for preconcentration and isolation of HA, whereas the PEG-dextran-H₂O system is preferable for HA fractionation. The extractability of HA in ABS increases with increasing the MW of HA molecules. Peat HA are extracted in ABS with higher distribution coefficients compared with chernozem and soddy-podzolic soil HA. It is consistent with higher hydrophobicity of peat HA revealed by hydrophobic interaction chromatography. ABS are promising for HA separation into fractions that differ in their hydrophobic/hydrophilic properties as well as for comparing HA of different origin by their relative hydrophobicity.     

Intramolecular Hydrogen Bonding Effects on the Reversed-Phase Retention of Substituted Acetophenones

Abstract

Reversed-phase 1 iquid chromatography (RPLC) is widely recognized as a valuable technique for the separation of compounds of varying 1 ipophil ic/hydrophobic nature. RPLC on hydrocarbon stationary phases has been used for the separation of homologues of various compound classes with outstanding success (1–3). Excellent correlations have been obtained between RPLC capacity factors and various parameters of the solvophobic theory (4–6). This theory suggests that solute-solvent interactions assume the primary role in the RPLC retention process (7–9). The creation of a suitable cavity in the mobile phase is a key factor affecting the retention of the solute. The size and shape of the cavity formed in the solvent depends on solute molecular volume and hydrophobic surface area as well as the dielective constant and surface tension of the solvent. Relationships between RPLC capacity factors, partition coefficients (10,ll) and biological activities (12) have been reported. These high correlations suggest RPLC can be a very useful tool for the study of molecular phenomena in solution.     

Identification of nitrates and sulphates with dynamic SIMS

Summary Sputter conditions are outlined for the identification of chemically sensitive salt compounds, such as nitrates or sulphates, in multicomponent samples of environmental origin using dynamic SIMS for depth-profiling with nanoscale resolution. Sputtering with 1 keV Xe⁺ has been found to be appropriate to enable both the emission of decisive molecular ions with enough intensity as well as substantial erosion for depth-profiling. The use of heavy projectiles reduces the destruction of chemical compounds in the surface of the solid and enhances sensitivity and identification power of SIMS. The method was applied to the analysis of urban outdoor aerosol particles to investigate the conversion of NaCl into Na₂SO₄ or NaNO₃ by the interaction of sea salt aerosol with the atmospheric pollutants NO_x and SO_x. Only NaNO₃ was found in the sea salt fraction.     

A new kind of “thiophilic” electron-donor-acceptor adsorbent

Four simple agarose derivatives have been synthesized: 3-(Z-pyridylsulfido)-2-hydroxypropyl- 3-(2-pyridyloxy)-2-hydroxypropyl- 3-(phenylsulfido)-2-hydroxypropyl-, and 3-(phenyloxy)-2-hydroxypropylagarose. Their affinities for serum proteins have been studied in the presence and absence of water-structuring salt. About 50% of the proteins, among them immunoglobulins and α₂-macroglobulin, but not albumin, were non-covalently adsorbed to the PyS-gel in buffer containing 0.5 M K₂SO₄. These proteins are the same as those adsorbed to the so-called thiophilic gel, T-gel (5), but there are some additional proteins found in the adsorbate that strongly indicate a specific influence on the protein adsorption exerted by the π-electron system of the pyridine residue. The thio-ether sulfur appears to be a necessary structural requirement for the characteristic adsorption behaviour. Thiophilic vs. hydrophobic ligand-protein interaction is discussed.     

Self-aggregation of nonionic surfactants in imidazolium-based ionic liquids with trifluoromethanesulfonate anion

Self-aggregation of polyoxyethylene (POE)-type nonionic surfactants in ionic liquids, 1-butyl- and 1-ethyl-3-methylimidazolium trifluoromethanesulfonate (bmimCF₃SO₃ and emimCF₃SO₃), was investigated by means of ¹H-NMR chemical shift, dynamic light-scattering (DLS), and surface tension measurements. The surfactants showed no definite aggregate formation in bmimCF₃SO₃. This shows a remarkable contrast to the previous observation in bmimBF₄ and bmimPF₆, and demonstrates an importance of anion species to determine the property of ionic liquids as a solvent to support the self-assembly of amphiphilic compounds. On the other hand, the surfactants formed micelles in emimCF₃SO₃, which shows an importance of alkyl chain attached to imidazolium ring to determine the solvophobic interaction between surfactant hydrocarbon chains in imidazolium-based ionic liquids. The low solvophobicity of the surfactants to the ionic liquid composed of imidazolium cation with long alkyl chain is attributed to an affinity of the surfactant hydrocarbon chain to the imidazolium alkyl chain. The values of micellization parameters and surface adsorption parameters obtained for the surfactant solutions in emimCF₃SO₃ are reported.     

Application of aqueous mixtures of polypropylene glycol or polyethylene glycol with salts in proteomic analysis

Partitioning of Bovine serum albumin (BSA), β-lactoglobulin (β-LG) and zein as model proteins in aqueous two-phase systems (S) containing polypropylene glycol (PPG425) or polyethylene glycol (PEG 6000) and salts (MgSO₄, (NH₄)₂SO₄, Na₂SO₄) is presented in this paper. The effects of different factors such as tie-line length, salt type and polymer type on the partition coefficient and recovery percent of proteins were analysed. The model proteins were separated by these systems (S) and directly used for gel electrophoresis without separating the target proteins from phase-forming reagents. The results revealed that the S, studied in this work could be used as a novel prefractionation method in proteomic analysis and could separate proteomic proteins in multigroup by one step extraction.     

Aqueous biphasic systems formed by nonionic polymers I. Effects of inorganic salts on phase separation

The effect of salts KSCN, KI, KBr, KCl, KClO₄, KF, K₂SO₄ and NH₄Cl, LiCl, NaCl, KCl, CsCl on the binodials of the phase diagrams for aqueous biphasic dextranpolyvinylpyrrolidone, dextran-polyvinyl alcohol, dextran-ficoll and dextran-polyethylene glycol systems was studied. It is established that the K-salts present at the concentrations of 0.1 and 0.5 mol/kg alter the binodials of the phase diagrams for the above systems. The effect of a salt is found to be related to the lyotropy of the salt quantified by the salt molal surface tension increment. It is assumed that phase separation in an aqueous polymer biphasic system is affected by the presence of a salt mainly due to the effect of the salt on the structure and/or state of water in the system.     

Interaction Between Two Similar Plane Parallel Double Layers for (NH4)2Fe(SO4)2 or (NH4)2Cu(SO4)2 Type Complex Salt Electrolytes at Positive Surface Potential

Interaction energies between two similar plane parallel double layers for (NH₄)₂Fe(SO₄)₂ or (NH₄)₂Cu(SO₄)₂ type complex salt electrolytes at positive surface potential were expanded in a power series and accurate numeral results were given for 0.1 ≤ y _e  < y ₀ ≤ 20. The general expressions were given for the interaction energies of A _ν +B _ν′ +C_ν? type complex salt electrolytes at y > 0. The interaction energies for simple salts NaCl, CaCl₂, Na₂SO₄, FeCl₃, Na₃PO₄, Mg₃(PO₄)₂, Al₂(SO₄)₃, and complex salts (NH₄)₂Fe(SO₄)₂ or (NH₄)₂Cu(SO₄)₂ at y ₀ = 1 were compared. There was hardly difference between these simple salts and this complex salt for the interaction energies. The interaction energy for complex salt (NH₄)₂Fe(SO₄)₂ was close to that for simple salt Na₃PO₄.

Supplemental files are available for this article. Go to the publisher's online edition of the Journal of Dispersion Science and Technology to view the free supplemental file.     

Hydrophobic interaction chromatography of proteins. I. The effects of protein and adsorbent properties on retention and recovery

The contributions of protein and adsorbent properties to retention and recovery were examined for hydrophobic interaction chromatography (HIC) using eight commercially available phenyl media and five model proteins (ribonuclease A, lysozyme, alpha-lactalbumin, ovalbumin and BSA). The physical properties of the adsorbents were determined by inverse size exclusion chromatography (ISEC). The adsorbents examined differ from each other in terms of base matrix, ligand density, porosity, mean pore radius, pore size distribution (PSD) and phase ratio, allowing systematic studies to understand how these properties affect protein retention and recovery in HIC media. The proteins differ in such properties as adiabatic compressibility and molecular mass. The retention factors of the proteins in the media were determined by isocratic elution. The results show a very clear trend in that proteins with high adiabatic compressibility (higher flexibility) were more strongly retained. For proteins with similar adiabatic compressibilities, those with higher molecular mass showed stronger retention in Sepharose media, but this trend was not observed in adsorbents with polymethacrylate and polystyrene divinylbenzene base matrices. This observation could be related to protein recovery, which was sensitive to protein flexibility, molecular size, and conformation as well as the ligand densities and base matrices of the adsorbents. Low protein recovery during isocratic elution could affect the interpretation of protein selectivity results in HIC media. The retention data were fitted to a previously published retention model based on the preferential interaction theory, in terms of which retention is driven by release of water molecules and ions upon protein-adsorbent interaction. The calculated number of water molecules released was found to be statistically independent of protein retention strength and adsorbent and protein properties.     

Composite Water Sorbents of the Salt in Silica Gel Pores Type: The Effect of the Interaction between the Salt and the Silica Gel Surface on the Chemical and Phase Compositions and Sorption Properties

The influence of the inorganic salt-silica gel surface interaction on the chemical and phase compositions and sorption properties of composites of the salt in silica gel pores type is studied. Two possible interaction mechanisms are considered: (1) the ion-exchange adsorption of metal cations on the silica gel surface from a solution of a salt (CaCl₂, CuSO₄, MgSO₄, Na₂SO₄, and LiBr) and (2) the solid-phase spreading of a salt (CaCl₂) over the silica gel surface. The adsorption of metal cations on the silica gel surface in the impregnation step affords ≡Si-OM ⁿ⁺¹ surface complexes in the composites. As a result, two salt phases are formed in silica gel pores at the composite drying stage, namely, an amorphous phase on the surface and a crystalline phase in the bulk. The sorption equilibrium between the CaCl₂/SiO₂ system and water vapor depends on the ratio of the crystalline phase to the amorphous phase in the composite.     

Synergistic improvement of foam stability with SiO2 nanoparticles (SiO2-NPs) and different surfactants

Foam fluids are widely used in petroleum engineering, but long-standing foam stability problems have limited the effectiveness of their use. The study explores the synergistic effects and influencing factors of SiO₂ nanoparticles (SiO₂-NPs) with different wettability properties and three different surfactants. The paper investigates the foaming performance of different types of surfactants and analyzes and compares the stability of foam after adding hydrophilic and hydrophobic SiO₂-NPs from macroscopic as well as microscopic perspectives, and the effects of temperature and inorganic salts on the stability of mixed solutions. The experimental results show that: 1) hydrophilic nanoparticles can significantly enhance the foam stability of amphoteric surfactants, with a small increase in the foam stability of anionic and cationic surfactants; 2) The concentration of nanoparticles did not have a significant effect on the stability of the cationic surfactants and this conclusion was verified in the experimental results of the surface tension measured below;3) The cationic surfactants showed better temperature resistance at temperatures of 50–90 °C. Both amphoteric surfactant solutions with the addition of hydrophilic SiO₂-NPs or hydrophobic SiO₂-NPs significantly improved the temperature resistance of the foam at high temperatures. The anionic surfactant solution with hydrophobic SiO₂-NPs did not enhance the solution temperature resistance; 4) The surface tension of the surfactant solution gradually increases with increasing concentration of hydrophilic or hydrophobic SiO₂-NPs and then levels off; 5) the hydrophilic SiO₂-NPs had a significant effect on the salt tolerance of the anionic and amphoteric surfactant solutions. The salt tolerance of cationic surfactant solutions with hydrophobic SiO₂-NPs was better than that of surfactants with hydrophilic SiO₂-NPs.     

Mechanism of electroless deposition of Ni–W–P alloys by adding surfactants

This paper describes an electroless deposition method for the formation of a thin metallic film containing mainly nickel with significant amounts of tungsten (up to 25%) and phosphorus (5–10%). The film was deposited from an aqueous electrolyte that contained sodium tungstate as a source of tungsten, nickel sulphate as a source of nickel and hypophosphite as the reducing agent and a source of phosphorus. The surfactants were p‐hexyloxy‐p‐sodium sulphonate azobenzene (HSA) with the formula H₁₃C₆OC₆H₄N₂ C₆H₄SO₃Na and p‐hexylbenzyltriethanol ammonium chloride (HBC) with the formula H₁₃C₆H₄CH₂N⁺ (C₂H₄OH)₃Cl^?, added as stabilizers. In this study the process parameters of typical solutions, such as temperature, pH and concentration of tungstate salt and the concentration of different surfactants, were presented and discussed. Adsorption of the surfactants on a metal surface was dependent, among other things, on the structure of their hydrophobic and hydrophilic portions. The effect of adsorption of these surfactants on a metal surface was examined above and below the critical micelle concentration (CMC). The deposition process involves several reactions that occur simultaneously and are described in detail in this work. The mechanism for interaction of the surfactants with the steel surface was proposed through the isotherm for adsorption from aqueous solution. Furthermore, the surface properties of the surfactants were measured, particularly the CMC, the surface tension reduction and the maximum surface excess Γ_max. The tungsten percentage in the deposit layer was strongly influenced by the plating conditions and the critical concentration of each surfactant. The results were discussed according to the surface properties of the additive. The thin film of Ni–W–P achieved high crystal refinement and high hardness, it was smooth and uniform and it exhibited superior corrosion resistance. Copyright © 2003 John Wiley & Sons, Ltd.     

Effect of surface hydrophobicity distribution on retention of ribonucleases in hydrophobic interaction chromatography

The effect of surface hydrophobicity distribution of proteins on retention in hydrophobic interaction chromatography (HIC) was investigated. Average surface hydrophobicity as well as hydrophobic contact area between protein and matrix were estimated using a classical thermodynamic model. The applicability of the model to predict protein retention in HIC was investigated on ribonucleases with similar average surface hydrophobicity but different surface hydrophobicity distribution. It was shown experimentally that surface hydrophobicity distribution could have an important effect on protein retention in HIC. The parameter "hydrophobic contact area," which comes from the thermodynamic model, was able to represent well the protein retention in HIC with salt gradient elution. Location and size of the hydrophobic patches can therefore have an important effect on protein retention in HIC, and the hydrophobic contact area adequately describes this.     

Surface and Thermodynamic Parameters of Polymeric Surfactants from Recycled Poly(ethylene terephthalate)

Recycling of PET waste was carried out in presence of diethanolamine and manganese acetate as catalyst. The produced oligomers were reacted with stearic acid and polyethylene glycol, PEG, which have different molecular weights 400, 1,000, and 4,000 to produce nonionic polymeric surfactants having different hydrophilic‐hydrophobic balance (HLB). The surface tension, critical micelle concentration (CMC), and surface activities were determined at different temperatures. Surface parameters such as surface excess concentration Γ_max, the area per molecule at interface A_min and the effectiveness of surface tension reduction (Π_CMC) were determined from the adsorption isotherms of the prepared surfactants. From the adsorption isotherm, some thermodynamic data for the adsorption process are calculated and discussed.     

Effects of Immobilized Metal Ion on Retention Behaviors of Proteins in Metal Chelate Affinity Chromatography

The chromatographic behaviors of proteins on iminodiacetic acid (IDA) column with and without immobilized metal ion were examined in detail. Comparing the effects of pI, solution pH, and salt concentration on retention of proteins in cation‐exchange chromatography (CEC) and metal chelate affinity chromatography (MCAC), the retention mechanism of proteins was investigated in MCAC. By aid of observing the retention characteristics of proteins on naked IDA and metal chelate columns in high concentration salt‐out salt solution, the hydrophobic interaction in MCAC and the influence of metal ion on it were proved. In terms of the comparison of the thermodynamics of proteins in CEC and MCAC, the thermostability, the conformational change entropy Δ(ΔS⁰) and enthalpy Δ(ΔH⁰), compensation temperature β, the driving force and caloritic effect of proteins in MCAC were discussed. The identity of retention mechanism at protein thermal denaturation in CEC and MCAC was demonstrated by using the compensation relationship between ΔH⁰ and ΔS⁰.