**2. Separation of components by means of gas chromatography**

Chromatographic techniques are based on separation of substances between a stationary and a mobile phase. The mobile phase moves relative to the stationary one. Components of a mixture to be separated move together with the mobile phase due to their different interactions with the phases.

**Figure 1.** Separation of a compound on a chromatographic column

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.

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

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

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

Chromatographic techniques are based on separation of substances between a stationary and a mobile phase. The mobile phase moves relative to the stationary one. Components of a mixture to be separated move together with the mobile phase due to their different interactions

**2. Separation of components by means of gas chromatography**

detection as well as chemiluminescence testing are included.

of mixture ingredients are also described.

**Figure 1.** Separation of a compound on a chromatographic column

increasing extent.

100 Column Chromatography

with the phases.

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 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 separation on-line.

group of SET assays. The HAT mechanism is of lesser importance in those assays [2]. SET assays include: DPPH, TEAC, FRAP, CUPRAC, DMPD, Folin-Ciocalteu; HAT assays include: ORAC, TRAP, CBA, β-carotene – linoleic model system. Those classified as "other" in literature include: cellular antioxidant activity (CAA), chemiluminescence, electrochemiluminescence,

Chromatography in Bioactivity Analysis of Compounds

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

103

*) 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

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.

Total Oxyradical Scavenging Capacity Assay (TOSCA) and others [3].

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

*3.1.1. 22-diphenyl-1-picrylhydrazyl (DPPH•*

resonance (EPR) spectroscopy.

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