John C. Ford

Conformational effects in the reversed-phase liquid chromatography of ribonuclease A.

This paper examines the reversed-phase liquid chromatographic behavior of ribonuclease A (RNase) using an n-butyl chemically bonded phase and a gradient of 10 mM H3PO4 and l-propanol. At a column temperature of 25 degrees C, a broad band followed by an overlapped late-eluting sharp peak is observed. As the temperature is raised, the sharp peak grows at the expense of the broad band until at 37 degrees C, only a single narrow-eluting band is found. Using an absorbance ratio of A288/A254, it is demonstrated that the broad band represents a folded or native state of RNase and the late-eluting band a denatured state. Based on postcolumn absorbance ratio changes in the denatured state as a function of time and the known behavior of the protein, reversible refolding or renaturation is proposed to take place in solution. RNase is denatured upon adsorbing to the bonded phase, and upon migration down the column, reversible refolding takes place in the mobile phase. The relaxation time for native state formation is assumed to be comparable to the time spent by RNase in the mobile phase. As temperature is raised, both the native and denatured states exist at equilibrium in solution, thus slowing the refolding process, until at 37 degrees C only the denatured peak appears. Changes in peak shape with flow rate provide further evidence for this model. The use of HCl or H2SO4 instead of H3PO4 yields similar results except that the temperature at which only the denatured peak is observed follows the order of salt stabilization of the native state.

Multisolute adsorption equilibria in a reversed-phase liquid chromatography system

Abstract Multisolute adsorption equilibrium data are reported for benzyl alcohol, 2-phenylethanol and 2-methyl benzyl alcohol in a reversed-phase system. The adsorption data acquired exhibit very good thermodynamic consistency. The thermodynamic functions of adsorption were derived from the single-solute adsorption data measured at different temperatures. The values of the isosteric heats of adsorption suggest a nearly homogeneous surface for the adsorbent (octadecyl bonded silica) under consideration. A nonideal adsorbed solution theory model is applied to the experimental data. The model provides an excellent prediction of the multisolute adsorption equilibria based on the parameters derived from the single-solute adsorption data. Liquid–liquid equilibrium models were also used to describe the experimental data. In this case, the best representation of the experimental data is obtained when using 1-decanol to represent the stationary phase. Although the partition model provides a satisfactory representation of the experimental data at low concentrations, it fails to represent the curvature of the equilibrium data at higher concentrations of the solutes. The latter model is also unable to describe the temperature dependence exhibited by the single-solute adsorption data.

Gas chromatographic detection and identification of aromatic and aliphatic hydrocarbons in complex mixtures by coupling photoionization and flame-ionization detectors

A technique has been developed that is based on analyzing the effluent from a gas chromatograph with both photoionization (PID, with 10.2-eV lamp) and flame-ionization (FID) detectors. The relative molar response per mole of carbon (RMR) for the FID is similar for many types of carbon atoms, e. g., aromatic and aliphatic and therefore the FID is used to measure the relative levels of a particular hydrocarbon regardless of the degree of saturation. The RMR for the PID, however, increases with unsaturation. A ten-fold difference is observed between aromatics and alkanes, and the alkene response on the PID is midway between these two classes of compounds. When the PID and FID responses (i. e., peak areas) are normalized to any alkene (e. g, n-hexane) and are compared, those compounds with a normalized PID/FID ratio of 2–4 are alkenes, those with a ratio of 5–10 are aromatics and those with similar ratios (<2) are alkanes. Preliminary work on this technique was started with simple hydrocarbon mixtures, then extended to high-molecular-weight heteroatom molecules such as chlorinated and sulfur-based pesticides. Finally, a practical application of this approach was demonstrated with the successful analysis of aromatics in a light hydrocarbon feedstock for a synthetic natural gas plant. This technique should prove useful for analyses (nanogram levels) and for the identification of complex hydrocarbon mixtures.

Comparison of selected retention models in reversed-phase liquid chromatography.

Retention models are usually compared by how well the model equation fits retention data for one solute taken over a range of mobile phase compositions. Even when retention data for multiple solutes are used, the quality of the fit is often judged by the statistical goodness-of-fit alone. This study compared four different RPLC retention models, encompassing three distinct mathematical forms. Each model was fit to the retention data of multiple solutes and the sets of best-fit parameters were examined in terms of the underlying physico-chemical assumptions of the models. Next, for the linear and quadratic models, some of the model parameters were calculated a priori and the rest of the model parameters were then obtained in subsequent fittings. The sets of best-fit parameters obtained in this manner were more consistent with the underlying assumptions of these models than were the sets of parameters obtained entirely through regressions to the experimental data. Thus, the extraction of parameters by fitting a model to the retention data of a single solute may result in unreliable values for those parameters, even in the case of a fit that would be considered good when judged by conventional statistical criteria. That is, although parameters extracted in such a fashion may be suitable for optimization or similar uses, they may not be suitable for determining the appropriateness of the underlying assumptions of retention models.

Synthetic peptide purification by application of linear solvent strength gradient theory

Abstract The purification of crude synthetic peptide by isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) requires time to determine suitable chromatographic conditions, which limits the use of isocratic RP-HPLC as a purification procedure for the newer, multiple-peptide synthetic techniques. An empirical or semi-empirical procedure to determine the appropriate chromatographic conditions based on a knowledge of the peptides sequence or molecular weight would allow direct purification of the crude synthetic peptide without the initial exploratory work. Using linear solvent strength gradient theory, a formula was derived which predicts the optimum gradient time from the number of residues in the sought-after peptide and certain chromatographic parameters. Using a variety of crude synthetic peptides of approximately twenty residues and this gradient time, the mass capacity of various commercially available RP-HPLC columns was determined. Typically, the described procedure was capable of isolating

Comparison of methods for extracting linear solvent strength gradient parameters from gradient chromatographic data

The linear solvent strength (LSS) theory of gradient elution is useful in the optimization of separations in high-performance liquid chromatography. While the fundamental parameters of this theory are defined in terms of isocratic behavior, gradient operation has been used previously to estimate those parameters to allow rapid optimization of the separation. In this study, various methods of extracting the LSS parameters from gradient retention data were examined. Sets of synthetic retention data were calculated directly from the equations of the LSS theory. When realistic experimental uncertainties were incorporated into these data sets, the LSS parameters used to generate the synthetic data were not recovered accurately unless special precautions were taken. For large molecules, an approximate LSS expression could be used to determine the solvent strength parameter with an error of less than 13%, which is comparable to or better than those for the other methods evaluated.

Modeling the effect of solvation on solute retention in reversed-phase liquid chromatography

The retention of a homologous series of alkylbenzenes was determined on octyl and octadecyl reversed-phase columns in several polar organic liquids. Free energies of transfer were calculated by the SM5.0R classical solvation model for each organic liquid tested and for several alkanes. The relationships between the measured retention factors and the calculated free energies of transfer were then investigated. Although the natural logarithms of the retention factor and the calculated free energies of transfer were linearly correlated, the obtained free energies of transfer of the solutes did not completely explain the retention behavior of the solutes. Nonetheless, even in these pure organic liquids, the energetics of RPLC retention behaved very similarly to those of partitioning.

The Instability of Silver Alkoxides

Abstract The reaction of silver ion with methoxide ion in methanol or with ethoxide ion in ethanol at 25°C affords highly unstable intermediates (presumably the silver alkoxides) which decompose rapidly to give metallic silver and unidentified oxidation products. Silver salts of tert-butyl alcohol and phenol decompose somewhat more slowly, while phenols with electron-withdrawing groups afford stable silver salts.

On-Line Detection in Slab Gel Electrophoresis Using Thermal Lens Spectrophotometry

Thermal lens spectrophotometry was used to detect hemoglobin and cytochrome c within an acrylamide gel matrix both during electrophoresis and in static experiments in which each protein was incorporated in the matrix during polymerization. For cytochrome c, the detection limit was 0.02 mg/mL for static experiments (approximately 0.5 ng in the laser beam volume), while a detection limit of 180 ng (5 ng in the beam volume) was found for electrophoresis runs.

Reversed-phase liquid chromatography of site-specific mutagenized staphylococcal nuclease : Gradient retention and correlations with amino acid-based predictive scales

Comparison of the gradient reversed-phase chromatographic retentions of twelve Staphylococcal nuclease mutants and the naturally occurring protein showed that the chain location and the chemical nature of the substituted amino acid(s) were equally significant in determining the retention. Correlations between the retention times of these nuclease mutants and of previously published data for interleukin 2 mutants and insulin variants with nineteen amino acid-based predictive scales revealed retention time to be significantly correlated to several scales. The use of mutagenized proteins allowed a more sensitive analysis of the individual amino acid contributions to retention than can be achieved by utilizing a more diverse set of proteins.