High-throughput determination of octanol/water partition coefficients using a shake-flask method and novel two-phase solvent system.

A high-throughput method for determining the octanol/water partition coefficient (P(o/w)) of a large variety of compounds exhibiting a wide range in hydrophobicity was established. The method combines a simple shake-flask method with a novel two-phase solvent system comprising an acetonitrile-phosphate buffer (0.1 M, pH 7.4)-1-octanol (25:25:4, v/v/v; AN system). The AN system partition coefficients (K(AN)) of 51 standard compounds for which log P(o/w) (at pH 7.4; log D) values had been reported were determined by single two-phase partitioning in test tubes, followed by measurement of the solute concentration in both phases using an automatic flow injection-ultraviolet detection system. The log K(AN) values were closely related to reported log D values, and the relationship could be expressed by the following linear regression equation: log D=2.8630 log K(AN) -0.1497(n=51). The relationship reveals that log D values (+8 to -8) for a large variety of highly hydrophobic and/or hydrophilic compounds can be estimated indirectly from the narrow range of log K(AN) values (+3 to -3) determined using the present method. Furthermore, log K(AN) values for highly polar compounds for which no log D values have been reported, such as amino acids, peptides, proteins, nucleosides, and nucleotides, can be estimated using the present method. The wide-ranging log D values (+5.9 to -7.5) of these molecules were estimated for the first time from their log K(AN) values and the above regression equation.

Counter-current chromatographic separation of nucleic acid constituents with a hydrophilic solvent system.

Nucleic acid constituents such as nucleobases, nucleosides and nucleotides were separated by counter-current chromatography using type J coil planet centrifuge. The separation was performed with a hydrophilic solvent system composed of 1-propanol/800 mM potassium phosphate buffer (pH 7.4) (1:1, v/v) by eluting the lower aqueous phase at a flow-rate of 0.5 ml/min. Eight selected nucleic acid constituents (4.0 mg, 0.5 mg of each), uridine monophosphate (UMP), adenosine monophosphate (AMP), deoxyadenosine monophosphate (dAMP), uridine, urasile, deoxy uridine, adenosine and adenine were well resolved within 160 min.