Application of Second Order Derivative Spectroscopy in Determining Aromatic Amino Acid Content of Polypeptides in Size Exclusion HPLC with On-Line Photodiode Array Detection

Abstract

The ultraviolet (UV) absorbance and corresponding second order derivative spectra of various polypeptides are acquired in size exclusion high-performance liquid chromatography (SE—HPLC). Analytical columns are used, permitting detection at low microgram levels. UV-absorbance spectra of the polypeptide eluites are collected on-line and instantaneously with HPLC photodiode array detection techniques. These spectral data are automatically processed to yield second order derivative spectra, which are visually compared with second order derivative spectra of standards (i.e., phenylalanine, tyrosine, tryptophan, and their heterodipeptides) that have also been chromatographed in SE—HPLC. This comparison permits identification and corroboration of aromatic amino acids present in several different polypeptides of either known or unknown composition. Based on the features of the derivative spectra (e.g., presence, absence, amplitude, and wavelength of primary minima, secondary minima, and intersection of zero ordinate axis) flowcharts are designed that not only facilitate interpretation of the spectra in general but allow a more accurate identification of the aromatic amino acid content of the polypeptides.