Capillary electrophoresis-mass spectrometry using an in-line sol-gel concentrator for the determination of methionine enkephalin in cerebrospinal fluid.

In this study, a CE-MS method using a monolithic sol-gel concentrator for in-line solid-phase extraction (SPE) is evaluated for the analysis of methionine enkephalin in biological samples. Operational SPE parameters such as sample pH, loading volume, elution volume and composition have been studied. After optimization of the in-line preconcentration methodology, a 40-fold preconcentration was demonstrated for a methionine enkephalin test solution using a loading volume of 3200 nL. The method was linear in the range from 62.5 to 1000 ng/mL (R(2)>0.99). R.S.D. values for migration times and peak areas were 1.2% and 8.4%, respectively. Finally, the analysis of cerebrospinal fluid samples spiked with methionine enkephalin and deproteinized with perchloric acid (1:1, v/v) showed a detection limit (S/N=3) of approximately 1 ng/mL (ca. 5 nM). The recoveries of methionine enkephalin for three concentration levels (100, 10 and 1 ng/mL) were in the range of 74-91%, demonstrating the promising potential of the methodology for the analysis of biological samples.

Characteristics of particle-loaded monolithic sol-gel columns for capillary electrochromatography. I. Structural, electrical and band-broadening properties.

Particle-loaded (3 microm, C18) monolithic sol-gel columns have been prepared and selected characteristics measured. They have a surprisingly high permeability, allowing their operation in the microLC mode at pressures as low as 69 kPa where their efficiency is about 50000 plates per meter and the CEC mode where efficiency is at least 106000 plates per meter. These columns can withstand over 13.8 MPa pressure without compression or movement within the 75 microm capillary. Field strengths in the packed segments are approximately 50% greater than those in the open segments, due to the higher resistivity of the particle-laden regions. There is a relatively rapid loss of efficiency with increasing linear velocity in both the CEC and microLC modes, which may be due to a tortuosity effect in the inter- and intra-particulate voids. Chromatographic behavior is characteristic of conventional C18 particles, indicating that analytes have significant access to the surface within the pores of the immobilized bonded phase.

Study of protein binding to a silica support with a polymeric cation-exchange coating.

A silica-based, polyacrylate ion-exchange stationary phase has been prepared using Ce(IV) as the initiator. Analysis of the physical properties of the polymeric layer separated from the silica surface indicates that the polymeric coating is cross linked to some extent. The polymerization carried out at different concentrations of Ce(IV) demonstrated that the effective surface area can be increased by lowering the Ce(IV) concentration at higher monomer concentrations of the reaction mixture. These materials are quite reproducible and of high electrostatic binding capacity; 1.485 mumol/m2. The electrostatic binding capacity of a non-polymeric stationary phase reached the theoretical limit for a monolayer (0.16 mumol/m2). However, the covalent binding capacity of the same stationary phase was only 50% of the electrostatic binding capacity. The same trend was observed in all the polymeric stationary phases tested. This shows that the mechanism of protein binding in polymeric and conventional stationary phases is similar, and multilayer electrostatic binding is highly unlikely in these sorbents examined. Z numbers revealed that the contact area of the protein is independent of the polymeric character of the stationary phase and therefore, the increased loading of these polymeric stationary phases is due to the increased surface area.

Lateral interaction between electrostatically adsorbed and covalently immobilized proteins on the surface of cation-exchange sorbents.

This paper examines the nature of chromatographic separations on a weak cation-exchange material in which immobilized proteins coats 50% or less of the sorbent surface. It was found that although these sorbents still function as cation exchangers, covalently immobilized proteins frequently contribute to the ion-exchange behavior of some protein analytes. Chromatographic retention of analytes was equal to or greater on immobilized protein derivatized columns than underivatized sorbents. Anionic proteins, in contrast, were not adsorbed, indicating that immobilized proteins were acting synergistically with ionic stationary phase groups to enhance retention. It was concluded that electrostatic adsorption is a prerequisite for analyte protein/immobilized protein interactions of sufficient magnitude to impact ion-exchange separations. Large differences in protein resolution were observed on columns that were identical in all respects except for the immobilized protein, further confirming that analyte/immobilized protein interactions were unique to the interacting pair. The extent of interaction was also influenced by concentration of the immobilized protein in the case of lysozyme. Interactions between the analyte and immobilized protein were found to occur between both the same two proteins and dissimilar species. It was concluded that these phenomena are due to lateral interactions between immobilized proteins and analyte proteins subsequent to electrostatic adsorption of the analyte on the underivatized surface of ion-exchange sorbents.