_Applied_  
_sciences_  
_The Power of HPTLC-ATR-FTIR Hyphenation in Bioactivity Analysis of Plant Extracts_  
**[PERSON] 1,2,*(r) and [PERSON] 1,2(r)**  
1 Department of Pharmaceutical and Toxicological Chemistry Named after [PERSON] of the Institute of Pharmacy, I.M. Sechenov First Moscow State Medical University (Sechenov University), 119991 Moscow, Russia; [EMAIL_ADDRESS]  
2 School of Pharmacy and Biomedical Sciences, La Trobe Institute for Molecular Sciences, La Trobe University, Edwards Rd, Bendigo 3550, Australia  
* Correspondence: [EMAIL_ADDRESS]  
Received: 13 November 2020; Accepted: 18 November 2020; Published: 20 November 2020  
Given the simplicity in sample preparation and application, thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) as its most enhanced form are commonly used to separate and identify complex mixtures in solution. (HP)TLC has a long tradition of use in quality control of medicinal plants, complex plant extracts and natural products. HPTLC is based on the use of special layers of fine particles of stationary phase (\(\sim\)5 \(\upmu\)m, narrow particle size range) applied to a plate. The use of automated computer controlled HPTLC instrumentation has improved method sensitivity, reproducibility, separation efficiency and enabled quantitative determination of separated components. Through coupling with direct enzyme and enzyme inhibition bioassays whose response can be visualized on the chromatographic plate, (HP)TLC has been established as a targeted bioactivity screening method. These bioautographic assays provide not only phytochemical results by chromatographic separation, but also additional information about the activity of constituents in complex mixtures and extracts. However, one of the problems in (HP)TLC is the identification of separated compounds on the plate. In recent years, considerable effort has been devoted to the coupling of (HP)TLC with spectrometric methods due to the robustness and simplicity of (HP)TLC and the need for effective identification and determination of sample constituents. In instrumental analysis, compounds are usually been identified based on chromatographic-spectroscopic hyphenated techniques. The goal of hyphenation of two analytical techniques in (HP)TLC is to enable rapid and efficient chromatographic separation with subsequent identification of the separated fractions. Mass spectrometry (MS) is one of the most powerful identification techniques used in analytical chemistry and biochemistry. It has been successfully hyphenated with (HP)TLC as thin layer chromatography/mass spectrometry (TLC/MS) and thin layer chromatography/tandem mass spectrometry (TLC/MS/MS) [1, 2, 3, 4, 5, 6]. Although TLC/MS/MS offers additional benefits, it requires more expensive instrumentation and more challenging operational requirements. Also, HPTLC-MS and HPTLC/MS/MS systems are available to only a small number of research groups. The introduction of an elution-based TLC-MS interface has also allowed the possibility of expanding the connection with other detectors. The chromatogram bands can be eluted from the HPTLC plate with a suitable solvent and either transferred online to the mass spectrometer or collected in a sample vial for further offline analysis. Infrared (IR) spectroscopy is another useful spectroscopic method that can be coupled with (HP)TLC. The major advantages of IR spectroscopy is no (or minimal) sample preparation is required, and it is an environmentally friendly and reagent-free green tool, that provides results within minutes [7]. Most IR spectrometers are easy to use, portable, and relatively inexpensive. After separation and bioassay detection, eluted bioactive bands can be easily chemically characterized using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectral analysis (Figure 1). FTIR spectra are typically measured in the mid-IR region (4000 to 400 cm\({}^{-1}\)) of the electromagnetic spectrum.