In HPLC analysis, seperated of compounds, given overlapped peaks using DAD, on-line FRAP and peroxynitrite reaction

In this study, 16 phenolic acids (rosmarinic acid, benzoic acid, p-OH benzoic acid, protocatecuic acid, protocatecualdehyde, syringic acid, synapic acid, p-cumaric acid, ferulic acid, caffeic acid, gentisic acid, vanillic acid, vanilline, gallic acid, syringaldehyde, chlorogenic acid) were overlapped by changing solvent gradient and time at 25 °C. After the analysis of the overlapping peaks with DAD, the retention times of the phenolic acids were determined. The phenolic acids having overlapping peaks were resolved by spectral differences. For the overlapping phenolic acids, compositions were determined from the graphs of percentage composition versus absorbance ratios at selected wavelengths. The wavelengths at which resolution could be attained were determined, the standard mixtures with non-overlapping composition were prepared, and on-line FRAP activities of phenolic acids at ten different concentrations were determined. Qualitative seperation of chlorogenic acid ? p-OH benzoic acid and vanilic acid ? caffeic acid pairs was realised from the peaks at 595 nm as only one was active, i.e. chlorogenic and caffeic acids. Vanillin ? p-cumaric acid pair could not be resolved as both were inactive. Because both components of gentisic acid ? protocatecualdehyde and ferulic acid ? synapic acid pairs were active, %molar compositions of these pairs were calculated from the graphs of ratios of peak areas at 280 and 595 nm versus component percentage. Additionally, standards were reacted with peroxynitrite separately at pH 7.4 and after inactivating peroxynitrite by premixing with 1 M HCl and then injected to HPLC system, and peaks were resolved based on reactivities towards peroxynitrite with peak disappearings and peak transitions.