Room temperature deep eutectic solvents of (1S)-(+)-10-camphorsulfonic acid and sulfobetaines: Hydrogen bond-based mixtures with low ionicity and structure-dependent toxicity

Article abstract of DOI:10.1039/c5ra03932k

Twelve novel deep eutectic solvents (DESs) were prepared and characterized in this work. They are mixtures of (1S)-(+)-10-camphorsulfonic acid (CSA) and differently structured sulfobetaines (SBs) with aliphatic, aromatic and amphiphilic moieties. They are liquids at room temperature, their melting points span, in fact, from -5° to 19 °C, so we can name these mixtures RTDESs (room temperature deep eutectic solvents). These zwitterionic DESs were characterized in terms of their viscosity, conductivity (and therefore ionicity via Walden plots), density, surface tension and toxicity on eukaryotic model cells. The collected data suggest that the interaction between CSA and the SBs can be ascribed as a hydrogen bond instead of a proton transfer, therefore they are not ionic liquids. To our knowledge, their position on the Walden plot, in the left portion close to the diagonal, has not yet been observed for other DESs or ionic liquid systems and indicates the low ionicity of these mixtures. A FTIR-based bioassay was performed to determine the toxicity of these mixtures on eukaryotic model cells (Saccharomyces cerevisiae). The DESs showed merely a dehydrating effect on the cells, similar to that produced by CaCl₂, a low cell toxicity salt. This supports these DESs as promising green media. Amphiphilic SBs DESs showed a stronger effect on the cells and a structure-activity trend can be described for this class. A preliminary study on the use of these novel DESs as Bronsted catalyst media was accomplished by the use of one of them in chalcone synthesis, which showed promising catalytic and recycling capabilities.

Full text of DOI:10.1039/c5ra03932k

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Room temperature deep eutectic solvents of
(1S)-(+)-10-camphorsulfonic acid and
Cite this: RSC Adv., 2015, 5, 31772
sulfobetaines: hydrogen bond-based mixtures with
low ionicity and structure-dependent toxicity†
a
a
Fabio Cardellini, Raimondo Germani, Gianluigi Cardinali,ab Laura Corte,b
*
Luca Roscini,b Nicoletta Spretic and Matteo Tieccoab
Twelve novel deep eutectic solvents (DESs) were prepared and characterized in this work. They are mixtures
of (1S)-(+)-10-camphorsulfonic acid (CSA) and differently structured sulfobetaines (SBs) with aliphatic,
aromatic and amphiphilic moieties. They are liquids at room temperature, their melting points span, in
fact, from ꢀ5ꢁ to 19 ꢁC, so we can name these mixtures RTDESs (room temperature deep eutectic
solvents). These zwitterionic DESs were characterized in terms of their viscosity, conductivity (and
therefore ionicity via Walden plots), density, surface tension and toxicity on eukaryotic model cells. The
collected data suggest that the interaction between CSA and the SBs can be ascribed as a hydrogen
bond instead of a proton transfer, therefore they are not ionic liquids. To our knowledge, their position
on the Walden plot, in the left portion close to the diagonal, has not yet been observed for other DESs
or ionic liquid systems and indicates the low ionicity of these mixtures. A FTIR-based bioassay was
performed to determine the toxicity of these mixtures on eukaryotic model cells (Saccharomyces
cerevisiae). The DESs showed merely a dehydrating effect on the cells, similar to that produced by CaCl2,
a low cell toxicity salt. This supports these DESs as promising green media. Amphiphilic SBs DESs
showed a stronger effect on the cells and a structure-activity trend can be described for this class. A
preliminary study on the use of these novel DESs as Brønsted catalyst media was accomplished by the
use of one of them in chalcone synthesis, which showed promising catalytic and recycling capabilities.
Received 5th March 2015
Accepted 26th March 2015
DOI: 10.1039/c5ra03932k
www.rsc.org/advances
which includes: low vapour pressure, high thermal stability and
non-ammability. Moreover, they have broad liquid ranges and
very high solubilizing capabilities.5,6 Unfortunately, ILs were
Introduction
The development of new reaction media plays a key-role within
the green chemistry framework. The goal of obtaining novel
media with low environmental impact represents, in fact, a
challenge in modern chemistry, considering the toxicity and the
high vapour pressure of traditional organic solvents.1–3 Ionic
liquids (ILs) are surely the most studied and developed solvents
in this eld in the last decades. ILs are formed by organic
cations and organic or inorganic anions, and theyꢁare liquid at
room temperature or at temperatures under 100 C.4 ILs have
important physico-chemical advantages in terms of “greenness”
compared to typical organic solvents, a non-exhaustive list of
found to be expensive and toxic.7–10
For these reasons bio-based mixtures such as Bio-ILs11 and
Bio-based solvents12 have gained relevance in literature more
recently. The Bio-based solvents class includes all the mixtures
with natural and biomass-derived substances, e.g. glycerol,13
lactic acid,14 g-valerolactone,15 limonene,16 and water solutions
of carbohydrates.17,18
Among these systems, deep eutectic solvents (DESs) repre-
sent a novel promising class of green solvents.19 DESs are a
family of solvents generally liquid at temperatures lower than
100 ꢁC. They can be prepared by simply mixing high-melting
point onium salts, such as ammonium or phosphonium, with
neutral compounds. The hydrogen-bond interaction that occurs
within the donor (HBD) and the acceptor (HBA) molecules
determines a considerable reduction of the melting point of the
mixtures, therefore a liquid system formation.20,21 For their
simplicity of preparation, the atom economy, and the low cost
of many starting products, DESs have gained particularly
attention as substitutes of conventional volatile organic
aCEMIN, Centre of Excellence on Nanostructured Innovative Materials, Department of
Chemistry, Biology and Biotechnology, University of Perugia, via Elce di Sotto 8,
I-06123 Perugia, Italy. E-mail: raimondo.germani@unipg.it
bDepartment of Pharmaceutical Sciences – Microbiology, University of Perugia, via
Borgo XX giugno 74, I-06121, Perugia, Italy
cDepartment of Physical and Chemical Sciences, University of L'Aquila, via Vetoio,
Coppito, I-67100, L'Aquila, Italy
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra03932k
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solvents in many chemical processes. DESs have found multiple
Liquid mixtures of sulfobetaines compounds with strong
applications in chemistry and in different technological appli- acids, such as sulfuric acid, are reported in literature.57–60 These
cations such as: electrochemistry,22–25 drug delivery,26 organic systems were obtained by mixing water solutions of these
synthesis,27–31 solubilization32–34 and bioconversion.35–37
molecules, then removing water by evaporation under vacuum.
The binary system choline chloride (ChCl)–urea was the rst The complete removal of water from ILs formed with mineral
DES developed, and it is the most widely studied and used.38–42 acids (e.g. H2SO4) can be very hard to obtain. Moreover, the
Other HBD compounds are able to form DESs in mixture with small difference of pKa values between the two sulfonic groups
ChCl such as phenols,43 carboxylic acids,44 glycerol,45 sugars,46,47 (sulfobetaines pKa z ꢀ2; sulfuric acid pKa1 ¼ ꢀ3, pKa2 ¼ 1.9)
and alcohols.48 In the cases of mixtures of natural compounds, suggests that the proton cannot be completely transferred from
such as amino acids, sugar and carboxylic acids, these systems one molecule to another, but indicates a more probable
are named Natural Deep Eutectic Solvents (NADES)49 because hydrogen bonding interaction formation.
derived from natural resources.
In this work we demonstrate that the interaction between
The interaction of the anion of the salt, which is usually a sulfobetaines and a sulfonic acid leads to a deep eutectic solvent
chloride, with the HBD molecule is the driving force that leads instead of IL. This is due to similar pKa values of CSA and sul-
to the reduction of the melting points of the mixtures, therefore fobetaines (e.g. ArSO3H pKa ¼ ꢀ2.5; PTSA pKa ¼ ꢀ2.8; MsOH
on liquid systems. Halides can be nucleophilic and oxidizing pKa ¼ ꢀ1.9; CSA pKa ¼ 1.2). We prepared sundry DESs from
agents, and they can lead to unwanted by-products such as differently structured sulfobetaines: aliphatic, aromatic and
halogenidric acids, which are also dangerous. For these reasons amphiphilic sulfobetaines with propyl or butyl spacer were
a halogen-free DES is strongly desirable. For this purpose synthesized. These DESs were characterized in terms of their
zwitterionic systems such as betaines are interesting molecules, conductivity, viscosity, ionicity (via Walden plot), density, and
because they do not have a counterion that can impact on DES surface tension. Their toxicity on eukaryotic model cells, such as
properties. Moreover betaines are cheap and have low Saccharomyces cerevisiae yeast, was determined with
a
toxicity.50,51 FTIR-based bioassay we developed in the recent years.61–63 The
Nowadays, there are few examples of deep eutectic solvents advantage of the use of these DESs as reaction media and
that are based on zwitterionic systems (carboxy- or sulfobe- catalysts was valued with a Claisen–Schmidt reaction performed
taines). A series of DESs obtainable from glycine betaine with in one of the novel DESs.
different neutral hydrogen bond donor compounds has recently
been reported.52 In our previous paper we have shown how
carboxybetaine trimethylglicine is able to form DESs when
mixed with carboxylic acids.53 The depression of the melting
Experimental
Materials
point was established by the onset of the hydrogen bond
between the trimethylglicine carboxylate with the acid hydrogen
of the carboxylic group. These DESs are liquid at temperatures
lower than 70 ꢁC, they present a good viscosity and conductivity
values, they derive from bio-renewable resources and above all
they are not toxic.
This work is a continuation of our previous investigations to
nd novel, different and tailor-made deep eutectic solvents,
Solvents of analytical grade, (1S)-(+)-10-camphorsulfonic acid
99%, 1,3-propanesultone, 1,4-butanesultone, N,N-dimethyl-
butylamine, tributylamine, N,N-dimethyl-cyclohexylamine,
N-methyl-N-octyloctanamine, 1-methyl-imidazole, 1-butyl-imida
zole, pyridine, N,N-dimethyl-tetradecylamine, N,N-diethyl-tetrade
cylamine, N,N-dipropyl-tetradecylamine, benzaldehyde, aceto-
phenone and surfactants sulfobetaines N-dodecyl-N,N-dimethyl-
3-ammonio-1-propanesulfonate (SB3-12) and N-tetradecyl-N,N-
without halides ions, with Brønsted acid properties, to use as
dimethyl-3-ammonio-1-propanesulfonate (SB3-14) were purch-
solvents and catalysts.53–56 The focus of this study reported, was
ased from Sigma-Aldrich, Fluka and Alfa Aesar. 1-methyl-
imidazole, 1-butyl-imidazole and N-methyl-N-octyloctanamine
were distilled prior to use. Surfactant sulfobetaines SB3-12 and
SB3-14 were puried by double recrystallization from acetone–
to develop a new class of DESs using an organic solid sulfonic
acid as HBD compound and zwitterionic sulfobetaine mole-
cules as HBA. As sulfonic acid, commercially available
(1S)-(+)-10-camphorsulfonic acid (CSA) was chosen, because of
methanol mixture prior to use.
ꢁ
its high melting point (198 C) and because of the absence of
hydration water that can generate solutions instead of true
eutectic systems (Fig. 1).
Synthesis of sulfobetaines
The sulfobetaines were synthesized from 1,3-propanesultone
(1,4-butanesultone for SB4-14) by reaction with the appropriate
tertiary amine to reux in acetonitrile or toluene for 2–10 h. All
the molecules were synthesized according to previous proce-
dures reported in literature and their recorded melting points
and 1H-NMR spectra agree with the same literature data.
1H-NMR spectra were measured at 25 ꢁC with a Bruker 200 MHz
instrument in D2O or CDCl3 solutions with tetramethylsilane
(Me4Si) as internal standard. Melting points were obtained on a
Fig. 1 (1S)-(+)-10-Camphorsulfonic acid (CSA) structure.
Barloworld Scientic Stewart SMP3 micro melting point
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apparatus and are uncorrected. Agilent 6850 Series II Network
Gas Chromatography instrument (column DB-35MS l ¼ 30 m,
d ¼ 0.32 mm, lm ¼ 0.25 mm) was used for GC analysis. Values
of the critical micelle concentration (c.m.c.) were determined
from plots of surface tension vs. – log [surfactant]. No minima
could be observed in these plots. Surface tensions were
measured on a Fischer, du Nouy type tensiometer.
DESs melting points determination
In a 10 mL one-necked, round-bottomed ask, equipped with a
thermometer used as stopper, weighed amounts of sulfobetaine
and (1S)-(+)-10-camphorsulfonic acid were introduced. The
binary systems were heated until they became homogenous
liquids, and then were gradually cooled rst to room tempera-
ture, then immersed in a water–ice bath (or ice–NaCl mixture
for lower temperatures) for melting point determination.
The characterizations of all the molecules are reported here:
SBE3-14:64 Yield 80%, m.p. 134–135 ꢁC, 1H-NMR (200 MHz
CDCl3) d ¼ 3.59–3.72 (m, 2H, N+CH2), 3.28–3.46 (m, 4H,
N+(CH2CH3)2), 3.05–3.20 (m, 2H, RCH2N+), 2.92 (t, 2H,
CH2SO3ꢀ), 2.09–2.28 (m, 2H, N+CCH2C), 1.58–1.78 (m, 2H,
RCH2CN+), 1.38 (t, 6H, 2 CH3), 1.20–1.39 (m, 22H, 11 CH2), 0.89
(t, 3H, CH3), c.m.c. ¼ 2.55 ꢂ 10ꢀ4 M; SBP3-14:64 Yield 75%, m.p.
Viscosity measurements
The viscosity of the eutectic mixtures was measured by using a
Fungilab Expert L viscometer, tted with a thermostatic jacket
and a temperature probe. The viscometer jacket was connected
to an external thermostated bath. The viscosity measurements
were obtained using a spindle attachment. Readings were taken
aer 20–25 minutes for each temperature.
1
ꢁ
124–125 C, H-NMR (200 MHz CDCl3) d ¼ 3.55–3.66 (m, 2H,
N+CH2), 3.10–3.30 (m, 6H, (CH2)3N+), 2.92 (t, 2H, CH2SO3ꢀ),
2.10–2.30 (m, 2H, N+CCH2C), 1.58–1.89 (m, 6H, (CH2C)3N+),
1.20–1.40 (m, 22H, 11 CH2), 1.04 (t, 6H, 2 CH3), 0.89 (t, 3H, CH3),
c.m.c. ¼ 1.99 ꢂ 10ꢀ4 M; SB3-8 bis:64 Yield 94%, m.p. 157–158 ꢁC,
1H-NMR (200 MHz, CDCl3) d ¼ 0.89 (t, 6H, 2CH3); 1.29–1.62 (m,
24H, 12CH2); 2.20 (m, 2H, N–C–CH2–C–SO3); 2.90 (m, 2H,
–CH2–SO3); 3.11 (s, 3H, CH3); 3.22 (m, 4H, (R–CH2)2N–); 3.51–
3.66 (m, 2H, –N–CH2–C–C–SO3); SB3-MIM:65 Yield 98%, m.p.
218–219 ꢁC, 1H-NMR (200 MHz D2O) d ¼ 9–7 (s + d, 3H, Ar); 4.1–
4.2 (m, 2H, CH2N+); 3.7 (s, 3ꢀH, CH3N); 2.7–2.8 (t, 2H, CH2SO3ꢀ);
2.0–2.1 (m, 2H, CH2CH2SO3 ); SB3-BIM:66 Yield 95%, m.p. 176–
177 ꢁC, 1H-NMR (200 MHz, D2O) d ¼ 9–7 (m, 3H, Ar); 4–4.1 (dt,
4H, (CH2)2N+); 2.7–2.8 (t, 2H, CH2SO3ꢀ); 2.1 (m, 2H, CH2CH2-
SO3ꢀ); 1.6–1.7 (m, 2H, CH2CH2CH3); 1.1–1.2 (m, 2H, CH2CH3);
0.7–0.8 (t, 3H, CH3); SB3-Cy:67 Yield 98%, m.p. 115–116 ꢁC,
1H-NMR (200 MHz, D2O) d ¼ 3.2–3.5 (m, 3H. CH2N+ + CHN+);
2.7–2.8 (m, 8H, (CH3)2N+ + CH2SO3ꢀ); 2.0 (m, 2H, CH2CH2-
Conductivity measurements
Conductivity measurements were performed on an Analytical
Control conductometer (model 120) equipped with a platinum
cell (cell constant ¼ 1.05 cmꢀ1). Sample temperature was
maintained constant by a glass-jacked beaker connected to a
thermostatic bath. To ensure correlation between the results,
the same sample was used for conductivity and viscosity
experiments. The readings were taken aer 20–25 minutes for
each selected temperature.
Density measurements
The density values at eutectic ratio at different temperatures
were obtained by measuring the weight of the sample in a
2.00 mL volumetric ask. The asks were held in a thermo-
stated bath for 1 hour then brought to volume by eliminating
the amount of liquid with a Pasteur pipette. The ask was then
thermostated at room temperature for 1 hour and then weighed
on analytical balance.
ꢀ
SO3 ); 1.1–1.8 (m, 10H, CH2Cy); SB3-Py:68 Yield 97%, m.p. 277–
278 ꢁC, 1H-NMR (200 MHz D2O) d ¼ 9–7 (m, 5H, Ar); 4.5 (m, 2H,
CH2N+); 2.8–2.9 (t, 2H, CH2SO3ꢀ); (m, 2H, CH2CH2SO3ꢀ); SB4-
14:51 Yield 85%, m.p. 273–274 ꢁC, 1H-NMR (200 MHz CDCl3) d ¼
3.6 (m, 2H, N+CH2RSO3ꢀ), 3.1–3.3 (m, 8H, N+(CH3)2
+
N+CH2C13), 2.9 (t, 2H, CH2SO3ꢀ), 1.9 (m, 4H, CH2CH2CH2SO3ꢀ),
1.6–1.8 (m, 6H, N+CH2CH2C12 + N+(CH2CH2CH3)2), 1.3 (m, 22H,
Surface tension measurements
(CH2)11CH3), 0.85 (t, 3H, (CH2)13CH3), c.m.c. ¼ 2.80 ꢂ 10ꢀ4 M;
Surface tension of eutectic mixtures was measured at 25 ꢁC
using a Sigma 700 Force Tensiometer with a platinum ring
(diameter 1.9 cm).
1
SB3-4:64 Yield 98%, m.p. 270–271 C, H-NMR (200 MHz, D2O)
ꢁ
d ¼ 3.1–3.3 (m, 4H, (CH2)2N+); 2.9 (s, 6H, (CH3)2N+); 2.7–2.8 (t,
2H, CH2SO3ꢀ); 2.0–2.1 (m, 2H, CH2CH2SO3ꢀ); 1.5–1.6 (m, 2H,
CH2CH2CH3); 1.1–1.2 (m, 2H, CH2CH3); 0.8 (t, 3H, CH3); SB3-4
Tris:69 Yield 96%, m.p. 219–220 ꢁC, 1H-NMR (200 MHz, D2O)
FTIR bioassay measures
d ¼ 3.0–3.2 (m, 6H (CH2)3N+); 2.7–2.8 (t, 2H, CH2SO3ꢀ); 1.9–2.9 In order to test the activity of the compounds used in this study,
(m, 2H, CH2CH2SO3ꢀ); 1.5–1.6 (m, 6H (CH2CH2CH3)3); 1.1–1.3 the yeast strain Saccharomyces cerevisiae CMC 520 was employed
(m, 6H, (CH2CH3)3); 0.7 (t, 6H, CH3).
as target. It was obtained from the internal collection of the
Microbial Genetics and Phylogenetics Laboratory of the
Department of Pharmaceutical Sciences (University of Perugia)
and it is also deposited in the collection of the Centraalbureau
DESs preparation
(1S)-(+)-10-Camphorsulfonic acid (CSA) was dried under voor Schimmelcultures (CBS) as CBS 13873. The D1/D2 domain
vacuum over P2O5 prior to use. The sulfobetaine and CSA, at of the LSU (26S) gene showed that this strain is 99.8% similar to
proper molar ratio, were directly weighed in a ask tted with the species type strain, thus allowing to identify it as an
a stopper. The solid mixture was magnetically stirred and authentic S. cerevisiae strain70 (data not shown).
heated at 90 ꢁC until a liquid was formed (typically 20–30
minutes).
Pre-culture was inoculated at OD600 ¼ 0.2 in 200 mL of YEPD +
chloramphenicol medium (Yeast Extract 1%, Peptone 1%,
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Dextrose 2% – chloramphenicol 0.5 g Lꢀ1 – Difco Laboratories,
Detroit, MI, USA) and grown 18 h at 25 ꢁC, under shaking at
150 rpm. Cell suspension was centrifuged at 4143g (4500 rpm) for
3 min, resuspended in distilled sterile water, divided in fourteen
50 mL tubes, washed twice with distilled sterile water and pel-
leted. A 2 g weight for each DES and for anhydrous CaCl2 (Sigma
Aldrich), was sampled in thirteen 50 mL tubes and the previously
prepared cell pellets were added to these tubes. The control
consisted of pelleted cells. The same experiment was conducted
with CaCl2$2H2O, leading to the same results (data not shown).
The tubes were incubated 2 min at 25 ꢁC, then 5 mL of
distilled sterile water were added in each tube, centrifuged
3 min at 4143g, washed twice with distilled sterile water and
re-suspended in 1.5 mL HPLC grade water in polypropylene
tubes. A 105 ml volume was sampled for three independent FTIR
readings (35 ml each, according to the technique suggested by
Manfait and co-workers71), while 100 ml were serial diluted to
determine the viable cell counting of tests and control
suspension, in triplicate, on YEPDA plates. The biocidal effect of
the compounds was tested as cell mortality induced by
these compounds. The cell mortality (M) was calculated as
M ¼ [(1 ꢀ Cv)/Ct] ꢂ 100.
Chalcone synthesis procedure
0.33 mmol of benzaldehyde and 0.33 mmol of acetophenone
were added to SB3-Cy–CSA DES (1 mmol CSA, 0.7 mmol SB3-Cy)
under magnetic stirring. The reaction was conducted at 90 C
ꢁ
for 16 h. The mixture was then cooled to room temperature and
then transferred into a separator funnel and washed ve times
with ethyl acetate–H2O. The organic phase was dried under
Na2SO4 and then ltered. The solvent was evaporated under
vacuum to give a orange solid. The product was puried via
chromatographic column with H2O–EtOH 80 : 20 mixture
as eluent. A pale yellow solid was obtained. Yield 70%, m.p.
56–58 ꢁC; 1H-NMR (200 MHz, CDCl3) d ¼ 8.0 (d, 2H, H-b + H-40);
7.8 (d, 1H, H-a); 7.6 (d, 2H, H-20, H-60); 7.5 (dt, 4H, H-2, H-6,
H-30, H-50); 7.4 (m, 3H, H-3, H-4, H-5).
The conversion was determined in a separate experiment via
addition to the reaction mixture of a specic amount (approx-
imately 1 : 1 of molar ratio with reagents) of tert-butylbenzene
(Sigma-Aldrich, 99%) as internal standard aer 16 h. The
conversion was determined via gas chromatography analyses.
Results and discussion
DESs preparation
The FTIR experiments were carried out with a TENSOR 27
FTIR spectrometer, equipped with HTS-XT accessory for rapid
automation of the analysis (BRUKER Optics GmbH, Ettlingen,
Germany).
The synthesis of several sulfobetaines was the rst step in DESs
preparation. The HBA molecules were synthesized via reaction
of tertiary amines with 1,3-propanesultone. The reaction
scheme is reported in Fig. 2.
The reaction is a SN2 substitution, which for ring opening
leads directly to the formation of the sulfobetaine without the
production of any side-product salt. Twelve differently struc-
tured sulfobetaines were synthesized, with aliphatic, aromatic
and amphiphilic moiety (Table 1). 1,4-Butanesultone was used
for the synthesis of 4-(tetradecyldimethylammonio)butan-1-
sulfonate, which has a tetramethylene spacer between the
ammonium and the sulfonate groups.
FTIR measurements were performed in transmission mode.
All spectra were recorded in the range between 4000 and
400 cmꢀ1. Spectral resolution was set at 4 cmꢀ1, sampling 256
scans per sample. The soware OPUS version 6.5 (BRUKER
Optics GmbH, Ettlingen, Germany) was used to carry out the
quality test, baseline correction, vector normalization and the
calculation of the rst and second derivatives of spectral values.
IR data analyses
The second step in DESs preparation was eutectic melting
point determination of SBs–CSA mixtures. The mixtures were
prepared by simply mixing the compounds at 90 ꢁC, and
resulted amber liquids for all the twelve betaines at this
temperature. The melting points of the DESs were determined
by varying the molar fraction (x) of the (1S)-(+)-10-Cam-
phorsulfonic acid. In Fig. 3 are reported the proles of melting
point vs. molar fraction x of SB3-Py–CSA and SB3-14–CSA
mixtures (see ESI† section for all the proles).
A lowering of the melting point (compared to the ones of CSA
and of the SB) was observed for 0.2 molar fraction of CSA–SB
mixtures for all the betaines. A further stronger decrease was
observed for 0.5 molar fraction. Little variations were observed
for subsequent additions of acid until 0.7 molar fraction.
The script MSA (Metabolomic Spectral Analysis) employed in
this study was developed in “R” language to carry out the
statistical analysis on the matrices of spectral data exported as
ASCI text from OPUS 6.5. The analytical procedure consisted in
normalizing raw spectra as suggested by Goodacre and coll.,72
calculating the Response Spectra (RS) as described by Corte and
colleagues61 and nally by calculating the correlations between
normalized spectra and between RS. RS are dened as the
difference between the average spectrum of the cells challenged
by the tested compound and that of the cells resting in water for
the same time. In order to highlight the differences exerted not
only by single compounds but also by their class of appurte-
nance (i.e. amphiphilic and non-amphiphilic SBs DESs), the
single RSs of the members of the same class were averaged
producing the Average Response Spectra (ARS). Amphiphilic
SBs–CSA mixtures: SB3-14, SBE3-14, SBP3-14, SB3-12, SB4-14,
SB3-8 bis–CSA. Aromatic–aliphatic SBs–CSA mixtures:
SB3-MIM, SB3-MIM, SB3-4, SB3-4 Tris, SB3-Cy, SB3-Py–CSA. The
spectral regions (W1, W2, W3, W4, W5) are reported as sug-
gested by Naumann and colleagues.73
Fig. 2 Preparation of sulfobetaines.
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Table 1 Structures of sulfobetaines (SBs) and eutectic properties of their mixtures with (1S)-(+)-10-Camphorsulfonic acida
Entry
1
Sulfobetaine structure
Acronym
SB3-12
DES eutectic ratio
1.5
SB m.p. (ꢁC)
DES m.p. (ꢁC)
Dt (ꢁC)
259
ꢀ1
ꢀ3
260
2
3
SB3-14
1.5
1.5
256
134
259
130
SBE3-14
4
3
4
5
SBP3-14
1.5
1.5
124
157
121
162
SB3-8 bis
ꢀ5
6
7
SB3-MIM
SB3-BIM
1.5
1.5
219
177
ꢀ4
223
177
0
8
SB3-Cy
1.5
116
9
107
9
SB3-Py
SB4-14
SB3-4
1.5
2.0
2.0
278
273
270
19
ꢀ5
13
259
278
257
10
11
12
SB3-4 Tris
2.0
219
9
210
a
m.p. of CSA ¼ 196 ꢁC.
In Table 1 are reported: the molar eutectic ratios of the DESs, provoke a folding of the chain that determines an increasing of
the melting points of the sulfobetaines (SBs), the melting points interactions between the positive and the negative charges so a
of the DESs and the Dt of melting point between the SBs and the higher amount of CSA is needed to form the DES. This partial
DESs observed for all our twelve mixtures.
neutralization of the charges has also impact on the solubility in
All the mixtures showed a eutectic ratio of 1.5, while SB4-14, water of SB4-14 sulfobetaine (0.022 M) that is much lower
SB3-4 and SB3-4 Tris–CSA mixtures (entry 10, 11, 12) showed a compared to SB3-14 molecule (0.92 M).64
2.0 molar eutectic ratio value. For SB3-4 and SB3-4 Tris–CSA
There is a strong depression of the melting point of the
mixtures, these values could be due to the small dimensions mixtures (Dt from 107 to 273 ꢁC) in all the realized systems: this
and to the symmetry of the sulfobetaines. These can impact on is indicative of strong interactions between the SB and the
their packing capability, so they need a higher amount of CSA to CSA.19 Moreover the decrease of entropy, therefore a lower Dt, is
form a DES. SB4-14 sulfobetaine has a longer spacer and it can generally related to the structure of the crystal lattice of solid SB.
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lower symmetry than SB3-4 Tris–CSA mixture (entry 11, 12) but
the three butyl chains are very bulky and lead to a more difficult
packing. SB3–Cy mixture (entry 8) presented a lower value of Dt
than the linear alkyl SB realized in this work, because of the
lower van der Waals interactions in the cyclohexyl group
therefore lower lattice symmetry. SB3-Py–CSA mixture (entry 9)
led to a great Dt value, due to the p–p interactions in SB
structure. This DES has the highest melting point in our set
ꢁ
(19 C) but it is close to the melting points of the other DESs.
The melting points of the DESs are quite similar for all the
mixtures realized in this work, and they span between ꢀ5 ꢁC to
ꢁ
19 C, so we can name these mixtures RTDESs (room temper-
ature deep eutectic solvents). This could indicate that the
driving forces that determine the liquid formation are the same
for all the DESs prepared, because they involve the same
portions of the molecules for all the mixtures.
Fig. 3 Melting points of SB3-Py and SB3-14 with CSA as a function of
acid molar fraction x. Legend (hollow triangles) SB3-Py–CSA mixture,
(solid circles) SB3-14–CSA mixture.
Viscosity
The viscosity of the DESs is exponentially dependent on the
temperature following the Arrhenius equation (eqn (1)).
The greater the symmetry of the lattice is, the greater the
decreasing of Dt is provoked.
In the SBR3-14–CSA mixtures series (entry 2, 3, 4) an increase
of steric hindrance of alkyl residues (R ¼ Me, Et, n-Pr) on
ammonium led to a decrease of Dt. The increase of the bulki-
ness of the ammonium group of sulfobetaines leads, in fact, to a
lower interaction between the positive and the negative charges.
In this case the coulomb interaction is lower and the related
entropic gain is smaller. This effect is relevant also for SB4-14–
CSA mixture (entry 10) where a higher Dt decrease is present:
the opposite charge vicinity provokes a higher increase of
entropy gain. In SB3-8 Bis, SB3-12 and SB3-14 mixtures a
decrease in chain length led to a reduction of Dt. SB3-8 Bis–CSA
mixture showed a smaller Dt than SB3-12–CSA (entry 1, 5)
because it has a lower symmetry of the SB, due the two bulky
octyl chains. The same reasons can be considered for SB3-MIM
and SB3-BIM mixtures (entry 6, 7). The butyl chain caused lower
symmetry in the sulfobetaine lattice of SB3-BIM and lower
entropy change in presence of CSA. SB3-4–CSA system have
Eh
ln h ¼ ln h0 þ
(1)
RT
Viscosity is related to the radius of the vacancies in the
structures compared to the radius of the molecules.74,75 Natural
logarithm of h vs. 1/T proles of our DESs are reported in Fig. 4.
The DESs from aliphatic and aromatic SB are reported in panel
A, the ones from amphiphilic SB in panel B. The viscosities (h)
and viscosity activation energies (Eh) measured for our DESs are
reported in Table 2.
Viscosity values observed in SBR3-14 series followed a good
linear correlation with Arrhenius law. The most viscous system
in this class is the SB3-14–CSA mixture (2161 cP, entry 2), while
SBE3-14–CSA and SBP3-14–CSA mixtures showed values of
1030 cP and 1673 cP respectively (entry 3 and 4). The two small
methyl groups on ammonium in SB3-14 mixture provoked a
strong interaction and therefore very low vacancies radius. The
Fig. 4 Plot of ln viscosity vs. reciprocal of temperature for SB–CSA mixtures. Legend Panel A (aromatic–aliphatic SB): (solid squares) SB3-4 Tris–
CSA mixture, (solid circles) SB3-4–CSA mixture, (hollow triangles) SB3-Py–CSA mixture, (hollow squares) SB3-Cy–CSA mixture, (hollow circles)
SB3-MIM–CSA mixture, (solid triangles) SB3-BIM–CSA. Panel B (Amphiphilic SB): (hollow squares) SB3-12–CSA mixture, (hollow triangles) SB3-8
bis mixture, (solid squares) SB3-14–CSA mixture, (solid triangles) SBE3-14–CSA mixture, (hollow diamonds) SBP3-14–CSA mixture, (solid dia-
monds) SB4-14–CSA mixture.
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Table 2 Viscosity and viscosity activation energy of SB–CSA DESs
hindrance led to an increase of Eh. The presence of two alkyl
chains in SB3-8 bis (entry 5) increased the Eh value that became
similar to 14-chain sulfobetaines. In the aromatic SB mixtures
(entry 6, 7, 9), imidazolium DESs presented similar values of Eh,
with a higher energy for SB3-BIM due to the alkyl chain, while
the pyridinium SB gave a higher Eh value. SB3-Cy–CSA mixture
(entry 8) showed an activation energy higher than the ones with
linear chain. The cyclohexyl-, in fact, can provoke good side–side
packing in the liquid structure. As well as its high viscosity, SB3-4
DES (entry 11) presented Eh ¼ ꢀ98 kJ molꢀ1, the highest value in
our set. The value of Eh for SB3-4 Tris (entry 12) is lower than SB3-
4 mixture. The presence of three butyl-chains could provoke a
decrease of symmetry. SB4-14–CSA mixture (entry 10) showed an
Eh value similar to linear chain SB which was between SB3-14
and SBE3-14 values.
Entry
SB
ha (cP)
Ehb (kJ molꢀ1
)
1
2
3
4
5
6
7
8
SB3-12
SB3-14
3025
2161
1030
1673
1488
6556
2305
2617
3472
1268
5371
3735
ꢀ38.2
ꢀ41.4
ꢀ48.2
ꢀ48.8
ꢀ48.0
ꢀ30.5
ꢀ36.8
ꢀ56.0
ꢀ60.9
ꢀ44.7
ꢀ98.8
ꢀ48.5
SBE3-14
SBP3-14
SB3-8 bis
SB3-MIM
SB3-BIM
SB3-Cy
SB3-Py
SB4-14
SB3-4
SB3-4 Tris
9
10
11
12
a
Values obtained at 85 ꢁC. b Energy of activation extrapolated at 25 ꢁC.
The viscosities of these novel DESs are quite high, maybe due
to the strong interaction between the SB and the CSA, and are
higher than the viscosities of other trimethylglicine/carboxylic
acids DESs that we developed recently.53 However these values
are lower than sugar DESs in literature such as ChCl/Xylitol and
ChCl/Glucose (5230 cP and 34 400 cP respectively).20 Viscosity
activation energies values are similar to the ones of other DESs
in literature.19,20,53
two ethyl groups in SBE3-14–CSA mixture are smaller than bulky
n-propyl in SBP3-14 DES, but the propyl can lead to van der
Waals interactions. The imidazole-based mixtures (SB3-MIM
and SB3-BIM–CSA) showed a non-linear correlation. The plot
showed two parted linear trends for both the mixtures, probably
due to system rearrangements. The values of viscosity observed
for this class were higher than the ones of SBR3-14 series
(6556 cP and 2305 cP respectively for SB3-MIM–CSA and
SB3-BIM, entry 6 and 7). This is due to the aromatic imidazo-
lium ring that determines a tighter packing of the molecules
and smaller vacancies. The butyl chain of SB3-BIM determines a
lower symmetry and higher radius of vacancies and therefore
lower viscosity than methyl of SB3–MIM mixture. SB3-4–CSA
mixture became more viscous than SB3-4 Tris at temperatures
Conductivity
Electrical conductivity of the DESs was measured between
35–100 ꢁC. The proles of natural logarithm of conductivity vs.
1/T are reported in Fig. 5, the conductivity of the mixtures at
75 ꢁC and the conductivity activation energies at 25 ꢁC are
reported in Table 3. All the mixtures showed a good linear
correlation with Arrhenius law (eqn (2)).
ꢁ
higher than 85 C (5371 cP for SB3-4 and 3735 for SB3-4 Tris,
EL
entry 11 and 12) due to higher order in the liquid phase of SB3-4
for its smaller dimensions. SB3-Py–CSA mixture is more viscous
(3472 cP, entry 9) than SB3-Cy–CSA mixture (Table 2 entry 8)
probably due to the planarity of the system. SB3-MIM structure
has also a planar aromatic group, and the viscosities are similar
to the one of SB3-Py. The SB3-8 bis mixture showed a low
viscosity value (1488 cP, entry 5). This seemed surprising if
compared to the one of SB3-12–CSA (3025 cP, entry 1) for their
alkyl portions, but the two alkyl chains in SB3-8 bis have more
conformational degree of freedom compared to the longer one
of SB3-14. The lowest viscosity observed in our set was for
SB4-14–CSA mixture (1268 cP, entry 10). In this case the higher
dimensions of the spacer may give to the molecules higher
mobility, and consequently lower viscosity.
ln s ¼ ln s0 þ
(2)
RT
The highest value in our set was SB3-Cy (102 mS cmꢀ1, entry 8)
while the lowest was SB3-4 Tris–CSA mixture with a value of 8.7 mS
cmꢀ1 (entry 12). In SBR3-14 series (entry 2–4) the mixture SBE3-
Table 3 Conductivity and conductivity activation energy of SB–CSA
mixtures
Entry
DES
sa (mS cmꢀ1
)
EL (kJ molꢀ1
b
)
1
2
3
4
5
6
7
8
SB3-12
SB3-14
23.3
13.8
34.7
15.1
20.5
27.8
39.1
102.0
30.6
48.3
26.3
8.7
33.0
33.3
34.1
34.6
31.5
42.1
38.0
39.9
44.9
35.3
40.0
42.3
SBE3-14
SBP3-14
SB3-8 bis
SB3-MIM
SB3-BIM
SB3-Cy
SB3-Py
SB4-14
SB3-4
SB3-4 Tris
The activation energies were calculated at 298 K from eqn (1)
via linear correlation of the viscosity values. The four points at
lower temperatures were used for correlations of SB–CSA
mixtures which showed non-linear trends. The Eh is dependent
on the ratio R /Rh, where R is the ion radius and Rh is the radius
ꢃ
ꢃ
9
of the vacancies in the liquid. High Eh values mean difficult
movement of the molecules in the liquid due to the small
dimensions of the vacancies.75,76 As showed in Table 2, an
increase of chain length (SB3-12 and SB3-14, entry 1 and 2) led to
an increase of Eh values. In SBR3-14 series, the increase of steric
10
11
12
a
Values obtained at 75 ꢁC. b Energy of Activation extrapolated at 25 ꢁC.
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Fig. 5 Plot of ln conductivity vs. reciprocal of temperature for SB–CSA mixtures. Legend Panel A (aromatic–aliphatic SB): (hollow squares)
SB3-Cy–CSA mixture, (solid circles) SB3-4–CSA mixture, (solid diamonds) SB3-4 Tris–CSA mixture, (hollow triangles) SB3-Py–CSA mixture,
(solid triangles) SB3-BIM, (hollow circles) SB3-MIM–CSA mixture. Panel B (amphiphilic SB): (solid diamonds) SB4-14–CSA mixture, (hollow
triangles) SB3-8 bis–CSA mixture, (hollow squares) SB3-12, (solid triangles) SBE3-14, (hollow diamonds) SBP3-14–CSA mixture, (solid circles)
SB3-12–CSA mixture.
14–CSA showed the highest conductivity value (34.7, 13.8 and
In Table 3 the activation energies of conductivity (EL) for
15.1 mS cmꢀ1 for SBE3-14, SB3-14 and SBP3-14 respectively). In these mixtures are reported. This energy is related to the radius
the aromatic series SB3-MIM showed a conductivity lower than of the vacancies in the liquid structure, responsible of mass
SB3-BIM–CSA mixture (27.8 and 39.1 mS cmꢀ1, entry 6 and 7) transport.75,80 An increase of the alkyl chain length (entry 1, 2)
and SB3-Py showed an intermediate value between them led to an increase of EL in the same manner of activation energy
(30.6 mS cmꢀ1, entry 9). The alkyl residues on the side chain didn't of viscosity. An increase of the steric hindrance on ammonium
impact relevantly on the observed conductivity values. SB3-12– atom (entry 2–4) increased activation energy because of the
CSA mixture showed, in fact, a value of 23.3 mS cmꢀ1 (entry 1) lower mobility of the cation. SB3-8 bis–CSA mixture values are
while SB3-8 bis–CSA showed 20.5 mS cmꢀ1 (entry 5).
due to the two alkyl chains (entry 5). The cyclohexyl group in
The observed conductivity values for these novel DESs are SB3-Cy–CSA mixture (entry 8) led to less mobility of the mole-
low, even lower than other carboxy-betaine based uids53 and cules compared to the linear chain for its easier packing.
comparable with AcChCl–urea and ChCl–ZnCl2 mixtures, SB3-BIM showed the lowest value in aromatic mixtures (entry 6,
(17 and 60 mS cmꢀ1 respectively).20 This could be due to the 7 and 9): the linear side-chain decreases the ion mobility and
dimensions of the molecules in the DESs, and therefore to their decreases also the capability of p–p stacking. The increase of the
mobility (as also demonstrated by their high viscosity values), spacer length in SB4-14–CSA mixture (entry 10) led to an increase
but particularly to the strength and to the kind of interaction of EL due to an increase of ionic radius with consequent mobility
between the species. The interaction between the HBA and the loss. The ion mobility is responsible also for SB3-4–CSA mixture
HBD molecules is quite strong as proved by the Dt of the value that is lower than SB3-4 Tris (entry 11, 12). The values of EL
mixtures, and could be ascribed to a hydrogen bond instead of a observed are similar to the ones of other DESs reported in
complete transfer of the hydrogen from HBD to HBA molecules. literature.44,76
This is probably due to the small difference of pKa between the
species. This phenomenon is proved also by the values of
conductivity: a complete proton transfer causes a formation of
Density
The novel DESs developed showed a linear dependence of
density with temperature. The density, as well as viscosity and
conductivity, relies on the presence of vacancies in the DES
an ionic couple, and consequently a protic ionic liquid forma-
tion. However the conductivity values observed are too low. The
conductivity values of typical Ionic Liquids span, in fact, from
0.0027 to 11 S cmꢀ1, because of the presence of two discrete
ionic species.77 Our mixtures showed values between ve and
seven orders of magnitude lower (moreover at higher temper-
atures). This indicates the absence of ionic species. The use of
liquid mixtures dened as protic ionic liquids is reported in
literature, but their conductivity values and their small differ-
ences of pKa between the HBA and the HBD molecules suggest
that they also could belong to the class of deep eutectic solvents.
This is the case of ethanolammonium lactate78 and 1-ethyl
imidazolium H2PO4 79 mixtures that showed conductivity values
of 48 mS cmꢀ1 and 11 mS cmꢀ1 respectively.
network.76 The density variations were detected between 25 C
ꢁ
and 80 ꢁC. The values obtained are reported in Table 4, in
Fig. 2S in ESI† section are reported density/temperature
proles.
The highest density variation with temperature was observed
in SB3-Py–CSA mixture. The values are similar to the ones
observed for SB3-MIM–CSA system. This indicates stronger
intermolecular interactions in these mixtures compared with
SB3-BIM and SB3-Cy systems. These four systems have values of
r at 25 ꢁC of 1.33 g mLꢀ1, 1.33 g mLꢀ1, 1.23 g mLꢀ1 and 1.27 g
mLꢀ1 respectively. The SBR3-14 series showed density values
following the trend methyl > ethyl > n-propyl with r values at
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Table 4 Density values of SB–CSA DESs
SB3-12
ꢁC
SB3-14
ꢁC
SBE3-14
ꢁC
SBP3-14
ꢁC
SB4-14
ꢁC
SB3-8 bis
ꢁC
T
r g mLꢀ1
T
r g mLꢀ1
T
r g mLꢀ1
T
r g mLꢀ1
T
r g mLꢀ1
T
r g mLꢀ1
25
50
65
80
1.18
1.16
1.14
1.13
25
50
65
80
1.20
1.18
1.17
1.15
25
50
65
80
1.14
1.13
1.11
1.1
25
50
65
80
1.12
1.09
1.08
1.06
25
45
60
80
1.17
1.15
1.14
1.13
25
45
60
80
1.15
1.13
1.12
1.11
SB3-MIM
SB3-BIM
SB3-4 Tris
SB3-4
SB3-Cy
SB3-Py
T
ꢁC
r g mLꢀ1
T
ꢁC
r g mLꢀ1
T
ꢁC
r g mLꢀ1
T
ꢁC
r g mLꢀ1
T
ꢁC
r g mLꢀ1
T
ꢁC
r g mLꢀ1
25
45
60
80
1.33
1.32
1.31
1.29
25
45
60
80
1.23
1.21
1.20
1.17
25
45
60
80
1.22
1.21
1.20
1.19
25
45
60
80
1.25
1.24
1.23
1.21
25
45
60
80
1.27
1.26
1.25
1.21
25
45
60
80
1.33
1.31
1.29
1.25
25 ꢁC of 1.20 g mLꢀ1, 1.14 g mLꢀ1 and 1.12 g mLꢀ1 for SB3-14, (entry 12) showed a value of 42.1 mN mꢀ1, lower than SB3-4
SBE3-14 and SBP3-14 respectively. The increase of steric mixture (entry 11, 43.3 mN mꢀ1), conrming the role of the
hindrance suggests the increase of radius of voids and therefore dimensions of the molecules on surface tension values.
of the lowering in density. SB3-8 bis–CSA mixture showed a low
The surface tension values observed for these novel DESs are
density at 25 ꢁC (1.15 g mLꢀ1): even in this case this value could similar to ChCl–alcohol mixtures reported in literature.45,82
depend on the radius of the voids, increased by two octyl chains
presence in the structure. The same behaviour occurs in SB3-4
and SB3-4 Tris mixtures with 1.25 and 1.22 g mLꢀ1 r values. Ionicity
SB4-14–CSA mixture showed values of density similar to
The ionicity of DESs is an evaluation of the free charges fraction
at a specic temperature. This property is directly correlated to
the low vapour pressure of these liquids that is lower, in fact, for
higher charge fraction systems. Different techniques were used
SB3-12–CSA mixture (1.17 g mLꢀ1 and 1.18 g mLꢀ1 values
ꢁ
respectively at 25 C) and this could be ascribed to weak inter-
molecular interactions.
The density values observed are similar for all the DESs we
in literature to evaluate ionicity, such as thermal properties
developed, and they are analogous to the ones observed
changes,83,84 NMR,85,86 and IR spectroscopy.83 The Walden plot
in literature for other DESs such as ChCl–EG systems
represents a simple and qualitative method that can be used in
(1.12 g mLꢀ1 76 or ZnCl2–Acetamide mixtures (1.36 g mLꢀ1).81
)
this eld to evaluate the ionicity of a DES.87,88 It is based on the
assumption that strong electrolytes such as KCl follow Walden's
law: the molar conductivity (L) is linearly dependent on the
viscosity (h) (eqn (3)).
Surface tension
The surface tension is a fundamental property of a solvent, it is
directly related to the radius of voids and it is a measure of
cohesive forces in liquid on the surface.75 The surface tension
Lh ¼ Const
(3)
ꢁ
values observed for our SB–CSA mixtures at 25 C are reported
Angell and co-workers proposed to use the Walden plot as a
in Table S1 in ESI† section. All the values observed are lower qualitative estimation of the ionicity of the media.87 According
than water value (71.5 mN mꢀ1). The lowest value observed in to this, we made Walden plots of ln L/ln hꢀ1 of our twelve DESs.
our set is for SB3-12–CSA mixture (25.1 mN mꢀ1, entry 1). In In this chart the diagonal represents the theoretical behavior of
SBR3-14 series, SBE3-14–CSA mixture showed the lowest value KCl in water, considering it fully dissociated and with equal
(36.5, 33.6, 34.8 mN mꢀ1 for SB3-14, SBE3-14 and SBP3-14 mobility ions. The mixtures that have ln L/ln hꢀ1 values close to
respectively, entry 2, 3, 4). This could be due to van der Waals that diagonal, can be considered “good ionic liquids”, the ones
interactions in propyl group and small dimensions of methyl far from these values in the lower portion can be considered
group: the same phenomenon was also observed in viscosity, “poor ionic liquids”. Non-ionic liquids are in the lower portion,
density and conductivity measures in this work. In the aromatic very distant from the diagonal. Super-ionic liquids can be found
SB–CSA mixtures (36.5, 32.9, 33.4 mN mꢀ1, entry 6, 7, 10) the on the upper side of the plot.79,89,90
values followed the trend SB3-BIM < SB3-Py < SB3-MIM due to
In Fig. 6 are reported the Walden plots of the investigated
the same reasons. SB3-8 bis–CSA mixture (entry 5) showed a DESs. For comparison with our systems, in the same gure are
value of 37.5 mN mꢀ1, that is higher than the other single-chain reported in both panels [BMIM][BF4] and ChCl–EG DES as
SB. The tetramethylene spacer in SB4-14–CSA mixture (entry 8, model respectively for ionic liquid systems and a choline-based
33.8 mN mꢀ1) provokes a lowering in surface tension compared mixtures.82,87 All our twelve mixtures showed values close to
to the one of SB3-14–CSA mixture. SB3-4 Tris–CSA mixture reference line, so they can be considered “good ionic liquids”.
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Fig. 6 Walden plots of SB–CSA mixture. Legend Panel A (aromatic–aliphatic SB), (solid squares) SB3-4 Tris–CSA mixture, (solid circles)
SB3-4–CSA mixture, (hollow squares) SB3-Cy–CSA mixture, (hollow triangles) SB3-Py–CSA mixture, (solid triangles) SB3-BIM–CSA mixture,
(hollow circles) SB3-MIM–CSA mixture, (black star) [BMIM][BF4], (black cross) ChCl–EG mixture. Panel B (amphiphilic SB), (solid circles)
SB3-14–CSA mixture, (solid triangles) SBE3-14–CSA mixture, (hollow diamonds) SBP3-14–CSA mixture, (hollow squares) SB3-12–CSA mixture,
(solid diamonds) SB4-14–CSA mixture, (hollow triangles) SB3-8 bis–CSA mixture, (black star) [BMIM][BF4], (black cross) ChCl–EG mixture.
Ionic liquids are close to the reference line in the Walden times (data not shown). Preliminary microbiological tests
plot.91 Protic ionic liquids (PILs), formed by proton transfer, are showed a very rapid decrease of cell viability aer few minutes
commonly far from the reference line in the plot,79 and there- of exposition to the tested mixtures. We observed the same
fore they can be considered as “poor ionic liquids”. Some protic behavior in our previous study regarding trimethylglicine and
ionic liquids, however, are closer to the reference line when the carboxylic acids DESs53 so we developed FTIR measures in the
DpKa of the molecules forming them is high (about 10).91 On the same manner. It was hypothesized that the high concentration
contrary, in these novel DESs the pKa values of the two species of the DESs caused a very rapid exit of the yeast cell water and
are similar but the positions in the Walden plot are close to the thus their immediate inactivation. In order to test this
reference line. In our systems the overall charges are the same hypothesis, CaCl2 was chosen as a dehydrating agent known not
as the ones of an IL, but the molecules presented a lower to be toxic, in order to avoid the simultaneous presence of cyto-
mobility due to an interaction between them that could be toxic and dehydration effect.
ascribed to a hydrogen bond. The proton could be located, in
The effects exerted by all these mixtures were evaluated by
fact, between the HBA and the HBD molecules, and therefore means of average Response Spectra (ARS), calculated for
leading to partial charges formation. The low conductivity amphiphilic SBs–CSA and aromatic–aliphatic SBs–CSA mixtures
values showed by these mixtures also conrmed this phenom- (Fig. 7).
enon. The position of these novel mixtures in the plot, down on
ARSs of aromatic–aliphatic SBs DESs showed intensity very
the le, is also an evidence of their properties: their behavior similar to that of CaCl2 with a similar shape in the fatty acids
could be ascribed to highly diluted solutions of electrolytes and (W1, 3200–2800 cmꢀ1) and mixed region (W3, 1500–1200 cmꢀ1).
the charge transfer between the species is very low. Both PILs In the amide I region (W2, 1800–1500 cmꢀ1) aromatic–aliphatic
(with a complete charge transfer) and ILs (with discrete ionic and amphiphilic SBs DESs showed almost identical
species) are on the right portion on the Walden plot: this ARSs, whereas in the carbohydrates and typing region (W5,
indicates our mixtures to be DESs instead of these sorts of 900–700 cmꢀ1) the ARSs of the tested compounds diverged.
mixtures. To our knowledge it is the rst time that novel media Amphiphilic SBs DESs showed always no lower ARSs intensity
are shown in this le portion of a Walden plot.
than aromatic and aliphatic ones. The ARSs shapes of these two
compound classes were similar throughout the whole spectrum
except in W4 (1200–900 cmꢀ1) and W5.
FTIR bioassay
Saccharomyces cerevisiae cells showed
a metabolomic
Fourier transform infrared spectroscopy (FTIR) has been
applied in the last two decades in microbiological studies,73
initially to dene the relations among strains and species,92
then to dereplicate and to dene possible correlations between
FTIR and other molecular descriptors.93 The low cost in
consumables, the reproducibility and the rapidity were
considered key factors in this application.94
response in W1 region mainly when challenged by amphiphilic
compounds, with two peaks around 2920 and 2855 cmꢀ1 cor-
responding to asymmetric and symmetric stretching of CH2,
respectively.95 The intensity of these bands was positively
related to the amount of lipids in bacterial cells.96 In the amides
region (1800–1500 cmꢀ1) the two ARSs displayed a similar trend
with an almost identical metabolomic response. Relevant peaks
were observed at 1726 cmꢀ1, 1695 cmꢀ1 and 1626 cmꢀ1. Many
From a previous analysis, it has emerged that these
compounds brought 100% of cell mortality aer little exposure
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Fig. 7 Averaged response spectra (ARS) of all RS obtained challenging S. cerevisiae cells by amphiphilic and aromatic–aliphatic SBs DESs and
CaCl2. Legend. ARS of S. cerevisiae cells challenged by amphiphilic (red), aromatic–aliphatic (blue) DESs and CaCl2 (green).
authors attribute these bands to C]O of lipid esters, antipar- class of mixtures. The chain length, and therefore the amphi-
allel plated sheet and b-turns in Amide I and protein b-sheet, philic behaviour of the molecules, had an impact on the effect
respectively.95,97,98 It has been reported that an increase in the of the mixtures on the yeast cells. The 14-methylene chain
number of b-sheets in the cell proteins and in the number of compounds showed a high correlation among them and
strands in the b-sheets, is linked to protein denaturation,98 as different from the aromatic–aliphatic mixtures, while
expected by proteins undergoing a fast dehydration.99 This was 12-methylene chains and 8 bis-methylene chains showed a
also observed with a negative peak, common to all DESs, behaviour that was more similar to the non-amphiphilic sul-
detected at 1585 cmꢀ1, corresponding to the amino acids side fobetaines mixtures. In recent studies we observed the same
chain vibration.100 Another negative band was observed in the effect of the chain length in FTIR in N-alkyl-tropinium surfac-
mixed region (W3) around 1400 cmꢀ1 (C]O n sym of COOꢀ).101 tants.63,103 14-methylene, or longer,104 chains showed high
Amphiphilic SBs mixtures determined a more intense biocidal effects on Saccharomyces cerevisiae and Candida albi-
response than aromatic–aliphatic ones in carbohydrates region cans yeasts and Escherichia coli and Listeria innocua bacteria,
(W4), around 1200 cmꢀ1 (C–O–C vibrations), 1065 cmꢀ1 (DNA while 12-methylene-chain surfactants showed weaker effects.
and RNA C–C n skeletal cis conformation)95 and 1040 cmꢀ1 (C–O
stretch of nucleic acids).102
Taken together, these data suggest that these novel DESs
exert a dehydration effect as CaCl2, as shown by the similar
In Fig. 8 (panel a) are reported the average normalized shape of the rst three spectral regions. Remarkably, the effect
spectra from cells treated with DESs and CaCl2. In panel b of the of DESs was always superior to that of calcium chloride salt,
same gure are reported Pearson's correlations calculated indicating more affinity of these compounds to water. The fact
among average spectra.
that the ARSs shapes in the fatty acid region were almost
The Pearson's correlations of the spectra (panel b) showed identical for CaCl2 and DESs can be justied by the fact that this
again that the effects of the DESs on the yeast cells could be region refers to highly hydrophobic compounds, less likely to
grouped in two main classes, corresponding to amphiphilic react to different amounts of water in the cells. Vice versa the
SBs–CSA mixtures (SB3-14, SBE3-14, SBP3-14, SB3-12, SB4-14, overall shape similarity of ARSs in W2 and W3 regions indicates
SB3-8 bis) and aromatic–aliphatic SBs–CSA mixtures that the dehydration caused strong variations in the protein
(SB3-MIM, SB3-MIM, SB3-4, SB3-4 Tris, SB3-Cy, SB3-Py). For this asset, which is indeed very sensitive to free water levels. The
reason the two classes are boxed in the table.
differences found in W4 and W5 between CaCl2 and DESs could
The rst group showed correlations ranging between 0.9721 be ascribed to some post mortem effects caused specically by
and 0.9988 and from 0.9435 to 0.9750 with CaCl2. The aromatic– DESs. One possible explanation is that the violent dehydration
aliphatic SBs showed high levels of correlation among them- triggered by DESs broke part of the internal cell compartmen-
selves (ranging from 0.9994 to 0.9999) and with CaCl2 (from talization with subsequent fast release of small molecules such
0.9844 to 0.9875). These gures indicated that the mode of as carbohydrates of W4 and, probably, the free amino acids
action of the aromatic–aliphatic SBs is likely due to the rapid sometimes detected in W5.105,106
dehydration of the yeast cells, whereas the amphiphilic SBs
could challenge the cells with different mechanisms, including
the dehydration itself.
Sulfobetaine DES as reaction medium for chalcone synthesis
One of the most important applications of novel liquid mixtures
Amphiphilic sulfobetaines–CSA mixtures showed a different
is represented by their use as reaction media. In this eld DESs
behavior. Structure-effect considerations can be made in this
have been largely used in the recent years as effective solvents
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Fig. 8 Panel a: FTIR average normalized spectra of S. cerevisiae cells subjected to the DESs. Panel b: correlation matrix of average spectra.
Legend Panel a: Dashed black ¼ control, dashed red ¼ CaCl2, orange ¼ SB3-14–CSA, red ¼ SBE3-14, purple ¼ SBP3-14, dark red ¼ SB3-12,
yellow ¼ SB4-14, pink ¼ SB3-8 bis, grey ¼ SB3-MIM, light green ¼ SB3-BIM, brown ¼ SB3-4, green ¼ SB3-4 Tris, dark brown ¼ SB3-Cy, light
blue ¼ SB3-Py. Panel b: Pearson's correlation within average spectra of cells subjected to amphiphilic SBs DESs (boxed upper-left part) and
non-amphiphilic SBs DESs (boxed lower-right part). Red > 0.9873; blue < 0.9873.
for their properties.27–31 In order to determine the acid proper- 96%. Subsequent experiments were carried out to determine the
ties of the novel DESs presented in this work, we have tested one DES recycling capability. The DES is, in fact, highly soluble in
of our novel mixtures as solvent and Brønsted catalyst in chal- water, while the chalcone is poorly soluble in it, so the DES
cone synthesis from benzaldehyde and acetophenone as probe could be easily removed by washing the mixture with H2O then
reaction (Fig. 9).
These novel DESs have also a catalytic behaviour in this constant for over 4 consecutives reaction cycles.
Claisen–Schmidt condensation due to the acid properties of The same reaction conducted in acid ionic liquids as
drying it under vacuum aer each cycle. The yields remained
CSA composing it. The reaction, in fact, is usually carried out in reported in literature led to almost the same yields but with
basic or acid catalysis conditions, with the addition of e.g. higher temperatures (140 ꢁC) and into inert atmosphere.112,113
sodium or potassium hydroxide, alkali alcoholates,107,108 solid The use of other reaction media led to higher yields but the use
basic catalysers109,110 or concentrated sulfuric acid.111 of harmful acids or bases as catalysts was needed and therefore
SB3-Cy–CSA deep eutectic solvent was tested for this reaction.
their disposal.110,114 These are not necessary in the case of the
Temperature and reaction times were varied to optimize use of these DESs. These data promote the “greenness” of these
conversion and yield of the reaction. A temperature of 90 ꢁC and mixtures and suggest further investigations on their properties
a reaction time of 16 h were chosen as optimal aer a series of as promising reaction media. Further studies on a wide set of
experiments (data not shown). In these conditions we obtained acetophenones and benzaldehydes molecules are currently
a weighed yield of puried chalcone of 70% with a conversion of undergoing in our laboratories, in order to determine the
versatility of these novel media.
Conclusions
Twelve novel deep eutectic solvents were prepared and charac-
terized. They are formed by mixtures of (1S)-(+)-10-cam-
Fig. 9 Chalcone synthesis reaction scheme.
phorsulfonic acid and aliphatic, aromatic and amphiphilic
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sulfobetaines. Their melting points span from ꢀ5 to 19 ꢁC, this
indicates that the driving forces leading to liquid systems are
almost identical for all the mixtures. They are liquids at room
temperature, so we can name these mixtures RTDESs (room
temperature deep eutectic solvents). The DESs were character-
ized in terms of their viscosity, conductivity (and therefore their
ionicity via Walden plot), density, surface tension and toxicity
on eukaryotic model cells. The collected data suggest that the
interaction between CSA and the sulfobetaine can be ascribed
as a hydrogen bond. The low conductivity values, the liquid
formation even at 0.2 molar fraction of the acid and the small
differences of pKa values between the two species, in fact,
suggest that the proton is not completely transferred from HBD
to HBA molecules. For these reasons these mixtures cannot be
considered ionic liquids. To our knowledge, their position on
the Walden plot, in the le portion close to the diagonal, has
not yet been observed for other DESs or ionic liquid systems. It
indicates that these mixtures have ionicity similar to highly
diluted electrolyte solutions. The values of their viscosity, their
surface tension and their density are similar to other DESs
systems in literature.
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The use of these novel DESs as reaction media with catalytic
properties was accomplished by the use of SB-Cy–CSA DES in
chalcone synthesis, a well-known and studied acid-catalysed
reaction. This DES showed acid catalytic properties, yields of
70% and conversion of 96%, recycling capabilities in subse-
quent reactions and prevented the use of harmful acid catalysts.
These preliminary data promote the “greenness” of these
mixtures and suggest further investigations in this eld.
´
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Acknowledgements
`
The authors thank the Ministero per l'Universita e la Ricerca
Scientica e Tecnologica, MIUR (Rome, Italy) [PRIN “Pro-
grammi di Ricerca di Interesse Nazionale” 2010–2011, n.
2010FM738P] and Regione Umbria (POR FSE 2007–2013,
Risorse CIPE, Perugia, Italy) for fundings.
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