Temperature effects on retention and efficiency of butyl and lauryl acrylate porous polymer monoliths in capillary electrochromatography.

The development of organic porous polymer monoliths represents an alternative approach to stationary phase design. The use of these materials has helped to rekindle interest in capillary electrochromatography. Although a large number of investigations have explored different monolith recipes, polymerization conditions, and application challenges, few investigations have addressed the fundamentals of this separation mode with this type of material. This study addresses the thermodynamics of the reversed phase retention mechanism on 100% butyl acrylate and 1:3 butyl:lauryl acrylate (volume/volume ratio) porous polymer monoliths used in a capillary electrochromatography mode. Linear van't Hoff plots yield enthalpies of retention of -3.9 to -14.3 kJ/mol on two different, but related columns for five selected hydrophobic analytes across a thirty degree temperature range. Minimum plate heights were only moderately impacted over this temperature range.

Fluorescence correlation spectroscopy study of protein transport and dynamic interactions with clustered-charge peptide adsorbents.

Ion-exchange chromatography relies on electrostatic interactions between the adsorbent and the adsorbate and is used extensively in protein purification. Conventional ion-exchange chromatography uses ligands that are singly charged and randomly dispersed over the adsorbent, creating a heterogeneous distribution of potential adsorption sites. Clustered-charge ion exchangers exhibit higher affinity, capacity, and selectivity than their dispersed-charge counterparts of the same total charge density. In the present work, we monitored the transport behavior of an anionic protein near clustered-charge adsorbent surfaces using fluorescence correlation spectroscopy. We can resolve protein-free diffusion, hindered diffusion, and association with bare glass, agarose-coated, and agarose-clustered peptide surfaces, demonstrating that this method can be used to understand and ultimately optimize clustered-charge adsorbent and other surface interactions at the molecular scale.

Permeability of anti-fouling PEGylated surfaces probed by fluorescence correlation spectroscopy.

The present work reports on in situ observations of the interaction of organic dye probe molecules and dye-labeled protein with different poly(ethylene glycol) (PEG) architectures (linear, dendron, and bottle brush). Fluorescence correlation spectroscopy (FCS) and single molecule event analysis were used to examine the nature and extent of probe-PEG interactions. The data support a sieve-like model in which size-exclusion principles determine the extent of probe-PEG interactions. Small probes are trapped by more dense PEG architectures and large probes interact more with less dense PEG surfaces. These results, and the tunable pore structure of the PEG dendrons employed in this work, suggest the viability of electrochemically-active materials for tunable surfaces.

Dye diffusion at surfaces: charge matters.

Fluorescence correlation spectroscopy and single molecule burst analysis were used to measure the effects of glass surface interactions on the diffusion of two common fluorescent dyes, one cationic and one anionic. The effects of dye-surface interactions on measured diffusion rates as a function of distance from the surface were investigated. Use of a three-axis piezo stage, combined with reference calibration measurements, enabled the accurate acquisition of surface-distance-dependent transport data. This analysis reveals attractive interactions between the cationic dye and the surface, which significantly alter the extracted diffusion values and persist in the measurements up to 1.0 microm from the surface. The Coulomb attraction between the cationic dye and the surface also results in rare, long-lived association events that lead to irreproducibility in extracted diffusion values. In addition to an assignment of the association lifetime for these events, this paper demonstrates that, if experiments must be performed with cationic probes near a glass surface, the use of solution electrolytes can eliminate deleterious dye-surface interactions, as the dyes were tested in a variety of environments. Finally, our data demonstrate that a better dye choice is an anionic probe, which exhibits no depth dependence of diffusion characteristics above a glass surface.

Approach for the Simulation and Modeling of Flexible Rings: Application to the α-D-Arabinofuranoside Ring, a Key Constituent of Polysaccharides from Mycobacterium tuberculosis.

A number of lower organisms (bacteria, fungi, and parasites) produce glycoconjugates that contain furanose rings. Of particular interest to our group are cell wall polysaccharides from mycobacteria, including the human pathogen, Mycobacterium tuberculosis, which contain a large number of arabinofuranose resides. As part of a larger project on the conformational analysis of these molecules, we report here molecular dynamics simulations on methyl α-D-arabinofuranoside (1) using the AMBER force field and the GLYCAM carbohydrate parameter set. We initially studied the ability of this method to predict rotamer populations about the hydroxymethyl group (C4-C5) bond. Importantly, we show that simulation times of up to 200 ns are required in order to obtain convergence of the rotamer populations for this ring system. We also propose a new charge derivation approach that accounts for the flexibility of the furanoside ring by taking an average of the charges from a large number of conformers across the psuedorotational itinerary. The approach yields rotamer populations that are in good agreement with available NMR data and, in addition, provides insight into the nature of the puckering angle and amplitude in 1.

GLYCAM06: a generalizable biomolecular force field. Carbohydrates.

A new derivation of the GLYCAM06 force field, which removes its previous specificity for carbohydrates, and its dependency on the AMBER force field and parameters, is presented. All pertinent force field terms have been explicitly specified and so no default or generic parameters are employed. The new GLYCAM is no longer limited to any particular class of biomolecules, but is extendible to all molecular classes in the spirit of a small-molecule force field. The torsion terms in the present work were all derived from quantum mechanical data from a collection of minimal molecular fragments and related small molecules. For carbohydrates, there is now a single parameter set applicable to both alpha- and beta-anomers and to all monosaccharide ring sizes and conformations. We demonstrate that deriving dihedral parameters by fitting to QM data for internal rotational energy curves for representative small molecules generally leads to correct rotamer populations in molecular dynamics simulations, and that this approach removes the need for phase corrections in the dihedral terms. However, we note that there are cases where this approach is inadequate. Reported here are the basic components of the new force field as well as an illustration of its extension to carbohydrates. In addition to reproducing the gas-phase properties of an array of small test molecules, condensed-phase simulations employing GLYCAM06 are shown to reproduce rotamer populations for key small molecules and representative biopolymer building blocks in explicit water, as well as crystalline lattice properties, such as unit cell dimensions, and vibrational frequencies.