Separation and Characterization of Wenjin Tongluo San Essential Oil with a Comprehensive Chromatographic Separation

Abstract

The essential oil components of traditional Chinese medicine in-hospital preparation were complex, and one-dimensional chromatographic separation was difficult to completely separate them due to the limited peak capacity. This study was carried out to establish a comprehensive two-dimensional chromatographic separation and analysis method based on countercurrent chromatography (CCC) and gas chromatography (GC). In this paper, we focused on the separation of the essential oil of the traditional Chinese medicine in-hospital preparation Wenjing Tongluo San by CCC × GC, and explored the orthogonality between the two chromatographic techniques to provide the new technical support for the screening of the active ingredients. A solvent system composed of n-hexane-ethyl acetate-methanol-water (9.5:0.5:8.5:1.5, v/v) was chosen for the first-dimensional CCC separation. All the fractions collected from CCC were transferred to GC for plotting two-dimensional contours map. The calculated capacity of the two-dimensional separation system exceeded 3000, which was 8 times more than that of the one-dimensional separation system. High orthogonality (r = 0.42) and spatial coverage factor (70.42%) were obtained. Meanwhile, all the fractions were identified by GC-MS. Our research provided a new methodology for separating essential oils in traditional Chinese medicine as well as an approach for evaluating the quality of traditional Chinese medicinal in-hospital preparation based on two-dimensional chromatographic fingerprints.

1. Introduction

Screening the medicinal active ingredients from the natural products was an important way to conduct new drug research and discovery. Typically, chromatographic separation technology plays a crucial role in screening the active components [1]. Essential oils of traditional Chinese medicines often contained a variety of active ingredients [2]. However, due to the complexity of the compositions, it was difficult to completely separate the essential oil with the limited peak capacity of the one-dimensional chromatography [3]. Although capillary gas chromatography (GC) and comprehensive two-dimensional GC have provided powerful means for the essential oil composition analysis [4,5,6], GC separation was not suitable for screening the active ingredients because the components were difficult to recycle. Therefore, the development of new multidimensional chromatographic separation methods based on liquid chromatography was of great significance for the separation and analysis of Chinese medicine and the corresponding active ingredients [7,8].

Comprehensive two-dimensional chromatography combines multiple separation mechanisms or modes to realize the separation of various samples. It was especially suitable for the separation of components with similar polarity, similar properties, and similar structures and could significantly improve the peak capacity, selectivity, and degree of separation [9,10,11]. Currently, comprehensive two-dimensional chromatographic orthogonality studies have focused on conventional GC and liquid chromatography [9]. In traditional two-dimensional GC and liquid chromatography, the selection of columns with different separation mechanisms could significantly increase the orthogonality and peak capacity of the two dimensions. However, there were still some unavoidable shortcomings: (1) The irreversible adsorption led to the inability of the samples to be completely analyzed in the second dimension; (2) The compatibility of the mobile phases was difficult to achieve due to the differences of the separation columns between the two dimensions such as the ortho-phase and the reversed-phase. In order to fully utilize the separation potential of two-dimensional chromatography, the construction of chromatographic separation systems with a high degree of orthogonality has become a hot spot in the study of multidimensional chromatography [12,13,14,15,16]. Notably, orthogonality and peak capacity were important parameters for measuring the separation ability of the multidimensional chromatography and the independence level of the separation mechanisms [17].

Countercurrent chromatography (CCC) was an efficient distribution chromatography technique that emerged in recent years [18]. Its main feature was that the mobile phase and stationary phase were both liquids, so the stationary phase did not require a solid carrier [19,20]. Compared with traditional chromatography, CCC inferred the following advantages: (1) No irreversible adsorption occurred; (2) Low requirements for the sample injection, free from complex pretreatment; (3) Large sample injection volume because the conventional injection volume could reach grams. Accordingly, CCC was theoretically well complementary to the traditional gas chromatographic separation techniques. The peak resolution of CCC was always low due to the low separation efficiency, while higher theoretical plates were involved in GC. On the contrary, the sample fractions were better recovered in CCC. Therefore, if the two chromatographic techniques were combined, a higher resolution might be obtained, which further solved the problem of co-dissipation and peak overlap in the analysis of complex natural products. However, there were few reports investigating offline comprehensive two-dimensional chromatography based on CCC and GC [21,22,23,24].

Wenjing Tongluo San, a compound in-hospital preparation at Jinhua Hospital of Traditional Chinese Medicine, has been in use for nearly 40 years. Wenjing Tongluo San received official approval in 2010 under the regulatory approval number Zhe Yao Zhi Zi Z20100225. The ten herbs to form Wenjing Tongluo San were Lycopodii herba (18.6%), Artemisiae argyi Folium (14.8%), Perillae folium (14.8%), Cinnamomi ramulus (7.4%), Angelicas pubescentis Radix (7.4%), Cortex Erythrinae Seu Kalopanacis (7.4%), Angelica dahurica Radix (7.4%), Acanthopanacis Cortex (7.4%), Zanthoxylum bungeanum Maxim (7.4%), and Aurantii fructus (7.4%). The acquired in-hospital preparation was a grayish-brown powder with an aroma. It has the benefits of activating blood circulation, relaxing tendons, warming the meridians, dispelling wind, and dispersing cold. It was clinically used for treating all kinds of joint rheumatism and cold pain, contracture, and soreness of tendons. Patients were usually treated through the fumigation with the in-hospital preparation. Therefore, the compositional analysis of the essential oils in Wenjing Tongluo San was especially important. Commonly, the essential oil of Wenjing Tongluo San was a transparent, yellowish, aromatic-smelling extract. The oil has been used for many years to clear the meridians. The oil was highly fat-soluble and could be absorbed into the skin more quickly to achieve better medicinal effects. Therefore, the compositional analysis of the essential oils in Wenjing Tongluo San was especially important. The major components of the essential oils of these ten herbs have been well-documented. The essential oil of Lycopodii herba contained fatty acids (44.6%), alcohols (15.6%), aldehydes and ketones (7.6%), hydrocarbons (12.6%), phenols (8.86%), and esters (3.1%) [25]. The essential oil of Artemisiae argyi Folium contained eucalyptol (10.66%), thujone (2.4%), d-camphor (3.2%), myrtenol (6.5%), trans-caryophyllene (12.1%), caryophyllene oxide (10.2%), 3,3,6-trimethyl-1,4-heptadien-6-ol (2.1%), terpinen-4-ol (4.0%), and globulol (6.7%) [26]. The essential oil of Perillae folium contained asarone (23.91%), β-caryophyllene (10.15%), and 1-cyclohexane-1-carboxaldehyde (3.38%) [27]. The essential oil of Cinnamomi ramulus contained E-3-Phenyl-2-propenal (50.4%), 3-(2-Methoxyphenyl)2-propenal (4.7%), hexadecanoic acid (4.3%), 1-ethenyl-4-methoxybenzene (3.8%) and benzaldehyde (2.9%) [28]. The essential oil of Angelicas pubescentis Radix contained esters (36.3%), alcohols (15.7%), phenols (14.7%), hydrocarbons (13.6%), ketones (4.9%), acids (2.5%), ethers (1.1%), and aldehydes (0.8%) [29]. The essential oil of Angelica dahurica Radix contained dodecanol (43.2%), diisooctyl phthalate (7.3%), hexadecanoic acid (4.2%), and isopropyl linoleate (1.6%) [30]. The essential oil of Acanthopanacis Cortex contained cedrol (41.5%), methyl palmitate (20.3%), β-cedrene (10.9%), and methyl linoleate (9.2%) [31]. The essential oil of Zanthoxylum bungeanum Maxim contained 1-(2-hydroxy-4,6-methyl ethyl ketone)-ethyl ketone (39.8%), 3-methyl-6-(1-methylethylidene)-2-cyclohexen-1-one (5.2%), 4-methylene-1-(1-methyl ethyl)-cyclohexane (4.2%) and beta-phellandrene (3.2%) [32]. The essential oil of Aurantii fructus contained limonene (40.9%), linalool (13.1%), 2-undecanol (10. 8%) and 7-terpinen (8.7%) [33]. The main components of the essential oil were terpenoids, esters, aldehydes, and ketones with antibacterial, antioxidant, and anti-tumor effects. At present, there is no report on the separation and analysis of the essential oil of Wenjing Tongluo San, and there was a lack of separation methods for the deep analysis of the Chinese herbal compound prescription.

In order to investigate the constituents in the essential oil of Wenjing Tongluo San, the comprehensive two-dimensional CCC × GC approach was applied in our present study. The first-dimensional chromatography was CCC, and the second-dimensional chromatography was GC. The fraction collected from the first dimension was directly transferred to the second dimension. The orthogonality, peak capacity, and spatial coverage were then explored in the separation of the essential oil of Wenjing Tongluo San. The first dimension could be used to recover the samples for the subsequent activity studies, while the second dimension, coupled with MS, was used for further identification. The drawn two-dimensional contour maps helped to visualize the classification of different active ingredients.

2. Materials and Methods

2.1. Apparatus

A custom-made 105 mL CCC system was utilized in this research. The details of the custom-made CCC system were outlined in our earlier work [34]. The mainframe was a combination of two multilayer coils connected in series (PTFE tube: diameter of 1.8 mm; with a total volume of 105 mL) and a 20 mL sample loop. GC analysis was performed on a Neixs GC-2030 system (Shimadzu Corporation, Kyoto, Japan) with a SE-30 capillary column.

2.2. Reagents and Chemicals

The in-hospital preparation Wenjing Tongluo San was bought from Jinhua Hospital of Traditional Chinese Medicine. All solvents employed for sample preparation and CCC, which were of analytical grade, were bought from Shanghai Zhanyun Chemical Co. Ltd (Shanghai, China).

2.3. Sample Preparation

Essential oils were extracted through the steam distillation. About 200 g of Wenjing Tongluo San powder was added to 2000 mL of redistilled water. After heating and refluxing the powder, the yellowish essential oils were obtained with a yield of 0.73%. Petroleum ether was used to extract the oil, and anhydrous sodium sulfate was used to dry the product. Finally, the obtained essential oil was stored in a 4 °C for the subsequent experiments.

2.4. ¹D Countercurrent Chromatographic Analysis

The selected solvent system was n-hexane-ethyl acetate-methanol-water (9.5:0.5:8.5:1.5, v/v). The solvent system was poured into a separatory funnel and mixed thoroughly. The two phases were placed separately in the solvent bottles after layering. The two solvent bottles were degassed in ultrasonic conditions for about 20 min. The upper phase was used as the stationary phase, and the lower phase was used as the mobile phase. The upper phase was first fed into the spiral separation column. After the upper phase was discharged at the end, the speed of the column was set at 800 r/min. The mobile phase was pumped into the column at 1.5 mL/min. The sample injection volume was set as 30 mg. The detection wavelength was 254 nm. The retention rate of the stationary phase was 64.1%. Each tube of fraction was collected at one-minute intervals and stored at a low temperature. The isocratic elution mode was applied in 0~120 min, while the elusion extrusion mode was in 120~163 min. A total of 130 fractions were collected from the first-dimensional CCC. Each collected fraction from the CCC separation was transferred into a sealed brown vial to preserve its original concentrations. These vials were stored at a low temperature (2–10 °C) to prevent degradation. Each fraction was diluted 1:1 with the mobile phase to ensure compatibility with the GC system. After dilution, the fractions were directly injected into the GC for analysis. All fractions collected in the first dimension were transferred to the second-dimensional gas chromatographic analysis to plot the two-dimensional contour map.

2.5. ²D Gas Chromatographic Analysis

A SE-30 column was used in the GC analysis. High-purity nitrogen was used as the carrier gas with a flow rate of 1 mL/min. The injection volume was 1 µL. The split ratio was 2:1. The inlet temperature was 250 °C. The column temperature program was 50~280 °C, which started from 50 °C, held for 5 min, then increased to 70 °C at 3 °C/min, to 136.2 °C at 2 °C/min, to 136.3 °C at 0.01 °C/min, to 152 °C at 3 °C/min, to 167 °C at 2 °C/min, to 203 °C at 10 °C/min, and finally to the termination temperature of 280 °C at 5 °C/min, then held for 2 min. The total run length was 88.5 min.

2.6. GC-Mass Analysis

The analysis was conducted using an Agilent 8890 GC system interfaced with a 7000D triple-quadrupole mass spectrometer (Agilent, Santa Clara, CA, USA). EI mode was applied for the ionization. Mass spectrometry conditions: Full scan mode, mass scanning range m/z: 30–650 Da; ion source temperature: 230 °C; filament current: 35 µA; electron energy: 70 eV; scanning speed: 5 scans/s. The NIST14 database was used for the qualitative analysis, and the identification results were finally obtained.

2.7. Data Analysis

2.7.1. Chromatograms and Comprehensive Two-Dimensional Contour Map

The peak data of CCC were exported with the SePu3000 workstation (Hangzhou Puhui Technology, Hangzhou, China), and the peak data of GC were exported with Shimadzu GC-2030 Labsolution. The comprehensive two-dimensional chromatographic contour map was processed by Matlab (R2018a) software (Mathworks Inc., Natick, MA, USA). All of the chromatograms, linear correlation coefficient plots, and normalized plots were processed with Origin2018 software (OriginLab, Northampton, MA, USA).

2.7.2. Peak Capacity

Peak capacity is the total number of chromatographic peaks for which a high degree of separation was obtained under specific chromatographic conditions. Generally, the more the chromatographic peaks, the better the separation capacity was. Stoll [35] et al. mentioned the relevant formula for calculating the theoretical peak capacity of the system in two dimensions (¹n_c and ²n_c) according to Equation (1).

$${\mathrm{n}}_{\mathrm{C}}={\displaystyle \frac{{\mathrm{t}}_{\mathrm{r},\mathrm{l}\mathrm{a}\mathrm{s}\mathrm{t}}-{\mathrm{t}}_{\mathrm{r},\mathrm{f}\mathrm{i}\mathrm{r}\mathrm{s}\mathrm{t}}}{{\mathrm{w}}_{\mathrm{b}}}}$$

(1)

in which t_r,last, and t_r,first was the retention time of the last and first observed peaks in the separation space, and w_b was the average peak width of the measured 4σ peaks.

The peak capacity in the first dimension (¹n_c) and the second dimension (²n_c) were then multiplied together to determine the system’s maximum theoretical CCC × GC capability (^2Dn_c), according to Equation (2).

$${}_{}{}^{2\mathrm{D}}{\mathrm{n}}_{\mathrm{c}}={}_{}{}^1{\mathrm{n}}_{\mathrm{c}}\times {}_{}{}^2{\mathrm{n}}_{\mathrm{c}}$$

(2)

2.7.3. Spatial Coverage

Spatial coverage, related to the level of separation space utilization on a two-dimensional chromatogram, was an important parameter that reflected whether the two-dimensional chromatography successfully separated the compounds. High spatial coverage meant that the system had better separation capability and space utilization; thus, the components hidden in the overlapped peaks were more easily separated and detected. The evaluation of orthogonality has been extensively explored using various methods such as the box number technique, information theory, factor analysis, convex hull approach, and nearest neighbor distance method etc. Notably, the convex hull method was regarded as particularly effective and suitable for measuring orthogonality in two-dimensional chromatography. The area of the convex hull was determined by summing the areas of the individual triangles [36], and the calculation of separation space coverage was outlined in Equation (3).

$$\mathrm{S}\mathrm{e}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{s}\mathrm{p}\mathrm{a}\mathrm{c}\mathrm{e} \mathrm{u}\mathrm{s}\mathrm{e}\mathrm{d}=\left({\displaystyle \frac{\mathrm{A}\mathrm{r}\mathrm{e}\mathrm{a} \mathrm{o}\mathrm{f} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{v}\mathrm{e}\mathrm{x} \mathrm{h}\mathrm{u}\mathrm{l}\mathrm{l} \mathrm{u}\mathrm{s}\mathrm{e}\mathrm{d}}{\mathrm{A}\mathrm{r}\mathrm{e}\mathrm{a} \mathrm{o}\mathrm{f} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{r}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{s}\mathrm{p}\mathrm{a}\mathrm{c}\mathrm{e} \mathrm{u}\mathrm{s}\mathrm{a}\mathrm{b}\mathrm{l}\mathrm{e}}}\right)\times 100\%$$

(3)

In order to mitigate the impact of fluctuations in operating parameters or column dimensions, it was imperative to normalize the retention times in both dimensions. Initially, the retention time data for each connectivity component in the second dimension were collapsed. Next, the retention times of the components were normalized according to Equation (4).

$${\mathrm{R}\mathrm{T}}_{\mathrm{i}\left(\mathrm{n}\mathrm{o}\mathrm{r}\mathrm{m}\right)}={\displaystyle \frac{{\mathrm{R}\mathrm{T}}_{\mathrm{i}}-{\mathrm{R}\mathrm{T}}_{\mathrm{m}\mathrm{i}\mathrm{n}}}{{\mathrm{R}\mathrm{T}}_{\mathrm{m}\mathrm{a}\mathrm{x}}-{\mathrm{R}\mathrm{T}}_{\mathrm{m}\mathrm{i}\mathrm{n}}}}$$

(4)

in which RT_max and RT_min represented the longest and the shortest retention time of the components. The retention time RT_i was normalized to RT_{i (norm)} with values between 0 and 1.

Pearson correlation coefficient was a linear correlation coefficient that was used to reflect the linear correlation of two normal continuous variables [37]. The Pearson correlation coefficient was calculated using Equation (5).

$${\mathrm{r}}_{\mathrm{p}}={\displaystyle \frac{{\sum }_{\mathrm{i}=1}^{\mathrm{n}}({\mathrm{X}}_{\mathrm{i}}-\overline{\mathrm{X}})({\mathrm{Y}}_{\mathrm{i}}-\overline{\mathrm{Y}})}{\sqrt{{\sum }_{\mathrm{i}=1}^{\mathrm{n}}{({\mathrm{X}}_{\mathrm{i}}-\overline{\mathrm{X}})}^2{\sum }_{\mathrm{i}=1}^{\mathrm{n}}{({\mathrm{Y}}_{\mathrm{i}}-\overline{\mathrm{Y}})}^2}}}$$

(5)

The value of r was in the interval [−1, 1]. When r = 1, X and Y were perfectly positively correlated. When r = −1, X and Y were perfectly negatively correlated. When r = 0, the linear correlation between X and Y was insignificant.

3. Results

3.1. Optimization of Separation Conditions

3.1.1. Optimization of Separation Conditions of GC

The essential oil of Wenjing Tongluo San was analyzed by GC to optimize the inlet temperature, split ratio, and elution time. Here, based on the polarity of the identified chemicals, a weakly polar capillary column (SE-30) was selected to achieve a satisfactory response, symmetrical peaks, and suitable elution time. The oil could be totally detected within 90 min and the local peak separation with good response was also well achieved by optimizing the ramp-up procedure and split ratio. Figure 1 shows the gas chromatogram of the essential oil of Wenjing Tongluo San. The complexity of the samples are shown in Figure 1.

3.1.2. Optimization of Separation Conditions of CCC

Selection of a solvent system for CCC separation required the condition that the K-value is kept within a reasonable range. Larger K values may result in longer retention times, while smaller K values may result in poorer separation resolution. Since the essential oils were composed of less polar components, small polar solvent systems were mainly investigated. The application of the solvent system petroleum ether-methanol-acetonitrile-water (10:0.5:9:0.5, v/v) was found to exhibit low K values. When cyclohexane-acetonitrile-water (10:9:1, v/v) was applied, both K and α values were improved but still not up to the mark. It was found that the systems n-hexane-ethyl acetate-acetonitrile-water (9.5:0.5:9:1, v/v) and n-hexane-ethyl acetate-methanol-water (9.5:0.5:8.5:1.5, v/v) were acceptable, while the system containing methanol had a better separation factor for most high peaks. For comprehensive consideration, n-hexane-ethyl acetate-methanol-water (9.5:0.5:8.5:1.5, v/v) was chosen as the solvent system for the first-dimensional chromatography, and the detection results were shown in Figure 2.

3.2. Comprehensive Two-Dimensional CCC × GC Analysis of the Wenjing Tongluo San Essential Oil

After the elution by the first-dimensional CCC, a total of 130 eluted fractions were collected, and each fraction was analyzed by GC. Combining the two chromatograms, a comprehensive two-dimensional CCC × GC contour plot of the components was produced with Matlab (R2018a), as shown in Figure 3. The main components of the essential oil of Wenjing Tongluo San were successfully separated in the map of the comprehensive two-dimensional CCC × GC contour plot. The components that could not be effectively separated in one-dimensional chromatography showed good separation in the other dimension, which demonstrated the complexity of the essential oil of Wenjing Tongluo San and the complementary advantages of the two chromatographic techniques. For example, the elution time of A and B in GC was 20.843 min, which failed to be separated efficiently, while their retention time in CCC was about 100 min and 120 min, respectively, which were eluted efficiently. Similarly, the elution times of components C and D in CCC were both between 55 and 75 min, and the peaks overlapped, while their retention times in GC were about 25.649 min and 28.780 min, respectively, which achieved the effective separation. In brief, the combination of the two chromatographic techniques compensated for their respective separation drawbacks and significantly improved the orthogonality.

It was very interesting to note that the chemical composition of the essential oils was really complex and varied. However, when we grouped these chemicals into broad categories, we did manage to find some significant similarities. The main components of the essential oils were usually hydrocarbons, alcohols, esters, aldehydes, ketones, ethers, phenols, and terpenes. These compounds gave essential oils their unique aroma, flavor, and biological activity. A large number of structurally similar substances were identified in the essential oil of Wenjing Tongluo San by GC-MS. The compounds were categorized into six zones based on the CCC separation, as shown in Figure 3. The compounds in different zones were divided into the following groups: alkoxysilanes in the first zone, aldehydes, and alkoxysilanes in the second zone, alcohols and terpenoids in the third zone, alkoxysilanes and monoterpenes in the fourth zone, ketones in the fifth zone, coumarins and esters in the sixth zone (Figure 3 and

Table 1. Substance classification in zone1: Alkoxysilanes.

No.	Compounds	Formula	MF	RMF	CAS	ID	Retention Time (min)
1	Cyclotetrasiloxane, octamethyl	C8H24O4Si4	850	910	556-67-2	41370	13.003
2	Cyclopentasiloxane, decamethyl	C10H30O5Si5	881	901	541-02-6	296357	22.247
3	Cyclohexasiloxane, dodecamethyl	C12H36O6Si6	830	927	540-97-6	12776	33.26
4	Cycloheptasiloxane, tetradecamethyl	C14H42O7Si7	662	843	107-50-6	41361	43.72

,

Table 2. Substance classification in zone2: Alcohols and ketones.

No.	Compounds	Formula	MF	RMF	CAS	ID	Retention Time (min)
1	3,6-Heptadien-2-ol, 2,5,5-trimethyl-, (E)-	C10H18O	782	880	26127-98-0	9037	12.837
2	Bicyclo[3.1.1]heptan-3-ol, 6,6-dimethyl-2-methylene-, [1S-(1α,3α,5α)]-	C10H16O	708	901	547-61-5	17309	20.495
3	2-Cyclohexen-1-ol, 1-methyl-4-(1-methylethyl)-, trans	C10H18O	751	851	29803-81-4	17434	20.642
4	Camphor	C10H16O	828	904	76-22-2	18060	20.842
5	p-Mentha-1,5-dien-8-ol	C10H16O	586	808	1686-20-0	35243	21.181
6	endo-Borneol	C10H18O	877	906	507-70-0	85143	22.185
7	α-Terpineol	C10H18O	662	803	98-55-5	9091	23.833
8	Naphthalene, 1,2,3,4,4a,5,6,7-octahydro-4a-methyl	C11H18	659	758	13943-77-6	148898	24.949
9	2-Cyclohexen-1-ol, 2-methyl-5-(1-methylethenyl)-, cis	C10H16O	708	799	1197-06-4	21593	25.649
10	(Z)-3-Phenylacrylaldehyde	C9H8O	948	953	57194-69-1	143344	28.78

,

Table 3. Substance classification in zone3: Ketones and alcohols.

No.	Compounds	Formula	MF	RMF	CAS	ID	Retention Time (min)
1	Linalool	C10H18O	852	876	78-70-6	45273	18.376
2	Bicyclo[2.2.1]heptan-2-one, 1,7,7-trimethyl-, (1S)-	C10H16O	882	922	464-48-2	84807	20.843
3	(+)-2-Bornanone	C10H16O	897	922	464-49-3	18069	20.843
4	Terpinen-4-ol	C10H18O	849	888	562-74-3	11291	22.963
5	α-Terpineol	C10H18O	794	883	98-55-5	9091	23.845
6	Perilla ketone	C10H14O2	715	847	553-84-4	85137	27.694
7	2-Cyclohexen-1-one, 3-methyl-6-(1-methylethyl)-	C10H16O	906	933	89-81-6	21855	27.799
8	2-Penten-1-one, 1-(3-furanyl)-4-methyl	C10H12O2	801	838	34348-59-9	85362	30.961
9	Decanoic acid, 1,1a,1b,4,4a,5,7a,7b,8,9-decahydro-4a,7b-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-	C40H64O9	518	564	24928-17-4	9246	34.566

and

Table 4. Substance classification in zone4: Esters and coumarins.

No.	Compounds	Formula	MF	RMF	CAS	ID	Retention Time (min)
1	1,2-Benzenedicarboxylic acid, butyl octyl ester	C20H30O4	533	786	84-78-6	29683	71.39
2	Osthole	C15H16O3	687	814	484-12-8	260376	74.93
3	Hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester	C35H68O5	640	691	761-35-3	8529	81.01
4	Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester	C19H38O4	768	866	23470-00-0	8469	81.03
5	Octadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester	C21H42O4	636	795	621-61-4	8534	84.09

).

In summary, the compounds with different structural features in the essential oil were summarized, and the two-dimensional fingerprints of the essential oil of Wenjing Tongluo San were established, which helped clearly observe the main material composition of the essential oil.

3.3. Orthogonality Evaluation of Comprehensive Two-Dimensional CCC × GC Analysis of the Wenjing Tongluo San Essential Oil

The subsequent orthogonality evaluation of the comprehensive two-dimensional CCC × GC analysis of the Wenjing Tongluo San essential oil was based on three indexes, including peak capacity, linear correlation coefficient, and spatial coverage [37,38]. The peak number in the first CCC dimension was 8, according to Equation (1), while the 375 peaks were obtained in the second GC dimension. The results revealed that a total of 3000 theoretical peak capacities were obtained in the separation of Wenjing Tongluo San essential oil with the comprehensive two-dimensional contour plot. The comprehensive two-dimensional chromatographic separations greatly increased the separation capacity compared to the single-dimensional separations. There were several methods for calculating the spatial coverage. The common ones were the bin-counting method and the convex hull calculation method. The experimental results showed that the spatial coverage obtained via the convex hull method was 70.42%, as shown in Figure 4. The result showed that there was a high spatial coverage in the two-dimensional separation of Wenjing Tongluo San essential oil, which generated a powerful independent separation mechanism.

The linear correlation refers to the variability of the interaction of the separated solute with the two-dimensional stationary phase as well as the mobile phase during the two-dimensional chromatographic separation. It mainly reflected the difference in the solute retention behavior, which was caused by the differential isotropy of the separation mechanism. When the linear correlation coefficient r was 0, it indicated that the two-dimensional separations were very weakly correlated, and the two-dimensional chromatograms differed greatly in the solute selectivity. When the linear correlation coefficient r was 1, it indicated that the two-dimensional separations were strongly correlated, and the two-dimensional chromatograms were strongly similar for the solute selection, with almost no orthogonality. After normalizing the retention time in both CCC and GC, the linear correlation coefficient r between the two dimensions was 0.42 according to the linear fit, as shown in Figure 5. In general, the r value less than 0.5 was a low degree linear coefficient. The obtained r value indicated that the comprehensive two-dimensional CCC and GC in separation had low correlation and high orthogonality.

4. Conclusions

In this paper, the essential oil of the in-hospital preparation Wenjin Tongluo San was characterized through an offline comprehensive two-dimensional CCC × GC, and the orthogonality was evaluated. The solvent systems with small polarity were applied for the first-dimensional CCC separation, and each fraction collected from the first dimension was transferred to the second-dimensional CC analysis. High peak capacity (3000 peaks), high spatial coverage (70.42%), and high degree of orthogonality (r = 0.42) were obtained. The comprehensive two-dimensional CCC × GC technique was able to achieve the orthogonal separation effects by separating through two-dimensional columns, which further improved the separation purity and accuracy. It was interesting to note that the two-dimensional separation could intuitively divide the compounds in the essential oil into different component groups.

In the same conditions of the analysis time and detection limit, the peak capacity of comprehensive two-dimensional chromatography reached 8 times or even higher than that of traditional one-dimensional chromatography. In particular, the resolution improved significantly, and the effective separation of multiple components in complex samples was effectively realized. Particularly, when the method delineated herein is integrated with advanced MS detectors, it offers enhanced capabilities for the identification of unknown compounds. The research work provided new separation and analysis methods for complex systems such as a series of natural products. Meanwhile, due to the advantage of good compatibility of mobile phases, it provided ideas for constructing new comprehensive two-dimensional chromatographic methods with promoted functions.