# Selected Chromatographic Methods for Determining the Biological Activity of Substances
## 6 Bioassay  
Another way to test the biological activity of a substance in a single chromatographic step is biological detection. Biological detection methods are used to detect substances that exhibit biological activity. They can be divided into two groups: microbiological methods and microbiological methods. Microbiological detection methods test the activity of individual substances or mixtures of substances against microorganisms to determine whether they have the ability to inhibit the reproduction of certain microorganisms--specifically bacteria and fungi. Microbiochemical detection methods analyse the effects of substances or substance mixtures on biochemical reactions. Microbiochemical detection methods can be used to determine the inhibitory properties of enzymes and the antioxidant properties of substances or substance mixtures and to detect the hormonal effects of substances. The combination of thin-layer chromatography and biological detection is known as bioautography. Bioautography was first introduced as a detection method in chromatography by [PERSON] and [PERSON] in 1946 [116], who used paper chromatography with biological detection to estimate the purity of penicillin, and in combination with planar chromatography in 1961 by [PERSON] and [PERSON] [117] and [PERSON] et al. for the detection of antibiotics [118]. Nowadays, due to the variety of biological detection methods in chromatographic systems, the term bioautography is mainly used in relation to microbial detection, and when referring to microbiological detection, the term bioassay is used. Bioautography and biological tests are carried out in three stages:  
Stage I: Chromatography. This stage encompasses the preparation of the sample for analysis, application of the test samples to the chromatographic plate, and development of the chromatogram, resulting in a separation of the sample components. Stage II: Bioassays. These make it possible to determine which substances on the chromatographic plate exhibit a particular type of biological activity. Stage III: Identification of biologically active substances. This is based on a standard or on coupling of TLC with UV/VIS, MS, or NMR, IR detection. Three different types of bioautography can be implemented (Figure 7):  
TLC-CB (contact bioautography). This involves direct contact of the chromatographic plate with the corresponding microorganisms in a Petri dish after the development of the chromatogram with an agar layer. Substances from the chromatographic plate diffuse into the agar layer (a mirror image of the plate is obtained on the agar layer). Substances that exhibit a certain type of biological activity appear in spots that are a different colour than the background colour [119; 120; 121]. TLC-IBs (immersion/overlay bioassays). In this method, the surface of the chromatographic plate is covered with an agar layer containing the corresponding microorganism or enzyme after the chromatogram has been developed and dried. The substances from the plate diffuse into the agar layer. The plate with the agar layer is then incubated according to the requirements of the respective analysis. The biologically active substances become visible in the form of spots that are a different colour than the plate (background)--[121; 122]. In TLC-DBs (direct bioassays), after the chromatogram has been developed and dried, the chromatographic plate is sprayed or immersed in a solution containing microorganisms/enzymes and then subjected to an incubation process. If the microorganisms or enzymes do not show fluorescence or are not naturally coloured, the plate is sprayed with a suitable reagent to create a coloured background of the plate. The substances that exhibit a certain type of biological activity become visible on the chromatographic plate in the form of spots that are a different colour to the background. This variant of bioavailability is most frequently used in research [29; 121; 122; 123; 124; 125].