Evaluation and Comparison of Two Improved Techniques for the On-line Detection of Antioxidants in Liquid Chromatography Eluates

Two methods for the on-line detection in HPLC eluates of analytes possessing radical scavenging activity were improved and compared. The instrumental set-up of the method that is based on on-line inhibition of luminol chemiluminescence (CL) by antioxidants was improved using better quality syringe pumps, employing a diode array detector, and introducing a mixing/neutralisation coil and a pulse damper. Sensitivity of the HPLC-CL detection increased by a factor of 4. Post-column neutralisation of eluates improved compatibility of this detection method with acidified HPLC eluents. The second method, which is based on the post-column quenching of 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH*), was improved by readjusting composition and flow-rate of the reagent, mounting an additional pulse damper and detecting unreacted DPPH* with a detector equipped with a tungsten lamp. Purging of the DPPH* solution with He gas prior to analysis was introduced. This led to 30-fold better detection limits. The improved methods were compared with respect to limits of detection, the radical scavenging mechanism involved, compatibility with common HPLC solvents and pH range, and some technical aspects. The techniques described have high potential for the rapid identification of radical scavengers in complex samples like plant extracts.