Abstract
Introduction
Carvacrol and thymol exist in many aromatic plants and hence in a number of essential oils.1-3 These materials are structural isomers. They have the same structural backbone except for the positioning of the OH functional group. In thymol, the OH is ortho to the isopropyl group while in carvacrol it is ortho to the methyl group. These compounds fall under the classification of monoterpene phenols. These monoterpene phenols have a wide range of applications. Thymol has medicinal properties that include among others, wound healing, analgesic, antimicrobial, and antiinflammatory.4-8 It is an active ingredient in food flavorings, topical ointments, various soaps, toothpastes, shampoos, deodorants, and mouthwashes. 9 It can also aid in digestion by relaxing smooth muscles, reduces menstrual cramps, and alleviates respiratory difficulties.10,11 Carvacrol on the other hand can be used as a disinfectant, fungicide, and it has been found to have insecticidal activity.12-14 Furthermore, it is used in food as a flavor, an additive, a preservative, and in cosmetics as a scent. 10 These materials have been identified as constituents of essential oils using a gas chromatography-mass spectrometer (GC-MS) and gas chromatography-flame ionization detector (GC-FID).15,16 GC-MS has proven to be a valuable tool in the quantification of the constituents of essential oils, but this method has a limitation in that it is unable to distinguish between closely related compounds or isomers unless an internal standard is used. This calls for rapid methods that can distinguish and validate compounds that have been detected by the GC-MS.
Apart from GC methods, carvacrol and thymol isomers have been mainly characterized in the mid-infrared region.17-19 This has resulted in limited studies of these materials in the lower frequency range, commonly known as the far infrared/ terahertz range. For an unambiguous characterization or identification of these materials, it is fundamental to understand their properties over an extended frequency range. Far infrared/terahertz spectroscopy has been widely used in material characterization/identification and has been proven to be an essential tool in this aspect. This is supported by the extensive research in this part of the electromagnetic (EM) spectrum.20,21 THz waves are sandwiched between the infrared and microwave regions in the EM spectrum. This position indicates that the THz systems require a combination of optics and electronics, which can be employed in THz generation, detections as well as processing, but this still remains a challenge.22,23 In definition, 1 THz corresponds to 4.1 meV of energy. It is equivalent to 33.3 cm−1 and THz waves have a frequency of 1012 hertz (Hz). THz waves possess unique properties that allow for their application in almost any field of research.24-31 These waves have the capability of penetrating a range of materials in any form of matter.32-37 They have a low photon energy and hence they are nonionizing. THz radiation unlike other types of EM waves is very sensitive to various resonances that include torsional, rotational, vibrational, and translational therefore, making it an important tool for providing information on molecules. 38 Infrared spectroscopic techniques are simple, less costly, rapid, and nondestructive and have the capability for routine analysis. 17 On the other hand gas chromatography devices require complex sample preparation and have limited sensitivity. 39
This work presents a comparison between the spectra of thymol and carvacrol in the far infrared region with the aim of introducing a cheap and robust alternative method that can be used for the characterization of these materials. Density functional theory (DFT) calculations will be employed for the interpretation of the far infrared spectra of these isomers and spectral feature assignments will be attempted. A comparison will be made between this work and the literature.
Experimental
Carvacrol and thymol were purchased from Sigma Aldrich and used without further processing. The THz spectra of these materials were measured using the Bruker Vertex 70v vacuum spectrometer utilizing the single bounce diamond crystal attenuated total reflection (ATR) accessory. The resolution at which the spectra were collected was 1 cm−1 (0.03 THz). The number of scans collected for each spectrum was 512.0 and on average it took 13 minutes for each scan. A broadband beamsplitter was employed. The tungsten lamp was used as the source of radiation and the deuterated triglycine sulfate (DTGS) was used as the detector. The ratio of the spectrum collected with the sample in the beam path to the spectrum with no sample in the beam path gave the transmission spectrum. The DFT modeling within the Orca software package 40 was utilized in the calculations of the infrared spectra. The calculations involved the use of the hybrid PBE0 functional and the def2-TZVPP basis set41-47 for geometry optimization and calculation of the energies. The model made use of the empirical Van der Waals correction. 48
Carvacrol and thymol were analyzed using an Agilent 6890N capillary gas chromatograph connected to an Agilent G5977 mass spectrometer and a PAL 3 RSI auto-sampler (5301 Stevens Creek Blvd. Santa Clara, CA 95051, United States). The NIST library was used for the identification of compounds (NIST, Mass-spectrometry Data Center, 2017). Separation was achieved using a standard nonpolar HP-5MS capillary column (30 m length, 0.32 mm internal diameter, and 0.25 µm film thickness). Sample injections were made in a split mode using a general-purpose split/split-less liner packed with glass wool. The GC oven temperature program was started at 70 °C and held for 5 minutes, then increased to 300 °C at a rate of 5 °C min−1 resulting in a total run time of 51 minutes. Sample volumes of 1 µL were injected into the instrument using helium as a carrier gas at a split ratio of 100:1. The flow rate of helium was set at a constant flow rate of 0.5 mL min−1. The injector and mass transfer line temperature settings were 250 °C.
Results and Discussions
The molecular structures of carvacrol and thymol are presented in Figure 1. As can be seen, the molecular arrangements of these compounds are almost the same. The only difference is in the position of the OH functional group. The retention times for carvacrol and thymol are very close (18.5 for thymol and 18.8 for carvacrol) on the GC-MS such that it is difficult to use only the NIST library for identification without the reference standards. Figure 2 shows that the mass spectra are identical, which makes the characterization of these isomers by GC-MS challenging. Additionally, the characterization will be even more challenging in complex matrices like essential oils. Therefore, this work presents a spectroscopic method to augment the characterization of these isomers.
Molecular structures of (A) carvacrol and (B) thymol.
Ms structures of (A) thymol and (B) carvacrol from the NIST library.
The far infrared spectra of thymol and carvacrol isomers are displayed in Figure 3. The spectra are characterized by a number of distinct absorption bands of various intensities in the considered frequency range. The spectrum of carvacrol is made up of 10 spectral bands while that of thymol comprises 13 absorption bands. Carvacrol displayed two high-intensity bands at 462.4 cm−1 (13.9 THz) and 566.9 cm−1 (17.0 THz). The rest of the bands have moderate to weak intensities. Thymol displayed a strong band at 586. 8 cm−1 (17.6 THz). Four medium intensity bands appeared at 342.1 cm−1 (10.3 THz), 392.5 cm−1 (11.8 THz), 488.5 cm−1 (14.7 THz), and 525.3 cm−1 (15.8 THz). The rest of the bands can be classified as weak-intensity bands. There is evidence of bands appearing at similar energies for thymol and carvacrol. This correspondence can be ascertained by determining whether the common bands share the same vibration mode. Four common bands were established between carvacrol and thymol. These bands were assigned on the basis of the appearance of the bands at similar frequencies and also displaying the same vibration mode. In addition, thymol and carvacrol had four seemingly common absorption bands that however exhibited different vibrational modes. On the other hand, the differences in the spectra of these materials are noticeable. There were nine absorption bands that were unique to thymol and six to carvacrol. Even though there is a slight difference between the molecular arrangements of these compounds, there are marked differences in the absorption spectra. This attests that far infrared/THz spectroscopy can be successfully used in the identification of materials with close structural features. It is quicker to obtain measurements using this alternative method as there is no need for extensive sample preparation. Furthermore, this result suggests that this method can be used for quality control as each material has a unique spectrum in the considered spectral range. There will however be a limitation in identifying constituents in aqueous solutions as terahertz radiation is strongly absorbed by water.
Room-temperature spectra of carvacrol and thymol.
Figure 4 shows the comparison between the experimental and single molecule DFT calculations of thymol. The single molecule theoretical model produced frequencies that matched well with the observed experimental features. There was a discrepancy in experimental bands and the matching theoretical peak intensities. From the experiment, the band labeled H had a low intensity while the matching theoretical peak had a high intensity. The thymol experimental band labeled M had a high intensity and the matching theoretical peak had a low intensity. The incongruity in the intensities is attributed to single molecule calculations since intensities usually depend on crystalline fields. Absorption bands labeled C and F did not have corresponding theoretical peaks. The absence of these peaks suggests that they are a result of intermolecular interactions. The model gave two peaks at 277.4 cm−1 (8.3 THz) and 513.1 cm−1 (15.4 THz) both denoted x in Figure 4, which did not have matching experimental bands. This proposes that the technique used was not able to resolve these two bands. Terahertz spectra are usually associated with homogeneous and inhomogeneous broadening that leads to the concealment of spectra features at room temperature. 49 This can be counteracted by low-temperature measurements. Cooling of the samples reduces broadening and therefore can unmask spectral features that do not appear in room-temperature spectra. 50
Comparison between the room-temperature experimental and theoretical spectra of thymol. The vertical lines are the calculated energies and their relative intensities.
Figure 5 shows a good agreement between the experimental and theoretical frequencies of carvacrol. All the observed experimental bands had corresponding theoretical peaks except for one band denoted S. The model further resulted in three theoretical peaks denoted x in Figure 5, which did not appear in the experiment. These peaks appeared at 258. 5 cm−1 (7.8 THz), 286.4 cm−1 (8.6 THz), and 332.9 cm−1 (10.0 THz). Due to limited resources, low-temperature measurements that have the capability of resolving hidden spectral features could not be undertaken. The carvacrol experimental band denoted P displayed a low intensity while its corresponding theoretical peak had a high intensity. Moreover, the experimental bands denoted Q and M had medium intensities while their corresponding theoretical peaks had low intensities. This phenomenon is also observed for thymol.
Comparison between the room-temperature experimental and theoretical spectra of carvacrol. The vertical lines are the calculated energies and their relative intensities.
Tables 1 and 2 present the assignment of the absorption bands for thymol and carvacrol respectively. For vibration modes assignment, the molecules were visualized using the gOpenmol program. 51 The assignment was based on the vibration modes, together with approximate assignment by the vibrational frequency. From the assignment, it was discovered that thymol and carvacrol share four absorption bands. These bands denoted G, J, L, and M for both materials were found to have the same vibrational modes. Absorption bands denoted A and K for thymol seemed to correspond to carvacrol absorption bands denoted N and R, respectively. These bands, however, displayed different vibrational modes. Superficial correspondences in the absorption bands have been previously reported. 52 The peak assignment revealed that majority of the vibrational modes in the considered frequency range are due to intramolecular interactions that encompass much of the entire molecule. It has to be noted that the other modes for phenolic compounds like thymol and carvacrol that give rise to bands below 600 cm−1 (18.0 THz) may be due to intermolecular hydrogen bond stretching and bending modes. The hydrogen bonding in phenols is important in that it can be used as a model for biological systems. 53
Experimental and Theoretical Frequencies of Thymol.
Experimental and Theoretical Frequencies of Carvacrol.
A further comparison was made between this work and the literature as shown in Table 3. There was correspondence between some of the observed absorption bands and the related literature studies.54,55 The most outstanding observation is that the literature is dominated by mid-infrared studies. This has left the lower frequency range unexplored for these materials. It was found imperative to perform the low-frequency studies because the availability of an extended spectral database of these materials is crucial for a better understanding of their properties. This information will be valuable in the manufacture and identification of products derived from medicinal plants or essential oils in particular.
Comparison of Spectral Features of Thymol and Carvacrol with Literature.
Conclusion
The far infrared/terahertz spectra of carvacrol and thymol were measured using the FTIR-ATR technique. The difference in the spectra of these materials that have an almost similar molecular structure proves the robustness of far infrared/terahertz radiation in the identification and characterization of isomers. The absorption bands were interpreted using the DFT model. The model revealed four common absorption bands between carvacrol and thymol. The common bands were found to share the same vibrational modes. The model further suggested that there were some concealed absorption bands in the room-temperature spectra of these materials. Despite these similarities, there were peaks that were characteristic to each of the isomers. This highlights the ability of the DFT calculations in the analysis of the terahertz spectra of materials. The retention times of these isomers as shown by the GC-MS are very close and they have a similar mass spectral profile that could pose a challenge when identifying them in complex matrices. This study is important as it ushers in the possibility of characterizing compounds of similar structural features commonly found in essential oils using far infrared/THz spectroscopy as a complementary method to the conventional GC-MS. This alternative method is nondestructive, as there are no sample alterations during the analysis process. Furthermore, it offers a more rapid analysis and there is no intensive sample preparation.
Footnotes
Acknowledgments
This work was supported by the Botswana International University of Science and Technology.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Ethical Approval
Not applicable.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
Statement of Human and Animal Rights
This article does not contain any studies with human or animal subjects.
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There are no human patients involved in this article and informed consent is not applicable.
