2.5.1. Fourier-Transform Infrared Spectroscopy (ATR-FTIR) and Density Functional Theory (DFT) Calculations

The ATR-FTIR spectra were collected on an IRTracer-100 spectrophotometer. All spectra were measured between 4000 and 400 cm⁻¹ in the absorbance mode. The following spectrometer parameters were used: resolution: 4 cm⁻¹, the number of scans: 400, and apodization: Happ–Genzel. The sample was placed directly on the ATR crystal. Solid samples were pressed against the ATR crystal and the ATR-FTIR spectrum was scanned. All IR spectra were acquired and further processed with LabSolution IR software. The results were interpreted by comparing the FTIR peaks of pure samples with those of the prepared binary systems. Origin 2021b (OriginLab Corporation, Northampton, MA, USA) was used to analyze the acquired data. LabSolutions IR software (version 1.86 SP2, Shimadzu, Kyoto, Japan) was used to calculate the second derivative of spectra for S. baicalensis radix extract, baicalein, baicalin, and wogonin. The software performed calculations according to the Savitzky–Golay numerical algorithm. The smoothing parameter was 25 points. A derivative spectrum allowed us to identify the peak positions of the original spectrum and to separate multiple peaks, which were adjoining or on the shoulder. The minima of the second derivative corresponded to the extremes of the original ATR-FTIR spectrum.

The molecular geometries were optimized using the Density Functional Theory (DFT) method with Becke’s three-parameter hybrid functional (B3LYP), implemented with the standard 6-311G(d,p) as a basis set. Additionally, the calculations of the normal mode frequencies and intensities were also performed. The PL-Grid platform (website: www.plgrid.pl, accessed on 1 December 2021) with the Gaussian 09 package (Wallingford, CT, USA) [66] was used for all DFT calculations. The GaussView (Wallingford, CT USA, Version E01) [67] program was used to propose an initial geometry of the investigated molecules and for the visual inspection of the normal modes. The obtained data were analyzed by means of the Origin 2021b software.