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.     

Micellar proportion: a parameter to compare the hydrophobicity of the pseudostationary phases or that of the analytes in micellar electrokinetic chromatography

Micellar proportion, t(prop,mic) = t(mic)/t(m), a quantity expressing how much time is spent by the analyte in the micellar phase related to its whole migration time (t(m)) has been introduced by utilizing the micellar phase residence time (t(mic)). The t(prop,mic) values have been determined for analytes of different chemical structures (alkyl benzene and alkyl phenone homologous series, alcohols, strongly hydrophobic peptides) studied by micellar elektrokinetic chromatography (MEKC) using various cationic and anionic pseudostationary phases. A good linear correlation was obtained between t(prop,mic) and the calculated hydrophobicity (CLOGP) of the analytes for all pseudostationary phases (CLOGP = A.logt(prop,mic) + B). Considering a given pseudostationary phase, t(prop,mic) as a relative quantity is a suitable parameter to characterize and compare experimentally the behavior of the various analytes in MEKC. Applying a set of probe molecules with known hydrophobicity, the CLOGP(50) value (showing the value of hydrophobicity of a virtual molecule spending exactly 50% of its migration time in the pseudostationary phase) has been calculated for each pseudostationary phase applied here. This experimentally determinable numerical value (characterizing the pseudostationary phase) can be utilized to compare the hydrophobicity and hence retention ability of the pseudostationary phases. The t(prop,mic) value was found to be applicable to compare the methylene selectivity of the different pseudostationary phases as well: logt(prop,mic) = A.Z + B, where Z is the number of carbon atoms of the alkyl chain in the alkyl benzene homologous series.     

Ion-interaction CZE: the presence of high concentrations of ion-pairing reagents demonstrates the complex mechanisms involved in peptide separations

We have furthered our understanding of the separative mechanism of a novel CE approach, termed ion-interaction CZE (II-CZE), developed in our laboratory for the resolution of mixtures of cationic peptides. Thus, II-CZE and RP-HPLC were applied to the separation of peptides differing by a single amino acid substitution in 10- and 12-residue synthetic model peptide sequences. Substitutions differed by a wide range of properties or side-chain type (e.g., alkyl side-chains, polar side-chains, etc.) at the substitution site. When carried out in high concentrations (400 mM) of pentafluoropropionic acid (PFPA), II-CZE separated peptides in order of increasing hydrophobicity when the substituted side-chains were of a similar type; when II-CZE was applied to the mixtures of peptides with substitutions of side-chains that differed in the type of functional group, there was no longer a correlation of electrophoretic mobility in II-CZE with relative peptide hydrophobicity, suggesting that a third factor is involved in the separative mechanism beyond charge and hydrophobicity. Interestingly, the hydrophobic PFPA- anion is best for separating peptides that differ in hydrophobicity with hydrophobic side-chains but high concentrations of the hydrophilic H2PO4- anion are best when separating peptides that differ in polar side-chains relative to hydrophobic side-chains. We speculate that differential hydration/dehydration properties of various side-chains in the peptide and the hydration/dehydration properties of the hydrophilic/hydrophobic anions as well as the electrostatic attractions between the peptide and the anions in solution all play a critical role in these solution-based effects.     

Mixed micelles of series of monomeric and dimeric cationic, zwitterionic, and unequal twin-tail cationic surfactants with sugar surfactants: a fluorescence study

Steady state fluorescence measurements have been carried out for binary mixtures of a series of monomeric cationic (MC), zwitterionic (ZI), dimeric cationic (DC), and twin-tail cationic (TC) surfactants with sugar (beta-C8G and beta-C12G) over the whole mole fraction range using pyrene as fluorescence probe. The cmc values thus determined for all the binary mixtures have been further evaluated using the regular solution theory. The various micellar parameters, such as micelle mole fraction (X1), regular solution interaction parameter (beta), micropolarity, and mean micelle aggregation number (Nagg), have been determined for all these series of mixtures. Variation in all these micellar parameters demonstrates that mixed micelles of these surfactants with beta-C8G are mostly synergistic in nature and the synergism increases with the increase in hydrophobicity of the cosurfactant in each case. The mixtures of beta-C12G with various cosurfactants do not show this behavior and instead of it, they show an increase in antagonism with the increase in hydrophobicity of cosurfactants. This discrepancy has been attributed to a large difference in hydrophobicity between beta-C8G and beta-C12G, and the chain folding of the latter is considered to be the reason for the antagonism.     

Amphiphilic derivatives of poly(acrylic acid) as stabilizer in emulsion polymerisation

Poly(acrylic acid)s of various molecular weights and different degree of hydrophobic modification have been synthesized by a polymer analogous reaction with tetradecylamine or copolymerization with styrene. Testing these amphiphilic polymers as stabilizers for the emulsion polymerization of styrene reveals that well stabilized latices are obtained within a certain range of molecular weight and hydrophobicity. As an example, the addition of 1 wt.-% polymer with respect to the monomer allows the synthesis of polystyrene latices with a radius of about 100 nm in the absence of ionic initiators or polar comonomers.     

Structural information of mussel adhesive protein Mefp-3 acquired at various polymer/Mefp-3 solution interfaces

Mytilus edulis foot protein Mefp-3 serves as a primer in the formation of adhesive plaques that attach the mussel to solid surfaces in its immediate environment. The adsorption behavior of this protein on various materials of different hydrophobicity was studied using sum frequency generation (SFG) vibrational spectroscopy. By collecting SFG signals from side chains of these amino acids and from secondary structures of the protein, we have determined that this protein adopts different conformations at different interfaces, depending on hydrophobicity of the contact medium and specific chemical group interactions. We have also demonstrated that SFG has the potential to track the interfacial conformations of a single amino acid in a protein.     

Covalently linked, water-dispersible, cyclodextrin: reduced-graphene oxide sheets

Reduced-graphene oxide (rGO) sheets have been functionalized by covalently linking β-cyclodextrin (β-CD) cavities to the sheets via an amide linkage. The functionalized β-CD:rGO sheets, in contrast to rGO, are dispersible over a wide range of pH values (2-13). Zeta potential measurements indicate that there is more than one factor responsible for the dispersibility. We show here that planar aromatic molecules adsorbed on the rGO sheet as well as nonplanar molecules included in the tethered β-CD cavities have their fluorescence effectively quenched by the β-CD:rGO sheets. The β-CD:rGO sheets combine the hydrophobicity associated with rGO along with the hydrophobicity of the cyclodextrin cavities in a single water-dispersible material.     

Critical factors for high-performance physically adsorbed (dynamic) polymeric wall coatings for capillary electrophoresis of DNA

Physically adsorbed (dynamic) polymeric wall coatings for microchannel electrophoresis have distinct advantages over covalently linked coatings. In order to determine the critical factors that control the formation of dynamic wall coatings, we have created a set of model polymers and copolymers based on N,N-dimethylacrylamide (DMA) and N,N-diethylacrylamide (DEA), and studied their adsorption behavior from aqueous solution as well as their performance for microchannel electrophoresis of DNA. This study is revealing in terms of the polymer properties that help create an "ideal" wall coating. Our measurements indicate that the chemical nature of the coating polymer strongly impacts its electroosmotic flow (EOF) suppression capabilities. Additionally, we find that a critical polymer chain length is required for polymers of this type to perform effectively as microchannel wall coatings. The effective mobilities of double-stranded (dsDNA) fragments within dynamically coated capillaries were determined in order to correlate polymer hydrophobicity with separation performance. Even for dsDNA, which is not expected to be a strongly adsorbing analyte, wall coating hydrophobicity has a deleterious influence on separation performance.     

Reversed-phase high-performance liquid chromatography of some ultra-heterogeneous and covalently-modified proteins from human hair

A new procedure for the fractionation of the heterogeneous cystine-rich proteins from human hair, utilizing reversed-phase high-performance liquid chromatography, is described. Of these proteins 27 fractions have been collected and analyzed for amino acid composition. There seems to be little correlation between the elution order and the hydrophobicity of the fraction constituents except for the late-eluting fractions. Based on the elution profiles and amino acid contents, these fractions appear to fall into four families. The effects of alkyl chain length, flow-rate and gradient slope, as well as various additives to the organic modifier on the separation have also been investigated. A low flow-rate (0.4 ml/min) and a shallow gradient were essential for the separation of these proteins as was the use of short alkyl chain (C4) or medium alkyl chain (C8) columns. However, with the C4 column reproducibility and recovery were excellent.     

Anion-exchange chromatography of metal cyanide complexes with gradient separation and direct UV detection

In the regulatory analysis of water samples, cyanide content is usually expressed in various forms as free cyanide, total cyanide, weak-acid dissociable cyanide, available cyanide, and cyanide amenable to chlorination. Concentration of individual metal cyanide complex, not furnished in any of these methods, is useful in meaningful assessment of toxicity due to cyanide. In the present work, two macroporous anion-exchange columns, having high and intermediate hydrophobicity, were evaluated to achieve separation of cyano complexes of silver, iron, gold, copper, nickel, and cobalt. On the QS-Al SC column, of high hydrophobicity, the monovalent cyano complexes of silver and gold eluted last while the multivalent cyano complexes, e.g. iron and copper, eluted early. It is suggested that the retention order on this column is due to relative hydrophobicity of the metal cyanide complex, and its affect on ion exchange. In contrast, on the QS-A2 SC column of intermediate hydrophobicity, with the exception of cyano complex with Fe, the separation of the cyano complexes of five other metals closely followed an anion-exchange mechanism. Under gradient conditions, the six metal cyanide complexes were well resolved on the QS-A2 SC column and the method with direct UV detection at 215 nm was accurate (spike recovery of 99.8-118.8%) and precise (RSD of 1.0-2.6%).     

Enantioselective synthesis of tetrafluorinated glucose and galactose

Polyfluorinated carbohydrates have emerged as interesting probes to investigate "polar hydrophobicity" effect(s) in protein-carbohydrate interactions. A convenient enantioselective synthesis of tetrafluorinated analogues of two of the most important monosaccharides, D-glucose and D-galactose, is reported, as well as our first results regarding the glycosylation of these sugar analogues.     

Nano-colloidal functionalization of textiles based on polysiloxane as a novel photo-catalyst assistant: processing design

Due to the opposite surface charge of TiO(2) and silver nano-particles, at around neutral pH, it is expected that the interaction between these particles and cross-linkable polysiloxane (XPs) resin and thus their final properties would be affected by their processing technique. This paper has focused on the effect of processing design on the interaction, surface orientation and final properties of surface nano-colloidal functionalization. The results disclosed the key role of the applied process on the properties of the treated fabrics which have been well discussed through the modeling of this effect on orientations of nanoparticles on the surface. The developed models are interestingly verified by various characterizations. Applying a premixed TiO(2)/XPs colloid as an after treatment on Ag treated samples caused more enhanced stain photo-degradability and UV protection properties, while the reduction of enhanced hydrophobicity, washing durability, and stain-repellency were observed as compared to applying Ag/XPs premixed colloid on TiO(2). The role of processing on XPs stabilizing efficiency and its co-photo-catalytic function on TiO(2) nanoparticles has been concluded and deeply discussed. The appropriate processing design can be tailored in order to accomplish desirable hydrophilicity/hydrophobicity with a granted bioactivity. The results reveal that ideal bioactivity, stain photo-degradability, self-cleaning, UV protection, anti-staining properties, and washing durability can be achieved by applying a mixture of silver and XPs as an after-treatment on TiO(2) treated fabrics.     

Adsorption behavior of nicotine on periodic mesoporous organosilicas

In this study, we focused on the adsorption of nicotine from aqueous solution such as water and simulated body fluids (SBFs), where SBF has ion concentrations approximately equal to those of human blood plasma. We prepared periodic mesoporous organosilica (PMO) materials as adsorbents from 4,4-bis(triethoxysilyl)biphenyl (BTES-biphenyl), 1,4-bis(triethoxysilyl)benzene (BTES-benzene) and bis[3-(trimethoxy silyl)propyl]amine (BTMS-amine) as precursors and investigated on their adsorption behavior of nicotine as a guest material under different solvent conditions. For this work, two different kinds of SBF, c-SBF and r-SBF, have been chosen, where c-SBF is a transitional SBF solution, and r-SBF is a modified SBF solution that is closer to human blood plasma. Adsorption of nicotine on PMOs has been characterized by a UV-Vis spectroscopy. The adsorption behavior was strongly dependent on the isoelectric point and hydrophobicity of the PMO as well as the hydrophobicity of nicotine.     

Hydrophobicityand collectorless flotation of inorganic materials

Hydrophobicity and floatability of solids have been analyzed from the standpoint of properties of solid-water and solid-water vapors interfaces, chemical bonds, bulk properties, crystal structure of the solid, and reactivity of the solid with water. Although the hydrophobicity results from complex interactions in the solid-water-air system, simple equations and rules for predicting hydrophobicity and floatability are presented. The applicability of the Gaudin-Miaw-Spedden theory which states that molecular and sheet crystals, if their structure is controlled by the residual bonds across their basal planes, are floatable was confirmed. It was also shown that elements and compounds with different degrees of ionic-covalent and metallic-nonmetallic characters of bonds in the absence of residual bonds can be either hydrophilic, hydrophobic, or change their properties from hydrophobic to hydrophilic and vice versa. For some materials, hydrophobidty was found to be time-dependent. Decreasing hydrophobicity occurs with the oxidation and hydroxylation of the surface (oxides, metals), while increasing hydrophobicity takes place due to non-dissociative adsorption of water vapors on the surface (noble metals). Increased hydrophobicity can also be due to the formation of hydrophobic species such as sulfur species on the surface of Sulfides. It was demonstrated that the potential hydrophobicity of solids, expressed as the contact angle formed between the three involved (solid, water, and air) phases, can be evaluated from the Hamaker constants.This work supplements the Gaudin-Miaw-Spedden theory by showing that not only molecular crystals (paraffin, I₂, S₈, As₄O₆, As₂S₂) and non-ionic sheet crystals (MoS₂, Sb₂S₃, talc, graphite, As₂S₃, boric acid, BN) but also elements and crystalline compounds without residual bonds can be hydrophobic and floatable. A partial list of such materials includes Hg, Ge, Si, SiC, AgI, CaF₂, and diamond (whose hydrophobidties are already well known) as well as BaSO₄, FeTiO₃, In, and Sn (whose hydrophobidties have been established in this work). It was also demonstrated that the hydrophobidty of some solids changes as a result of reaction of the surface with constituents of the air.     

Development and characterization of poly ethyl metha acrylate–iron oxide(III) based hydrophobic liquid nanocomposite films

Select applications of hydrophobic nanocomposites include preparation of robust self-cleaning surfaces, water-repellent glass surfaces, and waterproofing textiles. Various nanocomposites have been reported in the literature; however, the relationship between the nanocomposite surface morphology and its hydrophobicity needs to be understood better. In the present work Fe₂O₃ nanoparticles and poly ethyl metha acrylate (PEMA) were used in varying proportions to obtain a series of model hydrophobic surfaces (spin-coated on glass substrate). The hydrophobicity of these surfaces was measured by static contact angle; a maximum of 103° was obtained at highest loading of iron oxide nanoparticles. These surfaces were also characterized using AFM. The contact angle and characterization data were used to test some of the models which have been proposed in the recent literature on prediction of contact angle for composite surfaces. It is proposed that the hydrophobicity of the iron oxide–PEMA surface is due to the physical roughness causing air entrapment as well as the chemical heterogeneity. Based on the experimental studies and the simulations using the recent models on contact angle, some general features of relationship between a composite surface morphology and its hydrophobicity is proposed.     

Copolymerized polymeric surfactants: characterization and application in micellar electrokinetic chromatography

An achiral monomeric surfactant (sodium 10-undecenyl sulfate, SUS) and a chiral surfactant (sodium 10-undecenoyl L-leucinate, SUL) were synthesized and polymerized individually to form poly-SUS and poly-SUL. These surfactants were then copolymerized at various molar ratios to produce a variety of copolymerized surfactants (CoPSs), possessing both achiral (sulfate) and chiral (leucinate) head groups. The CoPSs, poly-SUS, poly-SUL, and sodium dodecyl sulfate were characterized using several analytical techniques. The aggregation numbers of the polymeric surfactants and the partial specific volumes were determined by the use of fluorescence quenching and density measurements, respectively. These polymeric surfactants were investigated as novel pseudostationary phases in micellar electrokinetic chromatography (MEKC) for the separation of chiral and achiral solutes. Solute hydrophobicity was found to have major influence on the MEKC retention of alkyl phenyl ketones. In contrast, hydrogen-bonding ability of benzodiazepines is the major factor that governs their retention, but hydrophobicity has an insignificant effect on MEKC retention of benzodiazepines.     

Plasma surface functionalization of poly[bis(2,2,2-trifluoroethoxy)phosphazene] films and nanofibers

Polyphosphazenes are a class of hybrid organic-inorganic macromolecules with high thermo-oxidative stability and good solubility in many solvents. Fluoroalkoxy phosphazene polymers also have high surface hydrophobicity. A method is described to tune this surface property while maintaining the advantageous bulk materials characteristics. The polyphosphazene single-substituent polymer, poly[bis(2,2,2-trifluoroethoxy)phosphazene], with flat film, fiber mat, or bead mat morphology was surface functionalized using an atmospheric plasma treatment with oxygen, nitrogen, methane, or tetrafluoromethane/hydrogen gases. Surface chemistry changes were detected by static water contact angle (WCA) measurements as well as X-ray photon spectroscopy (XPS). It was found that changes in the WCA of as much as 150 degrees occurred, accompanied by shifts in the ratio of elements on the polymer surface as detected by XPS. Overall this plasma technique provides a convenient method for the generation of specific surface characteristics while maintaining the hydrophobicity of the bulk material.     

Phase equilibrium and insulin partitioning in aqueous two-phase systems containing block copolymers and potassium phosphate

In this work, phase diagrams of aqueous two-phase systems (ATPS) containing PEO–PPO–PEO block copolymers and potassium phosphate as well as the partitioning behavior of insulin in these systems are presented. Experiments aimed at the identification of the effects of copolymer structure (by varying the number of EO units per polymer molecule), temperature (283.15 and 298.15 K) and pH (5.0 and 7.0) on the phase behavior of these systems were carried out. The results indicated the enlargement of the two-phase region upon increasing the temperature, pH and copolymer hydrophobicity (expressed as the PO/EO ratio in the copolymer molecule). Experimental measurements of the partitioning of human insulin in these ATPS also indicated the dependency of the partition coefficients on temperature, pH, and copolymer hydrophobicity, with the latter being the most influential factor. Finally, experimental data on the phase behavior and insulin partitioning were correlated using an excess Gibbs energy virial-type model modified in order to account for coulombic interactions and ionization equilibrium between the various forms of the phosphate ion.