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2 Limonene: Origin, Applications, and Properties

2 Limonene: Origin, Applications, and Properties

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Limonene as Green Solvent for Extraction of Natural Products


Fig. 5.1 Chemical structure

of d-limonene

Table 5.1 Relevant properties of n-hexane, toluene, and d-limonene





Empirical formula




Molecular weight




Boiling point (°C)




Heat of vaporization (kJ/kg)




Density (g/mL)








Environmental impact












compounds (VOCs). Solvents that are commonly replaced with d-limonene solvent

include methyl ethyl ketone, acetone, toluene, glycol ethers, and numerous fluorinated and chlorinated solvents. In industry, d-limonene solvent is typically mixed

with a surfactant, producing a solution containing 5–15% of d-limonene. The major

drawback of using d-limonene is its low viscosity and the higher energy consumption related to solvent recovery by evaporation due to its high boiling point (175°C)

compared to n-hexane (69°C). The chemical and physical properties of d-limonene

compared to n-hexane are illustrated in Table 5.1.

This molecule has been considered for many applications like insecticide, cosmetics, and food industry. This rising interest for d-limonene is due to its proved

cleansing and degreasing properties [3]. In this respect, that molecule has been designated as efficient alternative for halogenated carbon hydrates or conventional

degreasing agents used in industry and at households. In their attempt to develop an

industrial application for d-limonene, Liu and Mamidipally [4, 5] have indicated,

recently, the high suitability of this molecule as a solvent for rice bran oil recovery.

In parallel, citrus essential oil containing d-limonene is generally extracted by

hydrodistillation where Clevenger apparatus represents the most common system

that is used for decades to extract and evaluate essential oils in herbs and seed.

Although the hydrodistillation step requires several hours, it is interesting to note

that it allows extracting components at a lower temperature than their boiling point

(azeotropic distillation), which eliminate degradation risks at high temperatures. In

this essence, the introduction of electromagnetic energy as a heating source has

helped the improvement of the Clevenger system [6]. This technique has been

applied with success for the extraction of essential oils from orange peels in which


S. Chemat et al.

d-limonene constitutes more than 90% of the essential oil content. This approach

has permitted the reduction of processing time from 3 h (conventional conduction

heating system) to 30 min in the microwave-assisted system. In addition, the new

design is solvent-free, requires low capital investments, and consumes lower energy

compared to the conventional one.


Limonene as an Alternative Solvent for Soxhlet Extraction

For 130 years, Soxhlet has been the most common technique for the recovery of fats

and oils from biofeedstocks in teaching and research. However, the major drawbacks initiated with Soxhlet like long extraction time, energy consuming, and the

use of large amounts of petroleum solvents have called for an increased environmental concerns. For example, n-hexane, the solvent of choice for fats and oils

using Soxhlet extraction, is ranked on top of the list of the hazardous solvents. Over

the years, many researchers have concentrated their efforts to find alternative solvents [7].

This green approach has helped Chemat group to develop a new extraction system for fatty material using microwaves called “microwave-assisted Soxhlet” [8, 9]

that produces equivalent results to those obtained by standardized norms. This

green process, in which 90% of the solvent is recycled, has slashed time from an

exhaustive 8 h processing in conventional method to only 32 min. In this direction,

Virot et al. [9] proposed the combination of microwave-assisted Soxhlet as a process and d-limonene as a green extractant. This step is followed by a microwaveassisted distillation using Clevenger system. The proposed process has been applied

successfully to the recovery of olive oil. The comparison of both extracts has

revealed the superior quality of olive oil obtained by the new system. In order to

avoid the high boiling point required by d-limonene to be recovered, they proposed

an innovative approach capitalizing on the azeotropic distillation concept of essential oils used on steam or hydrodistillation using Clevenger. After Soxhlet extraction with d-limonene, distilled water was added to the mixture composed of

extracted oil and d-limonene. After azeotropic water distillation around 100°C with

a Clevenger system, d-limonene and extracted oil were recovered separately

(Fig. 5.2).

Yields obtained for both extractions showed that yields of d-limonene’s extracts

were slightly higher than those obtained using n-hexane (Table 5.2). This difference

is attributed to the slightly polar nature of d-limonene compared to n-hexane and to

a higher dissolving power of d-limonene for triglycerides. In addition, the higher

temperature used to boil d-limonene induced a better desorption rate of oil in the

matrix as a consequence of a lower viscosity.

The gas chromatography coupled to mass spectrometry (GC-MS) analysis data

of free fatty acid methyl esters (FAMEs) derivatives has revealed a good agreement

with literature data [10] in terms of qualitative composition where no significant

differences (P > 0.05) were detected for both methods. Nevertheless, we can note a


Limonene as Green Solvent for Extraction of Natural Products


Fig. 5.2 Extraction procedures function of solvent used (1, hexane, 2, limonene)

Table 5.2 Comparison of

main fatty acids’ composition

obtained from olive oil using

different solvent systems

(relative percent)

Fatty acids






n-Hexane (%)






d-Limonene (%)






higher level of free fatty acids, peroxide value, and conjugated dienes level in

d-limonene’s extracts. These results indicated the presence of traces of oxidized

products, which can be attributed to the higher temperature, used to extract oil.

The authors continued their investigations about measuring the ability of each

solvent system (n-hexane and d-limonene) to be recovered and recycled. In the system using n-hexane, recycling of approximately 50% of solvent could be achieved

compared to a recycling potential of almost 90% for d-limonene. This result confirms that d-limonene can be considered as suitable and effective as it complies with

standardization recommendation for solvents in which it should not be miscible

with water and must have a different density with it; needs to dissolve the analytes

for easier extraction, and finally, the solvent must be volatile enough to be easily

removed by evaporation.



S. Chemat et al.

Limonene as an Alternative Solvent for Dean-Stark


Moisture determination represents a key step in food analysis for which the ovendrying methods are commonly used. However, for matrix containing volatile compounds, the distillation method stands as the most suitable method. Several

distillation-based procedures have been tested in the late nineteenth and early twentieth century [11]. Dean and Stark developed the first continuous and refluxing method

in 1920 [12]. This innovation was followed by a development of different types of

receivers in an attempt to adapt the collecting trap to the material to be analyzed [13].

As a consequence, the Dean and Stark distillation procedure became the reference

method for water determination in food products containing volatile compounds [14].

It was later on used in food industry for water determination in herbs and spices [15,

16] and to tap the petroleum industry [17]. The recommended solvent for Dean and

Stark distillation was toluene. Due to increasing environment concerns and the spreading of green chemistry principles, such solvent has to be avoided as much as possible.

Toluene exerts toxic properties (Table 5.1) and detrimental health effects, especially

on the nervous system, the liver, and on the auditory function [18, 19].

In their attempt to alleviate this environmental and health concerns, the research

community set up 12 principles for a green chemistry in order to develop more environmentally acceptable experiments [20]. It involves the design of less hazardous

chemical syntheses (using substances with little or no toxicity) or the use of safer

solvents and reaction conditions. In this direction, d-limonene, which is the major

by-product obtained from the citrus fruit processing [1, 21], was used by Veillet et al.

[22] in order to replace toluene in the Dean-Stark procedure (Fig. 5.3).

Fig. 5.3 Dean-Stark apparatus


Limonene as Green Solvent for Extraction of Natural Products

Fig. 5.4 Kinetics of water

distillation of onions using

two different solvents (■)

d-limonene and (♦) toluene


% water









Time (min)



The low health risks and the very low environmental impact of d-limonene have

granted this molecule a green label. Despite the fact that its boiling point (175°C) is

higher than toluene (111°C), the azeotropic distillation concept based on the ability

of d-limonene to form an azeotropic mixture with water at 97.4°C is in favor of its

application as an alternative solvent to toluene. The Dean and Stark procedure is

based on the ability of the solvent to form an azeotropic mixture with the water

contained in the food matrix; thus, due to the difference in boiling points, it is

expected that different solvents would result different kinetic patterns.

The results of Veillet et al. [22] showed two similar kinetics of distillation with

minor variations could be observed (Fig. 5.4). They noticed at the beginning that the

recovery of water was delayed for about 3 min using d-limonene, mainly due to a

higher distillation temperature. However, the water recovery process was accelerated once distillation started. The higher temperature of the azeotropic mix in the

case of d-limonene could explain this phenomenon where bulk temperature is very

close to the water boiling point, rendering water more susceptible to volatilize than

at lower temperatures. In this situation, the slope of the distillation curve increased

for the system using d-limonene compared to toluene; thus, the total time required

to achieve 100% water recovery was shorter. It is interesting to note here that the

excess of energy required at the beginning of the experiment was balanced by a

shorter processing time.

The data extended to a wide range of products such as garlic, carrots, and leeks

revealed a comparable moisture value for methods using toluene and d-limonene

[22]. Aromatic plants such as rosemary, sage leaves, and mint were also tested


S. Chemat et al.

giving similar moisture content for both solvents (toluene and d-limonene) as follows:

64.1 ± 0.6 and 65.1 ± 1.8; 71.1 ± 1.0 and 70.4 ± 2.2; and 88.2 ± 1.8 and 87.6 ± 1.7,


According to these results, the moisture determination in food matrices using

d-limonene can be considered as a reference in student laboratory practices when

teaching green chemistry.


Limonene as an Alternative Solvent for Extraction

of By-Products

The positive effects on health that lycopene is offering has initiated an increased

interest on this carotenoid which is present in tomato and is largely used as simple

food dye.

Generally, extraction of lycopene from food sources is performed using pure

organic solvents such as dichloromethane or mixtures of polar and nonpolar extractants (e.g., acetone-chloroform (1:2) or hexane-acetone-ethanol (50:25:25)).

Conventional extraction methods for lycopene consume large volumes of organic

solvents, which are toxic, expensive, and hazardous. In addition, traces of the extractant can contaminate the final product, making it unsuitable for food, pharmaceutical, or cosmetic uses. New findings by Chemat et al. [23] suggested the suitability

of a major component limonene present in citrus rind oil for the recovery of lycopene from tomato. Taking into account the costs associated with environmental

compliances and insurances, the use of limonene is more competitive than dichloromethane (Table 5.1).

Chemat et al. [23] proposed a green cycle starting from obtaining d-limonene

from biofeedstocks (Valencia late orange: Citrus sinensis L. Osbeck peel) by steam

distillation. Then, a deterpenation process follows this step using a thin film evaporator in order to recover pure limonene. According to Leenaerts [24], the thin layer

technique allows a very short residence time under reduced pressure, a significant

heat-transfer surface, as well as a mixing potential that match heat and mass transfer

requirements. Next, limonene is used as the extracting solvent for lycopene from

tomato fruit as a substitute of dichloromethane.

They reported that the deterpenation achieved a recovery of 98.8% of limonene

after 30 min under optimal conditions (temperature = 65.4°C, flux = 0.036 kg⋅h–1,

pressure = 100 mmHg). After deterpenation, limonene was used as a solvent for

extracting lycopene from tomato fruit as a substitute of dichloromethane.

The results reported important lycopene yields for dichloromethane

(3.84 mg/100 mg of fresh tomato) representing 19.2% of lycopene total amount

compared to those obtained by d-limonene (2.44 mg/100 mg of fresh tomato) representing less than 13.1% of total lycopene content. On the other hand, dichloromethane

stands as a toxic and harsh organic solvent whereas d-limonene is recognized as

GRAS agent. Thus, the proposed approach using d-limonene is useful and can be

considered as an attempt to reduce toxicity for human and environment.


Limonene as Green Solvent for Extraction of Natural Products



Combining Green Extraction Technique and Green Solvent

Recently, an improved Clevenger apparatus using microwave energy has been suggested [11] and applied to extract essential oils and so, limonene (which represents

more than 90% of orange peels’ essential oil). This microwave extraction can be

considered as an effective approach since it offers, among others, short extraction

times (only 30 min against 3 h in conventional method), low cost, low coextraction

of by-products (compared to conventional distillation) and stands as environmentally friendly process. Virot et al. [8] developed a new Soxhlet assisted by microwave energy called microwave-integrated Soxhlet (MIS). This new device has been

set up to ensure a rapid, efficient, and green extraction procedure for fatty acids

recovery from olive oil. The aim of their approach was to evaluate the possible

extraction of fats and oils from olive seeds using d-limonene as solvent in combination with microwave heating for both the extraction and the cleaning steps. The

extraction step of oils from olive seeds was thus performed using the MIS, and then

the microwave Clevenger system performed the solvent elimination step (Fig. 5.5).

The yield and composition of fatty acids were compared with those obtained by

conventional Soxhlet and MIS extraction procedures using n-hexane. The data indicated similar results compared to conventional Soxhlet extraction in terms of gravimetric and fatty acids composition. This is true since limonene properties are quite

similar to hexane in terms of polarity and thus affinity for fats and oils. In addition,

the processing time was reduced to only 32 min as compared to the exhaustive 8 h

Fig. 5.5 Proposed extraction procedure using limonene: microwave-integrated Soxhlet extraction

followed by microwave Clevenger distillation


Table 5.3 Fatty acid

compositions of olive oil for

different procedures and


S. Chemat et al.



MIS-MC procedurec




Fatty acids (%)
























Conventional Soxhlet extraction using n-hexane


MIS extraction using n-hexane


MIS extraction using d-limonene followed by microwave

Clevenger distillation

required in the reference Soxhlet procedure. Since the possibility to recycle the

solvent (limonene) is up to 90% and the energy used is reduced, this method is considered as a promising green technology.

The results indicate an improved olive oil yield for MIS procedure using

d-limonene (44.9%) compared to systems involving n-hexane like conventional

Soxhlet (40.3%) and MIS system (39.1%). This result is attributed to the higher dissolving ability of limonene for triglycerides, which can, at higher temperature, be

used to boil this solvent, produce a lower viscosity of the analytes in the matrix, and,

as a consequence, a better diffusion rate of the solute from the solid phase to the


Virot et al. [8] continued their investigation to assess the influence of the method

on the chemical composition and relative amounts of fatty acid (Table 5.3). They

noted comparable results for the three methods, and that the main fatty acids

extracted using the new proposed procedure were oleic (C18:1), palmitic (C16:0),

and linoleic (C18:2) acids. These three fatty acids represent more than 90% of the

total fatty acid composition of the extracted oil. Other fatty acids such as palmitoleic

(C16:1), stearic (C18:0), linolenic (C18:3), or arachidic (C20:0) acids were also

noted with a less predominant peak area. Myristic (C14:0), pentadecanoic (C15:0),

margaric (C17:0), margaroleic (C17:1), nonadecyclic (C19:0), gadoleic (C20:1),

and behenic (C22:0) acids were found in trace levels. In addition, their data reported

that the sum of saturated, mono-, and polyunsaturated fatty acids was also in line

with those reported for olive oils in the literature [25]. Thus, the use of microwave

energy and limonene as solvent did not involve extraneous effects on the composition

of the extracted oils.

The proposed investigation revealed a green aspect that can be pointed out in

two points:

First aspect: Previous investigation dealing with MIS device [19] has shown that

extractions permit time reduction compared to conventional extraction procedure.

The solvent recycling possibilities using MIS instead of Soxhlet apparatus have

also been pointed out. Microwave energy is, in addition of that, the only heating

source used to perform extraction. Therefore, the extraction step of the proposed

procedure is clearly advantageous in term of time, solvent, and energy saving.


Limonene as Green Solvent for Extraction of Natural Products


Second aspect: The microwave Clevenger apparatus is presented as a green process

since it allows reduction of time and energy required for limonene distillation

step. Currently, energy that can be used to eliminate limonene from the distillation flask is reduced by using azeotropic distillation technique (vaporization temperature diminished from about 175°C to less than 100°C).

The proposed approach using a green solvent to perform extraction is useful and

can be considered as a good alternative to conventional petroleum solvent where

toxicity for both operator and environment is reduced. Furthermore, the use of a

by-product of the industry as solvent, its possible recycling, and life-cycle extension

is original and of increasing interest for many chemistry experiments. This useful

and safe procedure may lead to numerous investigations and/or alternatives to conventional chemistry procedures that are often hazardous.


Future Trends

Innovative and sustainable extraction, which typically involves less energy and

renewable solvents, is currently a dynamically developing area in applied research

and industry. Up to now, however, there are only a few reports that mentioned the

replacement of petroleum solvents by renewable solvents such as d-limonene, the

major essential oil component of a by-product from orange juice industry. The main

advantages of using renewable solvents for extraction includes: increase production

efficiency and contribute to environmental preservation by reducing the use of

solvents, fossil energy, and generation of hazardous substances. Extraction using

alternative and green solvent such as d-limonene will be of great interest in the near

future in the area of natural products.


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5. Liu SX, Mamidipally PK (2004) First approach on rice bran oil extraction using limonene. Eur

J Lipid Sci Technol 2:122–125

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Chem Soc 2(60):229–242

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Soxhlet extraction. An advantageous tool for the extraction of lipids from food products.

J Chromatogr A 1174:138–144


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9. Virot M, Tomao V, Ginies C, Visinoni F, Chemat F (2008) Green procedure with a green solvent

for fats and oils’ determination: microwave-integrated Soxhlet using limonene followed by

microwave Clevenger distillation. J Chromatogr A 1196:57–64

10. Di Bella G, Maisano R, La Pera L, LoTurco V, Salvo F, Dugo G (2007) Statistical characterization of Sicilian olive oils from the Peloritana and Maghrebian zones according to the fatty acid

profile. J Agric Food Chem 55:6568–6574

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12. Dean EW, Stark DD (1920) A convenient method for the determination of water in petroleum

and other organic emulsions. J Ind Eng Chem 12:486–490

13. Fetzer WR (1951) Determination of moisture by distillation. Anal Chem 8(23):1062–1069

14. AOCS Official Method Ja 2a-46 (1993) American Oil Chemist’ Society. AOCS Press,


15. Balladin DA, Headley O (1999) Evaluation of solar dried thyme (Thymus vulgaris Linne)

herbs. Renew Energ 17:523–531

16. Brunnemann KD, Qi J, Hoffmann D (2002) Chemical profile of two types of oral snuff tobacco.

Food Chem Toxicol 11(40):1699–1703

17. Fleury M, Boyd D, Al-Nayadi K (2006) Water saturation from NMR, resistivity and oil-base

core in a heterogeneous Middle-East carbonate reservoir. Petrophysics 47:60–73

18. Hass U, Lund SP, Hougaard KS, Simonsen L (1999) Developmental neurotoxicity after toluene inhalation exposure in rats. Neurotoxicol Teratol 21:349–357

19. McWilliams ML, Chen GD, Fechter LD (2000) Low-level toluene alters the auditory function

in guinea pigs. Toxicol Appl Pharmacol 1(167):18–29

20. Anastas P, Warner J (1998) Green chemistry: theory and practice. Oxford University Press,

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21. Njoroge SM, Koaze H, Karanja PN, Sawamura M (2005) Essential oil constituents of three

varieties of Kenyan sweet oranges (Citrus sinensis). Flavour Fragr J 20:80–85

22. Veillet S, Tomao V, Visinoni F, Chemat F (2009) New and rapid analytical procedure for water

content determination: microwave accelerated Dean-Stark. Anal Chim Acta 632:203–207

23. Chemat-Djenni Z, Ferhat MA, Tomao V, Chemat F (2010) Carotenoid extraction from tomato

using a green solvent resulting from orange processing waste. J Essent Oil Bear Plant


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Chapter 6

Glycerol as an Alternative Solvent for Organic


V. Calvino-Casilda

Abstract Glycerol has been successfully employed as a versatile and alternative

green solvent in variety of organic reactions and synthesis methodologies. Using

this valuable green solvent, high product conversions and selectivities were achieved

affording innovative solutions to the substitution of the conventionally used volatile

organic solvents. Besides solubility of the reactants and the catalysts and easy separation of the products, glycerol offers several other benefits such as catalyst recycling, microwave-assisting reaction, and emulsion mode. This chapter summarizes

selected examples of potential uses of glycerol in organic reactions as well as the

advantages and disadvantages of such a green methodology.

Furthermore, because of economical and environmental considerations nowadays, the possibility of directly using crude glycerol produced by the biodiesel

industry has significantly increased.



The impressive and fast development of the vegetable oil industries, mainly for

nonfood application, generates annually a great amount of crude glycerol as byproduct (around 1 million tons expected in 2010) which is nowadays in urgent

requirement of chemical exploitation. Accordingly, most scientists agree on the

transformation of glycerol to more valuable chemicals such as monoglycerides,

glycerol ethers, acrolein, acrylonitrile, polymers, etc. However, these high-tonnage

glycerol-based processes have been the focus of strong discussions for their environmental and economical viability.

V. Calvino-Casilda (*)

Instituto de Catálisis y Petroleoquímica, CSIC, Catalytic Spectroscopic Laboratory,

Marie Curie 2, 28049 Madrid, Spain

e-mail: vcalvino@icp.csic.es

A. Mohammad and Inamuddin (eds.), Green Solvents I: Properties

and Applications in Chemistry, DOI 10.1007/978-94-007-1712-1_6,

© Springer Science+Business Media Dordrecht 2012


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