**1. Introduction**

Chromatographic techniques have led to considerable development in mixture ingredient analysis. With the development of stationary-phase and high-pressure module technologies, new possibilities have emerged for the separation of complex systems whose components are characterised by similar structures and properties. Modern detection systems now allow detection and identification of individual ingredients. Data on their electrical properties, characteristic ways of molecule ionisation, and their ability to absorb or emit electromagnetic waves are used for this purpose (among others). The possibilities offered by chromatographic techniques are also used to isolate mixture ingredients for further analysis or to compose mixtures with predesigned properties. Appropriate tests preceded by isolation of analysed ingredients from the biological matrix are applied to determine the characteristics of compo‐ nents of analysed biological samples. In some circumstances this can be very arduous because the separation process itself is time- and cost-consuming or it is necessary to have appropriate infrastructure. Preparative-scale chromatographic separation can be conducted using the techniques of preparative thin-layer chromatography (prep-TLC), preparative column chromatography (prep-LC) as well as preparative-scale high-performance liquid chromatog‐ raphy (prep-HPLC). Isolated fractions or purified compounds can serve as material for determination of their biological properties. Currently, researchers are greatly interested in methods of analysis and identification for compounds which are antioxidants or inhibitors of specific transformations in the biochemistry of living organisms. Ingredients isolated from the material are purified by solid-phase extraction (SPE), thin-layer chromatography (TLC), preparative column chromatography (prep-LC) and preparative-scale high performance liquid chromatography (prep-HPLC). The obtained compounds or fractions are the subject of research to determine their biological properties in tests with DPPH, ABTS, AAPH radicals, the Folin-Ciocalteu reagent, etc. This procedure is cost-, time- and labour-consuming and the obtained results may be uncertain. This uncertainty is connected with the fact that during

© 2013 Czaplicki; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Wertheim, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

isolation, compounds are exposed to environmental factors such as oxygen, light, increased temperature. As a result the isolated compound may not have such a chemical structure as before isolation. This can cause a change in the biological activity of the examined substances. A combination of chromatographic ingredient separation methods with the detection of biochemical properties provides great possibilities for examination of the compounds present in complex biological systems. Methods have emerged which use the advantages of a solution to determine the ingredients of analysed mixtures with regard to both quality and the antioxidant activity detection. These methods have become very useful, e.g. during identifi‐ cation of the biological activity of plant extracts. The search for chemical compounds with desired biological properties by coupling chromatographic methods with biochemical detection has immense possibilities. The merits of this solution are currently used to an increasing extent.

Depending on the technique applied, the stationary phase can be: column packing (column chromatography), thin layer of active substances put onto a plate (alumina or predominatingly silica gel) (thin-layer chromatography) or absorbent paper (paper chromatography). A mobile phase is a liquid which moves relative to the stationary phase. To be separable by this technique, components of a mixture must be soluble in the mobile phase. Depending on the interactions between the components and individual phases, the components move faster or more slowly together with the mobile phase along the stationary phase. The speed of move‐ ment depends on the strength of interactions between the components and each of the phases.

Chromatography in Bioactivity Analysis of Compounds

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Such interactions cause sorption and desorption of the components from the mobile to the stationary phase. For the separation to be effective, the speeds of movement of components relative to the stationary phase must be different. The simplest solutions are based on free flow of solvent, but the system effectiveness can be improved by using pressurised flow. Upon leaving the chromatographic system, the separated components flow to detectors, where their specific properties are used to observe their presence, amount, and sometimes event to identify them. UV-Vis detectors, measuring absorbance of the solution which leaves a chromatographic column, are among the most widely-used devices. Components of a solution are observed as increased light absorption by the solution flowing through a quartz flow cell. With a detector of this type, it is possible to observe elution of individual mixture components on the "presentabsent" basis. An analyst is presented with better capabilities when using a version of the detector with a photodiode matrix. Owing to a photodiode detector (PDA), it is possible to observe light absorption within the UV-Vis range at different wavelengths simultaneously. Moreover, it is possible to conduct observations of the absorption spectrum within a selected part of the UV and visible range. Owing to that property, it is possible to conduct simultaneous observation of elution of compounds which absorb light to a different extent at different wavelengths and, additionally, the detector is more selective. It is very important that identification of the separated compounds can be conducted based on the spectra. This is possible because individual compounds have specific absorption spectra depending on the bond structures and function groups. A less popular detection technique makes use of the ability of compounds to emit light. Fluorescence detectors are used where separated com‐ pounds specifically emit energy after their excitation. These detectors are highly selective and sensitive, which is essential when other mixture components are co-eluted with the substances being determined. In such cases, the detection parameters are set to make the excitation or emission wavelength match the analysed compounds. Mentioned detectors make use of the ability to absorb or emit light, but other detection techniques are also used depending on the properties of the analysed compounds. These include the following types of detectors: refractometric – the signal is measured as a change of the light refraction coefficient caused by optically active substances; electrochemical – recording a change of the electric potential; detector of dispersed light – measuring the intensity of dispersion of a laser beam by molecules of the substance being separated; mass detectors – analysing compounds following their ionisation [1]. Apart from those mentioned here, which are the most popular, other techniques of detection are also applied, with different selectivity and using different properties of the analyte and with different degrees of sample degradation. If liquid chromatography is used to obtain mixture components with a view to further analysis, a method of detection must be

This paper presents analysis methods for compounds to determine their general biological activity. Characteristics of the methods in model systems using different antioxidant reaction mechanisms are also described. Both colorimetric methods and those with fluorometric detection as well as chemiluminescence testing are included.

The main element in the paper is presentation of the possibilities for using the liquid chroma‐ tography technique for screening compounds with regard to their biological properties. Examples of different uses of chromatographic methods in on-line analysis of the bioactivity of mixture ingredients are also described.
