**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, 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

#### *4.2.1. HPLC-DPPH• stable radicals decolorization*

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

biological activity [50].

110 Column Chromatography

protective effect on β-carotene [56].

DPPH•

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

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

**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

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

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 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.

with the curve prepared for Trolox enables the activity of compounds to be expressed as the

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

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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].

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

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


Trolox equivalent.

*4.2.3. On-line HPLC-CUPRAC assay*

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

unit, i.e. the equivalent of a standard antioxidant, e.g. Trolox.

antioxidant activity of the separated compounds relative to H2O2 and O2

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

**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 the substances as equivalents of the antioxidant.

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

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

with the curve prepared for Trolox enables the activity of compounds to be expressed as the Trolox equivalent.
