Surfactant-mediated hydrophobic interaction chromatography of proteins: gradient elution

Addition of 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulphonate (CHAPS) to mobile phases in gradient elution hydrophobic interaction chromatography (HIC) on SynChropak Propyl causes changes in observed elution times for nine globular proteins. The nine proteins showed different percentage reductions in capacity factor, k', demonstrating the ability of CHAPS to change the selectivity of the separations. Three basic types of gradient experiments have been explored for surfactant-mediated gradient elution HIC. Type I gradients are conducted with constant salt and variable surfactant concentration. Type II gradients with variable salt and constant surfactant concentration, and Type III gradients with variable salt and surfactant concentrations. By the criterion of a linear relationship between gradient time and retention time the linear solvent strength condition applies to Type II and Type III gradients. Type III gradients, with the fastest re-equilibration time, are preferable for repetitive analyses. Type I gradients are relatively ineffective in making use of the solvent strength of CHAPS, and Types I and II gradients require long equilibration times due to large changes in surface concentration of CHAPS which occur during elution. The presence of CHAPS had a negligible effect on peak shapes of the proteins examined, except for bovine serum albumin which yielded a narrower, less distorted peak in the presence of CHAPS.     

Solvophobic acceleration of Diels-Alder reactions in supercritical carbon dioxide

The rate of the Diels-Alder reaction between N-ethylmaleimide and 9-hydroxymethylanthracene in supercritical carbon dioxide (scCO(2)) was determined by following the disappearance of 9-hydroxymethylanthracene with in situ UV/vis absorption spectroscopy. The reaction conditions were 45-75 degrees C and 90-190 bar, which correspond to fluid densities (based on pure carbon dioxide) ranging between approximately 340 and 730 kg m(-3). The measured reaction rate at low scCO(2) fluid densities was nearly 25x faster than that reported in acetonitrile at the same temperature (45 degrees C). An inverse relationship between reaction rate and fluid density/pressure was observed at all temperatures in scCO(2). The apparent activation volumes were large and positive (350 cm(3) mol(-1)) and only a weak function of reduced temperature. A solvophobic mechanism analogous to those observed in conventional solvents is postulated to describe (a) the rate acceleration observed for this reaction in scCO(2) relative to that in acetonitrile, (b) the observed relationship between reaction rate and pressure/temperature/density, and (c) the large, positive activation volumes. Solubility measurements in scCO(2), rate measurements in conventional solvents, and an empirical correlation are used to support this theory. Our results advance the general understanding of reactivity in supercritical fluids and provide a rationale for selecting reactions which can be accelerated when conducted in scCO(2).     

Use of the surfactant 3-(3-cholamidopropyl)-dimethyl-ammoniopropane sulfonate in hydrophobic interaction chromatography of proteins

Isocratic hydrophobic interaction chromatography of five proteins has been carried out using mobile phases containing the surfactant 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS). Linear relationships were found between log k' and ammonium sulfate concentrations for all the proteins with CHAPS in the submicellar concentration range. The slope of such a plot decreases monotonically as CHAPS concentration is increased. To a first approximation, the effect of CHAPS on protein retention can be explained in terms of a competitive binding model. However, CHAPS does show differential effects on the elution of proteins, substantially altering selectivity. The use of a normalized capacity factor, k'/k'o, proves useful for comparing retention times of different proteins as a function of CHAPS concentration. The magnitudes of k'/k'o were found to be inversely correlated with the slopes of plots of log k' vs. ammonium sulfate concentration in the absence of CHAPS. Adsorption isotherms for CHAPS were determined over the working range of ammonium sulfate. The binding of CHAPS to the SynChropak Propyl stationary phase and its effects on retention were found to be readily reversible. For each protein, plots of k'/k'o vs. surface concentration of CHAPS were superposable for data obtained at different salt concentrations. These findings support a competitive binding model. A simple geometric argument for stationary phase occupancy provides a qualitative explanation for the observed surfactant selectivity.     

Reactions of Charged Substrates. 7. The Methoxymethyl Carbenium Ion Problem. 2. A Semiempirical Study of the Kinetic and Thermodynamic Stabilities of Linear and Cyclic Oxo- and Thiocarbenium Ions Generated from Aldehyde Hydrates, Hemiacetals, Acetals, and Methyl Ribosides and Glucosides

Factors affecting the cleavage of the carbon-oxygen bond in linear and cyclic aldehyde hydrates, heimacetals, acetals, and methyl ribosides and glucosides have been investigated using semiempirical calculations (AM1 and PM3). (For some systems, low- and high-level ab initio energies are available for comparison with the semiempirical results. With one exception, the results obtained by the two methods show excellent agreement in relative energies and trends in reactivity.) The effects on reactivity and stability caused by substituting a sulfur for the alpha oxygen in the oxocarbenium ion were also studied. In general, systems that can have an antiperiplanar alignment of lone pairs on the leaving group and potential oxocarbenium ion oxygens undergo spontanteous cleavage. An examination of various conformers of the leaving group relative to the potential oxocarbenium oxygen shows, however, that lone pair repulsion and steric factors for MeOH as the leaving group are more important than the antiperiplanar effect for bond cleavage. All compounds in which the alpha-oxygen in the potential carbenium ion is replaced by sulfur undergo spontaneous cleavage regardless of the leaving group or structure of the compound. Energy profiles, DeltaH(), and DeltaH(R) values show that linear and cyclic thiocarbenium ions are much more stable than the corresponding oxocarbenium ions. Comparison of results for methyl ribosides and glucosides with results for corresponding pyridinium substrates suggests that both should hydrolyze through an A-1 mechanism. General-acid catalysis with hydronium as the acid was studied. With solution results, the computations suggest that substrates with either a good leaving group or stable oxocarbenium ion react with rate-limiting proton transfer from the acid to the leaving group but that substrates with both a good leaving group and stable carbenium ion react with concerted proton transfer and bond cleavage.     

Holographic projector using one lens

It is shown that the lens count in a Fourier holographic projector can be reduced by encoding the equivalent lens power in sets of Fresnel holograms. By using appropriately calculated Fresnel holograms in a reflective configuration to effectively share a lens between the beam-expansion and demagnification stages of a holographic projector, a reduction in lens count from four to one is demonstrated.