**Acknowledgements**

mine the activity of compounds relative to H2O2, a solution of luminol and H2O2 must be

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

People have made use of the properties of different compounds without realising their existence for a long time. Since science found ways to determine the nature of the effects exerted by bioactive compounds (e.g. medicinal plants) analytical methods have been perfected to enable more detailed analysis of the material. Analyses have focused on determination of the intensity of biological activity and on identification of the components responsible for the activity. Many materials have been analysed in search of bio-components. Mastering chroma‐ tographic methods has provided the possibility of high-resolution analysis of compounds, including their biological activity. The analytical methods which have been characterised here are the result of several dozen years of research into improving analytical methods in the search for biologically active compounds. The proposed classification is based on the mechanism of reaction observed in assays. The methods make use of reactions induced by the presence of radicals generated as initiators of oxidation reactions prevented by analysed bioactive compounds. It is not the only possible approach to looking for and analysing bioactive compounds. Various methods of analysing antioxidant activity which have not been men‐ tioned above have been applied on a marginal scale due to their drawbacks. Apart from that, there are methods of analysing biological activity which analyse substances capable of inhibition of/affinity to certain enzymes, e.g. acetylcholinesterase, phosphodiesterase, gluta‐ thione-S-transferase (EAD – Enzyme Activity/Affinity Detection), affinity of bioactive sub‐ stances receptors, e.g. estrogen receptor (RAD – Receptor Affinity Detection) [76, 77, 78, 79, 80,


prepared. The activity of the analysed components relative to O2

prepared for a standard antioxidant, e.g. Trolox.

**5. Conclusion**

114 Column Chromatography

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