*4.2.4. On-line HPLC Crocin Bleaching Assay (HPLC-CBA)*

The Crocin Bleaching Assay (CBA) in an off-line colorimetric version provided the base for developing the On-line HPLC-Crocin Bleaching Assay [74]. When a high-resolution method of compound separation is used, CBA can be used to determine the activities of each mixture component. The results are not affected by other mixture components, which improves the usability of the method for an objective assessment of bioactive components of mixtures. Like the off-line method described earlier, the authors of this method proposed crocin as the oxidation indicator and the AAPH reagent as the source of the radical. Antioxidants in a sample prevent oxidation of crocin by inactivating radicals generated by the AAPH reagent, as was the case in the colorimetric method. The signal recorded by the detector for 440 nm shows the antioxidant activity of each compound as chromatographic peaks with a surface area propor‐ tional to their activity. The mixture of crocin and the AAPH reagent was kept at 0ºC before being transferred to the system. The reaction mixture was combined on-line with eluate from the chromatographic column and the reaction between compounds ran during their flow through the reaction coil at 90ºC. The reaction parameters have a great effect on the interference caused by the detector; hence, the authors optimised the method, showing that the interference is affected by: instability of the reaction temperature, change of the AAPH:crocin ratio, the presence of air or nitrogen bubbles in the reaction coil and changes in the mobile phase composition [74]. Like other methods, it seems justified to express the results in a universal unit, i.e. the equivalent of a standard antioxidant, e.g. Trolox.

### *4.2.5. On-line chemiluminescence detection (HPLC-CL)*

A sensitive on-line chemiluminescence method, "on-line HPLC-CL", was developed by Toyo'oka and co-workers [75]. This method helps to determine with high sensitivity the antioxidant activity of the separated compounds relative to H2O2 and O2 -•. In order to deter‐ mine the activity of compounds relative to H2O2, a solution of luminol and H2O2 must be prepared. The activity of the analysed components relative to O2 -• is measured in the system of reagents containing a mixture of luminol and hypoxanthine as well as a mixture of xanthine oxidase and catalase. The solutions, fed with two pumps, are mixed in a mixer before being joined with a stream of chromatographically-separated components which leave the column. Combining the streams of reagents and the analyte starts the reaction of radical quenching of the analysed sample by antioxidants. A decrease in the amount of radicals results in a decrease in the luminol luminescence intensity recorded by the detector. The compounds able to quench the radicals cause the signal to deviate from the base line, which is observed as negative peaks. The surface area of the peaks is proportional to the antioxidant activity of the analysed compounds. As is shown in the description, the method requires proper apparatus. The HPLC set, necessary to separate the mixture components under analysis has to be fitted out with two separate pumps and a mixer. The stream of eluate from the analytic column and the reagents are joined in a mixing module, after which the mixture is transferred to the reaction coil. When it flows through the reaction coil, radical capture reaction takes place and the other radicals react with luminol. The radiation generated in this way is recorded by a photomultiplier. A negative chromatogram generated in this manner is used as the basis for assessment of antioxidant activity of the separated compounds. Like the methods described above, the assessment is based on a comparison of surface area of peaks with the calibration curve prepared for a standard antioxidant, e.g. Trolox.

81, 82]. Conjunction of the chromatographic methods of component separation with methods of analysis of biological properties provides great opportunities in their analysis. This has made the search for bioactive substances easier and will aid the future development of new

Chromatography in Bioactivity Analysis of Compounds

http://dx.doi.org/10.5772/55620

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This study was supported by the Grant N N312 466340 from the National Science Centre,

Chair of Food Plant Chemistry and Processing, Faculty of Food Sciences, University of War‐

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**Author details**

Sylwester Czaplicki

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**Acknowledgements**
