Study on Lavender Essential Oil Chemical Compositions by GC-MS and Improved pGC

Lavender essential oil from the aerial parts of Lavandula angustifolia Mill. was analyzed by GC-MS equipped with three capillary columns of different polarities, which were HP-1, HP-5 ms and HP-INNOWax. A total of 40 compounds were identified by GC-MS, accounting for 92.03% of the total essential oil compositions. Nineteen monomers were separated by column chromatography and improved preparative gas chromatography (pGC), six of which could not be retrieved from the NIST 14 (National Institute of Standards and Technology, USA; 14th edition) library database. Fifteen compounds were identified for the first time in lavender essential oil. The improved pGC not only doubled the efficiency but also greatly reduced the cost.


Introduction
Lavender (Lavandula angustifolia Mill.) is a perennial herb and a member of the Lamiaceae (Labiatae) family that is native to the Mediterranean region and has been grown all over the world because of the huge market for essential oils. Lavender essential oil is widely used in fragrances and commodities including colognes, skin lotions, soaps, food flavorings, perfumes and aromatherapeutic medicines [1]. Lavender is also widely cultivated in China, and its main producing area is in Yili, Xinjiang. The Lavandula genus is divided into 37 varieties according to the shape of the leaves, corolla morphology, calyces and bract in "Lavender: The genus Lavandula" [2]. Only the essential oils of three lavender species (Lavandula angustifolia, Lavandula latifolia and Lavandula hybrid) play an important role in the perfume and cosmetics industry [3]. The essential oils of lavender are mainly produced from glands on the surface of the flowers and the leaves [2].
Although the chemical compositions of lavender essential oil were studied by GC-MS in different literatures [4,5], there is no report on the separation and analysis of lavender essential oil by preparative gas chromatography (pGC), NMR and GC-MS equipped with three capillary columns of different polarities. This study could not only verify experimental results, but also the isolated monomer by pGC is of great help to the study of the activity of the single and composite component of lavender essential oil. The combination of these two methods can effectively solve the problem of identifying the accuracy of essential oil components by GC-MS without reference compounds. Therefore, the study has important analytical significance for identifying essential oil components.
Many reports published have shown lavender essential oil to possess high cholinesterase inhibitory activities [6,7] and other biological activities beneficial to human health, such as being antibacterial, antifungal [8], sedative [9], anti-depressive, effective for burns and insect bites [10], anticancer [11], anti-spasmolytic, anti-inflammatory [12], antioxidant [13], acaricides etc. [14]. The main component of essential oils are terpenoids, which are low-molecular and lipophilic compounds that can easily cross the blood-brain barrier [15]. The compounds separated from lavender essential oil could lay the foundation for subsequent drug research.

Chemical Composition of the Essential Oils
The essential oil was extracted by hydrodistillation from the fresh plants of lavender aerial parts, and the mean values of the oil yields were 1.3% (v/w; mL/g) based on fresh weights. The essential oil GC-MS results of three different polarity capillary columns, which were HP-5 ms, HP-1 and HP-INNOWax capillary columns, are presented in Table 1 and Figures S1-S3 (Supplementary Materials). The relative content of the essential oil components was normalized by peak area and expressed as a percentage. The integration method is that 0.8% of the maximum peak area was selected as the minimum integral peak. According to the above integration method, 40 peaks could be identified by comparing their real retention indices relative to n-alkanes (C 7 -C 30 ) and mass spectra with the NIST 14 (National Institute of Standards and Technology, USA; 14th edition) Mass Spectral Library, which accounts for 92.03% of the total amount of essential oil compounds. The ninth peak can be determined as the mixed peak of the cis and trans isomer of the linalool oxide according to the GC-MS results of the HP-1 and HP-INNOWax capillary columns.
The compounds that were separated by silica gel column chromatography and pGC were analyzed and identified by NMR ( Figures S4-S21) (Supplementary Materials). Twelve compositions, which were compounds 9, 10, 12, 13, 17, 18, 21, 27, 32, 38, 39, 40 and 43, were further identified by comparing the carbon spectrum data in the literature [16][17][18][19][20][21][22][23][24][25][26][27][28]. Compared with the method of determining compounds by GC-MS, six missing compositions, which were compounds 19, 29, 33, 41, 45 and 46, were identified by comparing the carbon spectrum data in the literature [29][30][31][32][33][34], and could not be retrieved from the NIST 14 database. By comparison with the literature [35], 15 compounds were newly identified. In this study, sulfur was isolated from essential oil for the first time, and was identified by GC-MS, whose mass spectrogram is shown in Figures S2 and S3 (Supplementary Materials). Therefore, it was proved that lavender essential oil contained a small amount of sulfur compounds. The sulfur compounds provide a new explanation for the insecticidal activity of lavender essential oil and provide a new research idea for later researchers. This finding may help to develop lavender essential oil into a new natural acaricides [36].

Improvements in the Fraction Collector of pGC
In the early stages of the separation of lavender essential oils by pGC, we found that the cost and separation efficiency of this instrument were too low. To solve this problem, we converted the liquid nitrogen cooling system of the fraction collector to a cold trap cooling system. This improvement not only increased efficiency but also greatly reduced costs.
By improving the preparation fraction collector of pGC without affecting the normal operation of the pGC, it was found that the improved pGC had a similar collection amount in the same time and temperature compared with the unmodified equipment. The liquid nitrogen cooling system of the preparative fraction collector of pGC was transformed into a cold well cooling system. This improvement doubled the efficiency of preparative gas chromatography, and the cost of the cooling system was reduced from RMB 675/day to RMB 25/day.

Plant Material and Reagents
The aerial parts of Lavandula angustifolia Mill. were collected in June 2019 in Yili, Xinjiang, China. The plant material was dried in the shade. The plant sample was identified by Dr. Chunfang Lu and the voucher specimen was stored in the Xinjiang Technical Institute of Physics and Chemistry, Urumqi, Chinese Academy of Sciences (voucher species man No. WY02260). Anhydrous sodium sulfate was purchased from Tianjin Hongyan chemical reagent factory (Tianjin, China), n-hexane, ethyl acetate, and acetic acid were purchased from Tianjin Yongsheng Superfine Chemical Industry Co., Ltd. (Tianjin, China). n-Alkanes (C 7 -C 30 ) were purchased from Sigma-Aldrich (Shanghai, China).

Extraction of the Essential Oil
The lavender essential oil was obtained from aerial parts of the lavender that were cut into about 2 cm by hydrodistillation for 3 h using a self-assembling Clevenger-type apparatus, and the ratio of material to liquid was 1:10. The essential oil was collected, centrifuged, and taken from the upper layer. The lavender essential oil was extracted three times with 1200 g of material each time. The yields were 12.9852 g, 13.9137 g and 11.5129 g, respectively. The upper layer (essential oil) was dried over Na 2 SO 4 and stored at −20 • C until required for analysis and separation.

Gas Chromatography Analysis
The analysis of the essential oil was first performed on an Agilent GC-QTOF-MS system consisting of a 7890B gas chromatograph equipped with HP-5 ms capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm) and 7693 autosampler, connected to a hybrid QTOF mass spectrometer (Agilent model 7200, Santa Clara, CA, USA), controlled by MassHunter Acquisition B.07.00 software (Santa Clara, CA, USA).
Helium was used as a carrier gas at a flow rate of 1 mL/min. The injector and detector temperatures were 250 • C. The oven temperature was programmed from 70 (10 min) to 100 • C at a rate of 2 • C/min, then from 100 to 200 • C at a rate of 5 • C/min. The injection volumes were 0.4 µL. The split injection was conducted with a split ratio of 1:40. The mass spectra were recorded at 70 eV (EI) and were scanned in the range 50-500 m/z.
The essential oil was analyzed on the Agilent GC-MS system consisting of a 7693 autosampler and 7890A gas chromatograph connected to a 5975C mass spectrometer (Santa Clara, CA, USA) (inert XL EI/CI MSD with Triple-Axis detector), controlled by 5975-7890GC-MS software (Santa Clara, CA, USA). The GC was equipped with HP-INNOWax (30 m × 0.25 mm i.d., film thickness 0.25 µm) and HP-1 capillary columns (30 m × 0.25 mm i.d., film thickness 0.25 µm). Nitrogen was used as a carrier gas at a flow rate of 0.8 mL/min. The injector and detector temperatures were 250 • C. The oven temperature was programmed from 60 to 180 • C at a rate of 8 • C/min, then from 180 to 240 • C (5 min) at a rate of 10 • C/min. The injection volumes were 0.8 µL. The split injection was conducted with a split ratio of 1:40. The mass spectra were recorded at 70 eV (EI) and were scanned in the range 30-500 m/z. The components were identified by comparing their real retention indices relative to the n-alkanes (C 7 -C 30 ) and the mass spectra with the NIST 14 Mass Spectral Library. The formula for calculating the retention index is as follows [36]: where T X is the component retention time; Z is the carbon number of n-alkanes component; T Z is the the retention time of n-alkanes that carbon number is Z; and T Z+1 is the the retention time of n-alkanes that carbon number is Z + 1.

Isolation and Structure Elucidation
The pGC was modified based on the Agilent 7890B gas chromatograph system (Agilent model 7200, Santa Clara, CA, USA). It was equipped with a HP-5 capillary column (30 m × 0.53 mm i.d., film thickness 1.0 µm), a G4513A autosampler, a flame ionization detector (FID), a modular analytical system and a preparative fraction collector (Gerstel Company, Mülheim, Germany) equipped with a home-made cold trap cooling system.