Fuel 235 (2019) 1123–1130

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Biodiesel purification by column chromatography and liquid-liquid T

extraction using green solvents
⁎
Sérgio S. de Jesus , Gabriela F. Ferreira, Maria Regina Wolf Maciel, Rubens Maciel Filho
Laboratory of Optimization, Design and Advanced Control, School of Chemical Engineering, University of Campinas, P.O. 6066, 13083-852 Campinas, SP, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Biodiesel purification through acid-catalyzed in situ transesterification of dry biomass of Chlorella pyrenoidosa
Biodiesel was evaluated by liquid-liquid extraction and column chromatography, using the solvents hexane, 2-methylte-
Green solvent trahydrofuran (2-MeTHF), and cyclopentyl methyl ether (CPME). The purified biodiesel were characterized
Microalgae through their chemical composition and physicochemical characteristics. The results showed that the purified
Liquid-liquid extraction
biodiesel presented a purity level higher than 96.5%, and the purities obtained with CPME were very close to
Column chromatography
those obtained with hexane. Concerning physicochemical characteristics, biodiesel purified with green solvents
showed higher densities and viscosities, but compliant with the European and North American quality standards.
The difficulty to evaporate residual water from biodiesel purified with 2-MeTHF after the liquid-liquid extraction
process required that biodiesel was left in decantation for 24 h, which resulted in a biodiesel with water content
exceeding 0.05%mass. Biodiesel purified with green solvents, mainly by column chromatography using CPME as
eluent, show great potential to replace hexane in the biodiesel purification process, and the price of these
solvents is the only factor limiting their large-scale use.

1. Introduction biodiesel, as well as other impurities, is of great importance for the

The concern regarding the replacement of petroleum-based fuels has
grown considerably in recent years. From the economic point of view,
the threat of shortages and price instability, and from the environ-
mental point of view, the need to reduce climate changes caused also by
the effects of their use and energy sustainability have promoted the
search for alternative renewable fuels that are less harmful to the en-
vironment [1]. As an alternative to fossil fuels, the production and use
of biofuels from plant and animal raw materials has become a priority.
Considered as the best alternative to meet a growing demand, biodiesel
from microalgae cultivation gained wider prominence in research and
government investments, mainly in the United States [2]. However,
large-scale production is still a limiting factor for the use of this biofuel,
mainly because of some technical problems related to the downstream
step. With the aim of reducing some steps of the process in order to
make it cheaper, in situ or direct transesterification of biomass has be-
come a promising and economically viable alternative [3–5]. In direct
transesterification, extraction and transesterification occur simulta-
neously, alcohol acts as extraction solvent and esterification agent
[3–5]. After the process, the biodiesel formed is separated from the
catalyst (acid or base), from the excess alcohol, glycerol and glycerides,
in addition to the residual biomass [4]. The separation of glycerol from
production of a fuel that meets the established technical standards,
ensuring higher performance in use, as well as protecting the engine
from possible damage. The most widely used purification procedure is
performed primarily through liquid-liquid extraction or column chro-
matography (at the level of laboratory) and involves the use of large
amounts of solvent, hexane is usually used in this step due to presenting
greater solubility in biodiesel, easy recovery and low-cost [6]. However,
hexane is a solvent of fossil origin, highly toxic, classified as category 2
(in a 1–10 scale) for human health and for the environment, being
strongly recommended its replacement with other less toxic solvent
[6,7]. As an alternative, a new class of solvents that are en-
vironmentally friendly and comply with the principles of “green
chemistry” has emerged recently. In addition to their high potential in
the use of organic syntheses, these solvents present properties that are
very similar to the solvents used in lipid extraction process. 2-methyl-
tetrahydrofuran (2-MeTHF) and cyclopentyl methyl ether (CPME) are
environmentally sustainable organic solvents and showed to be ex-
cellent alternatives as solvents for lipid and protein extraction [8,9].
However, these solvents have not been tested for biodiesel purification
and may present as an environmentally correct alternative, since the
purification process requires a considerable volume of solvent, which
generates a large amount of residue, even if part of the solvent used is

⁎
Corresponding author.
E-mail address: ssjesus@gmail.com (S.S. de Jesus).

https://doi.org/10.1016/j.fuel.2018.08.107
Received 23 May 2018; Received in revised form 19 August 2018; Accepted 24 August 2018
Available online 30 August 2018
0016-2361/ © 2018 Elsevier Ltd. All rights reserved.
S.S. de Jesus et al.
reused.
Biodiesel purification with the solvents 2-methyltetrahydrofuran
and cyclopentyl methyl ether is proposed in this work. Two different
methods were chosen for purification of the mixture obtained after
acid-catalyzed direct transesterification: a) liquid-liquid extraction; b)
column chromatography.
Purification with hexane was used for comparative purposes.
2. Materials and methods
2.1. Materials
Microalgae: Chlorella pyrenoidosa was purchased from Taiwan
Chlorella Manufacturing (Taiwan, China).
Chemicals: Hexane and ethyl acetate (Labsynth Ltda., Brazil), 2-
methyltetrahydrofuran and cyclopentyl methyl ether (Sigma-Aldrich
Co., Germany) were used as solvents for biodiesel purification.
2.2. Methods
2.2.1. Determination of lipid content in dry microalgal biomass
The lipid content in dry microalgal biomass (3.5 ± 0.2 wt%
moisture) refers to the total lipid extracted by petroleum ether after the
bound lipid hydrolysis [10].
Boiling water (6 mL) was added to 1.00 ± 0.05 g of biomass, vortex
mixed for 1 min and cooled; 1 mL of 25% ammonia solution was added
and vortexed for 2 min, followed by 7.5 mL methanol addition and
vortex for 2 min. Finally, 17 mL of diethyl ether was added, stirred
vigorously, and 17 mL of petroleum ether was added for extraction, and
stirred once more. The mixture was centrifuged and the top layer
containing lipids was separated from the aqueous phase. Solvent was
evaporated using a rotary evaporator (RV 10, IKA, Germany). The lipid
fraction was dried until constant weight in a vacuum oven at 100 °C.
The moisture content from the microalgae was determined grav-
imetrically with three replicates.
2.2.2. In situ transesterification
Dry C. pyrenoidosa (50 g) was mixed with 5.4 mL catalyst (20%
sulfuric acid in relation to biomass) and 150 mL methanol and sub-
mitted to ultrasound bath at 25 °C for 20 min. The mixture was heated
to 100 °C under reflux for 4 h. After transesterification, the product
obtained was left at rest for 1 h. The reaction mixture was filtered under
vacuum in a büchner funnel. Residual biomass was resuspended in
methanol and stirred for 1 h, the resulting mixture was separated and
the filtrate was added to the raw product obtained previously for the
subsequent purification step. This procedure was performed two more
times with the purpose of recovering any trace of fatty acid methyl
esters (FAME) contained in the residual biomass (Fig. 1A).
2.2.3. Purification of crude methyl esters
Methyl esters produced after in situ transesterification process were
purified with two different techniques, in order to verify the influence
of green solvents on the properties of purified biodiesel and its yield.
2.2.3.1. Liquid-liquid extraction. The resulting mixture obtained
previously (4.5 ± 0.5 g) was added in a separatory funnel, in
addition to 200 mL solvent (hexane or 2-MeTHF or CPME) and
200 mL distilled water that was stirred and left at rest for 15 min.
After this period the upper FAME-rich phase was separated from the
intermediate and lower phases (Fig. 1B), to these phases were added
200 mL solvent (hexane or 2-MeTHF or CPME) and left at rest for
additional 15 min, the upper phase was separated and added to
previously obtained fraction. This procedure was performed three
times. FAME-rich phase was washed with 50 mL distilled water (to
remove residues of catalyst and methanol) several times until the water
pH was neutral. After separation, we added the organic fraction,
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Fuel 235 (2019) 1123–1130
anhydrous sodium sulfate (∼1.0 g) to remove remaining water.
Sodium sulfate was separated by vacuum filtration and then the
solvent was evaporated in a rotary evaporator (RV 10, IKA, Germany)
(Fig. 1D). Biodiesel was dried at 80 °C for 24 h.
2.2.3.2. Column chromatography. Purification of the crude extract
obtained in direct transesterification reaction by column
chromatography consisted in adding the sample (4.5 ± 0.5 g) into a
chromatographic column packed with 10 g silica gel (0.035–0.07 mm,
60A) and 60 g alumina (aluminum oxide 90 neutral, 0.05–0.2 mm,
70–270 mesh ASTM) at 1:6 ratio, as eluent we used approximately
300 mL a mixture of hexane:ethyl acetate (19:1) or the green solvents:
2-MeTHF or CPME. The collected fractions were submitted to thin-layer
chromatography (TLC), using as mobile phase a mixture of petroleum
ether, ethyl ether and glacial acetic acid (80:19:1), and the
chromatogram was developed with iodine vapors. Oleic and
heptadecanoic were used as chromatographic standards [11]
(Fig. 1C). After separation, FAME was washed with 50 mL distilled
water according to the procedure described in the previous section and
represented in Fig. 1C.
The yield of each process of separation was calculated according to
the equation:
Biodiesel mass (g)
Biodiesel yield (%) = × 100%
microalgae mass (g) × oil content (%) (1)
The samples were encoded as: HXLL, MeTHFLL, CPMELL, HXCC,
MeTHFCC and CPMECC, where: HX, MeTHF and CPME represent the
solvents hexane, 2-MeTHF and CPME respectively; LL for purification
using liquid-liquid extraction and CC for purification by column chro-
matography.
All experiments were performed in triplicate.
2.2.4. Analytical techniques
2.2.4.1. Gas chromatography. Fatty acids methyl esters were analyzed
by gas chromatography (GC/FID Agilent 6850, USA) equipped with
capillary column DB-23 Agilent (50% cyanopropyl and 50%
methylpolysiloxane), 60 m × 0.25 mm internal diameter, 0.25 μm film
thickness. The operating conditions of the gas chromatograph were:
1 mL/min carrier gas flow (He), 24 cm/s linear speed, 280 °C detector
temperature, 250 °C injector temperature, 110–215 °C oven
temperature (5 °C/min heating), 215 °C for 24 min, 1.0 μL injection
volume, and 1:50 split ratio. For identification and quantification, a
standard FAME mixture (Supelco® 37 component FAME mix, Sigma,
Germany) and an internal C17:0 standard (Heptadecanoic – Sigma,
Germany) were used. Ester content was calculated through all peaks
between C14 and C24 and relating it with the internal standard.
Quantifications of free and total glycerol, mono, di and triglycerides
were conducted according to ASTM D6584, using butanetriol (Sigma,
Germany) as internal standard for quantification of glycerol and trica-
prin (Sigma, Germany) as internal standard for quantification of mono,
di and triglycerides.
2.2.4.2. Chemical and physicochemical analyses. Density measurement
was performed at 15 °C using the hydrometer method.
Kinematic viscosity at 40 °C was tested according to standard ASTM
D445.Water content was determined by standard ASTM 6304 and acid
value through ASTM D664.
Iodine number was calculated based on the composition of the re-
sidual oil obtained by GC. According to the official method of the
American Oil Chemist’s Society (AOCS) Cd 1c-85, iodine number is
obtained through the following equation:
IN = (%C16: 1 × 0.950) + (%C18: 1 × 0.860) + (%C18: 2 × 1.732)
+ (%C18: 3 × 2.616) + (%C20: 1 × 0.785) + (%C22: 1 × 0.723)
(2)
S.S. de Jesus et al. Fuel 235 (2019) 1123–1130

Fig. 1. Schematic representation of the in situ transesterification and purifications steps used in this study (Adapted from Ref. [4]).

where: IN is the iodine number, in centigrams of iodine absorbed per molar fractions of all fatty acids in the sample), 56.1 is the molecular
gram of sample; % Ca:b is the percentage of fatty acid with a carbons mass of KOH and 92.09 represents the molecular mass of glycerol.
and b unsaturations obtained by GC. The method for estimation of cetane index (CI) was determined
The saponification value was calculated based on the composition in using the values obtained for iodine number (IN) and saponification
fatty acids and according to method Cd 3a-94 of AOCS, as shown in Eq. value (SV) and according to the Krisnangkura equation [12]:
(3):
5458
CI = 46.3 + −0.225 × IN
3 × 56.1 × 1000 SV (4)
SV =
[(MMt × 3) + 92.09]−(3 × 18) (3)

where MMt corresponds to the mean molecular weight (sum of the

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S.S. de Jesus et al. Fuel 235 (2019) 1123–1130

2.2.5. Solubility parameters Purity

100 98.1 96.6 97.3 97.8 96.7 97.4
To predict the compatibility of the solvents under test (2-MeTHF
and CPME) with the solute (biodiesel), the Hansen solubility para-
meters (HSP) were studied for miscibility and solvation and were 80
Biodiesel
compared with the standard solvent (hexane, hexane:ethyl acetate).
The cohesive energy density is more conveniently treated in terms

Yields (%)
of the solubility parameter δ where δ2 = E/V, where E is the cohesive 60

energy and V is the molar volume. This gives us the classic formula for
Hansen Solubility parameters, where the total parameter, δt, is divided 40
into δD, δP and δH for dispersion, polar and hydrogen-bonding [13]:
δt2 = (δD )2 + (δP )2 + (δH )2 (5) 20
½
The units of solubility parameters are MPa .
For a solvents mixture, composite Hansen solubility parameter, 0
δi,mix, is calculated with: HXLL MeTHFLL CPMELL HXCC MeTHFCC CPMECC

δi, mix = φ1 δi,1 + φ2 δi,2 + … (6) Fig. 2. Effects of purification method on biodiesel yield and purity by in situ
transesterification of dried C. pyrenoidosa biomass.
Subscript i signifies one of the three components of the Hansen
solubility parameters, φ is the volumetric fraction of each solvent in the
mixture. purified product with water in order to remove any traces of con-
The HSP distance between two molecules (Ra) can be conveniently tamination by catalyst, this procedure was performed several times
viewed with a spherical representation. Hansen solubility parameters of until the neutral pH of the solution was reached, which resulted in a
the solute are in the center of the solubility sphere and the solubility small loss of product. As observed in Fig. 2 the yield was obtained in the
sphere radius (Ro) indicates the extent of the interaction for occurrence following order: CPME > 2-MeTHF > Hexane. The yield of the pur-
of solubilization [13,14]. The lower the Ra, the higher the probability ification process using these three solvents can be explained through
of them being compatible, that is, good solvents are within the solu- the Hansen solubility parameters (HSP). In this work, we used the data
bility sphere and bad solvents are outside it [13,14]. available for soy biodiesel [14] to represent the algae biodiesel. The Ro
value of 11.33 MPa1/2 [15] was used to indicate the marginal solubility
Ra2 = 4 × (δDS−δDP )2 + (δPS−δPP )2 + (δHS−δHP )2 (7) of the solute in the solvent and RED was calculated according to Eq. (8)
Subscript S is for the solvent and subscript P is for the solute. to predict the solubility of the solvent in the mixture of FAME. As ob-
If Ra < Ro, the solvent is within solubility sphere and the solute is served in Table 1, the three solvents used in this study showed RED
soluble in the solvent. The Relative Energy Difference (RED) is the value < 1, indicating that they are highly soluble in biodiesel, and the
numerical representation of this graphical visualization. The lower the RED values for 2-MeTHF and CPME were lower than those obtained for
RED value, the better the solvent is dissolved in the solute [13,14]. hexane and hexane:ethyl acetate (19:1). According to the theory ap-
plied to the HSP, the lower the RED value, the better the solvent for
RED = Ra/ Ro (8)
extraction/purification. By comparing the values obtained for RED
(Table 1) with the experimental results (Fig. 2) it can be observed that
2.2.6. Statistical analysis the “green solvents” presented better yields, in this case, the theoretical
All experiments were performed in triplicate. The standard devia- data were similar to the experimental data, especially when biodiesel
tion of the measures was calculated to verify the reliability of the re- was purified by column chromatography, with the percentage of pur-
sults. The differences in mean values were evaluated through the ification obtained for 2-MeTHF and CPME being very close. On the
method of analysis of variance (ANOVA). The differences associated other hand, it was observed that the use of hexane as eluent for chro-
with p < 0.05 were considered significant. matographic separation was not very effective, requiring increase in the
polarity of the eluent with the addition of ethyl acetate, which theo-
3. Results and discussion retically results in a lower value for Relative Energy Difference (RED)
from 0.9 to 0.8, which implies a greater capacity for solubilization with
3.1. Influence of purification methods on biodiesel yields and purity biodiesel. Concerning the difference between the yields obtained in the
liquid-liquid separation process, it was observed that after evaporation
Chlorella pyrenoidosa, with 12.6 ± 0.3 wt% of lipids and with
moisture content lower than 3.5 ± 0.2 wt%, was used for in situ
Table 1
transesterification reaction according to the protocol described in
Hansen solubility parameters for Hexane, 2-MeTHF, CMPE and soy biodiesel.
Section 2.2.1. The yields obtained after the transesterification and Ra was calculated with soy biodiesel as a solute and Ro = 11.33 MPa1/2.
purification process were calculated according to Eq. (1), and biodiesel
purity was defined as the percentage of mass of methyl esters in the Organic δD (MPa1/2) δP (MPa1/2) δH δt (MPa1/2) Ra RED
solvent (MPa1/ (MPa1/
final product after purification. Fig. 2 shows the yields obtained after 2
) 2
)
the transesterification process and the biodiesel purity level after pur-
ification. In all cases the percentage of purification and the purity were Hexane* 14.9 0.0 0.0 14.9 9.7 0.9
greater than 70% and 96%, respectively. Fig. 2 shows that liquid-liquid 2-MeTHF*** 16.4 4.8 4.6 25.8 5.2 0.5
CPME* 16.7 4.3 4.3 25.3 5.7 0.5
extraction provided higher yields than purification by column chro- Ethyl acetate* 15.8 5.3 7.2 28.3 – –
matography. In liquid-liquid extraction efficiency depends on the affi- Hexane:Ethyl 14.9 0.3 0.4 16.0 9.2 0.8
nity of the solute for the extraction solvent, on the ratio of the phases acetate
and on the number of extractions, which leads to lower loss, since the (19:1)
Soy 15.0 3.7 8.9 27.6 – –
procedure was repeated three times. In purification by column chro-
bio-
matography, part of the fractions containing the material of interest diesel**
(FAME) can be removed together with the fraction of unwanted ma-
terial. In the two techniques employed, we opted for washing the Retired from: *Ref. [13]; **
Ref. [15]; ***
Ref. [16].

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Table 2
Characterization of biodiesel obtained by in situ transesterification and purified by liquid-liquid extraction and column chromatography.
Property Purification by liquid-liquid extraction Purification by column chromatography

HXLL MeTHFLL CPMELL HXCC MeTHFCC CPMECC

Density at 15 °C [g/L] 0.87 ± 0.03 0.89 ± 0.06 0.89 ± 0.05 0.87 ± 0.05 0.89 ± 0.02 0.89 ± 0.04
Kinematic viscosity at 40 °C [mm2/s] 3.97 ± 0.50 4.23 ± 0.66 4.21 ± 0.05 3.96 ± 0.03 4.28 ± 0.06 3.98 ± 0.06
Acid value [mg KOH/g oil] 0.41 ± 0.06 0.45 ± 0.09 0.37 ± 0.08 0.39 ± 0.06 0.42 ± 0.01 0.37 ± 0.03
Iodine number [g I2/100 g] 119 127 125 74 117 120
Water content [%] 0.04 ± 0.01 0.09 ± 0.01 0.03 ± 0.01 0.04 ± 0.01 0.05 ± 0.01 0.03 ± 0.01
Saponification value [mg KOH/g oil] 199 200 199 203 198 197
Cetane index (CI) 47 45 47 57 48 47
Monoglycerides [m/m] 0.15 0.22 0.46 0.22 0.71 0.12
Diglycerides [m/m] 0.27 0.00 0.28 0.00 0.26 0.00
Triglycerides [m/m] 0.00 0.02 0.01 0.00 0.01 0.00
Free glycerol [m/m] 0.01 0.00 0.01 0.00 0.00 0.00
Total glycerol [m/m] 0.08 0.06 0.17 0.06 0.22 0.03

Fatty acid methyl ester composition, mass %

Myristic C14:0 1.18 1.18 1.01 1.61 1.14 1.20
Myristoleic C14:1 0.96 0.78 0.77 1.04 0.62 0.73
Palmitic C16:0 27.72 27.10 26.61 37.57 29.47 27.95
Palmitoleic C16:1 4.60 4.76 4.75 6.37 4.42 4.74
Stearic C18:0 6.80 1.28 0.69 0.93 6.81 7.11
Oleic acid C18:1n9c 11.04 10.66 10.91 14.9 11.04 11.09
Elaidic acid C18:1n9t ND 6.03 6.22 8.48 ND ND
Linoleic C18:2n6c 19.60 19.48 19.92 26.05 19.25 19.81
α-Linolenic C18:3n3 27.12 27.24 27.92 0.49 26.86 27.36
Arachidic C20:0 0.37 0.37 0.38 0.54 ND ND
Total saturated 36.07 29.93 28.69 40.65 37.42 36.26
Total unsaturated 63.32 68.95 70.49 57.33 62.19 63.73

ND: Not detected.

of 2-MeTHF (process described in Fig. 1D) a fraction of residual water
was dissolved in the mixture of FAME with the fraction of 2-MeTHF that
was not evaporated, and it was only discovered after drying the bio-
diesel in oven at 80 °C for 24 h. This water was only separated effi-
ciently after the sample decantation process, which was achieved in a
period of about 24 h, in which there was formation of two distinct
phases: the heavier containing water and the lighter composed of bio-
diesel. This additional step may have resulted in significant loss of
FAME, leading to a yield lower than that obtained with CPME. Differ-
ently from the other solvents used, 2-MeTHF shows solubility in water
of 14 g water/100 g 2-MeTHF (against 1.1 g water/100 g CPME) and a
different characteristic in relation to other solvents with respect to so-
lubility, due to being “inversely soluble” in water [17]. That is, its so-
lubility decreases as temperature increases, which facilitates its eva-
poration during the drying process. Although the theoretical results
were similar to those obtained experimentally, the HSP characteriza-
tions should be treated with caution, because algae biodiesel presents
some differences in composition in relation to soy biodiesel, which
results in distinct values for δD, δP and δH. In addition, the limited
availability of the Hansen solubility parameters prevents the complete
characterization of the solubility of a given solvent for the desirable
products (FAME) and undesirable products (glycerides and glycerol).
Fig. 2 shows the purity of the biodiesel obtained for each technique
and solvent employed in this study. The purity of the biodiesel re-
presents the percentage of triglycerides converted to methyl esters.
Selectivity is a term used to represent the efficiency of a solvent in
extracting the product of interest from other undesirable components.
The two purification techniques produced biodiesel with same purity
level, and purification with hexane showed better results. Studies using
hexane as solvent for lipid extraction showed that this solvent is highly
selective due to its highly hydrophobic nature and its limited solubility
in water (0.01 g water/100 g hexane); however, this solvent is very
soluble in glycerides, which is undesirable for biodiesel purification
[9,18,19]. Extractions using CPME also resulted in a biodiesel with high
level of purity (Fig. 2). CPME is a solvent that is little soluble in water
and very efficient for extraction of hydrophobic proteins [8], and little
selective for extraction of triglycerides [9,20], which can be considered
an advantage for extraction of biodiesel, because triglycerides not
converted to methyl esters and present in the mixture can be separated
more efficiently through the use of this solvent. Depending on the
concentration in which they can be present in biodiesel, non-reacted
glycerides can increase the viscosity of the fuel and, consequently, re-
duce the efficiency of combustion, clogging the fuel filter and forming
deposits in engine parts such as pistons, valves and nozzles [21]. Bio-
diesel purity levels obtained with purifications using 2-MeTHF were not
as high as those obtained with the other solvents, 2-MeTHF is a little
selective solvent due to its borderline solubility and may be used for
extraction of proteins, fatty acids and other types of lipids that do not
contain fatty acids such as waxes, phospholipids, carotenoids and other
pigments [8,21], in addition studies have shown that 2-MeTHF is an
excellent solvent for extraction of mono, di and triglycerides, which
compromises the purity of biodiesel [6,22].
The values obtained for biodiesel purity are in accordance with the
standard EN 14214, which stipulates a minimum acceptable purity
level of 96.5% in methyl esters [21].
3.2. Influence of purification methods on biodiesel properties
To ensure the quality of biodiesel, quality standards should be es-
tablished, aiming to establish limits on contaminants that will not im-
pair the quality of emissions from burning, as well as the performance,
engine integrity, and safety in transport and handling [21].
The chemical and physicochemical characteristics of algae biodiesel
depend mainly on the production and purification process. In addition,
some properties of biodiesel are related with the molecular structures of
its constituent alkyl esters [21].
3.2.1. Physicochemical properties
The density of biodiesel is directly associated with the molecular
structure of its molecules [21]. Biodiesels obtained in extractions with
green solvents showed the same density (Table 2). While biodiesels
purified with hexane presented densities that were a little lower than

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S.S. de Jesus et al.
those obtained with green solvents, probably because they present
higher level of purity. The fact that the density does not vary sig-
nificantly with the purification method and solvent shows that the
density of biodiesel depends mainly on the origin of the raw material
[23]. Concerning quality standards, the ASTM standard does not con-
sider it relevant, whereas the European standard EN 14214 establishes
density values from 0.86 to 0.90 g/L. Biodiesels obtained in this work
presented densities lower than 0.90 g/L, being compliant with the
European quality standards.
Biodiesel viscosity is another important factor that should be taken
into consideration, because high viscosity causes heterogeneity in bio-
diesel combustion, due to decreased efficiency of atomization in the
combustion chamber, causing the deposition of residues in the internal
parts of the engine [21]. Therefore, these contaminants can be mon-
itored indirectly through the determination of kinematic viscosity at
40 °C. Standard EN 14214 (analytical method EN ISO 3104) establishes
an acceptable range of 3.5–5.0 mm2/s for viscosity, while standard
ASTM D6751 (analytical method D445) allows a little wider range of
1.9–6.0 mm2/s. Table 2 shows that the biodiesel obtained for each si-
tuation studied is compliant with the standards EN 14214 and ASTM
D6751. However, we can observe that the purification with green sol-
vents resulted in a more viscous biodiesel, especially when purified
with 2-MeTHF. Table 2 shows that purification by column chromato-
graphy using 2-MeTHF as eluent produced a biodiesel with higher
viscosity (4.28 ± 0.06 mm2/s) mainly due to its composition pre-
senting a high percentage of non-reacted glycerides (mono-, di- and
triglycerides) and a higher percentage of impurities (Fig. 2) compared
with the other solvents used in this study. On the other hand, we can
observe that in the liquid-liquid extraction the viscosity of the biodiesel
obtained with CPME was very close to 2-MeTHF; the fact that CPME
presents in its composition a high percentage of glycerides (data
available in Table 2) may have been the most significant factor for the
viscosity being relatively equal to that of the biodiesel purified with 2-
MeTHF.
3.2.2. Chemical properties
Monitoring the acidity in biodiesel is of great importance during
storage, and changes in values in this period could mean the presence of
water [21]. Acidity is measured in terms of quantity of KOH needed to
neutralize the sample [23]. The method recommended by EN 14214 is
EN 14104, and ASTM recommends the potentiometric method D664
[21,23]. All standards described above established maximum limits for
acidity of 0.5 mg KOH/g. Table 2 shows that the acidity ranged from
0.37 to 0.45 mg KOH/g, thus being compliant with the limits estab-
lished by the standards EN 14214 and ASTM D6751. It can be observed
that the purification of FAMEs by column chromatography resulted in a
biodiesel with lower acidity. Predojević [23] observed that when using
silica gel to purify biodiesel from waste frying oils the acidity decreased
by 65%, and this method was more effective than using warm distilled
water or 5% w/w phosphoric acid. Biodiesel purified using 2-MeTHF
presented higher levels of acidity, which leads us to conclude that in its
composition there is a higher percentage of free fatty acids, compared
with the other solvents used.
The number of unsaturations not only has effect on the values of
density and viscosity of biodiesel, but is also of great importance in
oxidative stability [21]. Standard EN 14214 adopted the iodine number
to determine the number of unsaturations, while North American
standard ASTM D6751 does not mention the maximum allowed value.
The iodine number depends on the origin of the raw material and has
major influence on the tendency for oxidation of the fuel. Although the
official analytical method of standard EN 14214 is EN ISO 1411, there
are different methods to determine the iodine number; in this work we
used the official method adopted by the AOCS Cd 1c-85. As observed in
Table 2 the iodine numbers are in the range of 74–127 g I2/100 g,
purification through liquid-liquid extraction provided higher iodine
numbers, and extractions with green solvents were higher than the
1128
Fuel 235 (2019) 1123–1130
maximum value accepted in standard EN 14214 which is 120 g I2/
100 g. Algae biodiesel presents in its composition (as well as its oils)
high concentration of unsaturated fatty acids, mainly linoleic and li-
nolenic, which results in a biodiesel with high iodine number [5,18].
Water, in addition to promoting the hydrolysis of biodiesel resulting
in free fatty acids, is also associated with the proliferation of micro-
organisms, corrosion in storage tanks with sediment deposition. Since
biodiesel presents a certain degree of hydroscopicity, water content
should be monitored during storage [21]. Both the European standard
(EN 14214) and the North American standard (ASTM D6751) establish
a maximum limit of 0.05% (i.e., 500 mg/kg) of water in biodiesel. The
percentage of water obtained in this study ranged from 0.03 to 0.09%.
Table 2 shows that the moisture content in the biodiesel purified by
liquid-liquid extraction with 2-MeTHF was higher than 0.05%. The
difficulty to separate the water after the extraction process, as men-
tioned in Section 3.1, had great influence on the high content of
moisture, thus being noncompliant with the quality standards of EN
14214 and ASTM D6751. According to Predojević [23], the use of an-
hydrous sodium sulfate to remove residual water, after the biodiesel
washing step, is not a very effective method, which results in a high
water content in the purified biodiesel.
The saponification value represents the milligrams of potassium
hydroxide required for saponification of one gram of fat or oil [23].
This value is inversely proportional to the mean molecular weight of the
fatty acids of the present triglycerides. This value is important to de-
monstrate the presence of oils and fats of high proportion of fatty acids
and low molecular mass, in mixtures with other oils and fats. No
standard establishes the minimum or maximum limits for saponifica-
tion value, for this reason, we hardly find relevant studies with these
values for biodiesel. In this work the saponification value was de-
termined in order to calculate the cetane index, which was calculated
according to the methodology of Krisnangkura [12]. Table 2 shows that
the saponification value ranged from 197 to 203 mg KOH/g. Higher
saponification values mean that the biodiesel is composed mostly of
fatty acids of lower molecular mass. However, the correlation of the
saponification value with the quality of biodiesel is not yet clear.
The cetane number indicates the time of delay in fuel ignition for
diesel cycle engines, therefore, it reflects the quality of fuel ignition. A
higher cetane number means a shorter ignition delay time and more
complete combustion of the fuel charge in the combustion chamber.
The cetane number increases with the length of the unbranched carbon
chain, thus the hexadecane (cetane) is established as default value of
100 in the range of cetane number, while for 2,2,4,4,6,8,8-hepta-
methylnonane (isocetane) is assigned the value 15 [21]. Compared with
fossil diesel, biodiesel provides higher values of cetane number. In
Europe, both for diesel and biodiesel, the minimum acceptable cetane
number is fixed at 51 (method EN ISO 5165). In the North American
quality standard, the cetane number for diesel is 40, while for biodiesel
the cetane number is established at 47 (method D613). In this work, we
calculated the cetane index, as an alternative to the cetane number. The
cetane index is also an indicator of quality for fuel ignition and is re-
lated with the time between the injection of fuel into the cylinder and
the start of ignition [24]. The methodology described by Krisnangkura
[12], in which the cetane index (CI) is estimated, is based on saponi-
fication values and iodine numbers, being used only for methyl esters.
Table 2 shows that the cetane index was higher than 47, except for the
biodiesel obtained through purification by liquid-liquid extraction with
2-MeTHF, thus being noncompliant with both EN 14214 and ASTM
D675. The biodiesel obtained through the process of purification by
column chromatography using as eluent hexane:ethyl acetate (19:1)
was the only one that met the two quality standards. However, it is
worth noting, the results were calculated for the cetane index, through
the equation of Krisnangkura who used data from vegetable oils [12].
Glycerol is a by-product from the reaction of transesterification of
oils and fats. The determination of residual glycerol serves as parameter
to evaluate the efficiency of the process of purification of biodiesel.
S.S. de Jesus et al.
Table 3
. Solvent properties of hexane, 2-MeTHF and CPME.
Solvent Hexane 2-MeTHF
Price ($/kg) 0.5 5.0
Toxicity indication 6 4
Boiling point [°C] 69 80
Solubility of solvent in water (23 °C) [g/100 g] 0.01 14
Solubility of water in solvent (23 °C) [g/100 g] 0.01 4.4
Azeotropic point with water [°C] – 71a
Evaporate 1 kg solvent [kWh] 0.121 0.126
Masse CO2 (g) generated by kg of solvent 96 101
Log P 3.94 0.82
Azeotrope (wt%).
Adapted from Refs. [20,22,26].
a
Composition = 2-MeTHF: 89.4 H2O:10.6.
b
Composition = CPME: 83.7 H2O:16.3.
High concentrations of glycerol in biodiesel cause storage problems,
because when biodiesel is mixed with petroleum diesel there is se-
paration of glycerol in storage tanks [21]. Problems such as formation
of deposits, clogging of engine nozzles and emissions of aldehydes are
also related with the high concentration of glycerol in biodiesel [21].
Residual free glycerol can be easily removed by washing biodiesel.
Although it is practically insoluble in biodiesel, glycerol can be found
dispersed in the form of droplets. The presence of residual soaps can
interfere, increasing the concentration of glycerol in biodiesel due to
the formation of emulsions [21]. In Europe and in the United States, the
maximum permitted content of free glycerol in biodiesel is 0.02%mass
and its determination is performed through the chromatographic
methods EN ISO 14105 and EN ISO 14106, established by the European
standard, and ASTM D6584, by the North American standard. Table 2
shows the absence of free glycerol in the biodiesel obtained for each
situation studied.
Mono-, di- and triglycerides result from the incomplete reaction of
glycerides, thus this is an important parameter that can be used to
evaluate the efficiency of the conversion of oils and fats into biodiesel.
Depending on their concentration in biodiesel, non-reacted glycerides
can increase the viscosity of the fuel [21]. The European standard sets
maximum limits of 0.8, 0.2 and 0.2%mass for mono-, di- and triglycer-
ides respectively. While the ASTM standard does not stipulate values for
these glycerides. Total glycerol can be calculated through the con-
centrations of free glycerol and mono-, di- and triglycerides. The Eur-
opean standard establishes a maximum limit of 0.25%mass, while in the
United States the limit is 0.24%mass. Table 2 shows that all purified
biodiesel presented in their composition low concentrations of mono-
glycerides; however, in all situations studied we observed limits lower
than 0.8%mass. On the other hand, the biodiesel purified by liquid-liquid
extraction with hexane and with CPME, in addition to the biodiesel
purified by column chromatography using 2-MeTHF as eluent, pre-
sented a percentage of diglycerides above that established by the Eur-
opean standard. The presence of mono- and diglycerides mean that not
all glyceride present in the sample was reacted and that they were
formed as intermediates of the esterification reaction. The presence of
these glycerides is often caused by the experimental conditions, such as
molar ratio of reactants and reaction time, thus it is not a problem
caused mainly by the extraction method. In this study, significant
amounts of triglycerides were not observed. Total glycerol concentra-
tion was lower than that established by the standards EN 14214 and
ASTM D6751.
The FAMEs obtained in this study are presented in Table 2. As can
be observed, the biodiesel purified with hexane presented a different
composition compared with those obtained with 2-MeTHF and CPME.
Depending on the purification technique the composition of the FAMEs
varied significantly. Purification by liquid-liquid extraction using
hexane as solvent resulted in a biodiesel with higher percentage of
C18:0 than that obtained with 2-MeTHF and CPME; on the other hand,
Fuel 235 (2019) 1123–1130
the presence of C18:1n9t was not observed. In the purification by
column chromatography, it was observed that, using hexane:ethyl
CPME acetate, the biodiesel presented higher percentages of C16:0 and C18:1
than other solvents studied, in addition to a significant percentage of
11.0 C18:1n9t, which was not observed in the purification by liquid-liquid
4
extraction. However, there was a decrease in the percentage of C18:0
106
1.1
and C18:3n3, these being lower than 1%. Table 2 shows that the bio-
0.3 diesel presented mainly the methyl esters C16:0, C18:1n9c, C18:2n6c
83b and C18:3n3 (except in purification by column chromatography with
0.132 hexane:ethyl acetate). Studies of in situ transesterification using Chlor-
106
ella vulgaris obtained a biodiesel with higher percentages of C16:1,
1.59
C18:0, C18:1n9c and C18:3n3 [5], on the other hand, in studies with
wet biomass of C. vulgaris and under subcritical conditions, the com-
position of the FAME was mostly composed of C16:0, C16:1, C18:2n6c
and C18:3n3 [25]. Another study, using the microalgae C. pyrenoidosa,
obtained higher percentages of C16:0, C18:2n6c and C18:3n3 [18]. The
composition of the FAMEs may vary according to the methodology
employed and the conditions during the transesterification reaction, as
well as to the methodology used for purification. However, microalgae
biodiesel is composed mainly of methyl esters C16:0, C16:1, C18:1n9c,
C18:2n6c and C18:3n3.
Finally, we can observe that all methods employed produced a
biodiesel with a percentage higher than 50% of long-chain methyl es-
ters of fatty acids with low degree of unsaturation (C14:0, C16:0, C16:1
and C:18:1), which produces a biodiesel with better quality.
Table 3 presents the main characteristics of the solvents used in this
study. Price, boiling point, partition coefficient (log P), toxicity cate-
gory, and energy required for solvent evaporation are very important
parameters for the choice of extraction solvent in the industry [22].
Table 3 shows that green solvents present great potential to replace
hexane in extraction processes, because they present boiling point
lower than 130 °C, which is ideal for industrial use, moreover, they
consume only 5%–10% more energy and produce only 5%–10% more
carbon dioxide than hexane [22]. In addition, these three solvents are
lipophilic, with Log P > 0. However, although these solvents present
high potential for the extraction and purification of biodiesel, their
prices are still prohibitive; while 1 kg of hexane costs $0.5, 2-MeTHF
and CPME are 10 and 22 times more expensive. These values can also
mean a great barrier in the large-scale use of these solvents.
However, this technique shows a few advantages compared to other
biodiesel purification methods that have been recently developed, not
only in avoiding the use of fossil-based solvents, but also reducing costs.
In comparison with techniques currently available, purification with
deionized water shows several drawbacks such as high water con-
sumption, which generates large quantities of waste water and high
production costs, such as deionization of water. Also, biodiesel obtained
has high moisture content, not meeting the quality standards [27,28].
Although the membrane process generates few residues, the energy
expenditure and the cost of equipment installation are barriers to this
technique use on a large scale [28]. Purification by dry washing results
in biodiesel with a significant amount of methanol that does not meet
the standard EN 14214 [29], in addition to the intensive use of energy.
Also, the purification method Magnesol among other absorbers, de-
mands further costs with absorber, which is not regenerated after the
purification process [30,31]. Thus, the purification process developed
in this work is more attractive than those currently used.
It should be noted that although the price of green solvents is still
prohibitive, once they are not yet produced on a large scale, the de-
mand is still small because it is a recent technology, still under devel-
opment. Energy expenditure for solvent evaporation is required only in
the final stage of the process, in the drying step, where biodiesel is
heated to 75 °C and an exact amount of absorbent is added to the bio-
diesel and left for a period until later filtration [30]. Fixed-bed pur-
ification techniques also show a great disadvantage in relation to the
purification proposed in this work, because the biodiesel separation is
carried out in a column that is kept under heating and requires vacuum
1129
S.S. de Jesus et al.
application for the purification to occur [27,28,30].
4. Conclusion
Algae biodiesel purification using green solvents was evaluated as to
its physicochemical characteristics and chemical composition. Biodiesel
produced through acid-catalyzed in situ transesterification was purified
through liquid-liquid extraction and column chromatography, using the
green solvents: 2-methyltetrahydrofuran (2-MeTHF) and cyclopentyl
methyl ether (CPME). The solvent hexane was used for comparison.
According to the results it was possible to conclude that the biodiesel
purified with green solvents showed results that were similar those of
biodiesel purified with hexane. However, it was observed that in the
process of purification by liquid-liquid extraction using 2-MeTHF there
was need for an additional step of purification, which consisted in
leaving the material in decantation for 24 h to remove residual water
contained in the biodiesel. This additional step can be a limiting factor
for the use of this solvent in the liquid-liquid extraction process.
Although biodiesels purified with green solvents present higher visc-
osities and densities, they are all compliant with the quality standards.
Concerning purity, some specific improvements in the purification
process, especially when using 2-MeTHF as solvent, can be im-
plemented so biodiesel purity is equal to or greater than that obtained
using hexane. Some chemical properties obtained in this study may be
related to the methodology employed for in situ transesterification re-
action, of which we can mention the concentration above 0.2%mass of
diglycerides in biodiesel purified through liquid-liquid extraction with
hexane and CPME, and by column chromatography using the eluent 2-
MeTHF. Finally, we can conclude that 2-MeTHF and mainly CPME used
as eluent in purification by column chromatography have great po-
tential to replace hexane in the biodiesel purification process, and the
price of these solvents is the only factor limiting their large-scale use.
Acknowledgments
This study was supported by the Coordination for Improvement of
Higher Education Personnel (PNPD-CAPES), the State of São Paulo’s
Research Foundation (FAPESP, grant process no. 2010/03764-3 and
2015/19609-0), National Council of Technological and Scientific
Development (CNPq, grant process no. 166844/2017-9) and Espaço da
Escrita (Coordenadoria Geral UNICAMP) for the language services
provided.
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