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In this study, we describe the application of a new analytical procedure based on capillary electrochromatographic (CEC) techniques for the characterization of different basic and acidic peptides using isocratic eluent conditions containing acetonitrile and ammonium acetate buffers of different molarities between pH 3.8 and 5.2. In particular, 10 immunogenic peptide analogs with isoelectric points ranging from 3.7 to 10.1 were investigated; nine of these peptides, 1-9, were truncated analogs of the parent peptide, 10, which is a peptidomimetic related to a HIV-1 gp120 epitope. Several of these peptides have the propensity to form α-helical secondary structures in solution. Electrochromatographic separations of these peptides were achieved with packed fused silica capillaries (25 cm packed length, 100 µm i.d.) containing 3 µm n-octadecylsilica particles. The influence of temperature on the CEC elution behavior of these peptides, as well as the impact of changes in the eluent composition, e.g. pH, buffer concentration and acetonitrile content, were examined. The results confirm that improvements in the resolution and analysis of synthetic peptides by CEC procedures result from the increase in electro-osmotic flow (EOF) as the temperature is increased. These findings emphasize the dominant influence of the temperature-dependent viscosity parameter, η, on the EOF and thus on peptide resolution in CEC. Moreover, these investigations have shown that eluent properties can be specifically chosen to favor either electrophoretic mobility or chromatographic retention, with the overall CEC selectivity peptides of different sequence or composition reflecting the summated contributions from both separation mechanisms. Over the pH range 4.0-5.0, and using eluents with ionic strengths ranging from 6.2 to 15 mm ammonium acetate but containing a fixed volume fraction, ψ, of acetonitrile above ψ=0.40, the CEC retention behavior of peptides 1-10 correlated with a linear relationship linking the retention coefficient, κ_cec, and the differential frictional size-to-mass ratio parameter, ξ_fric, of these peptides. However, using eluents with a low acetonitrile content and low pH values, linear correlations were also observed between the incremental retention coefficient, Δκ_cec, and the product term [- 0.66(Δ∑χ_n) log(M_i/M_j)], which links the difference in intrinsic hydrophobicities and molecular masses of two peptides, P_i and P_j. This study thus demonstrates the power of CEC procedures in the analysis of synthetic bioactive peptides and provides a general experimental framework to evaluate, using CEC procedures, the influence of the key molecular attributes of peptides on their structure-retention dependencies. Finally, these studies provide additional, practical insights into the use of CEC procedures for the analysis, resolution and biophysical characterization of closely related peptide analogs derived from solid-state peptide synthesis under conditions of different eluent composition or temperature.

On diol-modified silica columns the retention of proteins is governed by a size exclusion effect, but superimposed on this are some secondary effects, i.e., ionic and diol-ligand interactions which can be controlled and adjusted reproducibly by varying the eluent composition. The eluent composition also affects the column efficiency and peak shape. Both dependences can be employed to obtain a better resolution of proteins than can be expected from size exclusion alone.

A new procedure has been developed to immobilize iminodiacetic acid (IDA) onto the surface of silica supports, such as LiChrospher Si-1000 and 1.5 µm nonporous silica, for use in high-performance immobilized metal affinity chromatography (HPIMAC) of proteins. This IDA immobilization method has been achieved through the synthesis of a new silylation reagent, 1-(iminodiacetic acid di-tert-butylester)-3-glycidoxy-propyltrimethoxysilane (IDA-silane). Various modified silicas of different ligand densities have been prepared by using mixtures between 1 and 100% of the IDA-silane diluted with the corresponding 3-glycidoxy-propyltrimethoxysilane (GLYMO-silane). Frontal analysis was used with the IDA-Cu(II)-Concanavalin-A and IDA-Cu(II)-lysozyme systems to evaluate the capacity and the association constants for these HPIMAC sorbents. With these metal chelate sorbents the specific binding capacity per unit area increased continuously with the ligand density for the nonporous sorbents but reached a maximum at about 50% of the maximum ligand coverage for the porous sorbents. The association constant for the chelate-protein complex was highest for both concanavalin-A (Con-A) and lysozyme (HEWL) at the highest ligand density and decreased with lower ligand density. These observations have been evaluated in terms of the accessibility of histidine residues on the surface of the two test proteins and their ability to act as binding sites for the copper ions in the coordination complex. The experimental data indicate that both steric and conformational effects result in multiple classes of binding phenomena with Con-A and HEWL at high ligand concentrations. These experimental results provide a useful guideline for the design of silica-based sorbents for application in the HPIMAC of proteins.Copyright 1993, 1999 Academic Press

A reduction of the mean particle diameter of silica-based packings in the size-exclusion chromatography (SEC) of proteins to about 5 μm generates the expected increase in column plate number over the traditional 10 μm SEC columns, as demonstrated for the Zorbax Bio Series GF 250 and GF 450 and TSK-Gel 3000 SWXL columns. The slightly lower column efficiency of the TSK-Gel 3000 SWXL compared with the GF 250 column is compensated by the fact that the phase ratio of the 3000 SWXL column is higher by a factor of two. Hence both columns show nearly the same peak capacity of about 20–30. When the ionic strength of the eluent was changed by varying the salt concentration, the elution volume of proteins was found to be affected differently on the two columns. These alterations are attributed to the differences in the surface compositions of the two types of packings.

B-MFI and MTN-type materials were synthesized via the fluoride route from dry powders in the complete absence of a solution phase. Amorphous precursors obtained by drying SiO2· Al2O3gels at 700°C were transformed into MTN-type material in the presence of dried NH4F and TMACl. Amorphous precursors obtained by drying SiO2gels at 700°C were transformed into B-MFI in the presence of dried NH4F, B2O3and TPABr. Water is formed as a reaction product. However, the water vapor pressure is appreciably below the water vapor saturation pressure under the given reaction conditions. In addition, syntheses using seed crystals were carried out successfully.

Diesel exhaust soot samples are examined with sedimentation field flow fractionation (SdFFF), a relatively new separations method for submicron particulates. These studies show that the density of the diesel soot samples is relatively inhomogeneous, ranging from 1.17 to 1.34 g/cm3 for all samples, based on weight average particle diameter values. While diesel soot particle ultimates are in the 0.05–0.15-μm range, weight average particle diameters of about 0.2–0.3 μm are estimated by SdFFF, suggesting that many of the soot particulates are relatively strong aggregates. These studies indicate that SdFFF is a useful tool for characterizing a wide range of submicron, environmentally important particulates.