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Chapter 5

Microwave-Assisted Extraction in Natural


Products Isolation
Abbas Delazar, Lutfun Nahar, Sanaz Hamedeyazdan,
and Satyajit D. Sarker
Abstract
Microwave-assisted extraction (MAE) or simply microwave extraction is a relatively new extraction technique
that combines microwave and traditional solvent extraction. Application of microwaves for heating the
solvents and plant tissues in extraction process, which increases the kinetic of extraction, is called microwave-assisted extraction. MAE has a number of advantages, e.g., shorter extraction time, less solvent,
higher extraction rate and lower cost, over traditional method of extraction of compounds from various
matrices, especially natural products. The use of MAE in natural products extraction started in the late
1980s, and through the technological developments, it has now become one of the popular and costeffective extraction methods available today, and several advanced MAE instrumentations and methodologies have become available, e.g., pressurized microwave-assisted extraction (PMAE) and solvent-free
microwave-assisted extraction (SFMAE). This chapter provides an overview of the MAE and presents a
number of specific protocols for natural products extraction.
Key words: Microwave extraction, Microwave-assisted extraction, Natural products, Closed system,
Open system

1. Introduction
During the World War II, Percy Spencer discovered the heating
properties of microwaves while experimenting with radar. Actually,
with a candy bar in his pocket, he leaned in front of the microwave
tube and the candy bar promptly melted. Thus, the heating property of microwaves was evident and this event led to the birth of
microwave ovens after the World War II.
Microwaves are nonionizing electromagnetic waves of frequency (electromagnetic radiations) with a frequency from 0.3 to

Satyajit D. Sarker and Lutfun Nahar (eds.), Natural Products Isolation, Methods in Molecular Biology, vol. 864,
DOI 10.1007/978-1-61779-624-1_5, Springer Science+Business Media, LLC 2012

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300 GHz. Because of their electromagnetic nature, microwaves


have electric and magnetic fields, which are perpendicular to each
other. The electric field offers heating by two simultaneous mechanisms, dipolar rotation, and ionic conduction (1). Microwaveassisted extraction (MAE) or simply microwave extraction is a
relatively new extraction technique that combines microwave and
traditional solvent extraction. MAE has been shown to have a
number of advantages, e.g., shorter extraction time, less solvent,
higher extraction rate and lower cost, over traditional method of
extraction of compounds from various matrices. One of the major
benefits in a modern MAE system is its solvent-free applications
offering environmentally friendly green extraction technology,
especially for essential oils and other volatile natural products. The
use of MAE in natural products extraction started in the late 1980s,
and through the remarkable technological developments, it has
now become one of the popular and cost-saving extraction methods available today, and several advanced MAE instrumentations
and methodologies have become available, e.g., pressurized microwave-assisted extraction (PMAE) and solvent-free microwaveassisted extraction (SFMAE) (17).
As the modern MAE techniques operate at a far higher temperature and pressure, and require shorter extraction time than
traditional Soxhlet or sonication technologies, it has greater extraction efficiency. In microwave extraction, samples are enclosed in
high quality Teflon vessels together with solvent and heated to a
controlled temperature with microwave power. MAE has enviable
flexibility in terms of vessel options, choices of sample size, temperature, pressure, amounts of solvent, and number of samples,
and this flexibility has made this extraction method an attractive
technology for high-throughput sample preparation as well as routine extraction of natural products. However, the open-vessel
options, depending on the matrix to be extracted, are also available
in MAE.
Microwaves are made up of two oscillating perpendicular fields,
i.e., electric field and magnetic field, and the former is responsible
for heating. The principle of microwave heating of samples is based
on its direct impact on polar materials or solvents and is dictated by
two fundamental phenomena, i.e., ionic conduction and dipole
rotation. In most cases, both phenomena take place simultaneously
(see Note 1). The resistance produced by the solution to the migration of ions, as a result of ionic conduction, generates friction that
finally heats up the solution. Dipole rotation means realignment of
the dipoles of the molecule with the rapidly changing electric field.
At 2.45 GHz frequency that is usually used in commercial microwave ovens, the molecules change their arrangement 2 2.45 109
times per second and thus heat is produced. Microwave energy
causes molecular motion by migration of ions and rotation of
dipoles (8). With the decrease of the field, thermal disorder is

Microwave-Assisted Extraction in Natural Products Isolation

91

Table 1
Dielectric constant and dissipation factor of some common
solvents used in extraction process
Solvents

Dielectric constant (g) Dissipation factor tan d


determined at 20C
(104)

Ethanol (EtOH)

1.96

2,500

Methanol (MeOH)

2.87

6,400

2-Propanol

1.66

6,700

Water

2.3

1,570

restored resulting in the release of thermal energy. Microwave


extraction process is generally influenced by solvent nature and
volume, extraction time, microwave power, matrix characteristics,
and temperature (9).
Solvents, depending on the amount of dissipation factor (tan d),
absorb microwave energy and convert it to heat. The value of tan
is determined from the following equation.
tan d = e / e
where, e is dielectric loss and shows efficiency of conversion of
microwave energy to heat, and e is the dielectric constant that
depends on polarizibility of a molecule. Table 1 shows values of dissipation factor and dielectric constant of some solvents. Although
the dielectric constants of the solvents like ethanol (EtOH), methanol (MeOH), and 2-propanol are less than that of water, their tan d
value are more than water. In other words, these solvents have the
better ability to convert microwave energy into heat than water.
Unlike classical conductive heating methods, microwaves heat
up entire sample simultaneously (Fig. 1). Microwaves, because of
molecular dipoles, cause rotation of the molecules, resulting in the
disruption of weak hydrogen bonds. Thus, high viscosity solvents
reduce rotation of the molecules and affect weak hydrogen bonds.
In addition, ion migration under the influence of microwaves
enhances solvent penetration into the plant matrix and thus dissolution of extractable components is facilitated.
Solvent choice for MAE depends on the solubility of the target
analyte, the interaction between solvent, extraction matrix (e.g.,
plant material) and the microwave absorbing properties of the solvent. The extraction yield is generally directly proportional to the
extraction time; the longer the extraction time, the better is the
extraction. Microwave power and irradiation time influence each
other significantly. A combination of low or moderate power with

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Fig. 1. Microwave heating up process.

longer exposure helps avoiding any degradation of compounds


present in the matrix. Particle size of the extractable and the status
in which it is subjected to microwave extraction usually has effect
on the recoveries of the compounds. Like any other extraction
techniques, the smaller the particle size (larger the surface area),
the better is the MAE extraction because of better penetration of
microwave energy. Microwave power and temperature are interrelated and need to be given special consideration particularly when
working with a closed vessel system, where the temperature may
reach well above the boiling point of the solvent. The nature of the
matrix in which the analytes of interest are bound can also have a
profound effect on the recoveries of the compounds; in many cases,
the matrix moisture improves the extraction recoveries.
This chapter deals with the basic methodologies of microwave
extraction, especially in the context of natural products isolation,
and incorporates specific examples with step-by-step protocols.
Extensive details on microwave extraction principle, operation, and
technology are available in the literature (1, 1018).

2. Materials
2.1. Microwave
Extractors

The microwave extractors comprise four major components, i.e., a


microwave generator or source also called as the magnetron, a waveguide, an applicator or extraction vessel, and a circulator. Microwave
extractors are generally of two types, e.g., a closed (or pressurized)
system and an open (or focused) system. Figures 2 and 3 show the
main components of microwave extractors. A dynamic MAE
system is shown in Fig. 4.

Microwave-Assisted Extraction in Natural Products Isolation

93

Extraction vessels

Diffused microwaves

Turntable

Fig. 2. A typical microwave-assisted extraction system: a closed (or pressurized) system.

Water outlet

Reflux system

Water inlet

Magnetron
Wave guide

Extraction
vessel

Solvent
Focussed
microwaves
Sample

Fig. 3. A typical microwave-assisted extraction system: an open (or focused) system.

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Extraction tank
Outlet

Microwave

Microwave

Microwave
Refrigerant

Valve

Inlet
Pump

Controller

Container

Magnetic stirrer

Fig. 4. A dynamic MAE system.

A closed system is composed mainly of extraction vessels, a


turntable, a closed chamber, reflectors, and microwave source. In
the case of a multimode system, the applicator is a closed cavity
inside which a random dispersion of microwaves takes place (15).
Beam reflectors or turntables help in bringing about a uniform
distribution of microwave energy inside the cavity, irrespective of
the position of placement of sample. On the other hand, an open
system comprises primarily a focused microwave source, extraction
vessel with solvent, and a water condenser. In this system, the
microwave wave-guide acts as the applicator and the extraction
vessel is placed directly in the cavity. Only a few inches of the bottom of the vessel is exposed to the microwaves and as glass is transparent to microwaves the upper region of the vessel remains cool.
Additional cooling is brought about by the addition of a water
condenser. Thus, an effective condensing mechanism is a result of
such an inbuilt design of the microwave.
The extraction in a closed extraction vessel or multimode
microwave oven is performed by controlled pressure and temperature. Whereas in an open microwave-assisted Soxhlet or solvent extraction system, only the part of the extraction vessel
containing the sample and solvent is focused for irradiation with
microwave. The closed vessel type as well as the open type is available commercially as multimode and single-mode. A multimode
system ensures random dispersion of microwave radiation within

Microwave-Assisted Extraction in Natural Products Isolation

95

the microwave cavity, so every zone in the cavity and sample is


irradiated uniformly.
Because of the increased pressure inside the extraction vessels
leading to elevation of boiling point of the extraction solvent, in a
closed system higher temperatures can easily be reached. There is
no significant loss of volatile substances in a closed system and less
volume of solvent is required. Repeated addition of solvents is not
required in this system resulting in lower risk of air-borne contamination. Multiple samples can be easily extracted simultaneously. Among the disadvantages of the closed microwave extraction
system, the risk associated with the use of high pressure is the
major one.
The open systems are much safer than the closed systems as
they can be operated at atmospheric pressure and the reagents can
be added at any time during the treatment. Large samples without
the requirement of a cooling process can be used in this system.
However, the open systems are less precise than the closed systems,
and they cannot process multiple samples simultaneously.
Closed systems are generally recommended for extractions
under drastic conditions, since the solvents may be heated up to ca.
100C above their atmospheric boiling point. Both extraction
speed and efficiency are enhanced in this procedure. Hazards occasioned by heating highly flammable solvents are overcome through
the use of recent security techniques, such as high capacity exhaust
fans, solvent vapor detectors, or pressure-burst safety membranes
placed on each vessel.
In order to overcome the nonhomogeneity of the field, the
extraction cells are placed on a rotating carousel as in a domestic
oven (Fig. 2). The solvents can be varied, and the pressure in the
vessels essentially depends on the volume and boiling point of the
solvents used. The temperature can be set at a fixed value by adjusting the microwave power. Typically, the extraction cells are made
of Teflon. In closed vessel systems, the maximal power delivered is
about 6001,000 W (see Note 2). The vessel must be cooled to
room temperature before opening; this is particularly important in
the presence of volatile solutes which can partition into the headspace. Furthermore, an additional filtration or centrifugation step
is necessary in order to remove the solid residue.
The temperature in the extraction container (open system,
Fig. 3) is determined by the solvent used for extraction (not by the
set point of an external oven or heater), but the temperature of the
solvent in the closed container can rise more than its boiling point.
As a result, extraction takes place faster than it does with convection
heating. Multiple samples can be simultaneously handled in a microwave oven, which means high sample throughput. Commercial systems have the capacity to treat up to 24 extraction vessels.
The open system operates at atmospheric pressure, and the
maximum temperature is determined by the boiling point of the

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Table 2
Manufacturers/suppliers of microwave extraction apparatus
Model

Manufacturer/supplier Contact information

CW-2000 ultrasonicmicrowave cooperative


extractor/reactor

Hangzhou Nade
http://zjnade.en.alibaba.com/
Scientific Instrument
Co., Ltd.

ETHOS One
Advanced Microwave
Extraction Labstation

Milestone

http://www.milestonesrl.com/analytical/
products-microwave-extraction-ethos-one.
html

Microwave Digestion System:


Multiwave 3000

Anton-Paar

Anton-Paar-Strae 20, 8054 Graz, Austria


http://www.anton-paar.com/MicrowaveDigestion-System-Multiwave-3000/
Microwave-Sample-Preparation/60_
Corporate_en?product_id=98&gclid=CJvxdayhacCFQEOfAodp2VDdg

Microwave extraction
machine for roots

Yantai Care Microwave Yantai Care Microwave System Co., Ltd.,


System Co., Ltd.
Room 10071, First avenue building.28
xisheng road, Yantai, Shandong, China
http://industrialmicrowave.en.alibaba.com/
contactinfo.html

Ted Microwave Dynamic


Chinese Herbal Extraction
Equipment (extraction
equipment)

Wenzhou Chengdong
Import & Export
Co., Ltd.

http://wzchengdong.en.alibaba.com/

Ultrasonic-microwave
extractor and reactor

Lab-Kits Lab
Instruments
and Facilities

Lab-Kits, Utherm International (H. K.) Ltd.,


905, Workingberg Commercial Bldg.,
4147 Marble Road, Hong Kong
http://www.lab-kits.com/index.
php?route=information/contact

extraction solvent used. The solvent is heated and refluxed through


the sample, and in this case the microwaves are focused on the
sample placed into the vessel allowing homogeneous and efficient
heating. The sample to be extracted can be placed into a Soxhlettype cellulose cartridge in order to avoid filtration steps, or may be
directly dipped into the solvent.
Compared to closed vessel extractions, open cells offer increased
safety in sample handling and, furthermore, they allow larger samples to be extracted. A typical system is the Soxwave 3.6 commercialized by Prolabo (Fontenay-sous-Bois, France), which operates
at percentage power increments from 10 to 100%, corresponding
to a maximum power of 250 W. Various other commercial manufacturers/suppliers for microwave extractors of various sizes and
purposes are listed in Table 2.

2.2. Solvents

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Solvents of different polarities starting from n-hexane to MeOH,


or even water, which are usually used for conventional extraction
of natural products, can be used in microwave extraction methods.
However, there are also solvent-free MAE (SFMAE) protocols
available today, where the use of any extraction solvent is not
necessary.
One of the major factors that affect the overall output of MAE
is the correct choice of extraction solvents. The matrix:solvent ratio
is also another important factor. The choice of the solvent should
be based on the solubility of the desired analyte, the solvent-matrix
interaction and the property of the solvent to absorb microwaves
(13). The chosen solvent should be
(a) highly selective toward the analyte of interest than the other
matrix components;
(b) well compatible with further chromatographic analytical steps;
(c) able to absorb microwave energy well to ensure fast heating up
leading to enhanced extraction;
(d) of sufficient volume to immerse the plant matrix completely in
the solvent throughout the entire irradiation process.
n-Hexane is an example of microwave transparent solvent, whereas
EtOH is an excellent microwave absorbing solvent. In order to get
optimum extraction yields, mixtures of high and low microwave
absorbing solvents can be used.
The amount of solvent needed for a single sample is often
within the range of 1030 mL. In some cases, solvent volume may
be an important parameter, especially when having a matrix that
will swell during the extraction process. Generally in conventional
extraction techniques, a higher volume of solvent will increase the
recovery, but in MAE a higher solvent volume may give lower
recovery.

3. Methods
In the case of MAE, the extraction process is based on exposing
the analytes to the solvent through cell rupture. Desorption of
components from plant matrix takes place in MAE. The free
water molecules present in the gland and vascular systems of plant
matrices are heated and this results in localized heating and
expansion of their walls. As a result, plant constituents flow outside the cells. The dielectric susceptibility of the solvent and
matrix is the factor that affects utilization of microwave energy in
the process. Use of additional extraction solvents increases the
efficiency of this extraction method. In order to avoid degradation

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of thermolabile compounds, the sample matrix is immersed in a


microwave transparent solvent like n-hexane. In addition to correct
choice of extraction solvents, the following factors may affect the
MAE extraction yield.
(a) Extraction time. An increase in extraction time increases the
extraction yield. However, it also increases the risk of degradation of thermolabile components. Therefore, a balance between
the extraction yield and the stability of the components must
be achieved to ensure a meaningful MAE.
(b) Microwave power. To optimize an MAE protocol, a combination of low or moderate microwave power with longer extraction time is a generally desirable.
(c) Matrix characteristics. The matrix characteristics, e.g., particle
size and the nature of the material, may affect the recoveries of
the compounds. Finer the particle size of the sample larger is
the surface area and better is the penetration of microwaves.
Therefore, finer particle size results in better extraction
efficiency.
An MAE process involves the following generic steps.
1. Choose a microwave extractor suitable for the intended
extraction.
2. Choose MAE process whether it is a closed MAE or an open
MAE.
3. Decide whether it is a solvent-free process or solvent-assisted
process.
4. Choose a suitable solvent (see Subheading 2.2).
5. Optimize all variables, e.g., temperature, microwave power,
and duration of extraction, associated with the intended extraction process by the help of the Orthogonal array approach (see
Note 3).
6. Select an appropriate collection and filtration (or centrifugation) method.
7. Store the extract safely for further analysis.
3.1. Optimization

It is absolutely vital to optimize extraction parameters in MAE to


achieve the best possible extraction yield. An optimization strategy
known as Orthogonal array, was reported by Taguchi (see Note 3),
where interaction of various factors on the experiment output were
studied (17, 18). This strategy is still valid for optimizing MAE
through trial and error method. Application of Taguchi method
can improve the product and process quality. The best extraction
output can be achieved by determining the appropriate combination of design factors in an experiment. Several multivariate
approaches, e.g., response surface methodology (RSM) (see Note 4)
can also be used for optimization.

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3.2. Natural Products


Extraction Using MAE

A large and growing body of papers has been published on extraction


of natural products applying MAE. Most of these papers deal with
the development and optimization of the extraction conditions
considering sample size, temperature, solvent use, and extraction
time as well as the yield of extraction of particular type of natural
products. Some examples of MAE extraction of natural products
are summarized in the following Subheadings.

3.2.1. Extraction
of Essential Oils by MAE

Detailed examination of the essential oil of aerial parts of Bupleurum


fruticosum L. was undertaken by Liu et al. (19). To differentiate
between the SFMAE and conventional hydrodistillation (HD)
methods, the essential oils were extracted with SFMAE for 30 min
using a Milestone DryDist microwave apparatus (power 500 W)
and also by means of conventional hydrodistillation in a Clevenger
apparatus for 34 h. This study showed an almost qualitatively and
quantitatively parallel chemical composition of the essential oils for
both techniques. Compared to the oil attained by conventional
hydrodistillation, SFMAE displayed lower content of the major
component, -phellandrene, followed by higher percentage of
oxygenated compounds. Despite the fact that MAE is considered
as a green technology, savings of costs, time and energy, it appeared
not suitable for the industrial isolation of the volatiles of B. fruticosum due to the low essential oil yields (0.21 and 0.25%) obtained
by this technique in comparison to those attained with conventional hydrodistillation (1.71 and 1.86%).
More recently, the essential oil of Zygophyllum album L. was
obtained by the microwave-accelerated distillation (MAD) extraction and compared with the hydrodistillation method (20). The
extraction was performed at atmospheric pressure with Milestone
Dry Dist batch reactor, a multimode microwave reactor of
2.45 GHz with a maximum delivered power of 1,000 W. The
essential oils were found to have distinctive differences in the
chemical compositions, due to the several factors, such as water
content in plant material, solubility of the essential oil components in
water and variety of dipolar moments in the compounds. The
major identified compounds obtained with the MAD were
-terpineol and carvone, and -damascenone was the most
enriched component in the HD oil. Considering the possibility of
compound degradations by hydrolysis, trans-esterification or oxidation during the time consuming HD method, MAD was suggested to be more reliable technique. Since the volatile fraction of
MAD was richer in oxygenated monoterpenes, MAD was considered to be suitable for extraction of species that are recognized for
their olfactory value and their contribution to the fragrance of the
essential oil.
Hattab et al. (21) analyzed volatile metabolites from the brown
alga Dictyopteris membranacea by three different methods, HD,
focused microwave-assisted hydrodistillation (FMAHD) and supercritical fluid extraction (SFE). A focused Discover microwave oven,

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equipped with a modulator of power and an infrared temperature


capture was utilized in the experiment. In the optimum conditions
of 56 mg/50 mL (crude algal extract/water), 180 W irradiation
power and 10 min of extraction, the oil yield was 13.0% in the
FMAHD technique. The oils were analyzed by GCMS. The findings of the study revealed a notable difference between the GCMS
data of the oils, a class of compounds being totally absent in the oils
obtained by HD and SFE. Though, sesquiterpenes to a great amount
(58.8%) were present in the essential oil obtained by FMAHD. The
changes in the extraction parameters (mass of crude extract for HD,
and equilibrium time, extraction pressure, extraction time, and
experiment temperature for SFE) did not permit to obtain the set of
sesquiterpenes and contained only the C11 hydrocarbons and sulfur
compound. This study demonstrated that FMAHD could be used
for preferential extraction of certain classes of compounds.
A comparative study of different techniques for the isolation of
rosemary essential oil was carried out by Presti et al. (22). Fresh
leaves of Rosmarinus officinalis L. were submitted to the traditional
HD, SFE, organic solvent extraction (SE), and water microwaveassisted hydrodistillation (MAHD) techniques to obtain the volatile
oils and analyze by GC and GCMS. The GCMS analysis of the
essential oils led to the identification of approximately 54 components for all techniques that refer to five chemical classes: monoterpene and sesquiterpene hydrocarbons, oxygenated monoterpene,
sesquiterpene, and aliphatic compounds. Nonetheless, statistical
analysis indicated the only difference was in their quantitative values.
Besides, the volatile rosemary oils were assessed for their chiral profiles derived from GC chiral analyses and six similar enantiomeric
pairs were determined. MAHD in comparison to other extraction/
distillation methods provided the highest analytical repeatability and
a high quality distillate characterized by a composition similar to that
reported by the European Pharmacopoeia in the monograph concerning rosemary oil of Tunisian and Moroccan origin. Additionally,
MADH has the benefits of less time-consuming and low environmental impact as no organic solvents are used in its processes.
In a similar study with a minor modification in the MAE,
microwave hydrodiffusion and gravity (MHG) method was applied
for isolation of essential oil from fresh R. officinalis leaves (23).
The protocol can be summarized as follows:
1. Use a DryDist microwave oven, which is a multimode microwave reactor 2.45 GHz with a maximum delivered power of
1,000 W variable in 10 W increments.
2. Monitor temperature by an external infrared sensor.
3. Perform MHG procedure at atmospheric pressure.
4. Heat 500 g of fresh plant material using a fixed power density
of 1 W/g for 15 min without addition of solvent or water (see
Note 5).

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5. A mixture of hot crude juice and steam (in situ water) should
move downward by gravity into a spiral condenser outside the
microwave cavity where it is condensed.
6. Collect the oily condensate is collected continuously in a
receiving flask (similar to separator funnel).
7. Essential oil will form a film on the surface of the water. Skim
the film off the top and collect the essential oil.
8. Dry collected essential oil with anhydrous sodium sulfate and
store at 4C until used.
3.2.2. Extraction
of Flavonoids
and Isoflavonoids by MAE

Zhang et al. (24) reported MAE of rutin and quercetin from the
stalks of Euonymus alatus (Thunb.) Sieb. and compared with conventional extraction techniques. MAE was performed with an
MSP-100E multimode microwave extraction system (maximal
power: 850 W) in the optimized conditions of 50% EtOH (v/v)
solution, 40 mL the extractant volume, 170 W microwave power,
and 6 min irradiation time. MAE was found to be the fastest of all
extraction methods compared, requiring just 6 min, with a higher
yield and lower solvent consumption for extraction of rutin and
quercetin in plants
An approach for automated, continuous and rapid extraction
of flavonoids from the dried cell cultures of Saussurea medusa
Maxim., utilizing a fully optimized dynamic microwave-assisted
extraction (DMAE) system, was described (25). The yield of flavonoids reached ~5% in 60 min under the optimum extraction
conditions: 1,200 W of radiation power, 50:1 (v/w) of the liquid/
solid ratio, and 50 mL/s of solvent flow rate. The dynamic MAE
displayed distinct advantages in short duration and high efficiency
to extract flavonoids without causing degradation of target components from the S. medusa dried cell cultures in comparison with the
dynamic solvent extraction without microwave assistance. The
protocol of this extraction can be outlined as follows:
1. Use a dynamic MAE system as outlined in Fig. 3.
2. Place a given amount of S. medusa dried cell cultures and 2.0 L
of 80% EtOH into the container, and mix homogeneously by
a magnetic stirrer.
3. Recycle the mixed extraction sample between the container
and three extraction tanks by the liquid bump.
4. Irradiate the suspension with 60 s of power ON to reach the
desired temperature of about 80C, and then irradiate periodically with microwaves in a presetting procedure (15 s of power
ON for heating followed by 15 s of power OFF for cooling
without allowing the suspension to super-boil).
5. Carry out qualitative and quantitative analyses of flavonoids by
HPLC.

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Later, Chen et al. (26) conducted a similar DMAE coupled


with online derivatization and ultravioletvisible detection for
determination of total flavonoids in Platycladus orientalis (L.)
Franco. A WGY-20 Microwave source with a maximum microwave
output power of 100 W was applied in the experiment. The extraction solvent, microwave output power, and extraction time, as well
as the extraction solvent flow rate, affecting the performance of
DMAE were assessed to reach the optimized conditions for best
extraction efficiency. In the optimized extraction and derivatization conditions, the maximum flavonoids recovery yield (1.72%)
was achieved using 80% aq. MeOH as the extraction solvent; 80 W
of microwave output power; 5 min of extraction time; and 1.0 mL/
min of extraction solvent flow rate. Allowing the automation of the
preliminary step of the analytical process, DMAE provided higher
extraction yield of total flavonoids in a shorter extraction time and
small quantity of extraction solvent.
A solvent-free MHG has recently been described for the recovery of antioxidant flavonoids from onion (Allium cepa L.) (27).
Effectiveness of this method in extraction of onion total phenolic
content, total quercetin, quercetin aglycone, quercetin 3,4-diglucoside,
quercetin 4-glucoside, quercetin 3-glucoside, kaempferol, and
myricetin was evaluated and compared with conventional solvent
extraction. This extraction method afforded significant yield
(81.5%) with 41.9% of flavonol contents, with better retention of
remaining flavonoids (55.9%) in residues of onions. The protocol
can be shown as follows:
1. Perform the extraction in a Milestone EOS-G microwave laboratory oven, which is a multimode microwave reactor 2.45 GHz
with a maximum delivered power of 900 W variable in 10 W
increments.
2. Use the easy-WAVE software package to control time, temperature, pressure, and power.
3. Use the extraction vessels made from Pyrex and have a capacity
of 1,000 mL.
4. During experiments monitor the temperature by the temperature sensor optic fibers, which should be inserted in the center
and outer layer of sample and also in the sample reactor.
5. Measure temperature variations in different parts of plant
material and reactor continuously and data should be saved
automatically. This feedback helps in controlling the temperature by microwave power regulator.
6. Perform MHG procedure at atmospheric pressure.
7. Heat fresh entire onion bulbs (500 g) of fresh entire onion
bulbs using a fix power density without addition of solvents or
water (see Note 5).

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8. Flavonoids should move naturally by diffusion along with hot


water or crude juice out of the cells of plant material, and move
downward under the gravity on a spiral condenser outside the
microwave cavity where it will be condensed.
9. Collect the crude juice ted continuously in a graduated
cylinder.
10. Continue extraction until no more juice is obtained or overheating is detected.
11. Freeze-dry extracted crude juice.
Application of MAE with various suitable modifications in
extracting flavonoids from various plant species has been well documented in numerous other published papers (6, 2831). Similar
methodology has also been applied for the extraction of isoflavonoids. Rostagno et al. (32) developed an MAE for extraction of
12 isoflavones in soybeans, e.g., genistin, daidzin, glycitin, and
their respective acetyl, malonyl, and aglycone forms. A microwave
extractor ETHOS 1600 (Milestone) 500 W, using magnetic stirring at 50% of nominal power was handled. Along with the method
development, several parameters were evaluated. The extraction of
all isoflavones derivatives increased up to 20 min but no more isoflavone recoveries achieved as the extraction time extended over
20 min. Owing to the isoflavones degradations at higher temperatures, 50C was selected for a reliable extraction without affecting
the isoflavone profile on the sample. Considering the results for the
most isoflavone derivatives, highest extraction efficiency was
achieved using between 20 and 30 mL of EtOH. Performing the
extraction under the developed conditions achieved a high reproducibility with no degradation of isoflavones. Nonetheless, the
MAE method compared to a developed ultrasound-assisted extraction method showed no significant differences for the recoveries of
isoflavones.
Elsewhere, isoflavonoid aglycones in soybeans were extracted
through optimizing MAE (33). An experimental design was
adopted to find the best extraction conditions for genistein and
daidzein in an MLS-1200 MEGA, Milestone Microwave. A microwave power of 600 W, an extraction time of 1 min, HCl concentration of 12 M and 3 mL of a mixture ACN/H2O (80/20, v/v)
were found to be the optimal extraction conditions followed by
extraction yield of 94 8% and 97 5% for genistein and daidzein,
respectively. Application of MAE in genistein and daidzein extraction from soybeans exhibited excellent extraction efficiency and
low consumption of sample, solvent, and time.
3.2.3. Extraction
of Coumarins by MAE

The feasibility of the MAE extraction for determination of the


content of coumarin, melilotic, and o-coumaric acids from Melilotus
officinalis (L.) in comparison with traditional extraction techniques

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A. Delazar et al.

was reported by Martino et al. (34). In the case of melilotic acid,


MAE was the best technique for extraction with respect to the differences in results between the MAE and ultrasound-assisted
extraction (p < 0.01). Regarding coumarin and o-coumaric acid,
comparison of the extraction methods revealed that ultrasoundassisted extraction and MAE produced comparable extracts. The
MAE protocol adopted in this study can be summarized as
follows:
1. Perform MAE on a mono-mode microwave apparatus using a
closed vessel system and the power at 100 W.
2. Place ground plant material (0.2 g) into the extraction vessel.
3. Add solvent up to volume of 4 mL.
4. Carry out extraction at different temperatures for different
time of irradiation.
5. Once the extraction is finished, allow the vessels to cool to
room temperature before opening.
MAE was applied for the extraction of furanocoumarins from
Archangelica officinalis Hoffm. and compared with different methods of extraction (35). MAE was completed with 80% aq. MeOH
using a Plazmotronika UniClever BMZ I bath with a 600 W generator either in an open or in a closed system. MAE in an open
system offered extraction yields of furanocoumarins comparable to
those obtained in the time- and solvent-consuming Soxhlet extraction method. However, considerable changes in the extracted
compounds were found using the MAE in a closed system. The
changes in the extracted compounds resulted from transformation
of some furanocoumarins, e.g., imperatorin and phellopterin,
under pressure and microwave irradiations in the closed systems.
Xiao et al. (36) analyzed isofraxidin from Sarcandra glabra by
the open MAE system, with a maximum delivered power of
1,000 W. With 60% (v/v) aq. EtOH aqueous, solid/liquid ratio of
1/10 (g/mL) at 65C for 20 min and at microwave irradiation
500 W, 1.2 mg of isofraxidin was obtained from 2 g of S. glabra.
Ethanol showed better extraction efficiency because of its appropriate microwave absorption properties and high solubility to isofraxidin. In general, in this study, the MAE was presented as a
simple, fast and efficient method for the separation and purification
of isofraxidin and other natural products.
Strong antioxidants, 5,8-dihydroxycoumarin and its glucoside
were successfully extracted from sweet grass (Hierochloe odorata L.)
by MAE (37). One-step MAE afforded the most concentrated
extract with 8.15% of 5,8-dihydroxycoumarin (extract yield 0.42%).
The protocol for this extraction is outlined as follows:
1. Perform MAE in the closed vessel unit MSP 1000.
2. Set the maximum oven power for this system at 1,000 W.

Microwave-Assisted Extraction in Natural Products Isolation

105

3. For the first series of experiments, place 3 g of the whole dried


and ground material in 100 mL PFTE (CEM) extraction
vessels.
4. Add appropriate amount of solvent (30 or 45 mL).
5. Record temperature profile extraction.
6. After extraction, leave the vessels for 30 min to cool down
below 35C.
7. Apply Orthogonal array experimental design (see Note 3) for
the optimization of extraction.
8. Construct an L8 array for experiment and investigate four different factors, e.g., solvent, extraction time, temperature and
liquidsolid ratio.
9. Use three medium polarity solvents, diethyl ether, acetone and
ethyl acetate, and a mixture of acetonehexane (1:1).
10. Perform extractions formed at 40 and 80C.
11. Filter each extract through filter paper and then wash the filter
with 5 mL of an appropriate solvent.
3.2.4. Extraction of Other
Phenolic Compounds
by MAE

The stability of phenolic compounds in skin and seeds of the Red


Globe table variety (red), Napoleon (red) and Moscatel (white)
during MAE was studied (38). An ETHOS-1600 (Milestone)
closed vessel oven system equipped with 10 perfluoro alkoxy (PFA)
vessels was used in this study. The study was performed at 500 W
with MeOH as the extraction solvent and various temperatures up
to 175C for extraction of different families of phenolic compounds, e.g., benzoic acids, benzaldehydes, cinnamic acids, catechins, coumarins, stilbenes, and flavonols. The mean recoveries for
the most of phenolic compounds were about 95% at 125C.
Spigno and Faveri (39) analyzed the black tea phenols using
MAE with an ordinary household oven operating at 2,450 MHz
and with adjustable microwave powers output (from 100 to
900 W). Considering the influence of microwave power and irradiation duration on total phenols concentration and antioxidant
activity, the MAE offered higher recoveries compared to conventional brewing technique, without altering the antioxidant potential of the tea extracts. All the same, higher phenols content could
be acquired by adjusting microwave power and time combination
that could optimize the application of this technique for industrialscale tea infusion productions.
A closed vessel system of MAE under controlled temperature
and pressure was employed for the extraction of thermolabile bioactive compounds, gastrodin and vanillyl alcohol from Gastrodia
elata Blume (40). In the case of gastrodin, the optimized MAE
conditions were 100C of extraction temperature for 20 min at
power of 700 W and 100C for 10 min for vanillyl alcohol in a
constant power of 700 W.

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A. Delazar et al.

Salidroside and tyrosol in the extracts of Rhodiola L. were


extracted using an MAE protocol (41). A microwave oven equipped
with a magnetron of 2,450 MHz with a nominal maximum power
of 700 W was modified for this purpose. A hole was made through
the top wall plate of the microwave oven, and through this a condensation tube was connected to the sample cartridge vessel located
in the microwave irradiation zone. The whole system was open and
run at atmospheric pressure. Among the nine ratios of MeOHwater mixtures, the recovery yield increased with water ratio up to
50%. Similarly, the other optimum conditions for the MAE were
achieved with soak time of 60 min, at microwave power of 400 W
and 5 min irradiation where yielded more than 94% average
recoveries.
Li et al. (42) utilized a focused MAE for the extraction of geniposidic and chlorogenic acids from Eucommia ulmodies Oliv. The
whole extraction system was open and a commercially available
microwave oven equipped with a magnetron of 2,450 MHz with a
nominal maximum power of 700 W at atmospheric pressure was
used. The optimum extraction condition was achieved using the
Orthogonal experimental design and graphical analysis by altering
irradiation time, solvent volume, solvent composition, and microwave power (see Note 3). The extraction condition for geniposidic
acid and chlorogenic acid, was 50% microwave power, 40 s irradiation, and 80% (v/v) aq. MeOH as extraction solvent (20 mL/g of
sample) for the former and 50% microwave power, 30 s irradiation,
and 20% aq. MeOH (20 mL/g of sample) for the latter, respectively. An addition of acetic acid to the solvent enhanced the recovery yields for the two organic acids since ionization of geniposidic
and chlorogenic acids might occur during focused microwave heating
process.
Hayat et al. (43) optimized the MAE of phenolic acids from
Citrus reticulata Blanco. With the microwave power of 152 W;
extraction time, 49 s; liquid to solid ratio, 16; and MeOH concentration 66% as the solvent offered the highest extraction efficiency.
The protocol used in this study can be summarized as follows:
1. Use the MAE parameters as follows: microwave power of 100
200 W; irradiation time of 3060 s; liquid to solid ratio of
1020; and MeOH level of 6080%.
2. Carry out preliminary experiments and select the range of
independent variables based on the values obtained in preliminary experiments.
3. Employ a 24 full factorial central composite design comprising
30 experimental runs and perform experiments in randomized
order according to the run number as arranged by the
software.

Microwave-Assisted Extraction in Natural Products Isolation

107

4. The responses to be obtained from the experimental design are


the sum of content of five phenolic acids, namely, gallic acid,
p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, and
ferulic acid.
5. Use a laboratory scale microwave extraction apparatus (CW2000; Xintuo Technology Company, Shanghai, China),
equipped with a digital controlled system for temperature,
time, and power, operated at atmospheric pressure with microwave frequency of 2,450 MHz for the extraction purpose.
6. The microwave power should be linearly adjustable from 10 to
800 W, the temperature limit of the apparatus is 1120C and
time range should be adjustable from 1 to 999 s.
7. Carry out extraction according to experimental design.
8. Filter the extracts through 0.45 m nylon filter and analyze for
free phenolic acid content by HPLC.
9. After method optimization, perform the extraction of Kinnow
peel powder under optimized conditions.
10. Extract five grams of peel powder with 80 mL of 66% aq.
MeOH and filter the mixture through Whatman filter paper
No. 4.
11. Keep the residue for the determination of insoluble-bound
phenolic acids and evaporate the filtrate orated under reduced
pressure at 30C until its volume is about 20 mL.
12. Make the final volume of the extract to 25 mL with the extraction solvent for further analysis.
The same group of researchers reported a few other similar
extraction protocols for plant phenolics (44, 45). MAE was developed for the extraction of ferulic acid from Radix Angelicae sinensis
(46). In the optimum extraction conditions of 850 W microwave
power, 9 min irradiation time, 90% aq. EtOH, 6:1 ratio of liquid/
solid, 140 min of herbal material soak time and particle sample size
250 9.9 m, 76.19% of ferulic acid was extracted.
3.2.5. Extraction
of Alkaloids by MAE

Pan et al. (47) extracted polyphenols as well as the xanthine alkaloid, caffeine, from green tea (Thea sinensis L.) leaves utilizing the
MAE method resulting in good extraction efficiency. A household
microwave oven was used in evaluating different experimental conditions to optimize the extraction. The extraction efficiency of
polyphenols and caffeine was increased with the increase of MAE
time and the pre-leaching time from 4 to 90 min, as well as the
increase in liquid/solid ratio. Among the various solvents examined for the extraction, acetone and MeOH obtained higher extraction values for polyphenols and caffeine, respectively.
Ionic liquid (see Note 6)-based microwave-assisted extraction
(ILMAE) has recently been developed for the extraction of three

108

A. Delazar et al.
R

N
R

Alkaloids

N-Nornuciferine

OMe

OMe

O-Nornuciferine

OH

OMe

Me

OMe

OMe

Me

Nuciferine

Fig. 5. Structure of alkaloids extracted by ionic liquid-based microwave-assisted extraction


(ILMAE).

alkaloids, N-nornuciferine, O-nornuciferine, and nuciferine (Fig. 5)


from lotus leaf (48). The protocol of this extraction can be
presented as follows:
1. Use a domestic MAE unit with a 2,450 MHz magnetron, and
add a water condenser to the system.
2. Run the whole system at atmospheric pressure and the maximum power of 700 W can be employed.
3. Use ionic liquid in the extraction.
4. Mix dried sample (0.5 g) with 10 mL of different ionic liquids
to obtain a suspension.
5. Extract the suspension by MAE.
6. Study systematically the optimum anion, cation, concentration
of selected ionic liquid, microwave power, irradiation time,
and solidliquid ratio.
7. After irradiation, cool the extracts down to the room temperature, and dilute to 50 mL with deionized water.
8. Filter the extract through a 0.45 m filter prior to HPLC analysis.
The structure of ionic liquids has significant influence on its
physicochemical properties, e.g., water miscibility, which may significantly affect the extraction efficiency of target analytes. The
effects of changing the anion and the alkyl chain length of the cation of 1-alkyl-3-methylimidazolium-type ionic liquids on the
extraction efficiency were studied in the above work (48). Among
the studied 1-butyl-3-methylimidazolium ionic liquids with four

Microwave-Assisted Extraction in Natural Products Isolation

109

different anions, e.g., Cl, Br, BF4, and PF6, the most efficient
one was Br, which demonstrated that the extraction efficiency of
alkaloids was anion-dependent (see Note 7). This study also evaluated the effect of alkyl side chain; with the same anion of Br, a
series of 1-alkyl-3-methylimidazolium cations, including C2mim+,
C4mim+, C6mim+, and C8mim+ were studied. It was observed that
the extraction efficiency increased significantly when the alkyl chain
length was increased from ethyl to hexyl. However, increasing alkyl
chain length from hexyl to octyl dramatically decreased the extraction efficiency (see Note 8). A similar protocol was also reported
for the extraction of alkaloids from Nelumbo nucifera (49).
The use of the nonionic surfactant 5% oligoethylene glycol
monoalkyl ether (Genapol X-080) as a solvent for a focused MAE
of alkaloids from Rhizoma Coptidis was found to enhance the
extraction efficiency significantly with a massive 92.8% recovery of
alkaloids in a one-step and fully optimized extraction (50).
Focused MAE of cocaine and benzoylecgonine from the leaves
of Erythroxylum coca var. coca was reported by Brachet et al. (51)
utilizing a Soxwave (Prolabo, France) model 3.6 at atmospheric
pressure and a frequency of 2,450 MHz. Different solvents, particle size, radiation time, and power were evaluated to study their
effects and optimize the extraction condition. Because of the ability of MeOH for absorbing microwave energy and solubilizing
cocaine, it was found to be the best extraction solvent. On the
contrary, no quantitative extraction of cocaine was established with
nonpolar solvents having low dielectric constants (hexane and toluene). Smaller particle size distribution provided an enhanced
extraction of cocaine, revealing an evident influence of particle size
on the extraction efficiency. Radiation time and the applied power
had no obvious effect on cocaine recovery. Under optimum conditions of 90150 m particle size, 5 mL of MeOH, 30 s of radiation
time and microwave power of 125 W, the cocaine content was
determined as 0.6800.004% by GCFID analysis and 0.100.02%
for benzoylecgonine.
3.2.6. Extraction
of Saponins by MAE

MAE technology has been applied successfully in the extraction of


various classes of saponins from plant sources (5256). Triterpene
saponins were extracted from the well known medicinal plant
Ganoderma atrum using an MAE protocol (53), which only
required 5 min extraction time and 90C of temperature. The protocol of this extraction is outlined below.
1. Use a closed vessel microwave extractor (max oven power
800 W) comprising a pressure display, a time controller and a
temperature controller.
2. For the first series of experiments place 3 g of the dried and
ground plant materials in 100 mL PFTE (CEM) extraction
vessels.

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A. Delazar et al.

3. Add appropriate amounts of extraction solvent (3075 mL).


4. Place the lid on the vessel, insert the temperature sensor into it
to measure and control the internal temperature.
5. When the temperature is just 23C under the desired temperature, start the program for heating preservation.
6. Shut the heating device off when the set temperature is
reached.
7. Pressure should have been pre-established at a safe limit, and
the pressure profile must be recorded to ensure that it does not
go beyond the limit during the whole run.
8. Perform the extraction for a few minutes at a definite power.
9. Use the following program: ramp to the temperature for 5 min
step-by-step, hold at temperature for a few minutes, microwave power at 800 W (100%) (see Note 9).
10. After extraction, leave the vessels for 30 min to cool down to
room temperature.
11. Filter each extract through filter paper and evaporate to dryness.
A fully optimized MAE of triterpene saponins from defatted
residue of yellow horn (Xanthoceras sorbifolia Bunge.) kernel,
employing an MASII Microwave Extraction Testing Equipment
(atmospheric pressure, microwave frequency of 2,450 MHz and
the microwave power is linear and adjustable from 300 to 900 W),
has recently been described (54). The extraction system was
equipped with a temperature regulator, stirring device (agitation
speed 0500 rpm/min), and a circulating water-cooling system.
The MAE offered much better extraction efficiency for tritepene
saponins than other conventional extraction methods, e.g., ultrasonic-assisted extraction (UAE) and heat reflux extraction (HRE).
The optimum extraction parameters for the extraction of these
compounds using MAE were as follows: temperature 51C, extraction time 7 min, microwave power 900 W, solvent/matrix ration
32 mL/g, extraction solvent 42% (v/v) aq. EtOH and duration
three cycles. Under these conditions, the highest extraction yield
of triterpene saponins reached 11.62 0.37% of defatted kernel.
Chen et al. (57) reported the extraction of glycyrrhizic acid
and liquiritin from the roots of Glycyrrhiza urulensis Fisch. applying a micellar MAE method that utilizes nonionic surfactant (Triton
X-100) as the solvent. In a focused MAE unit (Star System 2, CEM,
Matthews, NC, USA) various concentrations of aqueous nonionic
surfactant solutions were analyzed for the active ingredients
extracted from the licorice root. The extraction yield of glycyrrhizic acid and liquiritin reached the maximum value in the optimal
conditions, at 373 K with 5% (v/v) Triton X-100 for 35 min.

Microwave-Assisted Extraction in Natural Products Isolation

111

3.2.7. Extraction
of Endogenous Steroids
in Marine Mussels Mytilus
sp. (58)

A simple, robust, repeatable, and successful coupling between


MAE and other analytical techniques, e.g., solid phase extraction
(SPE), GC, and MS, to extract and analyze eight endogenous steroids (testosterone, androstenedione, 17estradiol, estrone,
pregnenolone, progesterone, dihydroandrostenedione, and dihydrotestosterone) in marine mussels Mytilus spp. has been reported
by Devier et al. (56). After an MAE, samples were further extracted
and purified by two successive SPE. Steroids were derivatized with
a mixture of NmethylN(trimethylsilyl)trifluoroacetamide/mercaptoethanol/ammonium iodide and identified by GCMS in selective ion monitoring mode.

3.2.8. Extraction
of Chlorogenic Acid
from Lonicera japonica
by MAE (59)

Chlorogenic acid was extracted from the flower buds of Lonicera


japonica Thunb, using the MAE technique (59). The MAE method
was developed on a household microwave oven with a magnetic
stirrer, water condenser, temperature measurement, and a power of
700 W. The best possible conditions for the chlorogenic acid recoveries were 50% aq. EtOH as extraction solvent, 1:10 (w/v) of the
solid/liquid ratio and 60C of extraction temperature that led to
the highest yield of chlorogenic acid (6.14%) extraction within
5 min. Additionally, the structural changes during the MAE comparing with heat-reflux extraction procedures were assessed by
SEM. The micrographs demonstrated the absolute cell rupture after
MAE treatment, revealing the enhanced extraction of the MAE
method to the greater extent of cell ruptures compared to the few
slight cell ruptures in the conventional heat-reflux extraction.

3.2.9. Extraction
of Paclitaxel from Taxus
baccata by MAE (60)

Paclitaxel, the well-known anticancer compound, was extracted


from the needles of Taxus baccata L. by MAE using a closed-vessel
system (60) on an MARS Xm (1,200 W, 2,450 MHz) Microwave
Accelerated Reaction System from CEM Corp. The extraction
conditions were evaluated using a factorial design process allowing
a synchronized evaluation of four formulation variables; extraction
time, temperature, methanol concentration in water (v/v), the
ratio of sample to 10 mL of solvent, and their interactions.
Optimum paclitaxel recoveries was reached at an extraction temperature of 95C, 7 min of extraction time and with 1.5 g samples
in 10 mL of solvent (90% aq. MeOH).

4. Notes
1. Ionic conduction refers to the electrophoretic migration of
ions under the influence of the changing electric field.
2. The chosen power has to be set correctly to avoid excessive
temperatures leading to possible solute degradation and overpressure problems.

112

A. Delazar et al.

3. Orthogonal array testing is a systematic and statistical way of


testing. It can be applied in user interface testing, system testing, regression testing, configuration testing, and performance
testing. All orthogonal vectors display orthogonality and following properties.
(a) Each of the vectors conveys information different from
that of any other vector in the sequence, which means that
each vector conveys unique information therefore avoiding redundancy
(b) On a linear addition, the signals may be separated easily
(c) Each of the vectors is statistically independent of the others
(d) When linearly added, the resultant is the arithmetic sum of
the individual components
4. The RSM, introduced by Box and Wilson in 1951, explores
the relationships between several explanatory variables and one
or more response variables. The main idea of RSM is to use a
sequence of designed experiments to achieve an optimal
response. A second-degree polynomial model has been recommended to do this. Although this model is only an approximation, it is easy to estimate and apply, even when little is known
about the process.
5. The direct interaction of microwaves with biological water,
i.e., steam produced from the water present in the fresh plant
material, favors the release of essential oils trapped inside the
cells of plant tissues.
6. Ionic liquids, also known as molten salts, are a new class of
nonmolecular ionic solvents with a melting point fixed at or
below 100C. The potential of these liquids in both academic
and industrial fields is related to their unique properties of negligible vapor pressure, high thermal stability, low or virtually
no volatility and ease of handling. Ionic liquids are therefore
regarded as an attractive alternative or replacement to conventional volatile organic solvents.
7. The anion dependency was probably because [C4MIM]Br
solution had stronger multi-interactions, including , ionic/
chargecharge, and hydrogen bonding with alkaloids.
Moreover, [C4MIM]Br solution had slightly higher acidity,
which might facilitate the extraction of target alkaloids from
lotus leaf.
8. The length of the alkyl chain dictates the water miscibility of
the Br ionic liquids, and this property may affect the extraction efficiency.
9. It is advisable to carry out an orthogonal array experimental
design to identify the most probable active factors and possible
interactions that may affect the overall efficiency of the extraction process.

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113

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