Effects of pore structure and molecular size on diffusion in chromatographic adsorbents.

Two computational approaches, namely Brownian dynamics and network modeling, are presented for predicting effective diffusion coefficients of probes of different sizes in three chromatographic adsorbents, the structural properties of which were determined previously using electron tomography. Three-dimensional reconstructions of the adsorbents provide detailed, explicit characteristics of the pore network, so that no assumptions have to be made regarding pore properties such as connectivity, pore radius and pore length. The diffusivity predictions obtained from the two modeling approaches were compared to experimental diffusivities measured for dextran and protein probes. Both computational methods captured the same qualitative results, while their predictive capabilities varied among adsorbents.

Rapid estimation of octanol-water partition coefficients using synthesized vesicles in electrokinetic chromatography.

Vesicle electrokinetic chromatography (VEKC) using vesicles synthesized from the oppositely charged surfactants cetyltrimethylammonium bromide (CTAB) and sodium octyl sulfate (SOS) and from the double-chained anionic surfactant bis(2-ethylhexyl)sodium sulfosuccinate (AOT) was applied to the indirect measurement of octanol-water partition coefficients (log Po/w). A variety of small organic molecules with varying functional groups, pesticides, and organic acids were evaluated by correlating log Po/w and the logarithm of the retention factor (log k') and comparing the calibrations. A linear solvation energy relationship (LSER) analysis was conducted to describe the retention behavior of the vesicle systems and compared to that of octanol-water partitioning. The solute hydrogen bond donating behavior is slightly different with the vesicle interactions using CTAB-SOS vesicles as compared to the octanol-water partitioning model. The AOT vesicle and octanol-water partitioning systems showed similar partitioning characteristics. VEKC provides rapid separations for determinations of log Po/w in the range of 0.5 to 5 using CTAB-SOS vesicles and 0 to 5.5 using AOT vesicles.

Determination of octanol-water partition coefficients of pesticides by microemulsion electrokinetic chromatography.

Microemulsion electrokinetic chromatography (MEEKC) was evaluated as a screening tool for the indirect measurement of octanol-water partition coefficients (log Po/w) of pesticide compounds. Over 80 pesticide compounds representing a variety of structural characteristics were studied, and good correlation of log Po/w with the logarithm of the retention factor was found. The microemulsion system studied allowed the separation of compounds in the log Po/w range of -1 to 7. In addition, a smaller set of simple organic molecules that vary in structural features was evaluated and compared to the pesticide log Po/w calibration. The pesticide and simple organic molecule log Po/w calibration lines were statistically similar. This suggests that a universal set of standard compounds may be employed for the log Po/w calibration to provide measurements for a variety of compounds with good accuracy.

Surface coverages of bonded-phase ligands on silica: a computational study.

A computational study of the packing of various bonded-phase ligands bound to chromatographic silica is presented. This is done with the intention of examing the type of surface structures that are typically found in real chromatographic systems. Utilizing the surface structure of the (111) face of the beta-cristobalite crystal, it is shown that the maximum surface coverages of dimethyloctylsilane, dimethyloctadecylsilane, triisopropylsilane, diisopropyloctylsilane, and diisopropyloctadecylsilane can be calculated that are in good agreement with experiment. The maximum surface coverages are also calculated for the (100) face of the beta-cristobalite crystal and for a set of random silica surfaces. The coverages for the latter two surfaces types are found to be significantly lower than the experimental values for chromatographic silica surfaces. These results further suggest that chromatographic silica surfaces may resemble crystalline surface sites similar to the (111) face of beta-cristobalite, as has been previously suggested in the literature. Hence, these structures can be reliably utilized in molecular simulations of bonded-phase chromatography where the atomic-level detail of the silica surface has been previously lacking.

High-performance capillary gel electrochromatography with replaceable media.

Capillary gel electrochromatography is evaluated with an entangled polymer solution which is pumped into the capillary and run under fritless conditions. The polymer used has an acid backbone with grafted hydrophobic segments, the polyacid giving the electroosmotic flow and the hydrophobe segments providing the retentive component. Experimental evaluation of this type of system reveals performance similar to capillary electrophoresis and other forms of electrochromatography. The analysis of plate height data demonstrates that zone broadening is primarily due to diffusion with little contribution from nonequilibrium zone broadening. Hence, operation at high velocities (high voltages) is most desirable as opposed to most chromatographic methods. Some of the advantages of this type of experiment include being able to replace the retentive media in a few minutes, fast and reproducible high-performance separation, and having a retention mechanism similar to reversed-phase liquid chromatography. Disadvantages include a low retentive phase concentration and hence low sample loadability and limited solvent compatibility of the polymer. A number of different separations are demonstrated including separation of alkyl benzoates, alkylphenones, alkylbenzenes, oxidation inhibitors, and PAHs.

Intermolecular interactions involved in solute retention on carbon media in reversed-phase high-performance liquid chromatography.

Carbon adsorbents for RPLC separations are greatly underutilized due to the poor chromatographic properties of the earliest commercially available materials and our limited understanding of solute interactions with the solid surface. Previously, we reported on the properties of a carbon surface prepared by vapor deposition on porous zirconia microspheres. The resulting material is a new type of carbon sorbent with considerably improved chromatographic properties. Here we present a fundamental study of the intermolecular interactions influencing solute retention on these novel carbon phases under RPLC conditions. Retention on seven unique carbon phases has been correlated with solute descriptors of dispersion, dipolarity/polarizability, and hydrogen bond basicity through the use of linear solvation energy relationships (LSERs). In stark contrast, conventional bonded phases do not show the large contribution from dipolarity/ polarizability, that is observed on these types of carbon. The presence of this interaction indicates a distinct difference between carbon and conventional bonded RPLC phases. Other results suggest that solvent sorption plays a significant role in controlling solute retention on carbon. In addition, we investigated the temperature dependence of retention on carbon and found typical RPLC-like behavior.

Viscous effects in capillary electrophoresis: theory and experiment.

Simple theory which relates the viscous effects of additives used in capillary electrophoresis (CE) to resolution and resolution per unit time are developed for small molecule separations in the absence of sieving effects. The resolution theory shows that there is no advantage to using viscous additives for resolution improvement unless either a binding energy difference exists between the solutes and additive, or viscous effects cause a reduction in the vector sum of electrophoretic and electroosmotic velocities when these are of opposite sign. In general, increasing the viscosity is shown to result in a loss of resolution per unit time. Theory demonstrates that in cases where only binding is considered to be present, the maximum resolution increase is found to be at relatively small amounts of additive. In addition, specific regions of the elecctrophoreogram may demonstrate resolution enhancement at a specific concentration while other regions exhibit a resolution decrease, as compared to no additive present. CE separations of the components in a Triton surfactant, conducted using a polyethylene glycol (PEG) additive, demonstrate that improved resolution for some of the peaks exists at a specific PEG concentration. These results are mostly explained through the reduction in electroosmotic flow velocity that takes place through additive adsorption to the capillary wall, although some binding is present.

Conformational study of biotinylated DNA oligonucleotides utilizing two-dimensional nuclear magnetic resonance and molecular dynamics.

The solution structure of DNA oligonucleotides containing a biotin group covalently attached through a linker arm to the 5' end has been studied by two-dimensional NMR and molecular modeling, in an attempt to determine whether the biotin end group is accessible to avidin binding. Such experiments are useful in suggesting that purification of synthetic DNA oligonucleotides by avidin-biotin affinity chromatography is carried out with a minimum amount of intramolecular association of biotin with potential binding sites within the DNA. Two DNA oligonucleotides that form hairpin structures have been proposed as possible worst case scenarios where biotin would not be available for avidin binding. These structures are d(XGCGCGTTTTCGCGC) and d(XGCGCGTTTTCGCGCAAAAA), where X represents the biotin and linker structures. No NOEs were detected between the biotin/linker portion of the molecule and the DNA hairpin. In addition, the magnitude of the NOEs between neighboring groups in the biotin/linker was approximately an order of magnitude smaller than that of the neighbors at a comparable distance within the DNA hairpin, suggesting that the biotin/linker undergoes considerable additional motion compared to the DNA hairpin. Molecular dynamics calculations also show that the biotin/linker undergoes a considerable range of motion. Thus, all data indicate that the biotin is not immediately associated with the hairpin and should be available for binding to avidin under chromatographically relevant solvent conditions.