*4.2.3. On-line HPLC-CUPRAC assay*

to the standard curve plotted for Trolox) is the basis for expressing the result as its equivalent. An apparatus which can be used to conduct such an analysis should – like the basic HPLC set – consist of the main pump (feeding mobile phase to the system), an injecting device (injecting the sample), a column (which may be placed in a thermostat), a detector and a recording device. However, additional equipment must be used apart from the HPLC set. A solution of the DPPH• is fed through an additional pump and, together with eluate, leaves the HPLC system to the mixer. Mixed substances are transferred to the reaction coil. A reaction coil, which is a capillary tube with a length ranging from 0.2 to 15 m, is where a reaction takes place between

the mixture components and the DPPH reagent.

112 Column Chromatography

the substances as equivalents of the antioxidant.

*4.2.2. On-line HPLC-ABTS•+ stable radical decolorization*

**Figure 6.** UV and DPPH radical quenching chromatogram of a plant methanolic extract.

Bartasiute and co-workers [66] analysed the method with capillaries of different lengths. The shortest capillary used in their experiment (0.2 m) ensured sufficient reaction time. However, specific properties of analysed mixtures should be taken into account, whose components may react differently. The last element is another detector which records the characteristic signal of the DPPH• solution [65, 66, 67, 68]. Fading of a radical signal after reaction with an antiox‐ idant (visible as a negative peak) is proportional to its oxidative force. Comparison of the surface area of the peak with the calibration curve prepared for Trolox shows the activities of

Using the ABTS•+ cation radical in the analysis of activity of mixture components in conjunction with HPLC was proposed in 2001 [69]. That method is similar to the HPLC-DPPH• assay described above, but its sensitivity is higher. Components separated on an analytical column are transferred with a solution of the ABTS•+ cation radical to the reaction coil. A capillary with a length ranging from 1.5 to 15 metres is placed where the flow-through reaction of the radical quenching by the oxidants present in the sample takes place. The capillary length is selected depending on the expected reaction time. The signal characteristics of the ABTS•+ radical solution are recorded at 734 nm [69, 70] and in subsequent modifications – at 720 nm and 747 nm [16, 71, 72]. The antioxidant activity of individual compounds is determined based on the size of negative peaks which show the compound's ability to inactivate the radical. Compar‐ ison of the surface area of a negative peak caused by the presence of the analysed compound An on-line HPLC-CUPRAC assay has been proposed by Çelik and co-workers. They used a chromatographic set with configuration used in on-line analysis of antioxidant properties with the DPPH• and ABTS•+ radicals. Unlike the methods with the DPPH• and ABTS•+ radicals, the assay is based on measurement of the growth of the solution colour intensity [73]. As in the off-line method, the Cu(II)-Nc reagent reacts with an antioxidant and is reduced to a yellow complex Cu(I)-Nc. The solution containing the complex has the absorbance with the maximum at 450 nm. The compounds separated chromatographically in the developed on-line method are mixed with the Cu(II)-Nc reagent in a mixer and are subsequently transferred to the reaction coil where a oxidation-reduction reaction takes place during its flow through the capillary. The antioxidant activity of individual compounds is observed as an increase in the signal on the detector at 450 nm. When a calibration curve is plotted with data obtained for Trolox, it is possible to express the compound's activity as an equivalent of Trolox activity [73].
