Retention properties of the fluorinated bonded phase on liquid chromatography of aromatic hydrocarbons.

The unique characteristics of a bonded-fluoroalkylsilane column, Fluofix, are described by comparison with those of a bonded octylsilane column, C8, in the reversed-phase chromatography of aromatic hydrocarbons. The selectivity of homologous aromatics on the fluorinated column varied with the methanol concentration in the eluent. At a larger proportion of methanol than 80:20 (v/v) the larger aromatics eluted faster than the smaller ones. However, with less methanol, the aromatics were eluted in order of their molecular size, as usually reported for a conventional hydrocarbonaceous column. A dependence of the retention mechanism on the composition of the eluent was suggested by the decrease in the entropy-enthalpy compensation temperature, beta, with increase of methanol concentration in the eluent. In order to explain the concentration dependence of the retention, the molecular interaction energy in the retention process was calculated by computer simulation. The interaction energy between aromatics and the stationary ligand on the Fluofix column was smaller than that on the C8 column and comparable to the interaction energy between the aromatics and methanol. At higher methanol concentrations, solute-fluorinated ligand complex formation was obstructed by the methanol molecules solvating the solute, reducing the retention of the larger aromatics.

Subsite structure of the beta-glucosidase from Aspergillus niger, evaluated by steady-state kinetics with cello-oligosaccharides as substrates.

The beta-glucosidase from a commercially available preparation from Aspergillus niger was highly purified. The Michaelis constant Km and the molar activity K0 for cello-oligosaccharide substrates Gn (n = 2-6) were obtained by steady-state kinetic analysis on the beta-glucosidase-catalyzed hydrolysis at 25 degrees C and pH 5.0. Stoichiometric production of Gn-1 by the beta-glucosidase reaction for Gn was confirmed by HPLC techniques. Based on Km and K0 for Gn, subsite affinities (Ai, i = 1-6) were estimated as follows (kcal/mol): A1 = 1.3, A2 = 5.2, A3 = 0.65, A4 = -0.10, A5 = -0.65, and A6 = -0.26, of which A1-A3 are much higher than those of the beta-glucosidase of Candida wickerhamii. The subsite structure is quite similar to that of the alpha-glucosidase of A. niger, whereas the dependence of k0 on n is highly characteristic for beta-glucosidase, and decreases with n, suggesting some interaction between the particular subsites.

Microbore packed-column anion-exchange liquid chromatography of nucleobases, nucleosides and nucleotides.

Low-capacity anion-exchange resin was packed into a 45 cm X 0.19 mm I.D. fused-silica tubing and applied to micro-column liquid chromatography of nucleobases, nucleosides and isomers of nucleotides. The effects of the chromatographic conditions on the elution behaviour of these compounds were studied. The efficiency of the microbore packed column was comparable with that of the conventional size column.

Studies of short-lived radicals in the gamma-irradiated aqueous solution of uridine-5'-monophosphate by the spin-trapping method and the liquid chromatography.

An aerated aqueous solution of uridine-5'-monophosphate was gamma-irradiated with 2-methyl-2-nitrosopropane as a spin-trapping reagent. Liquid chromatography was applied to separate the stable nitroxide radicals in the irradiated solution. The radicals were detected by U.V. and e.s.r. spectrometry. The e.s.r. detection showed four peaks in the chromatogram. The orcinol method for detection of the residual sugar moieties was applied before and after reduction of the base to determine the existence of the 5,6-double bond for the molecules in each fraction. From the combined results of the e.s.r. and orcinol methods, the short-lived radicals which were trapped by 2-methyl-2-nitrosopropane were identified as radicals of N-1 and C-6 positions of the base moiety and t-butyl radical which was the radiolytic product of the trapping reagent.