pubs.acs.

org/jchemeduc Laboratory Experiment

GC−MS Analysis of Essential Oil Extracted from Acori tatarinowii

Rhizoma: An Experiment in Natural Product Analysis
Chang-Dong Zhang,§ Xin-Yuan Hu,§ Huai-Song Wang,* and Fang Yan
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ABSTRACT: A comprehensive undergraduate experiment about

extraction, separation, and identification of essential oils (EOs)
from a natural plant is described. Acori tatarinowii Rhizoma (ATR)
was used as the raw plant material. Two devices (Soxhlet and
Clevenger) with different extraction principles were employed for
EO extraction from ATR, separately. The chemical compounds of
the extracted EOs were separated and identified by gas
chromatography−mass spectrometry (GC−MS). The differences
in the chemical components obtained by the two extraction devices
were compared. The experiment can give students a deep insight
into the difference between the extraction methods. Additionally, the experiment can enable students to learn how to use GC−MS to
identify the chemical structures of the extracted complex samples. The grouped students who performed this experiment gained a
deep understanding of extraction and detection techniques, as well as the benefits of teamwork.
KEYWORDS: Upper-Division Undergraduate, Laboratory Instruction, Hands-On Learning/Manipulatives, Separation Science,
Gas Chromatography, Mass Spectrometry, Analytical Chemistry, Natural Products, Drugs/Pharmaceuticals

■ INTRODUCTION
The study of natural products (NPs) provides a good strategy
for the discovery of new bioactive chemical components. Many
NP structures have been successfully applied in the
pharmaceutical field.1,2 The awarding of the Nobel Prize in
2015 highlights the importance of NPs in physiology or
medicine. W. Campbell, S. Omura, and Y. Tu were given this
recognition for their pioneering works in the use of NP drugs
to target parasitic diseases. Because of the broad structural
diversity of NPs, it is very important to separate and analyze
the potential bioactive components before studying their
physiological activities. Introducing the experimental content
related to extraction, separation, and detection of the chemical
compositions in the undergraduate laboratory will be
significant for their research interests, laboratory capability,
and graduate school preparation.
Essential oils (EOs, also termed volatile oils) are extracted
from plants obtained mainly by distillation processes (such as
hydrodistillation, steam distillation, and dry distillation) or
solvent extraction.3,4 Generally, the EOs of plants are
biosynthesized in different organs and parts (such as leaves,
flowers, fruits, and roots). EOs are aromatic low-molecular-
weight compounds, including aliphatics, aromatics, mono-
terpenes, and sesquiterpenes, plus their oxygenated derivatives,
mainly alcohols, aldehydes, and ketones, which are important
in food, cosmetic, and pharmaceutical industries. Most of the
EOs possess physiological and pharmacological properties,
© XXXX American Chemical Society and
Division of Chemical Education, Inc.
A J. Chem. Educ. XXXX, XXX, XXX−XXX
such as anti-inflammatory, antimicrobial, analgesic, and
wound-healing activities.5,6
Acorus tatarinowii Schott belongs to the family of Acoraceae
and is widely distributed in eastern and southern Asia, north
America, and eastern Europe.7 It has been used as a traditional
Chinese medicine (TCM) for thousands of years for the
treatment of many diseases, such as forgetfulness, dementia,
and apoplexy. Modern pharmacological studies demonstrated
that its extract showed a variety of biological activities such as
hypoglycemic, antioxidant, anti-Alzheimer’s disease (anti-AD),
antimicrobial, anticonvulsive, and antiepileptic properties. The
active components are mainly found in the EOs from its
rhizome.8,9
The laboratory described here is an undergraduate experi-
ment focusing on the extraction and detection of EOs from
Acori Tatarinowii Rhizoma (ATR) in the course “Natural
Product Analysis”. This experimental process enables students
to experience the discovery of natural chemical compounds,
applying principles from extraction, separation, and structure
identification sciences. The experiment is divided into two
parts (extraction and detection) and completed in 2 weeks
Received: April 19, 2021
Revised: July 26, 2021
https://doi.org/10.1021/acs.jchemed.1c00451
Journal of Chemical Education
Figure 1. Illustration of the extraction and detection of EOs in ATR by GC−MS.
Figure 2. Devices (left, Soxhlet extractor; right, Clevenger-type apparatus) used for EO extraction from ATR.
(two laboratory classes). The EO extraction experiment is
performed in the first week. Different extraction methods have
a great influence on the composition of the extracted EOs.
Therefore, two devices (Soxhlet and Clevenger) are used for
EO extraction from ATR, separately.4,10 That is, the EOs of
ATR are extracted by two different extraction principles:
solvent extraction with a Soxhlet extractor and hydrodistillation
with a Clevenger-type apparatus. In the second week of the
experiment, gas chromatography−mass spectrometry (GC−
MS) is used to identify compounds of the extracted EOs,11−13
and the differences of the chemical components obtained by
the two extraction methods are compared.
This lab course is designed to guide students to have a more
comprehensive understanding of the process of extraction and
chemical identification in NPs. These procedures are used in
many laboratories around the world. The main objectives of
the experiment include the following: (1) guide students to
apply the knowledge of analytical chemistry to studying a
chemical analysis process for NPs, (2) help students to
understand the influence of the sample pretreatment methods
on the results of chemical composition analysis, and (3) learn
the important aspects of GC combined with MS in the
pubs.acs.org/jchemeduc Laboratory Experiment
chemical structure identification. Students were encouraged to
investigate parameters of GC (especially the column temper-
ature parameters in the complex sample analysis) and MS (the
various scan modes with different ionization energies)
appropriately to obtain better separation and analysis results.
■
EXPERIMENTAL SECTION
Students were organized with 3−4 members per group,
working collaboratively. Each group was provided with ATR
samples, a Soxhlet extractor, a Clevenger-type apparatus, and
the EO extraction related reagents and devices. The procedure
is shown in Figure 1.
Reagents, Materials, and Apparatus
ATR, dried in a vacuum oven at 40 °C for 10 h, was crushed
into particles with size of 0.4−0.9 mm by an herb grinder.
Petroleum ether (60−90 °C), distilled water, sodium sulfate
(anhydrous), Soxhlet extractor with 100 mL flask and
condenser, and Clevenger apparatus with 500 mL flask and
condenser were used for EO extraction. The extracted EOs
were detected by GC−MS (GC-MS-QP2020, Shimadzu)
equipped with helium gas (99.9999% purity) as the GC
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J. Chem. Educ. XXXX, XXX, XXX−XXX
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carrier gas and a GC column (SH-Rxi-5MS, 30 m × 0.25 mm
× 0.25 μm film thickness) (Shimadzu).
Laboratory Period 1: EO Extraction
The EOs were extracted in the first week. Before the
experiment, students were required to preview the course
and to learn about chemical components in ATR through a
literature search. Also, students should possess the basic
knowledge of EO extraction. A brief discussion about these
points should be guided by the instructor before the start of
the experiment.
Two devices were employed to extract the EOs from ATR, a
Soxhlet extractor and a Clevenger-type apparatus, separately
(Figure 2). In each group, students cooperated with each other
to set up the two extraction devices. The two extraction
methods were carried out simultaneously.
Soxhlet-Based EO Extraction. A 6.0 g portion of crushed
dry ATR and 65 mL of petroleum ether (60−90 °C) were
placed in the Soxhlet extractor, separately. The extraction was
performed in a water bath at 80 °C for 4 h. The petroleum
ether in the extracted solution was removed by a rotary
evaporator at 50 °C. The obtained EO was weighed and then
redissolved in petroleum ether to 5 mL. The EO sample was
stored in the dark at 4 °C and further GC−MS analysis.
Clevenger-Based EO Extraction. Crushed dry ATR (60.0
g) and distilled water (300 mL) were placed in the Clevenger
apparatus, separately. Then, 1.0 mL of petroleum ether (60−
90 °C) was accurately added in the graduated tube. The
sample in the flask was refluxed for 4 h. The volume of the
petroleum ether with extracted EOs (VEO+PE) is recorded. A
control experiment was also performed. The volume and
weight of the extracted EO were recorded. The extracted EO
mixture was dissolved in petroleum ether to 5 mL and stored
in the dark at 4 °C until the samples were used for GC−MS
analysis.
Laboratory Period 2: GC−MS Analysis
The extracted EOs by the Soxhlet extractor and Clevenger-
type apparatus, separately, were determined by GC−MS in the
second week. Meanwhile, pure petroleum ether was also
determined by GC−MS in the control experiment. Before
starting the experiment, the instructor taught the students how
to use the GC−MS instrument and identified points for
attention.
The GC−MS (GC-MS-QP2020, Shimadzu) instrument was
used in this experiment. Vials, filled with EO samples extracted
by the Soxhlet extractor and Clevenger apparatus, separately,
were placed in the GC−MS autosampler, and the sequence of
sample injections was scheduled. The EOs were analyzed by
the GC−MS using a fused-silica capillary column with a weakly
polar stationary phase, SH-Rxi-5MS (30 m × 0.25 mm × 0.25
μm film thickness). The detailed GC and MS parameters were
listed in the Supporting Information (pages S6−S7). Students
were encouraged to optimize the oven temperature progress of
the GC to obtain better separation resolutions of the complex
EO samples. EI mass spectra were used to identify compounds
by computer matching with commercial mass spectral libraries.
Relative amounts of individual components were calculated on
the basis of their GC peak areas.
■
HAZARDS
During the whole experiment, standard safety precautions
should be used. Personal protective equipment (PPE) must be
worn during the experiment. Petroleum ether is extremely
C https://doi.org/10.1021/acs.jchemed.1c00451
Laboratory Experiment
volatile, has a very low flash point, and presents a significant
fire hazard. Exposure to petroleum ether must be minimized.
Petroleum ether may cause dizziness and drowsiness if inhaled,
and high concentrations may result in central nervous system
depression, and loss of consciousness.
■
RESULTS AND DISCUSSION
This experiment was designed for third- or fourth-year
undergraduate students in pharmaceutical analysis. The
students have completed the courses in the chemistry of
natural products, analytical chemistry, and organic chemistry
and have experience in operating chromatographic instru-
ments. In previous laboratory courses, students have had
experimental experience about extraction or enrichment of one
special compound in complex samples for determination and
have completed quantitative analysis experiments by gas
chromatography with a flame-ionization detector (GC-FID).
However, GC-FID is mainly used for quantifying the known
components with the corresponding standard solutions. With
the consideration of the advantages of GC−MS in separation
and analysis of complex samples,14,15 this experiment was
prepared to be included in the course “Natural Product
Analysis” from the year 2018 and then performed and
improved in the following two years. This experiment will
help students better understand how to extract chemical
components from complex samples and identify unknown
components by GC−MS.
The student assessment is mainly based on the lab reports
and teachers’ observation of students’ cooperation and
exploration in the practice. The evaluation criteria are as
follows (details refer to Table S1 in Supporting Information).
• Preview: Students’ understanding of the experimental
principle can be evaluated from the prelab questions and
discussions.
• Experimental operation: The experimental operation is
correct; there is careful observation of the experimental
phenomenon. The data record is correct, and occasional
failures are dealt with flexibly.
• Data processing and discussion: There is discussion
about the experimental results; the problems are found
in the process of the experiment. The practical
significance of the experiment is determined. Improve-
ment needed is in this experiment, other means are to
complete the experiment.
• Other: Teamwork ability and enthusiasm are demon-
strated in the experiment.
The prelab handout (Supporting Information, pages S9−
S16) was assigned to students 1 week before the laboratorial
course. At the beginning of the experiment, students discussed
the prelab questions based on their prior knowledge. In the
first week, they discussed the chemical components in the ATR
that had been reported, the techniques used for EO extraction,
and the factors that could potentially affect the extraction
results. In the second week, they discussed the potential
techniques for the determination of the extracted EOs, the
sample characteristics that were suitable for GC, and the
advantages of mass spectrometry as a GC detector. Students
were assessed with these questions by the instructor. These
questions could evaluate the basic knowledge of students, as
well as their preview situation.
In the experiment, experimental operation, data processing,
and conclusions are the most important parts of student
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assessment. The most basic requirement for students is extraction time could be prolonged appropriately to increase
independent operation. The data record is correct, and the the extracted amount of EOs in future practical applications.
observation of the experimental phenomena is careful. If According to the results of the student experiment, the EO
students can deal with the mistakes in time, discuss the volume extracted by the Soxhlet extractor and the Clevenger
experimental principles in depth, show good teamwork ability, apparatus was 0.42 ± 0.06 and 0.36 ± 0.05 mL, respectively.
and actively explore in the experiment, they will obtain higher Obviously, the EO extraction mechanisms of the Soxhlet
scores. extractor and Clevenger apparatus are different. The instructor
Furthermore, students should answer the follow-up ques- led the students to discuss the potential differences in chemical
tions in the laboratory handout during the discussion. For composition of the extracted EOs between the two methods.
example, compare the advantages (disadvantages) of the two Compared with the Clevenger apparatus, the Soxhlet extractor
EO extraction devices, the purpose for adding 1.0 mL of extracts EOs by direct dissolution. Compounds that are not
petroleum ether in Clevenger apparatus, and the differences volatile but have less polarity may also dissolve in petroleum
between “SCAN” and “SIM” in GC−MS. These questions will ether. Also, the EOs should be collected after the rotary
strengthen the knowledge that students have learned in this evaporation process, during which higher volatile components
experiment. may be lost. The EOs extracted by the Clevenger apparatus
EO Extraction from ATR only dissolved in a small amount of petroleum ether, and no
rotary evaporation is needed. However, the amount or volume
There have been various methods developed for EO extraction,
of the EO extracted by the Clevenger apparatus might be less
mainly including hydrodistillation, steam distillation, solvent
than that extracted by the Soxhlet extractor. Therefore, GC−
extraction, and supercritical fluid extraction.16,17 The compo-
sition and content of chemical compounds in the extracted MS with a strong qualitative ability plays an important role
EOs are varied according to the different extraction methods. here.
As to the therapeutic EOs, their effect on the disease treatment GC−MS/SCAN Analysis
may be potentially affected by the extraction methods. After a detailed introduction to the application of the GC−MS
Therefore, it is important to choose suitable methods for instrument, the students were required to analyze the two EO
extracting therapeutic EOs. In this experiment, the classical samples, separately. Typical GC spectra generated by students
solvent extraction and hydrodistillation were selected and were shown in Figure 3A. Since the solvent petroleum ether is
compared to make students aware of the role of the EO an organic mixture, students may have questions about the
extraction methods. possibility that petroleum ether molecules may also be retained
The chemical constituents of the EOs of ATR mainly and separated by GC column. Therefore, pure petroleum ether
contain lignans, phenylpropanoids, and terpenoids.18,19 Among was used as a blank sample and analyzed by the GC−MS. The
them, asarone-based phenylpropanoids (α-asarone and β- chromatography peaks of all samples from 3 to 40 min were
asarone) are the characteristic constituents. As a type of
solvent extraction device, a Soxhlet extractor (Figure S1) has
its own advantages in EO extraction.20 The instructor
introduced the mechanism of the Soxhlet extractor in detail
and guided students to think about the advantages of such
design in the EO extraction. The advantages of the Soxhlet
extractor include the simple design, continuity of the extraction
process, and less solvent dosage. Most importantly, the main
advantage is that pure solvent is fed inside the extractor, and
the extracts can be concentrated in the distillation flask after
many cycles.21
As a type of hydrodistillation device, the Clevenger
apparatus is widely used.10 There are two types of the
Clevenger apparatus for the extraction of EOs (Figure S2).
The relative density of ATR EOs is higher than 1.0, showing
that the Clevenger apparatus in Figure S2b is suitable for
directly extracting EOs. However, due to the small volume of
the extracted EOs and emulsification to different degrees, it is
difficult to read its volume with the graduated tube accurately.
Therefore, the Clevenger apparatus in Figure S2a was selected
in this experiment. Petroleum ether (1.0 mL) was precisely
added in the measuring tube to ensure that the EO layer stayed
on the top of water.
The members of each group cooperated to carry out the two
extraction experiments simultaneously. There are several
points about which the instructor needs to remind the
students: ensure the devices are connected tightly, ensure the
condensate water is supplied, and prevent explosive boiling
during heating. Due to the limitation of class hours, the Figure 3. GC−MS profiles of EO samples (A) and typical identified
extraction time for both of the two methods was 4 h in this chemical compounds (B): C1, methyl eugenol; C2, (Z)-methylisoeu-
experiment. The instructor explained to the students that the genol; C3, γ-asarone; C4, β-asarone; C5, α-asarone.

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Table 1. Chemical Identification Results of the EO Samples by GC−MS

Relative C ontent (% Area)
Code Name Chemical Formula Molecular Weight (g mol−1) Clevenger Soxhlet
1 Endoborneol C10H18O 154.25 0.21 0.05
2 Terpineol C10H18O 154.25 0.17 0.04
3 Methyl eugenol C11H14O2 178.23 1.18 0.37
4 β-Cedrene C15H24 204.35 0.37 0.21
5 (Z)-Methylisoeugenol C11H14O2 178.23 5.53 2.41
6 Acoradiene C15H24 204.35 0.23 0.12
7 Eremophila ketone C15H24O 220.35 9.05 3.49
8 γ-Asarone C12H16O3 208.25 7.22 6.53
9 Cedrol C15H26O 222.37 0.18 0.07
10 β-Asarone C12H16O3 208.25 48.9 61.3
11 α-Asarone C12H16O3 208.25 1.59 1.87

recorded. There was no obvious chromatographic peak in the and further analyzed (Figure 4). The mass spectrum showed a
pure petroleum ether, indicating that the molecules of molecular ion peak at m/z 178. In addition, other prominent
petroleum ether have been eluted from the column at the
void time.
The main task of the students was to analyze the
chromatogram behavior and to identify the chemical structures
of the main components. In the beginning, students discussed
the chromatogram results of the EO samples extracted by the
Soxhlet extractor and the Clevenger apparatus. According to
Figure 3A, the peak area (or peak height) of all peaks before
25.6 min in the EO sample extracted by the Clevenger
apparatus is larger (or higher) than that extracted by the
Soxhlet extractor. After 25.6 min, an opposite phenomenon
appeared. The instructor guided students to find out the
reasons for this phenomenon. This is mainly due to the
difference of the two extraction methods and the postprocess-
ing methods. The Clevenger method is mainly to extract the
components with a low boiling point which can be distilled
with water vapor. However, the Soxhlet extractor is mainly to
dissolve and extract the components with a low polarity.
Therefore, the sample extracted by the Soxhlet extractor
generally contains EO components, as well as a small amount
of nonvolatile components with low polarity. Due to a larger Figure 4. (A) Mass spectrum of methyl eugenol and (B) the major
amount of organic solvent used in the Soxhlet method, a fragments.
process of solvent removal by rotary evaporation is needed.
This resulted in the loss of some low-boiling components. In peaks at m/z 163, m/z 47, and m/z 91 were also identified.
the low-polarity GC column, the retention time of the Other typical results were shown in the Supporting
compounds depends mainly on their volatility. Therefore, the Information (pages S30−S34). Additionally, it is necessary to
relative quantity of each component of the EOs obtained by remind students that chemical identification errors may occur
the two extraction methods is different. An oven temperature by simply computer matching with mass spectral libraries,
progress during GC separation was used in the experiment.22,23 especially in the cases of low similarity. Analyzing fragmenta-
The instructor encouraged students to optimize the oven tion spectra, as well as employing reference substances, is
temperature process to obtain ideal separation results. required to further confirm the structures of the chemical
Another important task is to identify the structure of the compounds.
extracted compounds. All data were obtained by acquiring full- At the end of the experiment, students wrote lab reports,
scan mass spectra (SCAN) in the scan range 30−500 m/z. The including an introduction, experimental section, discussion
ionizing energy was 70 eV. Relative amounts of individual about their results, and conclusion. The discussions were
components were calculated on the basis of their GC peak mainly about the effects of extraction methods and solvents on
areas. Compounds were identified using the computer the components of EOs, the advantages and disadvantages of
matching with mass spectral libraries. The chemical the applied extraction methods, some problems that occurred
components mainly identified from ATR in recent laboratory with the use of the GC−MS instrument, as well as some
courses were summarized in Table 1. Also, the typical suggestions for improvements in the experiment. The results
therapeutic compounds (such as methyl eugenol, (Z)- show that the students have been successful in extracting EOs
methylisoeugenol, γ-asarone, β-asarone, and α-asarone) are from the natural plant and identifying the chemical structures
shown in Figure 3B. It is also very important to guide students of the main components by GC−MS.
to use their chemical knowledge to analyze the mass spectra. For further assessing whether the pedagogical goals were
The mass spectrum of methyl eugenol was selected as a model achieved, each student’s score for the lab course was calculated
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by the instructor according to the assessment criteria (Table
S1). Every part of the experiment has its own scoring criteria.
The preview, experimental operation, data processing,
discussion, and other items (teamwork ability and activity),
respectively, accounted for 10%, 25%, 25%, 30%, and 10% of
the total score. The student’s scores from 2018 to 2020 were
shown in Figure S8 (Supporting Information). According to
the results, most of the students can complete the experiment
well. On the whole, the scores students earned on the prelab
questions are higher than those earned on postlab discussion
(Figure S9, Supporting Information). This shows that students
have a deeper understanding of the experimental principle after
completing the experiment. To further identify problems
students had in the lab course, the instructor analyzed the
scores of each experiment part. It was found that there are
some problems that often appear in the experiment. In
particular, there are often no signals in the GC−MS
chromatograms. Most of this problem is because of incorrect
injection volume, high split ratio, wrong temperature
programming, and nonstandard instrument operation and
other reasons. Students usually have a sense of achievement
after solving these problems.
Student evaluation of the course is also a very important
part. Students can provide evaluation through the school’s
website. The students commented favorably on the following
aspects of the lab course: (1) They understood the principle of
the two extraction methods through the operation of this
experiment. (2) The teamwork improved the capacities of
communication and interaction among them and with
instructors. (3) The most interesting thing they learned was
the qualitative method by GC−MS. Students also put forward
some suggestions. For example, the EO extraction time should
be optimized, and more opportunities are needed to investigate
the GC−MS conditions. After the course, students still have
opportunities to consolidate and develop what they have
learned. For example, they can participate in “training
programs for innovation and entrepreneurship” in the
laboratories of the college.
■
CONCLUSION
This experiment enables students to learn essential skills about
natural product extraction and identification of chemical
structures. Components are important as they give the EOs
characteristic natural odors and therapeutic activities. The
proportion of the components is maintained by the extraction
methods. By comparing the extraction methods, students can
understand the importance of method selection in the process
of natural product extraction, separation, and analysis. This
experiment expands students’ use of GC−MS to the detection
of the chemical components. This lab could be utilized in the
classes of Natural Product Analysis, Analytical Chemistry,
Pharmaceutical Analysis, and Food Analysis. Also, the lab
could also be modified by quantitation methods in the GC−
MS analyzing process.
■
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available at https://pubs.ac-
s.org/doi/10.1021/acs.jchemed.1c00451.
Notes for instructors, student laboratory handout week
1, student laboratory handout week 2, instructions for
F https://doi.org/10.1021/acs.jchemed.1c00451
Laboratory Experiment
the experiment, and examples of the students’ results
(PDF, DOCX)
■
AUTHOR INFORMATION
Corresponding Author
Huai-Song Wang − Department of Pharmaceutical Analysis,
China Pharmaceutical University, Nanjing 210009, China;
orcid.org/0000-0001-7892-018X;
Email: wanghuaisong@cpu.edu.cn, wanghuai1234@
gmail.com
Authors
Chang-Dong Zhang − National Experimental Teaching
Demonstration Center of Pharmacy, China Pharmaceutical
University, Nanjing 210009, China
Xin-Yuan Hu − Department of Pharmaceutical Analysis,
China Pharmaceutical University, Nanjing 210009, China
Fang Yan − Department of Pharmaceutical Analysis, China
Pharmaceutical University, Nanjing 210009, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jchemed.1c00451
Author Contributions
§
C.-D.Z. and X.-Y.H. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We would like to express appreciation to the colleagues in the
Department of Pharmacy and National Experimental Teaching
Demonstration Center of Pharmacy (China Pharmaceutical
University) for their contributions to the experimental
platform over the years. We also thank the students who
have participated in this experiment. This work was supported
by the National Natural Science Foundation of China
(21705165), and the Priority Academic Program Development
of Jiangsu Higher Education Institutions.
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