**4.1. TLC-methods for antioxidant activity analysis**

Analysis of the biological activity of extracts by the methods presented above provides a researcher with a pooled result of the activities of all the components of a mixture. When analysing extracts of evening-primrose and starflower, Wettasinghe and Shahidi [50] made use of the experience gathered by Amarowicz and co-workers in fractionation of plant extracts [51, 52, 53] to achieve more precise characteristics of components of the extracts under analysis. They separated extracts by column chromatography with Sephadex LH20 column packing. As a result, they obtained six fractions, which they further analysed to determine their biological activity [50].

tyofconductingscreeninganalysesinwhichmanyextractscanbeanalysed.Thisenableseffective

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**4.2. The use of high performance liquid chromatography as a tool for bioactivity analysis**

When seeking a tool which could be used to determine the biological activity of mixture components in a more precise way, researchers directed their attention towards high-per‐ formance liquid chromatography (HPLC). Its advantage over TLC is its higher resolution,

and cheap searching for bioactive substances in unknown samples [58, 59, 60, 61].

which helps to avoid false results caused by the co-elution of different compounds.

**Figure 5.** Instrumental setup for on-line HPLC radical scavenging assay

 *stable radicals decolorization*

a chromatographic column react with a radical in a reaction coil.

Initially, the use of HPLC in analysis of antioxidant properties with the DPPH• radical was restricted to chromatographic analysis of the radical content in solution. An assay was per‐ formedinwhichasolutionoftheradicalwastreatedwiththeextractunderanalysis.Thereaction ran in a reaction tube and the remainder of the radical after the reaction was analysed chroma‐ tographically.Acomparison ofthe radical contentin the blank sample andin the extract sample showed the amount of radical that was quenched by antioxidants in the analysed sample [62, 63].However,themethoddidnotprovidemoreinformationthanthecolorimetricmethod.Much better results are obtained in a post-column on-line reaction in which substances separated on

A detector records a signal at 515 or 517 nm [64, 65]. Depending on the antioxidant activity of the separated substances, greater or smaller signal fading can be observed, resulting in a negative peak. The surface area of the peak, proportional to the antioxidant activity (compared

*4.2.1. HPLC-DPPH•*

Separation of analyte fractions by column chromatography requires time, labour and money and the results show only properties of the properties of compounds in individual fractions. Using thin-layer chromatography in analysis of antioxidant activity of mixture components made it unnecessary to isolate them prior to analysis. Researchers from Kansas State University made use of the experience gathered by Marco [54] and Taga and co-workers [55] and proposed a method of determination of antioxidant activity of individual components of mixtures using the β-carotene bleaching assay for substances previously separated by TLC. They sprinkled β-carotene solution with linoleic acid on substances separated on a plate. They exposed the prepared plates to light and observed the disappearance of the orange colour of β-carotene. Spots with antioxidants were visible as ones with more intense colour because of their protective effect on β-carotene [56].

**Figure 4.** Compounds resolved on the TLC plate after spraying with DPPH methanolic solution.

Glavind and Holmer proposed a method of determination of antioxidants by TLC using the DPPH• radical. They sprinkled a plate with separated substances with methanol solution of the radical and observed discoloration where substances able to quench radicals were present [57]. The TLC-DPPH assay allows a researcher to access the analysed substances and to assess the biological activity of individual compounds. Another advantage of the method is the possibili‐

tyofconductingscreeninganalysesinwhichmanyextractscanbeanalysed.Thisenableseffective and cheap searching for bioactive substances in unknown samples [58, 59, 60, 61].
