ABSTRACT
An important diagnostic tool in this regard is the use of on-line Ultraviolet (UV) spectroscopy to assess conformational states and purities of chromatographic peaks. This approach can either involve the examination of the absorbance ratio at two separate wavelengths or the second derivative spectrum, in both cases at the apex or across the peak. For both types of measurements rapid wavelength scanning is required, and this conventionally means the use of a photodiode array spectrometer. Classically, UV spectroscopy has been employed to assess conformational changes of proteins as a function of alteration in conditions, e.g., temperature, pH, organic solvent. Increases in the intensities of the aromatic amino acids — phenylalanine, tyrosine and tryptophan — occur upon exposure of these residues to the aqueous solvent. As a general rule, unfolded species are eluted later than folded species in a chromatogram in reversed-phase chromatography and hydrophobic interaction chromatography due to the greater contact area of the former species with the adsorbent surface.
