Improved separation of poly(butylene glycol) oligomers on a C-18 column bygradient reversed-phase liquid chromatography and fine-tuning of the organic modifier system

Poly(butylene glycol) (PBG) samples of widely differing molecular weight (M ), either in the native form or as the corresponding alpha, omega-bis(1-nap hthyl)urethane derivatives, have been subjected to reversed-phase highperfo rmance liquid chromatography (gRPHPLC) on an octadecylsilyl (C-18) silica g el stationary phase with mobile phase gradients comprising binary eluent sy stems prepared from tetrahydrofuran (THF) and water and from acetone and wa ter, and with ternary systems prepared from acetonitrile, THF and water and from methanol, THF and water. Because of the lack of a chromophore in the PBG they were detected both by evaporative light-scattering detection (ELSD) and, after conversion to the alpha, omega-bis(1-naphthyl)urethane derivatives with 1-naphthylisocyanate, by UV monitoring at 280 nm. In general, sufficient resolution of the oligomers of low-M samples, i.e. P EG 650 and PEG 1000, was achieved with all the gradient systems used, and r esolution increased in the order acetone-water < THF-water < methanol-THF-w ater < acetonitrile-THF-water. Whereas the difference between chromatograph ic resolution (R-S) was relatively moderate for PEG 650 and PEG 1000 for al l the solvent-non-solvent combinations used, values were much larger for th e high-M samples PEG 2000 and PEG 3000 either in the native form or as thei r alpha, omega-bis(1-naphthyl)urethane derivatives. No separation of the hi gh-M oligomers of PEG 3000 was obtained with acetone-water and THF-water gr adients and a common signal envelope was observed for these sample constitu ents. In contrast, acetonitrile-THF-water and methanol-THF-water ternary gr adients effected satisfactory resolution of large amounts of oligomers and more than 70 oligomer peaks were recognizable. Chromatographic resolution ( R-S) of high-M oligomer was markedly better with acetonitrile-THF-water tha n with methanol-THF-water. Use of the alpha, omega-bis(1-naphthyl)urethane derivative of PEG 2000 as m odel component for fluorescence detection, with excitation and emission wav elengths of 232 and 358 nm, respectively, resulted in either improved detec tion sensitivity or much cleaner chromatograms and thus better selectivity compared with measurement of UV response at 280 nm. The effect of the different mobile-phase systems on the separations of thes e solutes is explained in terms of distribution of the salute molecules bet ween the bulk mobile phase and the hydrophobic network of C-18 chains, boun d to the surface of the silica gel base material. In this respect, the elue nt strength of an organic modifier increases with increasing solubility and /or solvation characteristics, in particular for the high-M fractions. It h ad nevertheless, to be taken into consideration that solvents that were too "good" afforded efficient sample elution on the one hand but markedly impa ired oligomer resolution on the other, as shown by separation of the high-M sample constituents by binary THF-water and acetone-water gradients.