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• A mass-based total pore blocking method is described for porosity measurements. • Porosity measurements of superficially porous particles are performed. • The Young-Laplace equation is used to describe the pore blocking stability. • Calculations suggest that larger pores can be blocked at extremely low flow rates. Experimentally determined total, interstitial and intraparticle porosity values are necessary to equate theory, simulation and experimental column performance. This paper reports a study of a mass-based technique for determining total, interstitial and intraparticle porosity measurements based on the total pore-blocking (TPB) method. Commercially available superficially porous particle (SPP) columns, in a variety of small-pore and wide-pore materials, with both hydrophobic and hydrophilic surfaces, are utilized as samples. The results are compared with previously determined literature values for a number of columns and contrasted with HPLC-based elution methods. This method uses only a high-precision balance and an HPLC pump. A simple theoretical analysis of the TPB method using the Young-Laplace equation shows the pressure bounds and flow rate constraints of the method which ensure pore blocking stability. The results suggest that particles with small-pore diameters can be analyzed over a range of solvent clearing pressures and flow rates. However, wide-pore materials, typically with pore diameters in excess of 400 Å, have very low critical pressures and are difficult to determine without losing the pore blocking component. Small mass differences between clearing solvents are shown to present a challenge for measuring the interstitial volume. [ABSTRACT FROM AUTHOR]

Abstract: Strong cation exchange (SCX) mode, an alternative to reversed-phase (RP) mode, was described in this paper to obtain symmetrical peak shape and high sample loading for the separation of basic compounds. The retention mechanisms on SCX modified silica sorbents have been demonstrated including electrostatic and hydrophobic interactions. The effects of eluent ionic strength, apparent pH on retention were investigated and the cation-exchange capacity was also characterized. Good efficiency (>56,000platem−1) and excellent peak shape (Tf<1.2) for various basic probes were obtained on the SCX column. Furthermore, good peak shapes and separation can be maintained under high concentration injections, indicating good potential in preparative use. These results were also compared with those obtained on two conventional RP stationary phases. The overall results demonstrated that SCX stationary phases can be used as alternatives to RP stationary phases in the separation of basic compounds, especially in the purification of basic compounds. [Copyright &y& Elsevier]

Abstract: Hydrophilic interaction liquid chromatography (HILIC) represents a modern MS-friendly approach to the analysis of polar compounds. To date especially silica-based HILIC stationary phases are utilized. Recently the papers concerning retention of polar analytes on unmodified titania and zirconia as well as a polybutadiene modified zirconia stationary phase under HILIC conditions have been published. In this work the investigation of a highly hydrophobic carbon-coated zirconia column under HILIC conditions was reported. The influence of buffer concentration, buffer type, pH and temperature on the elution and chromatographic efficiency were studied. The processes participating on the retention of polar compounds showed a multimodal character. The retention was governed rather by surface adsorption than phase partition in high ACN mobile phases. The ligand exchange remained an important retention force although the access of the analytes to the active surface was significantly hindered by the carbon coating. Besides ligand exchange and hydrophilic interactions also the hydrophobic carbon layer was involved in the retention of carboxylic acids and especially xanthines in the organic rich mobile phase. [Copyright &y& Elsevier]

Abstract: Selection of a first-dimensional separation method is very important in proteomic studies performed with two-dimensional liquid chromatography (2D-LC), since reversed-phase chromatography (RPLC) is usually chosen as the second-dimensional separation method in most studies. In this paper, we assess the potential use of mixed-mode reversed-phase/weak cation-exchange (RP/WCX) chromatography in 2D-LC proteomic studies. First, a new RP/WCX mixed-mode stationary phase (named C18WCX) was synthesized based on the polar-copolymerized approach, and a C18WCX column with separation efficiency comparative to conventional C18 columns was developed. This new mixed-mode column primarily provides hydrophobic interactions under acidic condition, but can offer hydrophobic and cation-exchange interactions under neutral and weak basic conditions. An off-line 2D-RP/WCX–RPLC system was established using the C18WCX column with a mobile phase of pH 6.5 in the first dimension and a C18 column with a mobile phase of pH 3.0 in the second dimension. The orthogonality of this two-dimensional system, evaluated through the separation of 123 tryptic peptides, was shown to be higher than that of the conventional 2D-RP–RPLC approach. Applying this 2D-LC method to rat brain samples, we identified 1031 proteins and 4397 unique peptides. In addition, this new two-dimensional method improved the identification of basic peptides. Therefore, we propose that this novel 2D-RP/WCX–RPLC system can be used as an alternative approach for the two-dimensional separation of peptides. [Copyright &y& Elsevier]

Abstract: Aqueous two-phase systems (ATPS) that are formed by mixing a polymer (usually polyethylene glycol, PEG) and a salt (e.g. phosphate, sulphate or citrate) or two polymers and water can be effectively used for the separation and purification of proteins. The partitioning between both phases is dependent on the surface properties of the proteins and on the properties of the two phase system. The mechanism of partitioning is complex and not very easy to predict but, as this review paper shows, some very clear trends can be established. Hydrophobicity is the main determinant in the partitioning of proteins and can be measured in many different ways. The two methods that are more attractive, depending on the ATPS used (PEG/salt, PEG/polymer), are those that consider the 3-D structure and the hydrophobicity of AA on the surface and the one based on precipitation with ammonium sulphate (parameter 1/m*). The effect of charge has a relatively small effect on the partitioning of proteins in PEG/salt systems but is more important in PEG/dextran systems. Protein concentration has an important effect on the partitioning of proteins in ATPS. This depends on the higher levels of solubility of the protein in each of the phases and hence the partitioning observed at low protein concentrations can be very different to that observed at high concentrations. In virtually all cases the partition coefficient is constant at low protein concentration (true partitioning) and changes to a different constant value at a high overall protein concentration. Furthermore, true partitioning behavior, which is independent of the protein concentration, only occurs at relatively low protein concentration. As the concentration of a protein exceeds relatively low values, precipitation at the interface and in suspension can be observed. This protein precipitate is in equilibrium with the protein solubilized in each of the phases. Regarding the effect of protein molecular weight, no clear trend of the effect on partitioning has been found, apart from PEG/dextran systems where proteins with higher molecular weights partitioned more readily to the bottom phase. Bioaffinity has been shown in many cases to have an important effect on the partitioning of proteins. The practical application of ATPS has been demonstrated in many cases including a number of industrial applications with excellent levels of purity and yield. This separation and purification has also been successfully used for the separation of virus and virus-like particles. [Copyright &y& Elsevier]

Abstract: The development of mixed-mode stationary phase to achieve multiple separation capabilities in one column is very important for high performance liquid chromatography. In this paper, a new specific stationary phase based on grafting N-methylimidazolium to a monolithic silica column was successfully prepared for performing capillary liquid chromatography. The characteristics of the column were evaluated by the separation of different types of compounds including inorganic anions, aromatic acids, nucleotides, polycyclic aromatic hydrocarbons, alkylbenzenes, and phenols. The mechanisms for the separation of these compounds were investigated and appeared to involve the mixed interactions including anion-exchange, hydrophilic, π–π, dipole–dipole, and hydrophobic interactions. [Copyright &y& Elsevier]

Abstract: A major challenge in the development of affinity adsorbents is the design of specific adsorbents for target molecules. In this paper, a two-step strategy was used to design a specific adsorbent for oligopeptides. Based on the structural characteristics of target peptide DFLAE (DE5), the affinity ligand CDenHis bearing hydrophobic inclusion and electrostatic interaction sites was prepared by grafting histidine onto β-cyclodextrin (CD) using ethylenediamine; ligands with single hydrophobic inclusion or electrostatic interaction sites (CDen and HisOMe) were used as reference ligands. Results indicated that the binding affinity (K a) of CDenHis with DE5 was 6.23×104 M−1, 23- and 61-fold higher than that of CDen and HisOMe, respectively. Computer simulations were used to further optimize the steric configuration of CDenHis. It was found that the optimized ligand CDdnHis exhibited a much improved binding affinity for DE5 (K a =1.02×105 M−1). Moreover, the corresponding adsorbent A-CDdnHis not only showed much better adsorption ability compared with A-CDenHis, but also excellent adsorption specificity for DE5-containing peptides. Kinetic analysis and adsorption mechanism studies suggested that the configuration matching of CDdnHis with DE5 and the cooperation of multiple interactions led to the fast and selective adsorption of DE5-containing peptides to A-CDdnHis. [Copyright &y& Elsevier]

Abstract: A novel form of reversed-phase liquid chromatography (RPLC) by the dynamically modified hydrophilic interaction monolithic column has been described in this paper. A porous poly(SPMA-co-PETA) monolith with strong cation-exchange (SCX) was prepared and the resulting monolith showed a typical hydrophilic interaction chromatography (HILIC) mechanism at higher organic solvent content (ACN%>50%). The good selectivity for neutral, basic and acidic polar analytes was observed in the HILIC mode. In order to increase the hydrophobic interaction, the monolith with SCX was dynamically modified with a long-chain quaternary ammonium salt, cetyltrimethylammonium bromide (CTAB), which was added to the mobile phase. CTAB ions were adsorbed onto the surface of the SCX monolithic material, and the resulting hydrophobic layer was used as the stationary phase. Using the dynamically modified SCX monolithic column, neutral, basic and acidic hydrophobic analytes were well separated with the RPLC mode. [Copyright &y& Elsevier]

The concept of hydrophobicity has been addressed by researchers in all aspects of science, particularly in the fields of biology and chemistry. Over the past several decades, the study of the hydrophobicity of biomolecules, particularly amino acids has resulted in the development of a variety of hydrophobicity scales. In this review, we discuss the various methods of measuring amino acid hydrophobicity and provide explanations for the wide range of rankings that exist among these published scales. A discussion of the literature on amino acid interactions is also presented. Only a surprisingly small number of papers exist in this rather important area of research; measuring pairwise amino acid interactions will aid in understanding structural aspects of proteins. [Copyright &y& Elsevier]

A preferential interaction quadratic isotherm model for hydrophobic interaction chromatographic systems is presented in this paper. In this isotherm, the nonlinear effect of salt on the capacity factor is described using the preferential interaction model developed by Perkins et al. [J. Chromatogr. A, 766 (1997) 1]. This is then coupled with a quadratic nonlinear isotherm to describe nonlinear adsorption behavior at high solute concentrations. The resulting preferential interaction quadratic isotherm is examined for its ability to describe solute adsorption behavior under both linear and nonlinear conditions over a wide range of salt concentrations in HIC systems. The results indicate that this isotherm is well suited for predicting nonlinear adsorption behavior in HIC systems for both proteins and low-molecular mass HIC displacers. [Copyright &y& Elsevier]

• Theoretical investigations show 2 µm open tube capillaries produce high performance. • The performance is compared with normal, slip-flow boundaries and electrochromatography. • These techniques are suggested to not be worth the effort, compared to gradient elution operation. • The zone standard deviations are shown to be similar for these different flow profiles. • The number of plates for the different flow profiles are also shown to be similar. Modifications to the flow profile used in open tube capillary liquid chromatography (OT-CLC) include using slip-flow walls and using electroosmosis as a fluid pump as practiced in electrochromatography. These modifications are implemented experimentally by changing the capillary surface and solvent conditions which results in the change of boundary conditions at the capillary wall. In this paper we employ a theory-based study and compare the zone broadening of simple solutes using parabolic flow from a liquid pump, slip-flow from a highly hydrophobic inner surface with water eluent, and electroosmosis for the conditions of pure water and dilute salt utilizing 2 µm inner diameter OT capillaries. In general, the two types of flow other than parabolic exhibit thin zones in the early part of the chromatogram, consistent with previous studies of slip-flow and electroosmotic flow used in electrochromatography. Electrochromatography is shown to yield higher efficiency and less zone broadening than parabolic and slip-flow conditions used in this study. Nonetheless, it is found that the zone standard deviations are shown to be similar for these flow profiles as is the number of plates for these different flow profiles under the conditions utilized here. It is revealed that these modifications do not warrant the effort to maintain the special solvent conditions when compared to gradient elution OT-CLC, which gives a nearly constant peak width throughout the chromatogram, is easiest to implement, and is the method of choice for complex analysis. [ABSTRACT FROM AUTHOR]