Adsorption of cations onto positively charged surface mesopores.

Uwe Neue developed a theoretical treatment to account for the adsorption of ions on mesopores of packing materials the walls of which are bonded to ionic ligands but left this work unfinished. We elaborated upon this treatment and refined it, based on the equivalence that he suggested between charged surface particles and a membrane that separates two ionic solutions but is impermeable to one specification. He had written that the electro-chemical potentials in both ionic solutions are equal (Donnan equilibrium).

Improved reversed-phase gradient retention modeling.

An empirical nonlinear equation is used to describe the relationship between chromatographic retention and solvent composition in both isocratic and gradient chromatography. The integration of the gradient equation can be solved with our equation, allowing a simple transition between isocratic and gradient conditions. Precise interpolation between experimental data points and reasonable extrapolation outside the directly measured data range are possible.

The determination of the plate count for large molecules under reversed-phase gradient conditions.

The true performance of HPLC columns in the gradient separation of macromolecules can be assessed by measuring the true plate count, if we know the retention factor of the analyte at the point of elution. We are demonstrating in this short communication how this can accomplished in a straightforward fashion. The procedure used here is a significant simplification over previous approaches, and enables the chromatographer to do so without complex algebra or additional experiments.

Influence of pressure on the retention of sugars in hydrophilic interaction chromatography.

Pressure can influence the retention of analytes in hydrophilic interaction chromatography as well as in RP chromatography. We demonstrate that the retention of sugars in hydrophilic interaction chromatography decreases with pressure, and interpret the observation as a gain in solvation, or more specifically hydration, as the sugar molecules enter the water-rich stationary phase.

Further investigations of the effect of pressure on retention in ultra-high-pressure liquid chromatography.

In this study, we investigated further the large increases in retention with pressure that we observed previously in RP-LC especially for ionised solutes. These findings were initially confirmed on a conventional silica C(18) column, which gave extremely similar results to the hybrid C(18) phase originally used. Large increases in retention factor of approximately 50% for a pressure increase of 500 bar were also shown for high MW polar but neutral solutes.

Effects of extra-column band spreading, liquid chromatography system operating pressure, and column temperature on the performance of sub-2-microm porous particles.

The effects of extra-column band spreading, LC system operating pressure, and separation temperature were investigated for sub-2-microm particle columns using both a conventional HPLC system as well as a UPLC system. The contributions of both volume- and time-based extra-column effects were analyzed in detail. In addition, the performance difference between columns containing 2.

Investigation of the effect of pressure on retention of small molecules using reversed-phase ultra-high-pressure liquid chromatography.

The effect of inlet pressure on the retention of a series of low molecular weight acids, bases and neutrals, was investigated at constant temperature in reversed-phase liquid chromatography using a commercial ultra-high-pressure system (Waters UPLC instrument). For neutral compounds, relatively small increases in retention factor of up to approximately 12% for a pressure increase of 500bar were noted; the largest values were obtained for polar solutes, or solutes of higher molecular weight. Ionisable acids and bases gave much larger increases in retention with pressure, in some cases as high as 50% for a pressure increase of 500bar.

Estimation of the extent of the water-rich layer associated with the silica surface in hydrophilic interaction chromatography.

The possible presence of a mobile phase layer rich in water on the surface of silica columns used under conditions typical in hydrophilic interaction chromatography was investigated by the injection of a small hydrophobic solute (benzene) using acetonitrile-water mobile phases of high organic content. Benzene does not partition into this layer and is thus partially excluded from the pores of the phase up to a water content of about 30%, after which hydrophobic retention of the solute on siloxane bonds is observed. In 100% acetonitrile, the retention volume of benzene was smaller than that estimated either by pycnometry or by calculation from the basic physical parameters of the column.

The application of novel 1.7 microm ethylene bridged hybrid particles for hydrophilic interaction chromatography.

An un-derivatized 1.7 microm ethylene bridged hybrid (BEH) particle was evaluated for its utility in retaining polar species in hydrophilic interaction chromatography (HILIC), and was compared to a 3 microm un-derivatized silica material. Retentivity as a function of mobile phase pH, polar modifier and ACN content was examined.

Peak capacity in unidimensional chromatography.

The currently existing knowledge about peak capacity in unidimensional separations is reviewed. The majority of the paper is dedicated to reversed-phase gradient chromatography, covering specific techniques as well as the subject of peak compression. Other sections deal with peak capacity in isocratic chromatography, size-exclusion chromatography and ion-exchange chromatography.

Peak capacity in gradient reversed-phase liquid chromatography of biopolymers. Theoretical and practical implications for the separation of oligonucleotides.

Reversed-phase ultra-performance liquid chromatography was used for biopolymer separations in isocratic and gradient mode. The gradient elution mode was employed to estimate the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.

Stationary phase characterization and method development.

The properties of stationary phases and their characterization methods are reviewed. New and significant developments have occurred in the last few years, and new methods for stationary phase characterization have become available. The characterization methods are discussed, and the differences between the different methods are pointed out.

The application of small porous particles, high temperatures, and high pressures to generate very high resolution LC and LC/MS separations.

The effect of combining sub-2 microm porous particles with elevated operating temperatures on chromatographic performance has been investigated in terms of chromatographic efficiency, productivity, peak elution order, and observed operating pressure. The use of elevated temperature in LC does not increase the obtainable performance but allows the same performance to be obtained in less time. Increasing the column temperature did allow the use of longer columns, generating column efficiencies in excess of 100,000 plates and gradient peak capacities approaching 1000.

Selectivity in reversed-phase separations: general influence of solvent type and mobile phase pH.

The influence of the mobile phase on retention is studied in this paper for a group of over 70 compounds with a broad range of multiple functional groups. We varied the pH of the mobile phase (pH 3, 7, and 10) and the organic modifier (methanol, acetonitrile (ACN), and tetrahydrofuran (THF)), using 15 different stationary phases. In this paper, we describe the overall retention and selectivity changes observed with these variables.

Performance of idealized column structures under high pressure.

We have examined the influence of the longitudinal temperature and pressure gradients in columns operated under very high pressures on the coefficients of the van Deemter equation under the idealized condition of complete radial uniformity. These gradients change the diffusion coefficients over the length of the column, and the equation takes a new form, where the classical linear C-term is replaced by more complex forms that capture the effects of these axial gradients. The details of the derivations are shown and the implications are discussed.

Comparison of mixed-mode anion-exchange performance of N-vinylimidazole-divinylbenzene sorbent.

A newly synthesized copolymer based on N-vinylimidazole-divinylbenzene (NVIm-DVB) was evaluated as a mixed-mode anion-exchange sorbent for SPE, since the NVIm monomer apart from its hydrophilic properties can be protonated at a certain pH, and then performs as an anion-exchanger. To investigate the behavior of the NVIm-based sorbent, the SPE performance was evaluated under reversed-phase (RP), weak anion-exchange, and strong anion-exchange conditions. The results for the NVIm-DVB sorbent were also compared to commercial reference sorbents from each group: Oasis HLB, Oasis WAX, and Oasis MAX, respectively.

Changes in the pore character of silicas caused by surface bonding and polymer coating.

The pore character of packings for liquid chromatography, especially reversed-phase (RPLC) packings, has been studied by means of the nitrogen adsorption method (BET method). Micro-spherical silica gels with 9, 12, 30, 40 and 50 nm average pore diameter have been used as carriers. These silica gels have been modified with monochlorodimethyloctadecylsilane and several polymers (polyoctadecylmethacrylate-methylmethacrylate co-polymer, polyacrylamide gel, polyvinyl alcohol, poly-2-hydroxyethyl-methacrylate).

A detailed discussion of the influence of the amount of deposited polymer on the retention properties of polymer-coated silicas.

Problems of the modification of the surface of micro-spherical silica gel with layers of polymer and the influence of the quantity of the coated polymer on the chromatographic properties of the resulting sorbents have been considered. The polymer modification of the surface of wide-pore micro-spherical silica gel obtained by means of hydrothermal treatment of the meso-porous silica gel under autoclave conditions is described. The polymer layer itself is formed by an octadecylmethacrylate-methylmethacrylate co-polymer.

Selectivity in reversed-phase separations Influence of the stationary phase.

The selectivity difference between 15 different stationary phases was measured using a large number of analytes at 2 or 3 different pH values (3, 7 and 10) with acetonitrile and methanol as the mobile phase modifiers. The packings discussed include standard C(8) and C(18) packings, packings with embedded polar groups, a phenyl packing, a pentafluoro-phenyl packing, an adamantylethyl packing and others. The major selectivity differences observed are discussed in detail.

Peak compression in reversed-phase gradient elution.

Previous reports suggest that peak widths in linear gradient elution are consistently larger than predicted by theory; however, if gradient compression is ignored, experiment and theory are in reasonable agreement. This suggests that gradient compression might represent an incorrect or poorly understood concept. In the present study, an experimental program was carried out to better understand the role of gradient compression and the reason for past differences between experiment and theory.

Theory of peak capacity in gradient elution.

Peak capacity is the best measure of the performance of a gradient separation. In this paper, the theory of peak capacity for the standard operating conditions of reversed-phase and ion-exchange chromatography is outlined. The influence of the operating conditions on the peak capacity of a separation are discussed.

The combined effect of silanols and the reversed-phase ligand on the retention of positively charged analytes.

The nature of the interaction of positively charged analytes with the surface of reversed-phase bonded phases has been investigated as a function of both pH and volume fraction of organic modifier. Studies of the combined effect of both the parameters have been previously reported by us, and the data presented here further demonstrate a multiplicative interaction between pH and the concentration of organic modifier in the mobile phase. Fitting of the data as functions of pH and eluent composition clearly shows that the hydrophobically assisted ion-exchange process dominates over a purely reversed-phase or a pure ion-exchange retention mechanism.

Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography.

Column peak capacity was utilized as a measure of column efficiency for gradient elution conditions. Peak capacity was evaluated experimentally for reversed-phase (RP) and cation-exchange high-performance liquid chromatography (HPLC) columns, and compared to the values predicted from RP-HPLC gradient theory. The model was found to be useful for the prediction of peak capacity and productivity in single- and two-dimensional (2D) chromatography.