**3.1. Single Electron Transfer (SET) methods**

used which does not change the structure or properties of the compounds under analysis. The most popular one in such cases is a UV-Vis detector. Spots of substances being separated by paper or thin-layer chromatography are observed under visual or UV light, in their natural form or after transformation into a coloured compound. Both TLC and column chromatogra‐ phy are used in analysis of antioxidant compounds. In TLC, substances previously separated on the plate affect the intensity of colour of the radical placed on it. In liquid chromatography, tests of antioxidant activity of different components can be performed after they are separated in a pure state, by performing post-column off-line reactions or during the chromatographic

**Figure 2.** Schematic diagram of the High Performance Liquid Chromatography (HPLC) system

In search of bioactive substances, researchers have directed their interest towards substances found in plants. Parts of plants which have been used in natural medicine have proved to be a rich source of bioactive compounds; however, to make use of them, they have to be isolated and their properties determined. Using selective techniques of extraction has resulted in obtaining concentrated preparations of bioactive substances. To achieve comprehensive knowledge of their properties, it was necessary to develop methods of isolation of individual components and testing these methods. This could be done with chromatographic techniques. Isolated compounds were tested in order to show which of them (and to what extent) are responsible for bioactivity of plant preparations from which they were obtained. Due to the fact that many of the substances have the opposite effect, it is frequently impossible to use

*In vitro* tests, used in evaluation of antioxidant properties make use of the ability of antioxidants to quench free radicals. Based on this mechanism, the methods are divided into two groups: SET – single electron transfer, and HAT – hydrogen atom transfer. Reactions with antioxidants in assays with the DPPH radical, ABTS and the Folin-Ciocalteu reagent both operate according to the SET and HAT mechanism. Due to the kinetics of the reaction, they are included in the

**3. In vitro analysis of biological activity of substances.**

extracts without isolating individual compounds.

separation on-line.

102 Column Chromatography

#### *3.1.1. 22-diphenyl-1-picrylhydrazyl (DPPH• ) assay*

Analysis of antioxidant properties relative to the DPPH• radical involves observation of colour disappearance in the radical solution in the presence of the solution under analysis which contains antioxidants. A solution of extract under analysis is introduced to the environment containing the DPPH• radical at a specific concentration. A methanol solution of the DPPH• radical is purple, while a reaction with antioxidants turns its colour into yellow. Colorimetric comparison of the absorbance of the radical solution and a solution containing an analysed sample enables one to make calculations and to express activity as the percent of inhibition (IP) or the number of moles of a radical that can be neutralised by a specific amount of the analysed substance (mmol/g). In another approach, a range of assays are conducted with different concentrations of the analysed substance to determine its amount which inactivates half of the radical in the test solution (EC50). The duration of such a test depends on the reaction rate and observations are carried out until the absorbance of the test solution does not change [4]. If the solution contains substances whose absorbance disturbs the measurement, the concentration of DPPH• radical is measured directly with the use of electron paramagnetic resonance (EPR) spectroscopy.

The disadvantage of the method is the fact that there are numerous modifications in the literature. These include using radical solutions at different concentrations, different reaction times and sometimes even a different reaction environment. The effect of exposure to light, the presence of oxygen, pH and the type of solution on the stability of the DPPH• radical have been studied by Ozcelik and co-workers [5]. The study methods developed so far employ radical concentrations ranging from 6\*10-5 to 2.0\*10-4 mol/dm3 [6, 7, 8, 9, 10]. Depending on the method, absorbance of the reaction mixture is measured at the wavelength ranging from 515 nm to 550 [11, 12] after a specified reaction time, e.g. 5 min [13], 10 min [14], 16 min. [9], 20 min [7] 30 min [15], 60 min [16]. As Sánchez-Moreno and coworkers [17] found in their study, the time after which the absorbance of the analysed solution takes place depends on various factors, e.g. on the antioxidant concentration. For individual standards, they determined the time needed to reach the plateau at an antioxidant concentration of EC50, i.e. such that is necessary to achieve 50% inactivation of the DPPH• radical. In order to make the results comparable, researchers frequently express them as Trolox equivalent [18]. The application of the assay is limited by the fact that the DPPH• radical is non-polar and as such, it is soluble in organic solvents. Noipa and co-workers [19] proposed to modify the method by using a cationic surfactant, which enabled analysis of the antioxidant activity of hydrophilic antioxi‐ dants contained in water extracts in the micelles formed in a solution.
