## 5 Hyphenation with Spectroscopic Methods of Identification  
The hyphenation of (HP)TLC with spectroscopic and spectrometric techniques makes the characterization of bioactive substances possible. HPTLC combined with bioassays and linked to spectroscopic methods of identification, allows the quantitative detection of bioactive compounds (as bands) in samples and their further chemical characterization by mass spectrometry or other spectroscopic methods (Figure 1). Thus, highly efficient non-targeted screening with bioassays can be used to detect modes of action followed by the targeted chemical identification of newly discovered bioactive compounds. Non-targeted analysis is superior to target analysis, if the whole sample (botanical extract) is of interest.  
A broad range of bacterial and fungal strains have been investigated for biological activity using appropriate detection methods with planar chromatography. This is referred to as bioautography [12].  
\begin{table}
\begin{tabular}{l l l} \hline \hline
**Detection** & **Activity** & **Test** \\ \hline \multirow{3}{*}{Microchemical detection} & \multirow{3}{*}{Free radical scavenging} & DPPH free radical \\  & & ABTS radical scavenging test \\ \cline{2-3}  & Antioxidants & \(\beta\)-Carotene bleaching test \\ \hline \multirow{3}{*}{\begin{tabular}{l} Microbial detection \\ (bioautography) \\ \end{tabular} } & \multirow{3}{*}{Bacterial culture} & _Bacillus subtilis_ (antibacterial test) \\  & & _Aliiibrio fischeri_ or _Vibrio fischeri_ (toxicity test) \\ \cline{2-3}  & Fungal culture & \
\begin{tabular}{l} p-YES test (planar yeast estrogen screen) \\ estrogenic activity \\ \end{tabular} \\ \hline \multirow{3}{*}{Biochemical detection (bioassays)} & \multirow{3}{*}{Enzyme inhibition} &
\begin{tabular}{l} Cholinesterase inhibitors \\ \(\alpha\)-amylase inhibitors \\ \(\alpha\)- and \(\beta\)-glucosidase inhibitors \\ Xanthine oxidase inhibitors \\ Tyrosine inhibitors \\ \end{tabular} \\ \hline \hline \end{tabular}
\end{table}
Table 1: Effect-directed analysis of plant material.  
Figure 1: Overview of the HPTLC-EDA approach.  
Several HPTLC-enzyme inhibition assays have been developed. For example, \(\alpha\)-amylase [13], cholinesterase [14] and glucosidase enzymic inhibition [15] methods have been reported. Hyphenation with mass spectrometry (MS) and, more recently, with nuclear magnetic resonance (NMR) has enabled the rapid characterization of the separated compounds. In the past, unknown substances were scraped off from the plate, eluted into a vial and transferred into a MS. Now elution-based TLC-MS interfaces can be used to extract zones of interest. The manually operated TLC-MS interface is equipped with an elution head, where pump-driven elution solvent directs the eluted zone directly online into an MS or HPLC-MS, or into a vial for the off line structural identification of the organic compound using either HPLC-MS/MS, gas chromatography-MS or NMR [16]. Thus, structure identification methods can be run at the analytical level directly from the detected bioactive zone of interest on the chromatogram. The advantage of this new strategy is that only bioactive components are isolated and characterized.