Synthesis of novel non-toxic naphthenic and benzoic acid ionic liquids. Structure-properties relationship and evaluation of their biodegradability potential

Article abstract of DOI:10.1016/j.molliq.2019.111927

Protic Ionic Liquids (PILs) are promising solvents with desirable physicochemical properties for a variety of applications. They are considered as greener alternatives to common organic solvents but in order to efficiently be characterized as green their environmental impact has to be assessed. In the present study, the design, synthesis and an initial potential hazard assessment of 11 naphthenic and benzoic acid based PILs is presented. PILs’ “greenness” is enhanced considering that they are non-toxic according to the aquatic toxicity evaluation and most of them show satisfactorily to extremely high biodegradability level. Moreover, two of their most important physicochemical properties, viscosity and glass transition temperature (T_g), were determined for selected ILs. The effect of the aromatic and alicyclic ring on the anions, the size and the presence of a hydroxyl substituent at anions and cations has been also discussed for the studied environmental and physicochemical properties.

Full text of DOI:10.1016/j.molliq.2019.111927

Journal Pre-proof
Synthesis of novel non-toxic naphthenic and benzoic acid ionic liquids. Structure-
properties relationship and evaluation of their biodegradability potential
Andromachi Tzani, Dimitrios Skarpalezos, Achilleas Papadopoulos, Dionysia
Aravopoulou, Ioannis Kleidas, Efstathia Ioannou, Epaminondas Voutsas, Apostolos
Kyritsis, Anastasia Detsi
PII:
S0167-7322(19)33949-2
DOI:
https://doi.org/10.1016/j.molliq.2019.111927
MOLLIQ 111927
Reference:
To appear in: Journal of Molecular Liquids
Received Date: 15 July 2019
Revised Date: 9 October 2019
Accepted Date: 13 October 2019
Please cite this article as: A. Tzani, D. Skarpalezos, A. Papadopoulos, D. Aravopoulou, I. Kleidas, E.
Ioannou, E. Voutsas, A. Kyritsis, A. Detsi, Synthesis of novel non-toxic naphthenic and benzoic acid
ionic liquids. Structure-properties relationship and evaluation of their biodegradability potential, Journal
of Molecular Liquids (2019), doi: https://doi.org/10.1016/j.molliq.2019.111927.
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Synthesis of novel non-toxic naphthenic and
benzoic acid ionic liquids. Structure-properties
relationship and evaluation of their
biodegradability potential.
Andromachi Tzania, Dimitrios Skarpalezosa, Achilleas Papadopoulosa, Dionysia
Aravopouloub, Ioannis Kleidasc, Efstathia Ioannouc, Epaminondas Voutsasd,
Apostolos Kyritsisb, Anastasia Detsia*
aLaboratory of Organic Chemistry, Department of Chemical Sciences, School of
Chemical Engineering, National Technical University of Athens, 15780 Athens,
Greece
bDepartment of Physics, School of Applied Mathematical and Physical Sciences,
National Technical University of Athens, 15780 Athens, Greece
cSection of Pharmacognosy and Chemistry of Natural Products, Department of
Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis
Zografou, Athens 15771, Greece
dLaboratory of Thermodynamics and Transport Phenomena, Department of Process
Analysis and Plant Design, School of Chemical Engineering, National Technical
University of Athens, 15780 Athens, Greece.
1

ABSTRACT: Protic Ionic Liquids (PILs) are promising solvents with desirable
physicochemical properties for a variety of applications. They are considered as
greener alternatives to common organic solvents but in order to efficiently be
characterized as green their environmental impact has to be assessed. In the present
study, the design, synthesis and an initial potential hazard assessment of 11
naphthenic and benzoic acid based PILs is presented. PILs’ “greenness” is enhanced
considering that they are non-toxic according to the aquatic toxicity evaluation and
most of them show satisfactorily to extremely high biodegradability level. Moreover,
two of their most important physicochemical properties, viscosity and glass transition
temperature (Tg), were determined for selected ILs. The effect of the aromatic and
alicyclic ring on the anions, the size and the presence of a hydroxyl substituent at
anions and cations has been also discussed for the studied environmental and
physicochemical properties.
KEYWORDS Protic Ionic Liquids, Biodegradability, Toxicity, Viscosity, DSC
2

1. INTRODUCTION
Ionic Liquids (ILs) are organic salts with a wide range of liquid phase (in many cases near to
the ambient temperature) which, during the last few decades, have received great attention as
alternatives to commonly used volatile organic solvents (VOCs). They can be divided into
two broad categories: protic ILs (PILs) and aprotic ILs (APILs) [1]. PILs, in comparison to
APILs, are cheaper and easier to be prepared via a proton transfer reaction between a
Brønsted acid and a Brønsted base without byproducts formation.
PILs have been less studied compared to AILs but they have attracted the scientific and,
lately, the industrial community’s interest, due to their remarkable properties such as high
thermal and chemical stability, non-flammability, high solvation ability, high ionic
conductivity etc [1,2]. The ability of PILs to adopt desirable properties by the targeted
selection of ions, make them attractive candidates for use in numerous applications [1].
Among others, they have been used in: organic synthesis as solvents and catalysts [3],
metallic nanoparticles synthesis [4,5], biocatalysis [6,7], separation of azeotropic mixtures
[8,9], CO2 capture [10], extractions [11,12] etc.
The considerable interest, the increasing production and use of ILs, raised environmental
awareness issues, since their probable deposition in the environment is increased. In this
context, the design of new environmentally friendlier ILs as alternatives to the commonly
used hazardous VOCs consists a big challenge. Consequently, recent studies focusing on the
ILs environmental impact have emerged evaluating factors such as chemical degradation,
bioaccumulation, biodegradability and toxicity [13-19]. The use of biodegradable and non-
toxic or of low toxicity compounds, are in accordance with the principles of green chemistry
introduced in 1998 by Paul Anastas and John Warner [20]. Τoxicity and biodegradability are
crucial to be assessed together since toxicity may significantly affect the biodegradation
3

process. This is because the tested ILs could be toxic to the used microorganisms for the
biodegradation process resulting in lower biodegradation percentages [21,22].
Taking advantage of the ILs structure-properties relationship, the design of biodegradable and
non-toxic or of low toxicity ILs becomes a feasible objective. However, due to the numerous
ILs that can potentially be designed through targeted ions selection, it is important to consider
some factors that could affect their toxicity and biodegradation. The study of Boethling et al.
[23], on the design of biodegradable chemicals, consists an important tool and suggests some
guidelines that can be applied in the ILs design.
As a continuation of our study on the synthesis, characterization and structure-properties
relationship of PILs [24], we hereby present the synthesis of 11 PILs, based on naphthenic
and benzoic acids, the evaluation of their “greenness” and their structure-properties
relationship. The ions selection was targeted in an effort to enhance their environmentally
friendly character. Benzoic acids were selected due their aromatic character that, according to
the Boethling guidelines, could possibly positively affect the biodegradability. Naphthenic
acids were also selected since, they could be almost completely biodegraded and
experimental studies on choline-based naphthenic acid ionic liquids (NAILs) have shown
high biodegradability percentages [25].
Furthermore, the effect of modifications on the ILs’ anions and cations structures on their
environmental and some of their most important physicochemical properties has been
investigated. More specifically, the alicyclic vs aromatic character of anions, the presence of
hydroxyl groups on both ions and also the overall ILs’ carbon load effect on their
environmental (biodegradability and toxicity) and physicochemical (viscosity and Tg)
properties were examined.
4

2. EXPERIMENTAL SECTION
2.1. Materials.
All the reagents were used without further purification (Table 1).
Table 1. Chemicals Used, Synthesized Protic Ionic Liquids and their Purities
Compound
Source
Purity
Analysis method
GC b,c
(wt %)a
99.99%
Ethanolamine
Sigma
Aldrich
Merck
Diethanolamine
>98%
98%
GC b,c
GC b,c
GC b,c
2-Methoxyethylamine
Bis(2-methoxyethyl)amine
Alfa Aesar
Sigma
99%
Aldrich
Alfa Aesar
TCI
Cyclohexanecarboxylic acid
Cyclopentanecarboxylic acid
Benzoic acid
98%
98%
99%
99%
GC b,c
GC b,c
Alfa Aesar
Alfa Aesar)
GC b,c
2-Ηydroxy-benzoic acid
GC b,c
2-Methoxy-ethylammonium
cyclopentane carboxylate (1)
Bis(2-methoxy-ethyl) ammonium
cyclopentane carboxylate (2)
2-Methoxy-ethylammonium
cyclohexane carboxylate (3)
Bis(2-methoxy-ethyl) ammonium
Synthesized >98%
Synthesized >98%
Synthesized >98%
Synthesized >98%
1H NMRd
1H NMR
1H NMR
1H NMR
5

cyclohexane carboxylate (4)
Bis(2-hydroxy-ethyl) ammonium
cyclohexane carboxylate (5)
2-Hydroxy-ethylammonium benzoate
(6)
Synthesized >98%
Synthesized >98%
Synthesized >98%
Synthesized >98%
Synthesized >98%
Synthesized >98%
1H NMR
1H NMR
1H NMR
1H NMR
1H NMR
1H NMR
Bis(2-hydroxy-ethyl) ammonium
benzoate (7).
2-Methoxy-ethylammonium benzoate
(8)
Bis(2-methoxy-ethyl)ammonium
benzoate (9)
Bis(2-hydroxy-ethyl) ammonium 2-
hydroxy-benzoate (10)
Bis(2-methoxy-ethyl) ammonium 2-
Synthesized >98%
1H NMR
hydroxy-benzoate (11)
a
c
d
wt %: weight percent. b Given by the suppliers. Gas Chromatography. Proton Nuclear
Magnetic Resonance.
2.2. Methods
1H-NMR spectra were recorded on a Varian 300 MHz and 600 MHz spectrometers at the
Institute of Chemical Biology of the National Hellenic Research Foundation, using DMSO-d6
and CDCl3 as solvents. The depletion of dissolved oxygen (DO) for the biodegradability
assessment was measured with a Consort C1010 DO analyzer. Viscosities were measured on
a Brookfield DV1. DSC measurements, were carried out on a TA instruments DSC Q200,
6

with temperature accuracy +/−0.1°C and sensitivity 0.2 ꢀW. The water content was
determined in triplicate using a TitroLine Karl-Fischer Titrator.
2.3. General Procedure for the Synthesis of ILs
Method A. According to our previously applied procedure [24], the naphthenic acid based ILs
(NAILs) 1-5 (Fig. 1) were prepared by direct neutralization of equimolar amounts of the
corresponding amines and carboxylic acids.
Figure 1. Synthesized NAILs 1-5
Method B. The benzoic acid based ILs (BAILs) 6-11 (Fig. 2) were prepared according to
Method A, slightly modified. The amine was placed in a two necked glass flask. The organic
acid was added dropwise to the flask through a dropping funnel after dissolution with
appropriate amount of ethanol. The reaction mixture was stirred for 24 h at laboratory
temperature and then the solvent was removed by vacuum evaporation.
O
O
O
O
O
OH
O
O
O
O
H3N
HO
N
H2
9
6
7
O
O
O
HO
OH
O
OH
N
N
H2
H2
OH
O
10
O
O
O
H3N
N
H2
OH
8
11
Figure 2. Synthesized BAILs 6-11
7

All the produced ILs (Table 1) were dried under high vacuum at 50°C, for a period never
lower than 3 h in order to decrease the water content as much as possible. To confirm the
synthesized products structures 1H NMR were performed (Table 2).
2-Methoxy-ethylammonium cyclopentane carboxylate (1). Method A. 0.06 mol
cyclopentanecarboxylic acid and 0.06 mol of 2-methoxyethylamine were used to obtain a
yellow liquid.
Bis(2-methoxy-ethyl)ammonium cyclopentane carboxylate (2). Method A. 0.028 mol
cyclopentanecarboxylic acid and 0.028 mol of bis(2-methoxyethyl)amine were used to obtain
a dark orange liquid.
2-Methoxy-ethylammonium cyclohexane carboxylate (3). Method A. 0.017 mol
cyclohexanecarboxylic acid and 0.017 mol of 2-methoxyethylamine were used to obtain a
yellow liquid.
Bis(2-methoxy-ethyl)ammonium cyclohexane carboxylate (4). Method A. 0.014 mol
cyclohexanecarboxylic acid and 0.014 mol of bis(2-methoxyethyl)amine were used to obtain
a yellow liquid.
Bis(2-hydroxy-ethyl)ammonium cyclohexane carboxylate (5). Method A. 0.037 mol
cyclohexanecarboxylic acid and 0.037 mol of diethanolamine were used to obtain a light
orange liquid.
2-Hydroxy-ethylammonium benzoate (6). Method B. 0.008 mol benzoic acid diluted in 10 mL
ethanol and 0.008 mol of ethanolamine were used to obtain a light yellow solid. (m.p. 139–
141°C)
8

Bis(2-hydroxy-ethyl)ammonium benzoate (7). Method B. 0.104 mol benzoic acid diluted in 15
mL ethanol and 0.104 mol of diethanolamine were used to obtain a yellow liquid.
2-Methoxy-ethylammonium benzoate (8). Method B. 0.008 mol benzoic acid diluted in 4 mL
ethanol and 0.008 mol of bis(2-methoxyethyl)amine were used to obtain an orange liquid.
Bis(2-methoxy-ethyl)ammonium benzoate (9). Method B. 0.014 mol of benzoic acid diluted in
7 mL ethanol and 0.014 mol of bis(2-methoxyethyl)amine were used to obtain a dark orange
liquid.
Bis(2-hydroxy-ethyl)ammonium 2-hydroxy-benzoate (10). Method B. 0.053 mol of 2-hydroxy
benzoic acid diluted in 17 mL ethanol and 0.053 mol of bis(2-hydroxyethyl)amine were used
to obtain a very light yellow liquid.
Bis(2-methoxy-ethyl)ammonium 2-hydroxy-benzoate (11). Method B. 0.033 mol of 2-hydroxy
benzoic acid diluted in 15 mL ethanol and 0.033 mol of bis(2-methoxyethyl)amine were used
to obtain a brown liquid.
Table 2. 1H NMR of the synthesized PILs
PILs
1
1H NMR
1H NMR (300 MHz, CDCl3, 26 οC) δ/ppm: 3.53 (t, J=5.1 Hz, 2H, ‒CH2OCH3), 3.35
(s, 3H, ‒OCH3), 2.99 (t, J=5.1 Hz, 2H, ‒CH2NH3)), 2.68 – 2.57 (m, 1H, ‒CHCOO-),
1.87 – 1.55 (m, 8H, 4(‒CH2‒)).
ο
+
1H NMR (300 MHz, CDCl3, 26 C) δ/ppm: 7.71 (br s, 2H, ‒NH2 ‒), 3.56 (t, J=5.1
Hz, 4H, 2(‒CH2OCH3-)), 3.37 (s, 6H, 2(‒OCH3)), 2.95 (t, J=5.1 Hz, 4H, 2(‒
CH2NH3)), 2.75 – 2.65 (quint, 1H, ‒CHCOO-), 1.96 – 1.52 (m, 8H, 4(‒CH2‒)).
2
3
1H NMR (300 MHz, CDCl3, 26 οC) δ/ppm: 6.75 (br s, 3H, ‒NH3 ), 3.49 (t, J=5.1 Hz,
+
9

+
4H, 2(‒CH2OCH3-)), 3.32 (s, 3H, (‒OCH3)), 2.95 (t, J=5.1 Hz, 4H, 2(‒CH2NH3 )),
2.08 (tt, J=11.1, 4.2 Hz, 1H, ‒CHCOO-), 1.86 – 1.82 (m, 2H, ‒CH2‒), 1.71 – 1.57
(m, 3H, 2‒CH2‒), 1.40 – 1.13 (m, 5H, ‒CH2‒).
+
1H NMR (300 MHz, CDCl3, 26 οC) δ/ppm: 8.47 (br s, 2H, ‒NH2 ), 3.56 (t, J=5.1 Hz,
4
5
+
4H, 2(‒CH2OCH3-)), 3.35 (s, 6H, 2(‒OCH3)), 2.95 (t, J=5.1 Hz, 4H, 2(‒CH2NH2 )),
2.24 (tt, , J=11.1, 4.2 Hz, 1H, ‒CHCOO-), 1.93 (d, J=12.8 Hz, 2H, ‒CH2‒), 1.81 –
1.60 (m, 3H, ‒CH2‒), 1.49 – 1.24 (m, 5H, ‒CH2‒).
+
1H NMR (600 MHz, CDCl3, 26 οC) δ/ppm: 7.09 (br s, 4H, NH2 & 2(‒OH)), 3.85 (t,
+
J=4.5 Hz, 4H, 2(‒CH2OH)), 3.07 (t, J=4.8 Hz, 4H, 2(‒CH2NH2 )), 2.12 (tt, J=11.1,
3.4 Hz, 1H, ‒CHCOO-), 1.88 (d, J=11.5 Hz, 2H, ‒CH2‒), 1.75-1.62 (m, 4H, 2(‒
CH2‒)), 1.45 – 1.16 (m, 5H, ‒CH2‒).
1H NMR (300 MHz, DMSO-d6, 26 οC) δ/ppm: 7.87 – 7.84 (m, 2H, aromatic H), 7.36
– 7.26 (m, 3H, aromatic H), 3.56 (t, J=5.1 Hz, 2H, ‒CH2OH)), 2.81 (t, J=5.4 Hz,
6
7
+
2H, ‒CH2NH3 ).
1H NMR (600 MHz, DMSO-d6, 26 οC) δ/ppm: 7.93– 7.88 (m, 2H, aromatic H), 7.43
(t, J=7.3 Hz, 1H, aromatic H), 7.37 (t, J=7.4 Hz, 2H, aromatic H), 3.66 (t, J=4.8
+
Hz, 4H, 2(‒CH2OH)), 2.96 (t, J=5.4 Hz, 4H, 2(‒CH2NH3 )).
1H NMR (300 MHz, CDCl3, 26 οC) δ/ppm: 8.01 – 7.99 (m, 2H, aromatic H), 7.46 –
7.33 (m, 3H, aromatic H), 3.54 (t, J=4.8 Hz, 2H, ‒CH2OH), 3.28 (s, 3H, (‒OCH3)),
8
+
3.08 (t, J=5.1 Hz, 2H, ‒CH2NH3 ).
1H NMR (300 MHz, CDCl3, 26 οC) δ/ppm: 8.02 – 7.99 (m, 2H, aromatic H), 7.43 –
9
+
7.31 (m, 3H, aromatic H), 7.21 – 7.16 (br, 2H, ‒ΝΗ2 ‒), 3.61 (t, J=4.8 Hz, 4H, -2(‒
CH2OCH3)), 3.31 (s, 6H, 2(‒OCH3)) 3.09 (t, J=4.8 Hz, 4H, 2(‒CH2OCH3)).
ο
1H NMR (600 MHz, CDCl3, 26 C) δ/ppm: 7.66 (dd, J= 7.5 Hz, 1H, aromatic H),
10
7.15 (td, 1H, aromatic H), 6.64 – 6.59 (m, 2H, aromatic H), 3.67 (t, J=5.1 Hz, 4H,
10

+
2(‒CH2OH)), 3.02 (t, J=5.4 Hz, 4H, 2(‒CH2NH3 )).
1H NMR (600 MHz, DMSO-d6, 26 οC) δ/ppm: 7.66 (dd, J = 7.5 Hz, 1H, aromatic H),
7.14 (ddd, J=8.2, 7.2, 1.9 Hz, 1H, aromatic H), 6.64 – 6.58 (m, 2H, aromatic H),
3.59 (t, J=5.1 Hz, 4H, -2(CH2OH-)),3.27 (s, 6H, 2(‒OCH3)), 3.12 (t, J=5.4 Hz, 4H,
11
+
2(‒CH2NH3 )).
2.4. Biodegradability assessment
In order to determine the biodegradability potential of the synthesized ILs, the BOD5
parameter was calculated. BOD5 depicts the biochemical oxygen demand by suitable
populations of microorganisms to metabolize the organic substance within 5 days. The BOD
test starts with the carbonaceous stage, followed by both carbonaceous and nitrogenous stage.
In order to ensure that only the carbonaceous oxygen demand is measured at the BOD5 test,
allyl thiourea (ATU) is used, and the results are reported as carbonaceous BOD5 (CBOD5).
According to the method, in 300mL airtight bottles, a nutrient aerobic aqueous medium was
added (containing specific quantities of necessary nutrients and micronutrients for the
microorganisms development and activity), inoculated with microorganisms and an
appropriate amount of known IL concentration. The tests were carried out in triplicate. The
seed source of microorganisms was mixed liquor, collected from a secondary sedimentation
tank of municipal wastewater from the Psyttaleia sewage treatment plant in Athens, Greece.
The BOD5 is calculated as the difference between initial and final DO. The DO is measured
both initially and after the 5-day incubation at 20 ± 1 °C, in darkness, using the Consort
C1010 DO analyzer and corrected for uptake by the use of a blank inoculum run in parallel
and then expressed as a percentage of the theoretical oxygen demand (ThOD) to give the
biodegradation percentage.
11

In this study only the carbonaceous stage is taken into account since the nitrogenous stage
according to bibliographic data begins after about 6 days, as nitrifying bacteria typically do
not consume an appreciable amount of oxygen until 6-10 days have passed. In order to ensure
that the nitrification can’t occur within five days, ATU is added to the samples as an
inhibitor, and so the results relate to carbonaceous BOD (CBOD) [26,27] .
2.5. Toxicity assessment
Cysts of the brine shrimp A. salina were stored at ‒20 °C before use. Cysts were incubated
for hatching in an Erlenmayer flask containing filtered seawater (3.2% salinity), at 25 °C
under aerobic conditions. After 24 h, hatched larvae (instar 1) were transferred to a new
Erlenmayer flask with fresh filtered seawater and incubated at 25 °C for a further 24 h under
artificial light with air sparging. After 48 h from the start of the incubation, all larvae had
moulted to the instar 2–3 stages, which were used for the toxicity evaluation. The phototropic
nauplii were collected by disposable glass Pasteur pipettes from the illuminated side. Stock
solutions and series of serial dilutions (at least three different concentrations) of the PILs
were prepared in filtered seawater. For the toxicity evaluation, 2 mL of each dilution (or
filtered seawater in the case of the control) were added to each well of a 24-well plate and ten
A. salina nauplii were transferred in each well using disposable glass Pasteur pipettes. The
plates were covered and maintained in an incubator at 25 °C. After 24 h of exposure, the
number of surviving and dead nauplii in each well were counted under a stereoscope and
recorded. The nauplii were considered dead if they did not show any forward movement for
10 sec after poking with a pipette. In case that dead nauplii were observed in the control
wells, the data was corrected using Abbott’s formula: corrected lethality (%) = [1 - (treated
population/control population)] x 100.
12

The experiments were conducted in triplicate for each concentration. The LC50 values were
calculated using the probit method [28] and expressed as a meanꢁ±ꢁstandard deviation
(p<0.05).
2.6. Differential scanning calorimetry (DSC) spectroscopy
The thermal behavior some of the synthesized ILs was investigated using a TA Q200 series
DSC instrument with Tzero functionality. Helium was used as a purge gas (25 mL/min).
Empty furnace and sapphire runs were previously performed to calibrate the capacitance and
resistance values of the Tzero Heat Flow Circuit. Then, Indium was used for temperature and
enthalpy calibration. Ionic liquid samples of similar size, 7-12 mg, were sealed in Low mass -
Tzero Aluminum Hermetic pans while cooling and heating rates were fixed at 10 °C/min.
The usual protocol of the measurements consisted of heating from 20 °C up to 150 °C, then
cooling down to -150 °C and heating to 150 °C (second heating). The temperature accuracy
+/−0.1°C and sensitivity 0.2 ꢀW.
2.7. Temperature-Dependent Viscosity
The tested synthesized NAILs and BAILs were dried under high vacuum at 50°C, for a
period never lower than 3 h in order to decrease the water content. The water content was
determined with Karl Fischer titration and is reported in Table 3. Then, their viscosity was
measured in triplicate in a broad temperature range (20 – 90 °C) using Brookfield digital
viscometer (LV-DVI-E) using a sample adapter (chamber SC4-13R) and spindle SC4-18. The
IL’s were waited using a KERN balance with an accuracy of ±0.0001 g for all measurements.
The samples temperature was maintained to within ±0.01 °C using a Julabo F12 thermostat
bath. Each measurement was carried out at a constant time of about seven minutes to
investigate if there is any time dependency of the viscosity. Viscosities were measured with
an accuracy of ± 2%.
13

3. RESULTS AND DISCUSSION
In this work, eleven naphthenic and benzoic acid-based protic ILs were synthesized and
structurally characterized, via a simple proton transfer reaction between an organic carboxylic
acid and a substituted amine. The % water content (% wt) was determined in triplicate using
a TitroLine Karl-Fischer Titrator and calculated below 1% w/w. Table 3 shows the average %
wt of the tested ILs. The % wt of IL 7 (which is expected to be one of the most hygroscopic
ILs in this study) was also determined after the viscosity measurements in order to quantify
the % wt change. The measurements confirmed a slightly % wt increase after the viscosity
measurement (from 0.8483% to 0.9489%).
Table 3. % water content and Standard Deviation (SD) of the synthesized PILs
PIL
2
% wt
0.4547
0.1737
0.9247
0.8483
0.6487
0.7397
0.6577
SD
0.0021
0.0031
0.0038
0.0047
0.0078
0.0025
0.0060
4
5
7
9
10
11
3.1. Biodegradability assessment
The ILs biodegradability assessment is essential in order to possibly label them as green.
Biodegradation is the natural process of removal of organic substances from the environment.
According to the Organization for Economic Cooperation and Development (OECD)
14

standards, organic compounds are classified as “readily biodegradable”, when the percentage
of biodegradation level is equal or exceed the ≥60% in 28 days [26].
All BOD5 tests were run in triplicate and the average amount of ILs biodegradability potential
expressed as the ratio CBOD5/UCBOD (Table 4) where:
CBOD5: Oxygen demand by microorganisms for degradation of the organic compound
within five days (only carbonaceous stage).
UCBOD: Oxygen demand by microorganisms for the ultimate degradation of the organic
compound referring only to the conversion of organic carbon to carbon dioxide, water, and
new microbial cellular constituents (ultimate carbonaceous demand). The UCBOD coincides
with the ThOD for complete conversion of the organic carbon to carbon dioxide.
Table 4. (%) Biodegradability evaluation of ILs 1-11
PIL CBOD5/ UCBOD (%)
44.5 ± 0.6
24.5 ± 0.1
52.3 ± 1.5
52.8 ± 3.2
79.0 ± 13.7
91.5 ± 8.8
90.3 ± 4.0
73.7 ± 0.9
27.1 ± 3.9
33.1 ± 2.8
30.6 ± 0.58
1
2
3
4
5
6
7
8
9
10
11
15

From this study, it is concluded that the synthesized ILs are susceptible to biodegradation,
especially those whose biodegradation of organic carbon exceeds 50% and, in some cases,
reaches up to 90% even within the first five days. Through a more extensive examination of
the anions’ and cations’ effect on the biodegradability of the examined ILs, some important
information can be gathered.
3.1.1. Cation effect on the ILs biodegradability
According to the CBOD5/UCBOD results of the benzoic acid-based ILs (BAILs, Table 5), it
is clearly observed that the effect of the cationic part is considerable on the organic carbon
removal. More specifically, by comparing PIL 6 with 8, and 7 with 9 it can be easily
concluded that the hydroxyl groups favour the biodegradation process. This observation is in
accordance with other authors’ findings where it is remarked that the insertion of hydroxyl
groups into long chained ILs might be advantageous for biodegradability [29]. Moreover,
according to the study of Boetheling et al. [23], it is proposed that groups which consist
possible sites of enzymatic hydrolysis, like hydroxyl groups, may lead to increased aerobic
biodegradability of the examined substance.
Table 5. Effect of BAILs cation on their (%) biodegradability
BAILs
6
Structure
CBOD5/UCBOD (%)
91.5 ± 8.8
90.3 ± 4.0
73.7 ± 0.9
7
8
16

27.1 ± 3.9
9
Almost the same trend seems to be followed by the biodegradability rates of the
cyclohexanecarboxylic acid-based ILs 3-5 (Table 6). In particular, by maintaining the
cyclohexanoate anion, it is observed that the IL 5 shows an increased biodegradability up to
26% compared to 4.
Moreover, from these results it can also be assumed that the biodegradability is not affected
by the amine’s class since the biodegradability percentage from the primary amine-derived IL
3 is almost identical to secondary amine-derived IL 4. This observation agrees with the
results of ILs 6 and 7.
Table 6. Effect of cyclohexanecarboxylic acid based-NAILs cation on their (%)
biodegradability
NAILs
3
Structure
CBOD5/UCBOD (%)
52.3 ± 1.5
52.8 ± 3.2
4
5
79.0 ± 13.7
Comparing ILs 1 and 2 (Table 7), a different trend is observed. It seems that the primary
amine-derived IL 1 exhibits overall almost double biodegradation rate compared to the
secondary amine-derived IL 2.
17

Table 7. Effect of the cyclopentanecarboxylic acid based NAILs cation on their (%)
biodegradability
NAILs
1
Structure
CBOD5/UCBOD (%)
44.5 ± 0.6
24.5 ± 0.1
2
Regarding the 2-hydroxy-benzoic acid based ILs 10 and 11, similar biodegradation rates are
exhibited (Table 8).
Table 8 Effect of the 2-hydroxy-benzoic acid based BAILs cation on their (%)
biodegradability
BAILs
10
Structure
CBOD5/UCBOD (%)
33.1 ± 2.8
30.6 ± 0.58
11
3.1.2. Anion effect on the ILs biodegradability
The influence of the anion moiety of the ILs on their biodegradability is identified by
comparing the results presented in Table 9. More specifically, the effect of the aromatic ring
and the alicyclic ring on the biodegradation rate is examined, by maintaining the selected
18

amine constant each time. According to literature data, ILs derived from surrogate naphthenic
acids possess biodegradability levels up to 60% during 28 day incubation [25].
Table 9. Effect of NAILs and BAILs anion on their (%) biodegradability
CBOD5/
CBOD5/
ILs
1
Structure
ILs
7
Structure
UCBOD (%)
UCBOD (%)
44.5 ± 0.6
90.3 ± 4.0
24.5 ± 0.1
52.3 ± 1.5
52.8 ± 3.2
73.7 ± 0.9
27.1 ± 3.9
33.1 ± 2.8
2
3
4
8
9
10
79.0 ± 13.7
30.6 ± 0.58
5
11
It is observed that between ILs 8 and 3, as well as between ILs 7 and 5, the ILs with an
aromatic ring on the anion exhibit higher biodegradation rates (up to 20%) compared to
cyclohexane carboxylate ILs. It is possible that the aromatic character positively affects the
biodegradation process. This observation comes in agreement with other bibliographic data
[30] according to which the aromatic ring positively affects aerobic biodegradation.
However, this trend is not followed by ILs 9 and 4.
Moreover, the number of carbon atoms of the alicyclic ring seems to affect the ILs’
biodegradability. ILs 3 and 4 (derived from cyclohexanoic acid) produce higher
19

biodegradation rates (between 10-28%) compared to ILs 1 and 2 (derived from
cyclopentanoic acid).
Regarding the presence of the hydroxyl group as anion substituent of the BAILs, it seems that
biodegradability is strongly hindered, since IL 7 (derived from benzoic acid and
diethanolamine) presents a biodegradability of 90.3% while the biodegradability of IL 10
(derived from 2-hydroxy-benzoic acid and diethanolamine) is only up to 33.1%. On the
contrary, the presence of the hydroxyl groups on the cations has a positive effect on
biodegradation compared to ILs which have a methoxy group at the corresponding position in
the cation.
Overall, from the experimental data obtained, it is observed that both the anionic and cationic
part greatly contribute to the final biodegradability level of the whole series of the studied
PILs, which seems to be in agreement with other bibliographic references [23].
3.2. Toxicity Evaluation
The majority of ILs are water soluble so their aquatic toxicity is considered important to be
evaluated in case of a possible realize in the aquatic ecosystem. In this work, the brine shrimp
lethality assay was used to assess the toxicity of the synthesized PILs. The lethality exerted
on the nauplii of the brine shrimp A. salina has been widely used in laboratory bioassays as a
predictor of chemical toxicity on organisms in aquatic environments through the estimation
of the medium lethal concentration (LC50). The crustacean A. salina L. (Artemiidae), is an
invertebrate organism residing in saline aquatic ecosystems and has been widely used in
laboratory bioassays for toxicological applications [31]. This test has been already
successfully used in the estimation of ILs toxicity [24,32].
20

The toxicity results of the synthesized PILs on the brine shrimp after 24 h of incubation are
presented in Table 10 and it is worth noting that all the tested PILs, were non-toxic to the
nauplii of A. salina, while, most of them exhibited LC50 values above 1000 mM.
Table 10. ILs Toxicity on the nauplii of the brine shrimp A.salina
IL
1
LC50 (mM)
331.9 ± 15.7
>1000
2
283.8 ± 11.3
>1000
4
5
>1000
6
>1000
7
>1000
9
403.6 ± 14.6
>1000
10
11
3.3. Differential Scanning Calorimetry (DSC)
Many applications demand ILs with low viscosity values and high conductivity at room
temperature. Both these properties are correlated and strongly depended on glass transition
temperatures (Tg), so the Τg value determination is considered crucial. The thermal behavior
and Tg of the studied ILs have been investigated by DSC.
The (second) heating DSC plots of selected NAILs and BAILs are illustrated in Fig.3-4 and
o
Fig. 5-6 respectively and the estimated Tg along with heat capacity increment ꢂCp (J/g C),
are presented in Table 11. The temperature accuracy +/−0.1°C and sensitivity 0.2 ꢀW. Due
to the fact that aging effects contribute to all heating thermograms (overshooting in the
21

heating scans), the Tg was determined by using the fictive temperature method in Universal
Analysis 2000 software (TA Instruments) for all the ILs.
The Tg is indicative of the ILs cohesiveness. So, low cohesive energy means low Tg values.
The cohesion energy is reduced by the Pauli’s repulsion energy due to the orbits overlapping,
while is increased through Coulomb, van der Waals and hydrogen bonds attractive
interactions [33]. On the other hand, the glass transition temperature is also determined by the
agility of the structural units and the steric hindrances that may exist in their movement. The
combination of intermolecular interactions and mobility of structural units greatly determines
the Tg. Thus, the ILs with the lowest Tg values are characterized by greater mobility of their
molecules and, possibly, weaker intermolecular interactions. According to previous studies
on alkylammonium PILs, the Tg increases when the alkyl chain length increases [33] but also
increases with the presence of a hydroxyl group [24,33]. Low Tg values could be an
indication that the ILs are likely to have desirable physicochemical properties, such as low
viscosity and high ionic conductivity at room temperature [33,34].
Table 11. Tg (°C) and ꢂCp (J/g °C) of NAILs and BAILs 1-11.
IL
1
Structure
Tg (°C) ∆Cp (J/g °C)
−80.5
−89.4
−78.9
−61.8
−105.7
0.38
0.13*
0.46
2
4
0.54
5
0.10*
6
22

−49.9
−74.4
−46.1
0.54
0.50
0.53
7
9
10
−59.7
0.44
11
*Semi-crystalline structure
The ΝΑILs Tg values derived from cyclopentane carboxylic acid range between −80.5 to
−89.4 °C (Fig. 3). As for the IL 2, the curve during the second heating run, exhibits an
endothermic peak (ꢂH = 134.6 J/g) at 92.2 °C that possibly corresponds to melting effects.
Consequently, the reduced value of ꢂCp (=0.13 J/g °C) may be attributed to the semi-
crystalline morphology of the sample.
Figure 3. DSC behavior of NAILs 1-2
For the ILs 4 and 5 which possess a cyclohexane carboxylate anion, the Tg ranges between ‒
61.8 to ‒78.9 °C. In Fig. 4 is perceived the effect of methoxy group on the NAILs cations
23

compared to the hydroxyl group presence. It seems that the hydroxyl groups in the cationic
part favor the formation of hydrogen bonds within the IL and thus the IL exhibits higher Tg
values.
Figure 4. Comparison of DSC behavior of NAILs 4-5
This is an observation that is also confirmed by the results of our previous study [24] where
cyclopentane carboxylate ILs derived from ethanolamine and diethanolamine exhibited
higher Tg values (−68.5 and −66.8 °C respectively) compared to NAILs 1 (Tg= −80.5 °C) and
2 (Tg= −89.4 °C).
The same trend is followed by the BAILs (Fig. 5). Specifically, diethanolamine derived
BAILs that possess two hydroxyl groups on the cations scaffolds, like ILs 7 (Tg= −49.9 °C)
and 10 (Tg= −46.1 °C) respectively, exhibit higher Tg values compared to those that have one
hydroxyl group (IL 6, Tg= −105.7 °C) or no hydroxyl group (IL 11, Tg= −59.7 °C). Τhe
presence of a hydroxyl group on the anionic part also affects the Tg values. The effect is
slight in case of diethanolamine based BAILs (7 and 10) but seems to be more intense in case
of bis(2-methoxy-ethyl)ammonium ILs 9 and 11 where no hydroxyl groups are present on the
24

cation. Furthermore, considering all 4 ILs we observe that Tg increases with increasing total
number of OH-groups (TgIL9 < TgIL11 < TgIL7 < TgIL10).
Figure 5. Comparison of DSC behavior of BAILs 6-7 and 9-11
From the examined ILs, IL 6 is solid at room temperature. From the DSC analysis, the first
cooling scan gives two exothermic peaks at 65.7 °C and 29.9 °C with ꢂH values equal to
84.57 J/g and 4.53 J/g respectively (Fig. 6, left) indicating the ILs crystallization. Therefore,
in the following heating stage (second heating for the specimen), the IL is in a semi-
crystalline state something that is indicated from the very small glass transition step (ꢂCp).
The second heating scan, shows that IL 6 has a melting point at 133.4 °C giving an
endothermic peak with ꢂH = 134.6 J/g, while at 3.12 °C appears an exothermic peak due to
cold crystallization (ꢂH = 22.55 J/g) (Fig. 6, right). Thus, DSC measurements reveal the
strong tendency of IL 6 to crystallize.
25

Figure 6. DSC Diagram of 1st cooling cycle (left) and of 2nd heating cycle (right) of BAIL 6
3.4. Viscosity Measurements
Viscosity is an important factor of understanding ILs’ behavior as well as one of the most
useful properties to determine the possible applications of ILs. Viscosity is the result of
numerous interactions such as Coulombic, Van der Waals and hydrogen bonding and is
highly dependent on many parameters such as the shape and the size of the ILs ions [35-37].
It is common ILs to show a non-Arrhenius behavior, hence the variation of the viscosity as a
function of temperature is well described by the Vogel–Tamman–Fulcher (VTF) equation 1
[35].


B
n(T ) = no exp
Eq. 1




T − To
In this work, the viscosity of some of the synthesized ILs is studied with respect to chemical
structure variations on the anions and cations. More specifically, the effect of hydroxyl
groups’ existence on either ions of ILs on their viscosities is investigated. Viscosity was
measured at temperatures from 20 to 90 °C, and the results span a wide viscosity range (cP),
with viscosities decreasing as temperature increases (Table 12). For the studied ILs, viscosity
increases rather rapidly at low temperatures and decreases asymptotically to smaller values
towards higher temperatures.
26

The viscosity of ILs 4 and 5 and also ILs 7 and 10 was determined with an accuracy of ±2%
and reported at Table 12.
Table 12. Viscosity values of ILs 4, 5, 7 and 10 at atmospheric pressure (accuracy ±2%).
IL
T(°C)
20
4
5
7
10
n(cP)
87.8
63.6
46.2
26.7
16.7
-
-
-
-
-
6310.0
9860.0
3436.7
1406.7
646.0
327.3
183.5
109.6
-
25
3900.0 7360.0
1668.7 2644.3
30
40
779.7
398.2
217.5
126.5
78.2
1109.0
523.8
273.2
154.1
93.8
50
60
-
70
-
80
-
90
Concerning the structure-logarithm of viscosity dependence, the data of this study (Fig. 7)
indicates a significant potential effect on the viscosities of the ILs by the presence of hydroxy
group on both anions and cations. The effect of the hydroxyl groups in the cationic part is
apparently stronger than the effect evident by a hydroxyl group in the anionic part.
27

Figure 7. Logarithm of viscosity dependence of ILs 4, 5, 7 & 10 in different temperatures.
According to the data above, IL 5, which possesses two hydroxyl groups on the cation, has
almost twice the viscosity value of IL 4 that lacks a hydroxyl group. In the case of IL 10,
derived from diethanolamine and 2-hydroxy-benzoic acid, an even greater increase of
viscosity is observed compared to IL 7 (derived from diethanolamine and benzoic acid).
These observations can be considered reasonable since the increase of the hydrogen bonds,
due to the presence of the hydroxyl groups, leads to the increased cohesion energy.
Having determined the Tg of the examined ILs and utilizing the VFT equation, viscosity
values can be compared with each other on the basis of the Tg-scaled Arrhenius plots. In most
cases, the logarithm of viscosity on Tg/T is used to assess the fragility of the materials based
on whether viscosity follows the Arrhenius equation at temperatures higher than the Tg
(0<Tg/T<1). A greater deviation from the linear trend indicates increased fragility, which
means that as the temperature increases, the viscosity decreases at a faster rate than the
Arrhenius ratio. Therefore, strong liquids are those in which a linear increase in the logarithm
of viscosity as a function of Tg/T is observed, i.e. the viscosity follows the Arrhenius
equation. The fragility of liquids above the Tg is of great importance as the increased fragility
corresponds to a greater viscosity reduction as temperature increases [33,34].
28