## 3 Effect-Directed Analysis (EDA)  
EDA is a combined approach, linking biological and chemical analytical methods. The aim is to identify which compounds present in a natural sample are bioactive using a direct chemical analysis, and then identify only those compounds with significant target effect. After a specific biological effect is detected in a bioassay, chemical fractionation (i.e., chromatographic separation) is used to reduce the complexity of the sample (e.g., plant extracts), to bioactive chemical classes and, eventually, to individual compounds. Once fractionation has reduced the sample matrix so that the cause of the biological effect is isolated, the compounds present in the fraction are identified. Chromatographic separation of the sample allows for easier chemical analysis and better identification of the unknown compounds. EDA is an effect-directed nontargeted analysis, used in screening for natural products with bioactivity from complex samples, like botanical extracts, in drug discovery processes. The chemical characterization of plant components is usually linked to liquid chromatography, column or planar, to enable effective separation and isolation of its constituents. In recent decades the technical developments that have allowed hyphenations of planar chromatography have enabled the parallel biodetection, bioprofiling and chemical characterization of individual bioactive constituents from botanical extracts. High Performance Thin Layer Chromatography/Thin Layer Chromatography (HPTLC/TLC) and Effect-Directed Analysis (EDA)  
The most popular separation method for effect-related analysis is thin-layer chromatography (TLC). TLC is a relatively old, well established, chromatographic separation technique, that is ideally suited for the high-throughput screening of highly complex samples, such as plant extracts. The simple concept of TLC allows the parallel analysis of many samples in a single run (on the same plate) in contrast to high performance liquid chromatography (HPLC) or gas chromatography (GC) where samples must be analyzed sequentially. Thus, with TLC, multiple samples can be analyzed in parallel, under identical laboratory conditions, providing a parallel profiling of samples, which allows for enhanced recognition of spot similarity [1]. Profiling samples in this way also allows for the parallel evaluation of different effect-directed assays. TLC is a matrix tolerant method. Crude extracts can be directly applied on the plate with minimal or no sample preparation and possible loss of sample components [2]. Thus, all sample components separated on the plate can be directly evaluated using a bioassay, enabling sample bioactivity to be maintained and accurately assessed. The possibility of multiple detections (UV/Vis absorption and fluorescence measurements) without the need to repeat separation, provides more information about separated compounds. TLC is the only chromatographic method that offers the visual evaluation of the chromatogram. High-performance thin-layer chromatography (HPTLC) is an enhanced form of TLC, which consists of the automatization of instrumentation and increased chromatogram resolution. This advanced form of TLC enables the quantitative analysis of separated analyte components in sample mixtures. A combination of TLC chromatography with microbial tests and bioassays has led to the rapid and reliable characterization of unknown samples in terms of their mode of action and bioactivity profiles [3]. As an open chromatographic system, HPTLC is compatible with microbial (bacteria and yeast) and biochemical (enzyme) derivatization methods (Table 1). It is possible to run direct in situ bioassays on the plate without much effort, since the mobile phase evaporates from the HPTLC plate before the application of cells or enzymes, thereby improving their effectiveness [4] as cell viability and enzyme inhibitory assays can be affected by organic solvents present in the mobile phase.