Particle Beam LC/FT-IR Spectrometry Studies of Biopolymer Conformations in Reversed-Phase HPLC Separations: Native Globular Proteins

Abstract

A particle beam LC/FT-IR interface has been employed in the investigation of the effect of chromatographic conditions (e.g., mobile-phase composition, elution process, and stationary phase) on protein secondary structure in reversed-phase HPLC. The ability of the particle beam to obtain IR spectra which are highly representative of protein elution conformation from an HPLC column is demonstrated. Qualitative and semiquantitative measurements of major IR amide bands made from the band intensity and position enabled assessment of the secondary structure content of the eluting protein. Spectra obtained from gradient elutions with n-alkyl silica columns show a degree of randomization during separation. When deposits were redissolved into water and evaporated, however, they reverted to a structure close to the native structure. An n-octadecyl column preserved β-sheet structure, while an n-butyl retained more α-helix structure. IR spectra of lysozyme collected from isocratic elutions with varying concentrations of acetonitrile and 1:1 2-propanol/acetonitrile showed that, when pure acetonitrile was used as the modifier, a partial structural alteration from α to β at high organic concentrations occurred but reverted to α upon reevaporation. No change in conformation was observed with the 2-propanol/acetonitrile mobile phase. With both elution processes, secondary structure changes caused by the chromatography were reversible.