Rev Chem Eng 2019; aop

Ghada Ben Salha, Manef Abderrabba and Jalel Labidi*

A status review of terpenes and their separation

methods
https://doi.org/10.1515/revce-2018-0066 materials vary from highly complex mixtures to single
Received September 18, 2018; accepted June 4, 2019 chemicals called terpenes (Surburg and Panten 2016). The
term “terpene” originates from turpentine (balsamum ter-
Abstract: Terpenes or terpenoids are extracted or steam
ebinthinae). Turpentine is the viscous pleasant smelling
distilled for the recovery of the essential oils of specific
balsam that flows upon cutting or carving the bark and the
fragrant plants. These steam distillates are used to cre-
new wood of several species of pine tree (Pinaceae; Breit-
ate fine perfumes, to refine the flavor and the aroma of
maier 2006). In nature, terpenes occur predominantly as
food and drinks, and to produce medicines from plants
hydrocarbons, alcohols and their glycosides, ethers, alde-
(phytopharmaca). In recent years, consumers have devel-
hydes, ketones, carboxylic acids, and esters. These com-
oped an increasing interest in natural products, as most
pounds are referred to as terpenoids. The terms terpene
of these terpenoids have been identified as high value
and “terpenoid” are often used interchangeably (Qiu et al.
chemicals in food, cosmetic, pharmaceutical, biotechnol-
2017). These natural compounds are comparatively strong-
ogy, and industrial crops. Extensive chemical techniques
smelling organic compounds with characteristic, usually
and biological tests have led to the identification, biologi-
pleasant odors. They are therefore used in perfumes and
cal characterization, and extraction of major components
cosmetic products. Furthermore, aromas that develop
that are of wide interest, especially to the cosmetic and
during frying and baking are used as flavoring substances
industrial recovery of selective terpenes. The current sta-
to enhance the enjoyment of food. In contrast, flavor and
tus of the knowledge of their general structure, functions,
fragrance substances are indispensable for the survival of
and bioactive properties and the methods for their separa-
many organisms, which use chemoreceptors to find their
tion are covered in this review.
way, to hunt for and inspect food, to detect enemies and
Keywords: chemical techniques; functions; general harmful objects, and to find members of the opposite sex
structure; industrial crops; natural product; separation (pheromones; Surburg and Panten 2016). Among the ter-
methods. penes, monoterpenes (MTs) and sesquiterpenes and their
oxygenated derivatives are the largest group of chemi-
cal entities in an essential oil (EO). Most of the time, the

1 Introduction bioactivities of a particular EO are decided by either one

or two of its main components. However, sometimes the
overall activity cannot be attributed to any one of the
Since early antiquity, spices and resins from animal and
major constituents but is rather a consequence of the syn-
plant sources have been used extensively for perfum-
ergistic effects of several chemical constituents (Raut and
ery and flavor and to a lesser extent for their observed or
Karuppayil 2014). In recent years, there has been increas-
presumed preservative properties. Fragrance and flavor
ing interest worldwide in the use of single active species
or multiple active constituents of EO for modern medicine
*Corresponding author: Jalel Labidi, Chemical and Environmental and different industrial crops. However, quality-related
Engineering Department, University of the Basque Country, problems (lack of stability and efficacy) seem to be over-
Plaza Europa, 1, 20018 Donostia-San Sebastián, Spain, shadowing the potential benefits of various EO products.
e-mail: [email protected]
The terpene hydrocarbons do not contribute much
Ghada Ben Salha: Chemical and Environmental Engineering
Department, University of the Basque Country, Plaza Europa, to the flavor or fragrance of the oil, and because they are
1, 20018 Donostia-San Sebastián, Spain; Laboratory Materials, unstable to heat and light, they must be removed to stabi-
Molecules and Application, Preparatory Institute for Scientific and lize the final product. The oxygenated compounds provide
Technical Studies, 2070 Marsa, Tunisia; and Faculty of Sciences of much of the characteristic flavor strength of EOs and
Tunisia, University of Tunisia El Manar, Farhat Hached University,
consist mainly of alcohols, aldehydes, and ketones (Stuart
Campus PB 94 – Rommana 1068, Tunis, Tunisia
Manef Abderrabba: Laboratory Materials, Molecules and
et  al. 2001). Thus, it is common industrial practice to
Application, Preparatory Institute for Scientific and Technical remove some or all of the terpene hydrocarbons to concen-
Studies, 2070 Marsa, Tunisia trate the oxygenate fraction in the oil. These concentrated

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2      G. Ben Salha et al.: Terpene separation methods

oils are referred to as folded, terpeneless, or deterpenated as the true precursor of the terpene molecule, is the basis
oils (Brose et al. 1995). To obtain pure compounds or con- of the concept of the “isoprene rule” set out in 1953 by
centrates of interest, the fractionation of natural complex Ruzicka (1994). Figure 1 presents the categories of the
mixtures has been implicit in various studies, but gener- sequential combining of basic five-carbon units: C5 (hemi-),
ally they have not been made explicit and have not been C10 (mono-), C15 (sesqui-), C20 (di-), C25 (sester-), C30 (tri-),
presented in an integrated fashion. C40 (tetra-), and C(5)n with n > 8 (poly-).

2 General structure 2.1 Hemiterpenes (HTs)

Amid a bewildering array of structures, terpenes form the There are about 50 compounds known among the natural
largest group of natural products with more than 55,000 compounds found in plants that can be considered as HTs
different structures known at present in the literature (Breitmaier 2006). The most prominent HT is isoprene,
(Maimone and Baran 2007). The isoprene unit, considered which is emitted from the leaves of many trees (including

Figure 1: Categories of hydrocarbon terpenes (isoprenoids).

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 G. Ben Salha et al.: Terpene separation methods      3

conifers, poplars, oaks, and willows) and herbs (e.g. and is mainly found in pepper and some spices (Malecky
Hamamelis japonica). Other known hemiterpenoids found 2008).
in plants are angelic, tiglic, isovaleric, and senecioic acids
along with isoamyl alcohol (Ludwiczuk et al. 2017).
2.4 Diterpenes (DTs)

2.2 MTs In total, there are about 2700 DTs in nature. They are
formed from their precursor, geranylgeranyl pyrophos-
Approximately 1500 MTs have been documented. They are phate. These compounds are mainly present in higher
the simplest components of terpenes, which are primary, plants in the resins or gibberellins. Hydrocarbon phytane
and are the majority components of EOs (90%; Deans is the parent compound of about 5000 acyclic DTs, among
1999). They are mainly divided into three structural cate- which we find phytol, which is the best-known represent-
gories: linear MTs (acyclic), MTs with a single ring (mono- ative in chlorophyll or vitamins K and E. The rest, which
cyclic), and those with two cycles (bicyclic). Most of these represent the majority of DTs, are in cyclic form and are
are a result of a typical head-to-tail combination of the derivatives of cyclophytane. Retinol and retinal, two forms
isoprene units (Qiu et al. 2017). of vitamin A, are best known in this family (Brielmann
Acyclic MTs: Geranyl pyrophosphate (GPP), the first et al. 2006).
compound derived from mevalonate, is the precursor of
this category of MTs. In this group, geraniol is the most
widespread in the Plant Kingdom (Malecky 2008). 2.5 Sesterpenes
Monocyclic MTs: These compounds derive from neryl
pyrophosphate or GPP (Qiu et al. 2017) and are primarily About 150  sesterpenes, compounds C25, are well known
divided into two main groups. (Surburg and Panten 2016). Mevalonic acid is the precur-
C10H16 or C10H18 hydrocarbons: D-limonene, the phel- sor of this class. Sesterpenes are rare in nature. They are
landrenes, and the terpineol derivatives are the best arranged either in linear or cyclic form, with one, two,
known representatives of this family. three, or four rings, and they have been isolated from
C10H20 hydrocarbons: Menthane derivatives (menthol plants, fungi, and insects (Qiu et al. 2017).
and menthone) and those containing an oxide such as
cineole or eucalyptol are very abundant.
Bicyclic MTs: These compounds are found in many 2.6 Triterpenes (TTs)
EOs, particularly those from conifers. Bicyclic cyclopro-
panes, carane, thujane, bicyclic cyclobutane pinane, About 5000 naturally abundant TTs have been docu-
camphane, fenchane, α-pinene, and β-pinene are widely mented in the literature. Most of these are derived from
distributed in plants and are the most important skeletons squalane and squalene, with two farnesane units linked
of naturally occurring bicyclic MTs (Qiu et al. 2017). in a tail-to-tail manner. Majority of the compounds of
this family are tetracyclic or pentacyclic in form and are
derived from squalene and plant steroids. The acyclic
2.3 Sesquiterpenes form is very rare. Most TTs are alcohols, in free form or
glycosides (saponins) or ester (Malecky 2008).
Approximately 10,000 naturally abundant sesquiterpe-
nes, derived from the parent 2,6,10-trimethyldodecane
or farnesane, found in oil slate, are known (Breitmaier 2.7 Tetraterpenes
2006). This class of terpenes is divided into the following
structural categories: acyclic, monocyclic, bicyclic, tricy- About 200 naturally abundant tetraterpenes are known to
clic, and polycyclic. date and are referred to as carotenoids. The most typical
Acyclic sesquiterpenes are mostly derived from are the apocarotenoids, the diapocarotenoïdes, and the
trans-farnesyl pyrophosphate. The monocyclic sesquit- megastigmanes. All of them represent structural vari-
erpenes are divided mainly into four families: bisabolan, ants or degradation derivatives of “β-carotene” from the
germacran, eleman, and humulan. Among the polycy- Daucus carota (Umbelliferae) with 11–12 C-C-conjugated
clic sesquiterpenes, caryophyllene is the most important double bonds (Breitmaier 2006).

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4      G. Ben Salha et al.: Terpene separation methods

2.8 P
 olyterpenes (PTs) and prenylquinonoes the host plant (Vandermoten et al. 2012). These terpenes
play an important role as chemical communications for
Terpenes with more than eight isoprene units are classified algae in marine ecosystems too (Girola et al. 2015).
as PTs. These compounds are often found in two isomeric Furthermore, these volatile organic compounds
forms, cis and trans. cis-Polyisoprene is the main con- can cross the membrane to be released into the atmos-
stituent of natural rubber, whereas gutta-percha consists phere to influence air quality and climate changes, such
essentially of trans-polyisoprene. Moreover, a mixture of as ozone production and secondary organic aerosol
1:2 of the cis and trans isomers of PTs can be used to make formation (Jones et  al. 2014). The main biogenic pre-
a chicle. In contrast, prenylquinones are PTs with up to 10 cursors are myrcene, ocimene, linalool, terpinen-4-ol,
isoprene units. Among them, we find vitamins K1, K2, and β-caryophyllene, α-pinene, limonene, etc. (Pye and
E (Malecky 2008). Pouliot 2012).
In contrast, some MTs are useful as porogens in porous
silicon oxide dielectric film deposition processes. Diethox-
ymethylsilane is used as a silicon oxide precursor for the
3 Functions of terpenes manufacture of integrated circuits of electronic devices.
The two most commonly used porogens are α-terpinene
In general, terpenes are secondary metabolites. Majority and d-limonene (Bowen 2010).
of them are popular materials for making perfumes. The In bacteria, some terpenes decrease the adhesion and
most important terpenes used as fragrance and flavor sub- proliferation tendencies of a wide variety of bacteria. The
stances are the MTs, such as the acyclic alcohols geraniol, presence of the coating produced from terpinen-4-ol can
linalool, and citronellol and the monocyclic compound be effectively integrated into medical and other clinically
limonene, which is considered as the chief contributor to relevant devices to prevent bacterial growth and to mini-
the aroma of all the citrus cultivars (Rouseff and Perez- mize bacteria-associated adverse host responses (Bazaka
Cacho 2007, Qiu et al. 2017). et al. 2011).
However, some of them play important roles in basic Terpinen-4-ol, an active ingredient of the EO of
processes, including the growth and development of the majoram, shows a direct correlation with antifungal per-
side chain of the photosynthetic pigment chlorophyll, formance against Aspergillus niger (Ben Salha et al. 2017).
such as phytol. It is the most abundant terpenoid in plants It is also mentioned in the literature that certain oxygen-
(Davis and Croteau 2000). ated MTs, such as citronellal, citronellol, nerol, geraniol,
In natural ecosystems, due to their structural prop- borneol, and geranial, and several phenolic compounds,
erties, viz. that they are lipophilic molecules with low such as thymol, eugenol, and carvacrol, make a signifi-
molecular weight and vapor pressure at elevated tempera- cant contribution to the antifungal activity of certain EOs
tures, MTs and sesquiterpenes play a key role by serving (Morcia et al. 2012). In addition to their antifungal power,
as a means of communication between species, most of MTs, sesquiterpenes, and phenylpropanoids possess
which are plant-insect interactions (Langenheim 1994, notable antiviral properties, e.g. isoborneol and eugenol
Dudareva et  al. 2006). Some of the terpenes produced extract from clove oil. The first one inhibits the glycosyla-
by plants serve to attract specific insects for pollination tion of viral proteins, and the second one interferes with
or as defenses against herbivores, pathogens, and abiotic the development of herpes virus-induced keratitis and
stresses (Huang et al. 2010). They are good remote infor- thereby prevents viral activation (Astani et al. 2009, Raut
mation vectors, e.g. the cyclobutane MTs, which are sexual and Karuppayil 2014). The diverse potential biological
pheromones of various beetles, and some of them are activities of terpenes have drawn the attention of research-
aphid alarm pheromones, such as (E)-β-farnesene. This ers; they have been tested for anticancer activity, taking
last is the primary component of the alarm pheromone of advantage of the fact that their mechanism of action is
several economically important species of aphids. In fact, dissimilar to that of the classic cytotoxic chemothera-
in response to an attack by natural enemies, aphids secrete peutic agents (Rajesh and Howard 2003). The camphene
droplets of fluid from specialized structures, named isolated from Piper cernuum (Piperaceae) exerts an antitu-
cornicles, located on the upper, posterior surface of the mor activity in vivo by inhibiting the subcutaneous tumor
abdomen. These secretions contain an alarm pheromone growth of highly aggressive melanoma cells in a synge-
that induces behavioral responses in receiving conspecif- neic model, suggesting a promising role of this compound
ics. Typically, individuals receiving the alarm signal stop in cancer therapy (Girola et al. 2015). Some research has
feeding, move away from the signal, and often drop from indicated that MTs have multiple pharmacological effects

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 G. Ben Salha et al.: Terpene separation methods      5

on mevalonate metabolism, which could account for the 4.1 C

 hromatographic technique (CT)
terpene-tumor suppressive activity (Edris 2007).
Despite the importance of individual or mixture com- The use of the thin-layer CT known as “chromatostrip”
pounds in medicine, agriculture, and industry, still, com- or “chromatoplate” that we are familiar with today had
paratively little is known about the actual role of most its origins in the 1930s (Barker et  al. 2016). In the early
terpenes in nature (Gershenzon and Dudareva 2007). 1950s, Kirchner et  al. (1951) began to investigate the use
Therefore, there has been more recent research concen- of glass strips coated with a thin layer of adsorbent (Ad)
trating on the extraction of new active terpenes and the to analyze terpene derivatives. This technique consists
enhancement of their biological and pharmacological of forming a thin layer of Ad on a glass strip, adsorbing
activities. Some of these volatile compounds have been a small sample of the material to be resolved near the
isolated from fungi (Kramer and Abraham 2012, Xiao et al. bottom, and developing the column by immersing the
2013), seaweed (Pérez et  al. 2016), insects (Degenkolb bottom end in a test tube containing a small amount of
et al. 2011), and sponges (Saito et al. 2013), but the major- solvent (Kirchner and Miller 1952). Later, the adsorption
ity of these substances can be obtained from plant parts principle was applied to the production of terpeneless
(seeds, flowers, fruits, leaves, stems, roots, and rhizomes) oils (folded oils; Table 1). Most CTs involve the following
by various separation techniques. steps: the EO is adsorbed on a solid Ad such as silica gel
or alumina. The treated Ad is then eluted with a terpeno-
philic solvent such as hexane or dichloromethane or a
4 Separation methods mixture of solvents such as hexane ethyl acetate (Gressler
et al. 2011) or methanol (Girola et al. 2015), whereby the
Fractionation or separation of natural complex mixtures terpenic constituents are removed. Next, the Ad is eluted
to obtain pure compounds or concentrates of interest with an oxygenated organic solvent such as ethyl acetate.
has been the subject of extensive research. It is used to By removing the solvent from the resulting elute, there
produce EOs. A mixture of MTs and sesquiterpenes is is obtained the desired product containing mainly the
widely used in perfumes, toilet preparations, pharmaceu- oxygenated constituents, the EO substantially free from
ticals, therapeutic treatments, and as flavoring agents in undesirable terpenes (Rockland and Debenedict 1975).
food and drinks as well as for agriculture and crop pro- Another step for recovering hydrocarbon MTs can be
tection (Gañán and Brignole 2013). Hydrocarbon terpe- implemented by applying a pressure above atmospheric
nes suffer from hydrolysis reactions on exposure to heat pressure from a gas source without the use of a solvent.
or light, which degrades them, generating undesirable This purifying process can provide high hydrocarbon MT
compounds (Lago et al. 2011). To alleviate this difficulty, purity, for example, α-terpinene (90–95%), which can be
various procedures have been developed for the selective used as porogen species for the highest quality material
removal of MTs and the purification or enrichment of the on the market. In contrast, the oxygen fraction provides
more bioactive oxygenated terpenes (OT) to result in more a desirable property for commercial-scale separation and
effective, stabler, and water-soluble products. engineering scale-up of the commercial separation and

Table 1: Application of CT techniques to obtain terpenless EOs.

Raw material Initial data Results Exploitations References

Wood chips α-terpinene 90%; impurity: Recovering neat α-terpinene and Use of α-terpinene as a Bowen 2010
and citrus 1,8-cineol (3.45%) reducing the impurity of 1,8-cineol porogen
peels to 0.024%
Leaves of Crude EO; a large sequence of CT; Ad: Isolation of α-pinene, camphene, Camphene exhibits Girola et al. 2015
P. cernuum SiO2 and SiO2/AgNO3; solvents: limonene, carvol, thymol, myrcene, antitumor effects
(Piperaceae) CH2Cl2, CH2Cl2/MeOH and CH2Cl2/acetone p-cymene, α-terpineol, and linalool
Brazilian Crude EO of L. dendroidea; Ad: Isolation of two sesquiterpenes Sesquiterpenes used as Gressler et al. 2011
seaweed silica gel 60 F254 0.5 mm (Merck); derived from undecan-2-ol antioxidant agents
species solvent: hexane/ethyl acetate (95:5)
Citrus fruits Orange, grapefruit, lemon, clove, Reduction of the concentration Use of terpeneless oils for Kirchner and Miller
peppermint, petit grain, cedar wood EOs of undesirable hydrocarbon flavoring and perfuming 1952
terpenes of crude citrus EOs of industry products

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6      G. Ben Salha et al.: Terpene separation methods

production of MTs (Bowen 2010). Furthermore, it can yield proved that CT requires a huge number of sequences to
α-pinene, camphene, limonene, myrcene, and p-cymene isolate a compound of interest in case of varied mixture.
with purities higher than 98% from the crude EO of P.
cernuum (Girola et al. 2015) and two known sesquiterpene
derivatives of undecan-2-ol isolated from the EO of the red 4.2 Solvent extraction (SE)
seaweed Laurencia dendroidea collected on the Brazilian
coast (Gressler et al. 2011; Table 1). At the end of the 19th century, the use of liquid–liquid
All these techniques can be applied to the commercial extraction (LLE) or SE made possible the isolation of many
preparation of terpeneless citrus oils or more generally chemical species (Abe et  al. 2010). This technique, con-
to the separation of hydrocarbons from the oxygenated trolled by thermodynamics, is based on the difference in
constituents of any EO, as shown in Table 1 (Bowen 2010, the solubility of the solute in two immiscible phases. LLE
Girola et al. 2015). consists of two separate steps. First, passing the solute
One of several factors of critical importance with dissolved in a solvent (S1) from which it is difficult to sep-
regard to the repeatability and reproducibility of the results arate to another solvent (S2) from which it will be easily
of CT is the use of the same purity/grade of solvents for the isolable. Then, the solute is extracted from S2 to regener-
extraction process and for preparing solvent systems. In ate it and allow it to carry out a new extraction cycle (Arce
addition, the direction in which the silica coating of the et al. 2002, Seifi et al. 2013).
plate is applied should be oriented the same way for each LLE due to its simplicity of implementation was
analysis. Other critical factors include control of environ- applied in the petrochemical and petroleum industries,
mental conditions (e.g. relative humidity), overloading of which used huge decanters to extract the molecules of
samples spotted on plates, use of plates from different lots interest (Abe et  al. 2010, Seifi et  al. 2013). Today, LLE is
and/or manufacturers, and reuse of solvent systems for much used in pharmacology and toxicology for the puri-
multiple analyses (Barker et al. 2016, D’Souza et al. 2017). fication and separation of biological samples and/or the
The most severe problem is that the separation is not suf- concentration of mixtures of terpenes (Table 2; Arce et al.
ficiently sharp: some of the important oxygenated flavor 2002, Abe et al. 2010). To perform the SE, many different
components are eluted with the terpene components with physicochemical properties of solvents and solutes have
the result that the end product is deficient in these flavor to be taken into account. The selection of the solvent
components (Bowen 2010). In addition, Girola et al. (2015) depends on its miscibility with water, proticity, dielectric

Table 2: LLE methods for EO deterpenation and for compound extraction.

Raw material Initial and experimental data Results Solvent References

Citrus EO Mixture of limonene + linalool; Deterpenation process Water + ethanol Arce et al. 2002

T = 298.15 K requires low EO/solvent ratio
Orange Crude orange EO Deterpenation process of Water + ethanol Gonçalves et al.
citrus EO 2015
Citrus EO Mixture of limonene +  LLE provides high purity and 2-Butene-1,4-diol, ethylene glycol; Arce et al. 2006
linalool + solvent; agitation: 1 h; high linalool recovery 2-Butene-1,4-diol, ethylene glycol;
rest: 4 h; bath thermostat (Selecta 1-Ethyl-3 methylimidazolium
Ultraterm 6000383); T = 0.1 K methanesulfonate
Citrus EO Mixture of limonene and linalool; Use of an ionic liquid for 1-Ethyl-3-methylimidazolium Arce et al. 2007
T1 = 298.15 K; T2 = 318.15 K deterpenation process ethylsulfate
Bergamot EO Key constituents of crude EO: Oxygenated compounds: Inclusion in DCA Fantin et al. 2010
limonene (38.8%), γ-terpinene linalool (21.6%) and linalyl
(9.1%), linalool (16.6%) and acetate (55.7%); hydrocarbon
linalyl acetate (21.4%) MTs: limonene (10.9%) and
γ-terpinene (2.2%)
Tomato paste 1500 g tomato paste; Extraction and purification of UDA Seifi et al. 2013
ethanol (95%): dehydration; lycopene (tetraterpene) from
dichloromethane (1400 ml): tomato paste
dissolution of lycopene; 9 g UDA;
ether/n-hexane [1:1 (v/v); 750 ml]:
purify the lycopene-UDCA complex

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 G. Ben Salha et al.: Terpene separation methods      7

constant, dipolar moment, density, volatility and toxicity, MTs from bergamot EO by selective inclusion DCA (Fantin
the solute distribution ratio, the selectivity, and a com- et  al. 2010) and to extract lycopene (tetraterpene) from
parison to other solvents from the literature (Arce et  al. tomato paste by UDA (Table 2; Seifi et al. 2013). Although
2006). In the same way, the evaluation of the properties of the important features of this method are its simplicity,
the solutes, such as their structure, acidity constant, and green chemistry, moderate conditions, and recycling of
lipophilicity, together with the nature and complexity of the host without loss of substance, it still ends up con-
the matrix, will allow the optimization of the extraction taminating and making unusable the obtained fractions
process (Abe et  al. 2010). Solvents such as 2-butene-1,4- (Bertolasi et al. 2006, Perini et al. 2017).
diol, ethylene glycol (Arce et al. 2002, 2006), or a mixture
of ethanol and water (Arce et  al. 2008, Gonçalves et  al.
2015) are widely used to avoid environmental pollution. 4.3 Membrane separation
In the last years, ionic liquids have awakened a big inter-
est as potential substitutes for classic organic solvents in As described previously, the conventional LLE has been
many different applications, especially as reaction media investigated to recover the oxygenated compounds from
and separation agents (Arce et  al. 2006, 2007). They are oil matrices. However, the obtained fractions seem unfit
electrolytes with low melting temperatures, and they because of the contamination they undergo. Membrane
have interesting physical and chemical properties that are contactors therefore represent an interesting alterna-
attracting increasing attention from research and indus- tive for the selective extraction of OT from EOs (Dupuy
try (Arce et  al. 2006, Bogdanov and Svinyarov 2018). In et  al. 2011a). In LLE with membrane contactors, the
a fundamental study, Arce et  al. (2007) reported a suc- interface between phases is stabilized in a porous mem-
cessful demonstration of the unique advantage of ionic brane (Dupuy et al. 2011b). The pores are filled with one
liquid as providing the highest purity for terpeneless EOs or the other of the two immiscible fluids depending on
such as 1-ethyl-3-methylimidazolium methane sulfonate the hydrophilic or hydrophobic nature of the membrane.
and 1-ethyl-3-methylimidazolium ethyl sulfate. Still, the The porous material usually does not influence the mass
procedure outlined above has a problem due to its use transfer selectivity. It merely puts the phases without any
of organic solvents (volatile substances), which leads to dispersion in contact. In addition to the intensification of
significant operating costs due to losses by evaporation the process as a result of avoiding emulsions, the use of
(Gonçalves et al. 2015). membrane contactors affords a wide choice of solvents
A novel and effective approach known as the inclu- as the density difference between the phases (necessary
sion complex plays an important role in the overall effort for the decantation step) no longer matters (Dupuy et al.
of ensuring and providing high-quality EO products to 2011a).
consumers worldwide (Huie 2002, Bertolasi et  al. 2006). Dupuy et  al. (2011a) investigated the potential of a
This technique has two components: host and guest. The polypropylene hollow fiber membrane contactor (Liqui-
attraction between the host and the guest is based on Cel® 2.5 × 8  module with X30 fibers) for the selective
intermolecular noncovalent interactions, such as hydro- extraction of OT from lemon EOs. They evaluated the
gen bonds and van der Waals forces. The host forms stabilization of the interface between the liquids and
a cavity; in the case of a crystal, these are spaces in the found that the best results for the mass transfer of citral
shape of long tunnels or channels in which molecular (1.08 × 10−6 m/s) were obtained with a solvent containing
entities of the guest are located, and it can be separated 66% (v/v) ethanol. However, polymeric membranes are
from a solution mixture by simple filtration of the solid not efficient for the separation of oil-in-water emulsions
inclusion compound. The guest can then be recovered because of their tendency to easily foul. A study dem-
under mild conditions, and the host material is recycled onstrated that EO components could be concentrated
(Fantin et al. 2010). while maintaining acceptable flux levels using a ceramic
Ursodeoxycholic acid (UDA) and bile acid derivatives membrane (Dubreuil et al. 2017). Very few examples of a
such as deoxycholic acid (DCA: 3α,12α-dihydroxy-5β- membrane-based solvent applied to the selective recovery
cholanic acid) and cholic acid (3α,7α,12α-trihydroxy-5β- of OT from EOs have been reported in the literature and
cholanic acid) have specific ability for the inclusion of represented in Table 3.
guest molecules such as aromatic and aliphatic hydro- Membrane separation offers tremendous poten-
carbons, ketones, esters, alcohols, amides, epoxides, and tial to reduce energy consumption, to simplify product
natural pigments (Fantin et al. 2010, Seifi et al. 2013). The purification, and to recover valuable coproducts from
inclusion process was recently developed for removing complex liquids (Dubreuil et al. 2017). However, the main

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8      G. Ben Salha et al.: Terpene separation methods

Table 3: Experimental data and results of purification by membrane techniques.

Raw material   Initial experimental data   Results   References

Lemon EO   Six polymeric membranes   Two liquids/membrane   Dupuy et al. 2011a

combinations ensure a stable
interface in membrane based SE
Lemon EO (mixture of  A commercial polypropylene hollow fiber contactor and  Best results were obtained for   Dupuy et al. 2011b
citral and limonene) mixtures of water and ethanol as solvents [20–66% a solvent containing 66% (v/v)
(v/v) ethanol] were used ethanol required pressures above
0.5 bar
Commercial orange   Ceramic membranes: ZrO2 ultrafiltration (UF): 50.000   Best result of terpeneless oil was   Kim et al. 1998
oil (citrus juices) Da and 73 cm2 area; ZrO2 microfiltration (MF): 0.08 μm found with ZrO2-UF membrane
and 73 cm2 area; Al2O3-MF: 0.20 μm and 73 cm2 area
Aqueous solution   An organic polyamide membrane of molecular weight   Rich aromatic oxygenated EOs   Dubreuil et al. 2017
contains aromatic cutoff of 1000 and a cetyl (hexadecyl) pyridinium were obtained
alcohols chloride as cationic surfactant were used

drawbacks of this technology include a limited pressure deliciosa Tenore) EO at temperatures ranging from 80°C to
range to ensure a stable interface between the fluids, 90°C under 10 kPa pressure, where the temperature con-
additional resistance to mass transfer, and the limited dition would have had to have been 180°C at atmospheric
membrane lifetime (Pabby et  al. 2008). From the indus- pressure. The obtained results clearly demonstrate that
trial application viewpoint, the most serious limitations VFD is capable of separating the hydrocarbon terpenes,
of the use of membranes are the continuous decrease of which were removed from the top/stages of the column,
permeation flux that is caused by several factors, such as from the terpenes with other chemical functions, which
concentration polarization, gel layer formation, adsorp- remained at the bottom (Silvestre et  al. 2016). Similarly,
tion of surfactant molecules, and plugging of membrane the fractionation of Brazilian orange peel oil by VFD leads
pores (Kim et al. 1998). to a reduction in MT hydrocarbons, which increases sig-
nificantly the aroma of the concentrated product in com-
parison to the REO (Stüker et al. 2011). A recent study of
4.4 Distillation techniques biological activities of Origanum majorana L. was con-
ducted to prove the excellent antifungal activity of oxy-
Many distillation techniques have been applied to sepa- genated fractions compared to the REO obtained by VFD
rate and improve EOs, which have potential applications system (Ben Salha et al. 2017).
in the pharmaceutical, cosmetic, and food industries, as More studies proposed the use of the molecular dis-
well as for agriculture and crop protection (Gañán and tillation technique (MDT) known as short path distilla-
Brignole 2013). Distillation, unlike a chemical extraction tion and operating under high vacuum. This technique
separation, is a physical separation process based on is characterized by a small distance between the evapo-
the relative volatility of chemical components at various rator and the condenser (20–50 mm) and the short expo-
temperatures. Steam distillations are mainly used for sure of the distilled liquid to the operating temperatures
heat-sensitive chemical compounds (EOs), which can be (Morgan 1990, Xiong et  al. 2013). MDT was applied to
degraded or transformed by high-temperature exposure. remove phthalates from sweet orange oil [Citrus sinensis
Such methods include direct distillation and fractional dis- (L.) Osbeck], to increase the flexibility and extensibility
tillation at atmospheric pressure (Nam et al. 2016). Several of plastic products (Xiong et al. 2013), and to concentrate
works in the literature report the application of vacuum orange oil with oxyterpenes, which is a material of great
to the fractional distillation system to reduce the boiling interest for the aroma and industrial production of juice
temperature of the raw EO (REO) inside the column, which (Martins et  al. 2013). In a fundamental study, molecular
reduces the degradation of the components (Beneti et al. distillation of oregano EO was modeled with artificial
2011, Perini et al. 2017). The lower operating pressure also neural networks to obtain enriched thymol fractions with
increases the relative volatility of the compounds, allow- an increase in antioxidant properties and storage stability
ing a more efficient separation (Perini et  al. 2017). In a (Borgarello et al. 2015).
recent study, vacuum fractional distillation (VFD) was VFD and mild MDT are useful methods for the separa-
conducted to obtain Brazilian green mandarin (Citrus tion and purification of heat-sensitive materials as well as

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 G. Ben Salha et al.: Terpene separation methods      9

for those compounds with high molecular weight and to govern the suitability of chemical processes; second, its
eliminate the problems of alteration associated with other moderate critical temperature (31.2°C) is a key issue for
separation techniques (Morgan 1990, Martins et al. 2013). the preservation of bioactive compounds in extracts; and
The main drawback is the risk of implosion of the equip- finally, CO2 is readily separated from the oil: by reducing
ment in case of a room pressure higher than the pressure the pressure it resumes its gaseous state and can then be
of the system (Morgan 1990, Perini et al. 2017). recycled (Sánchez-Vicente et al. 2013, Da Silva et al. 2016).
Therefore, supercritical CO2 (SCO2) has been found to be a
good solvent for the fractionation of EOs to obtain concen-
4.5 Supercritical fluid (SF) fractionation trates or compounds of interest (Gañán and Brignole 2011,
Sánchez-Vicente et al. 2013). The deterpenation technique
SF extraction (SFE) has been used for many years for the using SCO2 fractionation increases the quality, stability,
extraction of volatile components, e.g. EOs and aroma and value of various EOs, such as Tagetes minuta, Salvia
compounds from plant materials, on an industrial scale officinalis, and Mentha piperita (Gañán and Brignole
(Huie 2002). It provides attractive features, overcoming 2013), Mandarin and citrus peel (Diaz et al. 2005, Daniel-
many of the limitations of conventional extractions as it ski et al. 2008), and Origanum (Köse et al. 2000; Table 4).
uses an SF with different physicochemical properties such Water, as a polar fluid, naturally solvates more polar
as density, diffusivity, viscosity, and dielectric constant compounds more readily than nonpolar compounds. The
(Da Silva et al. 2016). selectivity of subcritical water extraction (a preference for
This technique is based on the solvating proper- more polar organics at milder conditions) is opposite to
ties of SF, which can be obtained by employing pressure that of SCO2 (a preference for nonpolar organics before
and temperature above the critical point of a compound, polar organics; Kubatova et al. 2001).
mixture, or element. It depends also on some extrinsic Thus, working with subcritical water extraction has
features, such as the characteristics of the sample matrix demonstrated its ability to selectively extract different
and interaction with targeted analytes (Sharif et al. 2014). classes of compounds, with the more polar organics being
By proper control of the SFE parameters, before each run, extracted at lower temperatures and the less polar organ-
the extractive power of the SF can also be modified, which ics being extracted at higher temperatures. For example,
means that there are more tunable properties in SFE than the oxygenates carvone and eugenol have a solubility at
in the conventional methods (Lagadec et al. 2000, Kuba- 100°C (~65 bar), two orders of magnitude higher than the
tova et al. 2001, Sharif et al. 2014). nonoxygenated d-limonene (Kubatova et al. 2001).
SFE is regarded as a green process because it does not These trends in solubility correspond to studies
use chemical solvents with drastic environmental impacts demonstrating high recoveries of oxygenated flavor and
(Sharif et  al. 2014). Carbon dioxide (CO2) is the most fragrance components (Ozel et  al. 2003). Moreover, the
commonly used solvent for three major reasons: first, higher degree of freedom obtained in SF extraction make
it is nontoxic to human health and to the environment, this process more unique, sensitive, and specific in com-
respecting the sustainability criteria that increasingly parison to conventional extraction methods. Nevertheless,

Table 4: Application of SCO2 extraction to obtained deterpenated EOs.

Raw material   Initial conditions   Results   References

T. minuta and S.   Semicontinuous apparatus: T = 313 K; p = 80 bar; flow rate = 0.1 g/   OT-rich fraction was obtained   Gañán and
officinalis EOs min in the case of T. minuta oil Brignole 2013
Citrus reticulata   Countercurrent experiments were carried out at T = 50–60°C;   High separation between   Danielski et al.
peel oils from p = 8.5–10.0 MPa; CO2 purity: 99.95% total terpene and aroma 2008
Spain and Brazil components was achieved
Orange peel oil   Component  T (K)   p (MPa)   Molecular weight  Diameter (cm/mol)   Production of high-purity   Diaz et al. 2005
  CO2   304.2   7.280   44.00   3.129 aroma (98 wt.%)
  Limonene   660.0   7.750   136.2   5.603
  Linalool   630.5   2.388   154.4   6.171
Origanum EO   Dense CO2 used at T = 38–55°C, p = 70–85 bar   Deterpenated essential oils   Köse et al. 2000
(36.44% MTs and obtained were rich in MTs (48–89%
63.56% non-MTs) MTs and 52–11% non-MTs)

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10      G. Ben Salha et al.: Terpene separation methods

high extraction efficiencies of flavor compounds in some In the SE approach, the basic advantages are its sim-
EO matrices have required the addition of organic modi- plicity of implementation and the possibility of recycling
fiers called cosolvents to pure CO2 (Kubatova et al. 2001). the solvent (Bertolasi et al. 2006). However, this method
In the SFE experiments of De Melo et al. (2017), it was con- does not always use benign solvents: sometimes it contrib-
cluded that the addition of ethanol (2.5 wt.%) is important utes to environmental pollution and it frequently requires
to enhance the uptake of friedelin (a bioactive triterpe- an expensive evaporation/concentration operating step
noid) from Turkish oak (Quercus cerris) cork. Besides, for the recovery and decontamination of the solvent
the high operating pressure and the large flow rates of (Dupuy et  al. 2011a, Gonçalves et  al. 2015, Perini et  al.
SFE require robust equipment, with a costly construction 2017, Zuin and Ramin 2018). Despite this, it is largely used
and special security of the operation systems (Gañán and in industries to remove impurities as well as to provide
Brignole 2011, Perini et al. 2017). high-quality commercial deterpenated EOs and antioxi-
dant agents (Fantin et al. 2010, Seifi et al. 2013). Recently,
several SE models have been proposed to predict the best

5 C
 omparison among the cited solvents to be used in a specific case as ionic solvents and
the use of the inclusion complex technique as a green
separation methods chemistry (Bertolasi et al. 2006).
The main benefits of membrane extraction are that
The isolation of pure bioactive compounds from aromatic the consecutive phase separation step is no longer needed
and medicinal plants (AMPs) on a preparative or semi- and that the interfacial area between fluids at the mem-
preparative scale is frequently necessary for further phar- brane level remains constant regardless of operating flow
macological investigations. Yet, the isolation of terpenes rates, which reduces the energy consumption and provides
as natural compounds is normally a complex process high selectivity (Dupuy et al. 2011a, Dubreuil et al. 2017).
requiring a comprehensive range of separation techniques However, the main drawbacks of this technology include
that often present multiple challenges including irre- a limited pressure range to ensure a stable interface
versible adsorption, low yield, high cost, and low purity between the fluids, additional resistance to mass transfer,
(Marques et al. 2018). and limited membrane lifetime (Pabby et al. 2008). Data
Thus, CTs, SE, membrane separation, distillation concerning the industrial applications of membrane sepa-
techniques, and SF fractionation, as methods for the ration available in the literature mainly concern extrac-
extraction of terpenes from EO removed from AMP, have tions of aroma compounds and by-products during the
been compared in terms of extraction steps, advantages, concentration of industrial citrus juices (citral, linalool,
disadvantages, and applications. The results are reported etc.; Dupuy et al. 2011a,b, Dubreuil et al. 2017).
in Table 5. The most traditional method for isolating EO com-
Frequently, chromatography based on silica gel is pounds from plant materials is hydrodistillation.
used to isolate terpenes with high purity (Bowen 2010, However, this method is always accompanied by some
Gressler et  al. 2011, Girola et  al. 2015) and sometimes to disadvantages, including lengthy duration, low extraction
provide commercial and therapeutic products (Gressler efficiency, and loss of some components due to degrada-
et al. 2011, Girola et al. 2015). However, it is time consum- tion and isomerization under prolonged high temperature
ing, and in most cases, the major components in EOs treatment (Liu et al. 2019). To overcome those shortcom-
cannot be retained on silica gel (Dudareva et  al. 2006). ings, the application of vacuum and fractional distillation
Other complications arise from the use of a silica station- system eliminate the problems of alteration (Borgarello
ary phase in liquid chromatography (complicated oper- et  al. 2015, Da Silva et  al. 2016) and provide highly
ating systems) such as irreversible solute adsorption, enriched fractions (Martins et al. 2013, Sharif et al. 2014).
contamination, size exclusion, residual silanols, and pH However, in some cases it can lead to an implosion of the
limitations that affect yield and purity (Berthod et  al. equipment (Perini et al. 2017). Despite this risk, this tech-
2009, Bowen 2010, Barker et al. 2016, D’Souza et al. 2017). nique is widely applied in the food industry especially in
Usually, this method is used to produce high-purity MTs juice production (Stüker et al. 2011, Borgarello et al. 2015,
(α-terpinene and d-limonene) for use in electronic mate- Silvestre et al. 2016). These traditional methods cannot be
rials of construction for integrated circuits (Bowen 2010) used for preparing MTs as porogens in electronic devices:
but it is not effective when the raw material is a complex they are not effective for removing the impurities due to
mixture because it requires a huge number of sequences to the similar boiling points of the desired product and all of
separate the fractions and compounds (Girola et al. 2015). the impurities (Bowen 2010).

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 Table 5: Comparison of the separation techniques in terms of extraction steps, advantages, disadvantages, and applications.

Separation   Extraction steps   Advantages   Disadvantages   Applications

techniques

CT   1: EO was adsorbed on a solid Ad   High purity of the molecules of   Complicated operation systems,   Porogen species, commercial
2: Ad was eluted with a terpenophilic solvent to remove interest (Bowen 2010, Gressler time-consuming, and loss of production, and therapy use
terpenic constituents et al. 2011, Girola et al. 2015) product (Barker et al. 2016, (Bowen 2010, Gressler et al.
3: Ad was eluted with an oxygenated organic solvent D’Souza et al. 2017, Marques 2011, Girola et al. 2015)
4: Removing the solvent from the resulting elute to et al. 2018)
obtain the desired EO (Rockland and Debenedict
1975, Gressler et al. 2011, Girola et al. 2015)
SE   1: Passing the solute on a solvent (S1) from which it   Simplicity to implement, solvent   Environmental pollution of   Commercial deterpenated EO,
is difficult to separate to another solvent (S2) from recycling, and green chemistry organic solvents, expensive antioxidant agents, and removal
which it will be easily isolable of inclusion complex technique operating step for recovery, and of impurities (Huie 2002, Seifi
2: Decantation (Bertolasi et al. 2006) contamination (Dupuy et al. et al. 2013)
3: Extracted the solute from S2 2011a,b, Gonçalves et al. 2015,
4: Regeneration of S2 (Arce et al. 2002, Seifi et al. 2013) Perini et al. 2017)
Membrane   1: Interface between two immiscible solvents stabilized   High selectivity and reduction of   Unstable interface between fluids  Removal of toxic chemicals, aroma
separation in a porous membrane energy consumption (Dupuy et al. and short membrane lifetime compounds, and by-product of
2: Pores were filled with one or the other of the fluid 2011a, Dubreuil et al. 2017) (Pabby et al. 2008) industrial citrus juices (Dubreuil
depending on the nature of the membrane (Dupuy et al. 2017)
et al. 2011a,b)
Distillation   1: Bring the treated mixture to the boiling point   High purification and enriched   Degradation and risk of   Food industry and production
techniques 2: Vapors were condensed in a refrigerant fractions; no problems of implosion (Perini et al. 2017) of juice (Stüker et al. 2011,
3: Separation was based on the difference of density alteration (Martins et al. 2013, Borgarello et al. 2015)
if the mixture contains water and on the boiling Borgarello et al. 2015, Da Silva
temperature if it is an EO (Beneti et al. 2011, Perini et al. 2016)
et al. 2017)
SF fractionation  1: SCO2 extraction of EOs was performed at low   Green chemistry, high yield and   Costly, special security of the   Flavor and fragrance industrial
temperatures and moderate pressures selectivity, and special properties operation systems, large number products (Diaz et al. 2005, Gañán
2: CO2 was separated from the EO by reducing the of SF (Kubatova et al. 2001, of factors to adjust, and addition and Brignole 2013, Marques et al.
pressure Sánchez-Vicente et al. 2013) of organic modifiers (Gañán and 2018)
3: Gaseous CO2 could be recycled (Sánchez-Vicente Brignole 2011, Sharif et al. 2014,
et al. 2013) Perini et al. 2017)

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12      G. Ben Salha et al.: Terpene separation methods

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compounds. 1953. Experientia 1994; 50: 395–405. Ghada Ben Salha received her BA in mathematics in 2008. She
Saito K, Nishimori A, Kotsuki H, Nakano K, Ichikawa Y. A biomimetic obtained a Fundamental License in Chemistry and a Master’s degree
approach to terpenes isolated from marine sponges: a UGI in Desalination and Water Treatment in 2011 and 2014, respectively,
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24: 757–761. She has been carrying out her PhD in cotutelle between the Faculty
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3: 6065–6075. techniques related to chemical engineering.

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 G. Ben Salha et al.: Terpene separation methods      15

Manef Abderrabba Jalel Labidi

Laboratory Materials, Molecules and Chemical and Environmental Engineering
Application, Preparatory Institute for Department, University of the Basque
Scientific and Technical Studies, 2070 Country, Plaza Europa, 1, 20018 Donostia-
Marsa, Tunisia San Sebastián, Spain,
[email protected]

Jalel Labidi has a chemical engineering degree and a PhD in chemi-

Manef Abderrabba is currently the Dean of Preparatory Institute
cal engineering from the Polytechnic Institute of Lorraine (France).
for Scientific and Technical Studies IPEST (University of Carthage,
He has developed his research career in several centers: Ecole Poly-
Tunisia) and the Head of the Molecular Materials and Applications
technique of Montreal (Canada), CanmetENERGY (Canada), CNRS
Laboratory. He has been a professor in higher education since 1988
(France), University of Girona, and finally at the University of the
and a full professor since 1995. Prof. Abderrabba contributed greatly
Basque Country. Currently, he holds a position of senior researcher
to the advancement of the scientific research and has a great aca-
in the Department of Chemical and Environmental Engineering, Uni-
demic footprint. He is the author of more than 170 papers published
versity of the Basque Country. He is the head of the research group
in international peer-reviewed journals. His H-index reached 24 in
“Biorefinery Process” (BioRP). His research interest is focused on
the Scopus database. He is also the author and coauthor of four
the conversion and valorization of biomass.
industrial patents.

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