4-Hydroxy-3-methoxybenzoic acid (HVA) is one of the antibacterial components extracted from plants in nature. It is an important endogenous metabolite of adrenaline and norepinephrine, with anti-mutation and anti-cancer effects in the human body. It can also be used in food additives and pharmaceutical synthesis. 5-methoxysalicylic acid (5-MeOSA) is a natural product that serves as an effective matrix for mass spectrometry analysis of oligonucleotides when combined with spermine. It is also an important intermediate in organic synthesis in the chemical industry. 5-MeOSA and HVA are isomers, sharing the same functional group in their structures. Although these two isomers appear similar, their functions differ significantly. Many organic molecules exhibit intramolecular vibrations and intermolecular interactions in the THz range, making terahertz time-domain spectroscopy (THz-TDS) a useful method for identifying substances and studying the physicochemical properties of materials. Due to its low-photon energy and fingerprint properties, THz-TDS has become widely used for the qualitative detection and molecular interaction studies of isomers.

A transmission terahertz spectrometer is used to measure the terahertz spectra of the samples. The system employs the electro-optic sampling method, with a testing range of 0.5?7.0 THz and a spectral resolution of 0.008 THz. Solid-state samples of HVA and 5-MeOSA are prepared for experimental measurement by pressing them into tablets with a 13 mm diameter. Theoretical simulations are performed using the method of linear combination of atomic orbitals, employing the hybrid functional of B3LYP and the Grimme’s hexamethylcyclotrisiloxane dispersion correction, with a 6-311g (d, p) basis set. Vibrational modes are analyzed using Bayesian linear regression’s vibrational mode automatic relevance determination (VMARD) method to identify the sources of the THz absorption peaks. The weak interaction forces within the molecular systems are analyzed using energy decomposition analysis based on force field (EDA-FF).

As shown in Fig. 2, HVA exhibits six characteristic absorption peaks at 1.10, 1.63, 1.76, 2.19, 2.59, and 3.08 THz. 5-MeOSA displays four clear characteristic absorption peaks within the testing range, located at 1.30, 1.49, 2.20, and 3.24 THz. The THz experimental and theoretical spectra for HVA and 5-MeOSA are shown in Fig. 3. Theoretical calculations effectively reconstruct the experimental spectra, which provide a basis for explaining the vibrational modes of the absorption peaks. Table 1 shows the assignment of the THz experimental absorption peaks and theoretical optical modes for both isomers. Figures 4 and 5 show the vibrational modes of HVA and 5-MeOSA at specific frequency values. The results indicate that the vibrational modes of HVA in the THz low-frequency range are due to dihedral angle and bond angle bending, while the vibrational modes of 5-MeOSA mainly result from dihedral angle torsion and out-of-plane angle bending. Specifically, dihedral angle torsion contributes most to the vibrations and is the primary source of THz characteristic absorption peaks for both isomers. The absorption peaks of HVA and 5-MeOSA at 2.20 THz are mainly due to the vibration of methoxy functional groups, which are influenced by weak intermolecular interactions. Furthermore, Tables 2 and 3 show the interaction energy components for the HVA and 5-MeOSA fragments. Finally, Figs. 6 and 7 illustrate the total binding energy atomic coloring diagram and dispersion atomic coloring diagram for both isomers. The results show that the electrostatic attractions between Flag1-Flag2 and Flag3-Flag4 in HVA are -12.78 kJ/mol and -33.93 kJ/mol, respectively. A pair of hydrogen bonds (O₂₁—H₃₃…O₄) is formed between Flag1 and Flag2, and two pairs of hydrogen bonds (O₄₁—H₅₃…O₆₄ and O₆₄—H₇₅…O₄₂) are formed between Flag3 and Flag4. Dispersion forces contribute significantly to the total binding energy in the HVA system. In 5-MeOSA, a pair of hydrogen bonds (O₁₅—H₁₆…O₈₀ and O₅₅—H₅₆…O₄₀) are formed between Flag1 and Flag4 and between Flag2 and Flag3, with similar contributions of -24.44 kJ/mol and -24.54 kJ/mol to the electrostatic attraction between the fragments. The dispersion effect in 5-MeOSA mainly arises from the methoxy, carboxyl, and hydroxyl functional groups, as well as carbon atoms on the benzene ring.

In response to the difficulty of distinguishing isomers in the food industry, we apply terahertz time-domain spectroscopy to study the spectral information of HVA and 5-MeOSA in the THz region. The THz spectra of these two isomers in the range of 0.5?3.5 THz are obtained, revealing significant differences in their THz absorption peaks. Theoretical simulations based on density functional theory are conducted, and the theoretical and experimental spectra are found to match well, allowing us to summarize the origins of the measured THz absorption peaks. It is found that the THz peaks of HVA primarily originate from dihedral angle and bond angle bending, while the THz absorption peaks of 5-MeOSA mainly come from dihedral angle torsion and out-of-plane angle bending. In addition, the weak interactions in the molecular systems of HVA and 5-MeOSA are analyzed using EDA-FF, which shows that the weak interactions in both molecules are mainly dominated by dispersion forces, followed by electrostatic interactions. Three pairs of hydrogen bonds form in the molecular system of HVA, stacking in a spatial structure, while two pairs of hydrogen bonds are present in 5-MeOSA’s molecular system, with the structure being close to planar. Our research demonstrates that combining THz-TDS with DFT, VMARD, and EDA-FF methods is an effective approach for identifying isometric organic molecules. We also provide valuable reference data for the identification and characterization of HVA and 5-MeOSA, as well as for investigating their physicochemical properties.