Importance of Extraction

According to world health organization (WHO) survey, over 80% of the world’s population
depends on traditional forms of medicines for their health care and treatments. The use of
plants as therapeutic is older as 4000–5000 B.C. The first natural herbal preparation as
medicines was used by Chinese. Mostly large number and variety of drugs are derived from
plants. The medicinal plants are rich in essential oils and secondary metabolites of therapeutic
importance. The advantages of medicinal plants stated for therapeutic uses in a variety of
ailments are their safety besides being economical, efficient and their easy availability. Plants
contain several types of phytochemicals includes alkaloids, tannins, flavonoids, phenolic
compounds and lot of other chemicals that have disease preventive properties. For example,
Favonoids have antimicrobial, antidiarrhoeal, antifungal, anti-inflammatory, antibacterial
activities; polyphenols and tannins have antimicrobial, antidiarrhoeal, anthelmintic activities;
coumarins have antivira activity; terpenoids and essential oils have antimicrobial, antidiarrhoeal
activities; alkaloids have antimicrobial, antidiarrhoeal, anthelmintic activities; lectins and
polypeptides have antiviral activity; glycosides have antidiarrhoeal activity; saponins have
antidiarrhoeal, anticancer, anthelmintic activities; steroids have antidiarrhoea activity; quinones
have antimicrobial activity
In order to benefit from these properties of plants, it has to be extracted the necessary actives.
For this reason, many different extraction methods have been used since the past.

General information

The history of the extraction of natural products dates back to Mesopotamian and Egyptian
times, where production of perfumes or pharmaceutically active oils and waxes was a major
business [1]. The increasing interest in plants and their metabolites made it necessary to
expand and modify the traditional extraction techniques. Nowadays, plant-based extracts are
requested in food, flavor, cosmetics, and in the pharmaceutical industry. Moreover, the
consumer demand for products from natural sources increases continuously [1-3].

In general, the extraction of compounds from a solid plant matrix can be regarded as a five-
stage process:

1. Desorption of the compound from the active site of the plant matrix.
 2. Diffusion into the matrix itself.

3. Solubilization of the analyte in the extractant.

4. Diffusion of the compound in the extractant.

5. Collection of the extracted compounds.

Ideally, the extraction process is exhaustive, fast, simple, and inexpensive. Conventional extraction

techniques include maceration, Soxhlet extraction, percolation, and steam/hydro distillation.

These methods present major drawbacks, for example long extraction times, large amount of

organic solvents, unsatisfactory extraction efficiencies, and potential degradation of labile

compounds. For this reason, new extraction techniques were developed in recent years with

significant advantages. These new processes include supercritical fluid extraction (SFE),

microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), and pressurized-

liquid extraction (PLE) [2].

Moreover, major advantages are e.g. the reduction of solvent consumption, the decrease of the

extraction time, and the improvement in extraction efficiency and selectivity. Also conventional

methods, although effective for extraction, can lead to degradation of heat sensitive compounds as

well as leave traces of toxic solvents in the solute. This is a concern for food and medicinal

extracts, because of the increasing regulation of solvents used. On the other hand, supercritical

fluid extraction (SFE), with its advantages, attracts the interest of process engineers. With SFE

higher yields and better quality products can be achieved. One particular application, where the

process is proved to be useful, is the recovery of high-value and low-volume end products from

dilute process streams that are typical of many specialty chemical, pharmaceutical, and

biotechnology processes. In addition, SFE can be operated under a wide range of conditions to

selectively extract specific end products or new products with improved functional or nutritional

characteristics for use as building blocks in creating new formulated foods. Most of the

applications have been carried out in SFE of oilseeds, however, commercial SFE applications
involve decaffeination of coffee and tea, extraction of hops, flavors and spices where the products

have high economical values.

On the other hand, economical analysis of the processing of milk fat by supercritical carbon

dioxide (SC-CO2), shows that the operation is viable, if it is continuous with high capacity. The

extraction techniques are in conformity with the concept of “green extraction” [4-6]. The six

principles of “green extraction” are [4]:

1. Innovation by selection of varieties and use of renewable plant resources.

2. Use of alternative solvents (principally water or agro-solvents).

3. Reduction of energy consumptions by energy recovery and using novel processes.

4. Production of co-products instead of waste (principle of biorefinery).

5. Reduction of unit operations (favor safe, robust, and controlled techniques).

6. Aim for a non-denatured and biodegradable extract without contaminants.

Extraction methods

Mechanical expression

A common way to extract vegetable oils from seeds is mechanical expression. This method
is suitable for plant materials with high oil contents. It is the more economic technique
compared to solvent extraction, but over 5 wt% of the oil can remain in the raw material.
Nonetheless, mechanical expression has relatively low initial and operational costs. In
addition, the extracted oil is free of solvent residues and thus has a higher overall quality [7].
Mechanical expression is for example used for the extraction of citrus oil. Therefore, peels
from lemons, oranges or tangerines are abraded and pressed out. These extracts resemble
closely the original oil of the plant material as they are not exposed to heat during the
extraction process. However, high temperatures must be avoided as thermal degradation of
some compounds can be induced, which results in a modified scent of the extract [8, 9].

Steam and hydro distillation

Hydro and steam distillation are traditional methods to extract volatile compounds, especially
essential oils, from plants. However, the extraction procedure of both techniques is slightly
different [5]. The procedure of steam and hydro distillation is shown in Figure 2.1. Regarding
the hydro distillation, the plant material and water are mixed together in a flask.
Subsequently, this suspension is heated until boiling. In the case of steam distillation, the
steam is generated separately and then guided through the plant material.

Figure 2.1: Procedure of (A) steam and (B) hydro distillation of plant material to obtain the
essential oil.

In general, the steam takes the essential oil along to the condenser. There, the vapor is
liquefied and a water/oil mixture is gained. Subsequently, the mixture is collected in a
separator, where a two phase system is observed. There, water is the lower phase and the
upper phase represents the essential oil. Afterwards, the essential oil can be recovered
[5, 10]. The distilled and condensed water phase is called hydrosol. If this hydrosol is
recycled and re-used to carry out another steam or hydro distillation the process is called
cohobation [11].

The techniques of steam and hydro distillation merely work because of the coexistence of
two immiscible liquids (water and essential oil). The basic principle is that the immiscible
compounds form low-boiling azeotropes.
In a good approximation, a very low solubility of the essential oil in the water phase is
assumed. Thus, the involved substances are not mixed within the vapor phase. Thereby,
both mole fractions are equal to one. That is why the resulting vapor pressure of the system
is equal to the sum of the vapor pressures of the pure compounds. Consequently, the boiling
point of the mixture is below the boiling points of both, water and essential oil at atmospheric
pressure. For this reason, the essential oil can be extracted without reaching the boiling point
of the single compounds [5, 12, 13].

One of the disadvantages of steam and hydro distillation is that the essential oils can
undergo chemical alteration. Additionally, heat-sensitive compounds can easily be destroyed.
For this reason, the quality of the essential oil extracts can be extremely impaired. Moreover,
the scent of the extract is more akin to a cooked aroma, rather than a fresh fruit or vegetable
flavor [8, 14]. A further drawback is the high-energy and time consumption during steam
and hydro distillation [15].

Microwave-assisted distillation (MAD)

Microwave-assisted distillation (MAD) is a recently developed method to extract essential oil

from plant materials. MAD can be an alternative technique for steam and hydro distillation
[16].
Microwave-assisted distillation is performed by putting fresh plant material in a beaker placed
in a microwave reactor. However, no additional water or solvent is necessary. The microwave
radiation causes the heating of the water in the raw material. This results in the bursting of
plant cells, releasing the contained essential oil.

In addition, steam is produced in situ from water included in the plant matrix. The released
essential oil is transported by the generated steam to a condenser at the top of the microwave
oven. The vapor is subsequently condensed obtaining a two phase system (essential oil and
water). Finally, the excessive water is redirected to the plant material in the microwave oven
[16]. A further development of this extraction technique is called Microwave Hydrodiffusion
and Gravity (MHG). This process is performed in an “upside down” microwave alembic. The
plant material is also heated by microwave radiation, but the extract drops out of the reactor
by gravity. In difference to MAD, this method can be used to extract additionally non-volatile
compounds like pigments or antioxidants [7, 15, 16].
These microwave-assisted extraction methods have major advantages compared to traditional
techniques. In general, the total extraction time can be significantly reduced. Moreover, the
energy consumption and CO2 emission is decreased and a lower amount of waste water is
produced [16].
Nonetheless, whether these technologies can economically be transferred to the
manufacturing scale is questionable, since the technical complexity of applying microwaves
in a large-scale equipment is high [1]. Currently, pilot microwave reactors with a feeding
volume up to 75 L are feasible [17]

Solvent extraction

Solvent extraction is the most widely used technique for plant extractions. The principle is
based on the transfer of the compound from the sample to an organic solvent [8]. It is a very
important operation in numerous industries, such as the chemical, biochemical, food,
cosmetics and the pharmaceutical industries [7].

The organic solvent extraction gives better extraction yield, but the technique requires solvent
recovery through distillation which may degrade thermally labile compounds. Moreover, the
organic solvent extraction is non-selective and achieves the simultaneous removal of non-
volatile pigments and waxes, resulting in viscous, dark-coloured extracts contaminated with
solvent residues. This makes them difficult to handle without further refinement and may even
dictate the future commercial viability of the oil extract. Furthermore, the presence of traces of
residual solvent in the final product makes the process less attractive.

Maceration and correlated techniques

Maceration describes the soaking of a solid plant material in a solvent at room temperature
for a defined time. Thus, the solid is just in contact with the solvent without any motion.
Typically, the extraction time is long, whereas the efficiency is poor. This can be explained
by the fact that maceration is a process which induces equilibrium of the compound
concentrations in the plant material and the extraction solvent.
This means that the extraction takes place until a maximum concentration of the extracted
compounds is reached in the solvent. However, depending on the solubility properties, a
significant amount of the desired components may remain in the plant material. For a
complete extraction the repeated addition of fresh solvent is required, which can result in a
high solvent consumption. Maceration is especially used in the case of fragile molecules
because of the mild extraction conditions. If macerations are carried out at a constant elevated
temperature, the method is called digestion. Thereby, the establishment of the equilibrium
can be enhanced [1, 7, 18].

Another modification of maceration is called immersion. To this purpose, the plant material
is immersed in a solvent, while the mixture is stirred. This procedure ensures a very intense
contact between the phases [1].

A further extraction technique is infusion. Here, the solid is immersed in a heated solvent
without boiling, followed by the cooling of the suspension. The most prominent example of
infusion is the preparation of tea. In the case of a boiling solvent the extraction technique is
called decoction. However, these methods are only feasible for non-thermo sensitive
compounds. Nonetheless, this is a very fast and sometimes inevitable extraction method [7].

Percolation and Soxhlet extraction

In contrast to the previous described techniques, percolation is a method for the exhaustive
extraction of compounds from plant materials. To this purpose, the solid plant material is
stacked as a fixed bed while fresh solvent passes through. Generally, this process is driven
by gravity from the top to the bottom. Furthermore, adequate extraction efficiencies can be
achieved by recycling the extraction solvent and passing it several times through the plant
bed. It should be pointed out that a constant flow of the solvent has to be ensured.

An advantage of this technique is the relatively low mechanical stress on the solid material.
Moreover, no additional filtering step is required, as the extract is relatively free of solid
particles. The most prominent example of percolation is the preparation of coffee [1, 7, 18].
Another exhaustive method to extract compounds using a suitable solvent is the Soxhlet
extraction. In general, this technique is performed in lab scale in order to determine the total
content of compounds that can be dissolved in the used solvent. A sketch of the Soxhlet
apparatus is presented in Figure.
 Figure. Sketch of a Soxhlet extraction apparatus for the exhaustive extraction of plant
material.
Preliminary, the solid plant material to be extracted is filled in a porous extraction thimble,
mostly consisting of cellulose fibers. Afterwards, the thimble is placed in the chamber of the
Soxhlet extractor. The solvent in the bottom flask is then heated to boiling. The vapor fumes
through an ascending pipe to a condenser. There, the pure solvent condenses, drops into
the extraction thimble and comes into contact with the plant material. Once a certain amount
of solvent has been collected, the chamber is emptied through a special siphon arm.

The extract solution flows back into the bottom flask. Subsequently, a new extraction cycle
starts automatically. With this process, the plant material is extracted several times with fresh
solvent until, theoretically, all soluble components are extracted. The extracted compounds
are collected and concentrated in the bottom flask. Nonetheless, not all of the soluble
compounds may be extractable due to adsorptive or chemisorptive forces in the plant matrix.
Degradation of the components may also occur in the bottom flask, as the solvent is kept at
its boiling temperature for several hours [1, 8, 18, 19].

Selection of solvent

The selection of an appropriate solvent is the most important parameter of solid-liquid

extractions. The choice of the solvent often follows the principle “like extracts like”.
This means that the extraction efficiency is based on the polarity of both, the target
compound and the solvent. Plant ingredients can be classified into five main chemical groups:
fats, essential oils (terpenes) sugars, polyphenols, and glycosides. Figure 2.3 presents these
ingredients and their corresponding suitable extraction solvent in dependence of their polarity
[1, 3].

Figure 2.3: Selection of solvents which are suitable for different plant
ingredients [7].

 The solubility, which is a key feature in obtaining a crude solvent extract of plants, there
are additional criteria for solvent selection [1, 8]:

 Selectivity: A high selectivity of the solvent enables the reduction of purification steps
of the extract to obtain the target compound.

 Recoverability: The solvent should be easily recovered to obtain the crude extract.

 Solvents with low boiling points are preferred in order to remove it easily and prevent
the loss or degradation of compounds.

 Viscosity: Highly viscous solvents reduce the mass transfer efficiency and can lead to
difficulties with pumping or dispersion.
  Surface tension: A low surface tension of the solvent promotes the wetting of the
plant material. This ability is very important since the solvent must penetrate the plant
matrix.

 Thermal and chemical stability: The solvent should be thermal and chemical stable in
order to facilitate the recycling, for example the solvent recovery in an evaporator.

 Corrosivity: Corrosive solvents can increase equipment costs.

 Availability and costs: The solvent should be ready available. However, not the price of
the solvent is important, but the costs of inevitable losses during the process.

 Toxicity and flammability: Especially for food processing only nontoxic solvents will be
taken into consideration. In general, any associated hazard with the solvent will
require extra safety measures. For this reason, aliphatic solvent are preferred to aromatic
or halogenated ones.

 Environmental impact: The solvent should be environmental friendly and compatible

with downstream process steps in order to minimize losses due to evaporation,
solubility and carryover. Additionally, a full removal of the solvent from the residual
plant material should be achieved.

Especially, the last two aspects gained more and more importance in recent years. Therefore,
the HSE (health, safety and environment) profile of the solvent must also be taken into
account. A classification in regards of flammability, toxicity, and the HSP profile of
traditional and alternative organic solvents is presented in literature [25, 26]. Currently, there
is a strong tendency towards the use of “green” extraction solvents and agents which are in
conformity with the concept of “green extraction” [4-6].

An example for a new alternative solvent is 2-methyltetrahydrofuran (MeTHF). This solvent

was recently considered to be a green alternative for the petroleum-based n-hexane in
extraction processes. MeTHF is a biodegradable and plant derived solvent. It is produced by
hydrogenation of products obtained from carbohydrate fractions of hemicellulose from
various feedstocks. This solvent is in accordance with several principles of green chemistry
and sustainable or green extraction [27].

Enhancement of solvent extraction

There are different techniques to accelerate and enhance the previously described extraction
methods. Microwave- and ultrasound-assisted extractions are two promising techniques,
which will be described in the following sections.

Microwave-assisted extraction (MAE)

In recent years, microwave-assisted extraction (MAE) has been intensively studied and
successfully applied in solid–liquid extractions of plant materials. Microwave radiation can be
used for the fast extraction of several classes of plant compounds [16]. To this purpose, the
plant material is immerged in an organic solvent, which must not absorb microwave radiation,
such as n-hexane.

More precisely, the solvent must have a low dielectric constant, which makes it relatively
transparent for microwave radiation. Microwaves affect directly the water in the cells of
the plant matrix. These cells are ruptured by the caused high pressure in the cell,
releasing the plant compound. The substance is dissolved in the organic solvent and can be
further processed [6, 7, 16, 47]. Extractions can also be performed with a solvent which
absorbs microwave radiation. However, strong heating must be avoided in order to prevent
thermal degradation of the plant compounds. A binary solvent mixture is usually used for
extraction processes, containing only one solvent which absorbs microwaves [1].

Microwave-assisted extraction has several advantages compared to traditional techniques.

First of all, compounds can be extracted more selectively and more rapidly. In addition, MAE
enables equal or even better extraction yields by a minimal sample manipulation.
Furthermore, the consumption of energy and organic solvents is reduced [1, 2, 7].

Ultrasound-assisted extraction (UAE)

Another technique to enhance the extraction efficiency is ultrasound-assisted extraction
(UAE). The most common tools for UAE are ultrasonic baths, ultrasonic probes, and
cavitating tubes. The efficiency of the technique can be explained by the phenomenon of
cavitation [6, 7]. Preliminary, the plant material is immerged in a solvent and ultrasound is
applied to the sample. Fundamentally, two different effects of ultrasound on the cell walls can
be described. On the one hand, sonication can easily destroy the thin skin of glandular plant
cells releasing the cellular content into the surrounding solvent. On the other hand, ultrasound
can also accelerate the swelling and hydration of plant matrices, which results in an
enlargement respectively disruption of the plant cell walls. This behavior improves the mass
transfer rate, resulting in an increase of the extraction efficiency [1, 6, 7, 48]. The advantages
of ultrasound-assisted extraction are the shortening of extraction time and the enhancement
of extraction efficiencies. In addition, the energy and solvent consumption can be reduced
compared to conventional techniques [6, 48-50].

Supercritical fluids

The occurrence of a supercritical state was for the first time observed by Baron Cagniard de
la Tour in 1822. Thereby, a pure substance was trapped in a molten glass vessel and
subsequently heated. It was observed visually that the boundary between the liquid and
gaseous phase disappeared, obtaining one visual phase [51]. The term “critical point” was
introduced by T. Andrews about half a century later [52]. Only a few years later, the first
experimental evidence has been provided by Hannay and Hogarth in 1879 that supercritical
fluids enhance the solubility of solids. In addition, they were able to show that the dissolving
power of a supercritical fluid (SCF) is depending on the pressure [53]. This tunable solvation
power is the basis of modern-day research in the sector of supercritical fluids, for example
supercritical fluid extraction (SFE).

In the following sections, supercritical fluids are described with respects to their
characteristics and physical behavior. The main focus will be on supercritical dioxide and its
application in supercritical fluid extraction.

Supercritical state
In general, pure chemical substances can exist in different states of aggregation, depending on
the prevailing pressure and temperature conditions. Figure 2.4 presents the phase diagram of
carbon dioxide (CO2). By means of this diagram, it can be realized what state of the
substance is present under a given pressure and temperature. Particularly, four different
regions are present, in which the substance occurs as a single phase: solid (s), liquid (l),
gaseous (g) and supercritical (sc). Here, the pressure and the temperature can be changed
without the occurrence of a phase transition. These areas are limited by lines at which the
compound is in equilibrium with two different phases.

The sublimation curve represents the pressure and temperature conditions under which the
solid and gaseous states are present at the same time. The coexistence of the solid and liquid
phase is represented by the melting curve, whereas the vaporization curve indicates the
equilibrium of the gaseous and liquid states. The intersection of these three curves is called
triple point. Here, the substance is in equilibrium with the solid, liquid, and gaseous state [13].

Figure: Phase diagram of carbon dioxide. Data from [54 57].

Furthermore, the point at which the vaporization curve ends is called "critical point". It is
characterized by the critical temperature Tc and critical pressure pc. By exceeding these
critical values, the phase boundary between the liquid and gas vanishes and both phases
cannot be distinguished anymore. In this area, the substance is in a state, which is designated
as “supercritical”. Moreover, it is possible to transfer a supercritical fluid to a solid state, but
only at very high pressures [13]. In addition, critical opalescence can be observed before
reaching the supercritical state [58]. The critical point of carbon dioxide is at Θc = 31.1 °C
and pc = 73.8 °C [55].

Physical properties

The physical properties of supercritical fluids vary over a wide range depending on pressure
and temperature. These properties are in general between those of gases and liquids. In detail,
the densities of supercritical fluids can reach the values of liquids, whereas viscosities are
similar to those of gases. Moreover, the diffusion coefficients are intermediate between those
of gases and liquids. Precise values of density, viscosity, and the diffusion coefficient are
summarized in Table.

Table Physical properties of solvents in different states of aggregation [59].

The density of supercritical fluids is comparable to liquids, but it is strongly depended on

pressure and temperature. The density of the supercritical fluid can be varied above the
critical values without the occurrence of a discontinuity. It is fundamental that the solubility
of a substance is strongly influenced by the density of the supercritical fluid. Thus, it is
possible to adjust continuously the solvation power of the supercritical fluid by varying the
temperature respectively pressure. In detail, this means that increasing the pressure at a
constant temperature results in an increased solubility of the compound due to the rising
density [60, 61].
The relative dielectric constant εr and thus the polarity of the solvent also affects the
solubility of substances in supercritical fluids. The dielectric constant of carbon dioxide can
be slightly increased with pressure from a value of 1.0 at 20 bar up to 1.6 at 300 bar and
40 °C [62]. The modification of the dielectric constant at different conditions can be better
demonstrated with water. The dielectric constant of liquid water is 78.4 at ambient conditions.
However, supercritical water (Θc = 373.9 °C and pc = 220.6 °C) shows a dielectric
constant of 2 near the critical point. Thus, water is a polar solvent at ambient conditions and
a more or less nonpolar solvent in the supercritical state [63, 64].

The diffusion coefficient and viscosity represent the transport properties of the supercritical
fluid which affect the mass transfer rate. In general, the viscosity of a supercritical fluid is
similar to the gaseous state. On the other hand, the diffusion coefficient is at least one order
of magnitude higher compared to liquid solvents.

This means that the diffusion of a substance through a supercritical fluid will occur at a faster
rate than that obtained in a liquid solvent. This behavior implies that a solid will dissolve
more rapidly in a supercritical solvent. In addition, the surface tension is lowered, which
enhances the penetration of a supercritical fluid into a micro-porous solid structure, like a
plant matrix. The diffusion coefficient and the viscosity can be varied by the applied pressure
and temperature. In detail, the viscosity increases with rising pressure, causing a decrease of
the diffusion coefficient. This effect is less pronounced at high pressure values, as the
density becomes less sensitive to the pressure. In contrast, the diffusion coefficient generally
increases with an isobaric increase of the temperature. However, changing the temperature at
a constant solvent density has only a small effect on the diffusion [65].

Applications

These special and tunable physical properties of the supercritical fluids induced the invention
of several technical applications. Supercritical fluid chromatography (SFC) is a separation
method in which a solvent is used in its supercritical state as mobile phase. It can be
considered as an intersection between gas chromatography (GC) and liquid chromatography
(LC). The stationary phase consists either of a capillary column or packed column. The
transport velocity or separation efficiency depends on the distribution of the substances
between the mobile and stationary phase.
The solvent power of the mobile phase can be varied by the density of the supercritical fluid.
In addition, an organic solvent can be added in order to modify the mobile phase and facilitate
the separation of the compounds. The separation efficiency can be enhanced by varying the
pressure and temperature of the supercritical fluid. The importance of SFC is based on the
fact that it permits the separation of compounds which are usually not processed by LC or
GC. An advantage of SFC is the easy removal of the mobile phase from the separated
compounds. However, a disadvantage is that strongly polar and ionic molecules are not
soluble in supercritical fluids [65-67].

In addition, supercritical fluids can be used as solvent in chemical synthesis. In detail,

hydrogenations, oxidations, radical reactions, enzymatic reactions, polymerization, and
several other reactions can be performed in supercritical fluids [65, 68, 69]. Supercritical
carbon dioxide can also be used to dry hydrogels, in order to obtain aerogels. In detail,
aerogels are lightweight materials with outstanding textural properties (i.e. high surface area
and open porosity) consisting of silica.

Aerogels are obtained from wet gels by using the supercritical drying process. By means of
this method, it is feasible to avoid the pore collapse phenomenon and keep the porous texture
of the wet material intact [70-72]. Further applications of supercritical fluids are for example
dyeing [73, 74], impregnation [75,76] and the preparation of fine particles (“supercritical
fluid precipitation”) [77].

Supercritical fluid extraction (SFE)

Supercritical fluid extraction (SFE) is an interesting alternative technique to

conventional solid-liquid extractions with organic solvents. Amongst all supercritical fluids,
carbon dioxide is the most common and suitable solvent in SFE [2]. This fact is a
consequence of several advantages which offers CO2 in comparison to other supercritical
fluids. First of all, carbon dioxide exhibits moderate critical conditions. The critical point
of carbon dioxide is at a critical temperature of 31.1 °C and a pressure of 73.8 °C [55].
Consequently, thermal degradation of natural compounds during the extraction process can be
avoided [2].
Carbon dioxide shows only a low toxicity which results in a safer handling of the solvent.

Actually, the maximum workplace concentration (MAC value) of CO2 is 9 g/m3. Carbon
dioxide is also an environmental friendly and biocompatible solvent. In particular, the
concentration of CO2 in the atmosphere is 0.03% by volume and it can be degraded by
photosynthesis of plants. Moreover, carbon dioxide is relatively chemical inert and non-
flammable [78].

A further advantage of carbon dioxide is its low price [65, 79, 80]. In addition, CO2 is
available in large quantities and high purity. Carbon dioxide can be recovered as by-product
in the exhaust gases of power plants or the chemical industry, for example in the production
of lime, hydrogen, ammonia, and ethylene oxide. Also subterranean natural sources from
active or extinct volcanoes are feasible. The crude gas of the chemical processes contains
more than 95% of carbon dioxide. After the deposition of gaseous impurities and moisture,
the carbon dioxide is compressed and exhibits a purity of at least 99%. The liquefaction of
the gas is performed at pressures between 14 and 20 bar and temperatures of -40 to -25 °C
[81]. This liquefied carbon dioxide, with a vapor pressure of 57.29 bar at 20 °C, is used
directly in supercritical fluid extraction [82].

Procedure of SFE

Supercritical fluid extraction of plant materials consist of two main steps. Initially, the
extraction process itself, followed by the separation of the solvent from the extract [66]. A
simplified flow scheme of a SFE unit is presented in Figure 2.5. By means of this scheme,
the procedure of SFE is explained.

The process steps are the following. Preliminary, the high pressure extraction vessel is filled
with the plant material. Liquid carbon dioxide is supplied from a storage tank and guided
through a condenser in order to ensure the liquid state of the solvent. CO2 is compressed
and transported by a pump (mainly piston pump) to a heat exchanger in order to reach the
desired process conditions. Afterwards, the supercritical carbon dioxide passes through the
extraction vessel, usually from the bottom to the top. Here, the mass transfer of the solutes
into the fluid solvent takes place. A second pump can be used to add an organic solvent in
order to influence the extraction efficiency. Furthermore, the pressure in the vessel is adjusted
by a back pressure regulation valve. The loaded supercritical fluid is transported to the
separation vessel, where the extract is precipitated by adjusting the temperature and the
pressure. Generally, the carbon dioxide gets gaseous in this step and exits the separator.
Finally, the exhausted CO2 can be recycled by liquefaction in a condenser and can be
recirculated by the pump [1, 2, 66].

Figure Simplified flow scheme of a supercritical fluid extraction unit with the most
important sections.

Extraction

Extractions with supercritical carbon dioxide are especially applied for the recovery of
volatile (e.g. essential oil) and/or nonpolar (e.g. fats, waxes) compounds from plant materials.
There are many variables which have to be considered in order to optimize the extraction
yield and selectivity of SFE. These parameters include the values of pressure, temperature,
extraction time, solvent flow rate, particle size, packing density, and the addition of a
cosolvent [47, 83-85]. In the following, a few general statements regarding these variables for
plant extractions are discussed.

First of all, the pressure and temperature in the extraction vessel are the most important
parameters in SFE. By means of them, the density and thus the solubility of the target
compound can be adjusted. In general, it can be stated that the extraction of volatile
compounds is more favorable at low densities (~ 0.3 g/mL) respectively pressures (100 bar).
Otherwise, higher densities (~ 0.8 g/mL) of carbon dioxide enhance the extraction of by-
products with a high molecular weight, e.g. fatty acids, triglycerides, or waxes. Thus, the
selectivity of supercritical is impaired [83-85]. However, in most cases of plant extractions,
the diffusion of the target compound out of the matrix is usually the limiting step [86]. This
limit can be avoided either by increasing the temperature and thus the volatility of the
compound, or at high solvent densities. In order to achieve a good selectivity SFE, it is
significant to control the solvent density carefully [83].

The solubility of the target compound can also be limited by the nonpolar character of carbon
dioxide. Consequently, an (polar) organic solvent, called modifier or entrainer can be added
to the supercritical carbon dioxide in order to enhance the solvating properties. Volatile polar
solvents such as ethanol, methanol, acetone, or acetonitrile are preferred as cosolvent [2]. At
least, 17 different modifiers in SFE of natural products have been studied [87]. Among all the
modifiers, methanol is the most commonly used cosolvent for SFE because of its polar
properties and good miscibility with CO2. Usually, the extraction of polar compounds can be
increased. However, the selectivity of the extraction process gets impaired due to the
enhanced extraction of by-products [83]. Generally, longer extraction times increase the
extraction yield of most of the compounds. Thereby, it has to be distinguished between static
(no solvent flow) and dynamic (constant solvent flow) extraction. It has been shown that a
preliminary static extraction step can enhance the extraction yield, as the sample-solvent
contact is improved [84, 88].

The flow rate of the supercritical carbon dioxide through the extraction vessel has also a
strong influence on the extraction efficiency. In fact, slow flow rates enhance the penetration
of the plant matrix. However, if the mass transfer of the target compound in the supercritical
fluid is low, the yield is not influenced by the solvent flow rate [84, 85].

As a general rule it can be asserted that decreasing the particle size of the plant material
results in a higher surface area, which facilitates the extraction. However, excessive grinding
of the solid may hinder the extraction due to re-adsorption of the target compounds on the
matrix surface [84, 88].
Finally, a detailed discussion of the influences of different parameters on the extraction yield
and selectivity of SFE is presented in chapter 6.4.4. There, SFE was performed in order to
extract selectively odorants from iris rhizomes.

Separation

The second main step of SFE is the separation of the extract from the solvent. The
development respectively application of an appropriate collection technique for the target
compound is mandatory. To this purpose, three different separation approaches are commonly
used in supercritical fluid extraction [66, 83-85].

Liquid-solvent collection is technically simple and has been the most widely used technique
for natural products. Therefore, the supercritical carbon dioxide loaded with the extract is
guided through a solvent and depressurization of the supercritical fluid is performed directly
in the collecting solvent (e.g. ethyl acetate, acetone) [83, 84]. A major disadvantage of this
method is the additional recovery of the extract from the solvent.

Another technique for the separation of extracts from the supercritical fluid is the solid-phase
trapping method. It is performed by depressurization of the supercritical carbon dioxide and
the extract prior to the trap. Subsequently, the target compound is directly collected from the
gas phase. Suitable sorbents include silica gel, glass, or stainless steel beads. After SFE, the
extract can be recovered from the trap with a suitable organic solvent. A major advantage of
this method is that the selectivity of the extraction can be improved by selective trapping
coupled with selective elution of the target compound. However, the solvent has to be
removed in order to obtain the crude extract [66, 83, 84].

Furthermore, the collection of the extract in an empty vessel is another separation method.
For this technique, the solvating power and thus the density of the supercritical fluid need to
be decreased. This can be achieved either by increasing the temperature or decreasing the
pressure in the separator. In addition, a fractionation of the extract can be realized by two or
more separators, which are connected in series and operated at different conditions. The
major advantage of this technique is that the crude extract precipitates and can be recovered
without the addition of an organic solvent [66, 85].

Advantages & disadvantages compared to conventional techniques

SFE with carbon dioxide has several advantages compared to conventional extraction
techniques. In detail, the extraction time can be significantly reduced compared to traditional
solvent extraction or steam distillation. Typically, SFE is performed within a few tens of
minutes, whereas solvent extractions or steam distillation is carried out for several hours
respectively days. Moreover, the solvent power of the supercritical fluid can be adjusted by
the solvent density, which leads to a higher selectivity regarding the target compound.
Supercritical fluid extraction with carbon dioxide is performed at low extraction temperatures,
which prevents the degradation of volatile, thermal labile or oxygen-sensitive compounds.

The scent of a SFE extract is comparable to the original odor of the plant material, whereas
extracts of steam distillation can exhibit a rather different and “cooked” aroma. A further
advantage of SFE compared to solvent extraction, is the use of carbon dioxide, which is a
non-flammable and non-toxic solvent. CO2 can be easily removed by depressurization
obtaining the crude extract without any residual solvent. Thus, no further processing, like the
evaporation of an organic solvent, is needed [8, 47, 83, 84]. Finally, the energy consumption
of supercritical fluid extraction is significantly lower compared to traditional extraction
methods. In detail, about 0.8 kWh are consumed during SFE to obtain 1 kg of plant extract.

However, approximately 8 kWh are consumed in solvent extraction and 10-30 kWh in steam
distillation to obtain the same amount of extract [89]. Concluding, SFE is an
alternative method for plant extraction in regards to the principle of “green extraction” [5, 6].

Nonetheless, supercritical fluid extraction with carbon dioxide has some minor
disadvantages. In detail, CO2 can only be used to extract more or less nonpolar compounds.
However, this can be overcome by using modifiers like methanol or ethanol. Additionally,
the development of an extraction procedure is very time consuming in order to find the best
extraction conditions and balance the temperature, pressure, and solvent flow rate for each
type of plant. Further drawbacks of SFE are the high engineering requirements and
investment costs of the extraction unit due to the high required pressures. Finally, carbon
dioxide can react with water and form carbonic acid or amine groups in order to form
amidocarbonic acids and carbamates which can cause damage of the unit parts [1, 8, 90].

Applications

Supercritical fluid extraction of natural products from plants with carbon dioxide has become
one of the most important application areas [83]. One of the best-known application of SFE is
the decaffeination of coffee beans [91]. The process is based on a patent of K. Zosel
developed in 1970 at the Studiengesellschaft Kohle mbH (Mülheim) [92]. By means of
this method, green coffee beans are extracted with supercritical carbon dioxide at a
temperature of 40-80 °C and a pressure of 200-300 bar [93].

It is noteworthy that caffeine can be removed in a highly selective way. In addition,

compounds are not lost that contribute to the typical coffee aroma after roasting. The main
advantage of this technique is that no residual solvent is remaining in the coffee beans.
Formerly, toxic chlorofluorocarbons (dichloromethane, trichloroethylene) were used as
solvent for the decaffeination of coffee beans. In addition, the decaffeination of tea leaves
can also be performed with supercritical carbon dioxide [91].

Another application of SFE is the extraction of volatile essential oils or active compounds
(antioxidants, flavonoids, etc.) from plants [83-85, 94-99]. For example, the company
FLAVEX (Rehlingen/ Germany) distributes a large number of different natural extracts
obtained by SFE with carbon dioxide of plants like rosemary, oregano, ginger, hop,
cinnamon, iris, pepper, and juniper berry [100].
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