Synthesis and properties of gallate ionic liquids

Article abstract of DOI:10.1016/j.tet.2016.09.007

An efficient method for the synthesis of novel antioxidants in the form of ionic liquids (ILs) was described in the framework of this study. The ILs were obtained by the reaction of quaternary ammonium hydroxides with gallic acid as well as the protonatio

Full text of DOI:10.1016/j.tet.2016.09.007

Accepted Manuscript
Synthesis and properties of gallate ionic liquids
Kamil Czerniak, Agnieszka Biedziak, Krzysztof Krawczyk, Juliusz Pernak
PII:
S0040-4020(16)30901-2
10.1016/j.tet.2016.09.007
TET 28078
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 12 May 2016
Revised Date: 20 August 2016
Accepted Date: 5 September 2016
Please cite this article as: Czerniak K, Biedziak A, Krawczyk K, Pernak J, Synthesis and properties of
gallate ionic liquids, Tetrahedron (2016), doi: 10.1016/j.tet.2016.09.007.
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Synthesis and properties of gallate ionic liquids
Kamil Czerniak,*a Agnieszka Biedziak,a Krzysztof Krawczyk,b Juliusz Pernaka
aDepartment of Chemical Technology, Poznan University of Technology, 60-965 Poznan,
Poland
bVirology and Bacteriology Department, Institute of Plant Protection-National Research
Institute, 60-310 Poznan, Poland
*Corresponding author.
Tel: +48-61-6653541; e-mail: kamil.z.czerniak@doctorate.put.poznan.pl
Abstract
An efficient method for the synthesis of novel antioxidants in the form of ionic liquids (ILs)
was described in the framework of this study. The ILs were obtained by the reaction of
quaternary ammonium hydroxides with gallic acid as well as the protonation of amphoteric
betaine. The following physicochemical properties of the obtained ILs were characterized:
solubility in water as well as in polar and non-polar organic solvents, octanol-water partition
coefficient, thermal stability, and surface activity. The investigation of bactericidal activity
indicated that the synthesized gallates exhibited high efficacy against bacteria, especially
against Bacillus subtilis. The DPPH radical scavenging assay confirmed the antioxidant
properties of gallate ILs.
Keywords: Ionic liquids; Gallic acid; Antioxidant; Radical scavenging activity.
1. Introduction
Antioxidants are substances which are capable of preventing oxidation as a result of reducing
the concentration of free radicals or chelating metal ions. Some well-known antioxidants
include L
-ascorbic acid (vitamin C), α-tocopherol (vitamin E), carotenoids or β-carotene.1,2
Among natural antioxidants occurring in plants in the form of polyphenols, gallic acid (GA)
exhibits one of the strongest antiradical properties due to efficient protection against different
reactive species.3 GA has also exhibited other forms of biological activity, such as anticancer
properties, due to the inhibition of the growth of lung cancer or prostate cancer cells with
stem-like properties.4 This natural phenolic acid can be used as a reductant for the preparation
of highly reduced graphene oxide at room temperature,5 or can generate hydrogen peroxide in
situ from air in slightly basic media.6
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During recent years, ionic liquids (ILs) have been described as solvents with high chemical or
thermal stability and low vapour pressure.7 ILs have found wide application in the synthesis
of various forms of antioxidants. Primarily, they can be used as environmentally friendly
catalysts for the preparation of GA esters,8 or act as the reaction solvent in the galloylation of
catechins.9 ILs are applied as sustainable dual solvent-catalysts for the preparation of an α-
tocopherol derivative.10 Moreover, they have been used in the preparation of novel
compounds with antioxidant activity e.g. the synthesis of naphthoquinone - urazole hybrids.11
In addition, ILs have been used for the production of polypyrimidine-amide antioxidants by
polycondensation in a mixture of IL and triphenyl phosphite.12 Current research has
demonstrated that imidazolium ILs could be potential candidates for antioxidants capable to
the neutralize the hydrogen peroxide or radicals.13
The rapid progress of studies in this field contributed to a steady advance from first generation
ILs (with unique, designable physical properties), through the second generation (with
targeted chemical properties, combined with selected physical properties), to the third
generation (with targeted biological properties, combined with physical and chemical
properties).14 The third generation of ILs have been discussed as active pharmaceutical
ingredients.7c,15 Herbicidal ionic liquids were introduced in 2011.16 In recent years, widely
applied formulations of antioxidants were esters, such as propyl or lauryl gallates.17
Moreover, it is possible to prepare water soluble antioxidants in the form of cholinium-based
quaternary salts.18 The aim of this work was improve activity and properties of gallic acid by
turning it into multifunctional ILs. These compounds may have a wider range of tunable
properties than traditional forms of antioxidants. Except high water solubility, ILs can also
exhibit antibacterial properties and improved surface activity.
2. Result and discussion
2.1. Synthesis of ILs precursors
Quaternary ammonium salts and an amphoteric surfactant (Fig. 1) were applied as starting
materials in order to obtain ILs with the gallate anion. Each bromide salt was obtained by the
quaternization (Menshutkin reaction) of tertiary amines and long chain alkyl bromides
(Scheme S1, ESI). The time of the reaction was optimized to achieve full conversion. Optimal
time was determined with in situ IR spectroscopy (ReactIR 15, Mettler Toledo) and processed
by iC IR software. This method let us monitor the direct alkylation of the amine and define
the mechanism of the reaction.19 IR spectra of the reaction mixture at the beginning and end
of the synthesis are shown in Fig. S1-S4, ESI. In the case of [C10DMEA][Br], the
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disappearance of band intensity at 1035 cm-1 was mainly observed. This band can be assigned
to the C-O out-of-phase stretching vibrations of 2-dimethylethanolamine.20 During the
quaternization of 1-methylmorpholine, stretching vibrations at 1145 and 1122 cm-1 can be
observed which are attributed to C-N stretching vibrations.21 Slight differences between the
IR spectra were observed in the case of the reaction involving 2-[2-
(dimethylamino)ethoxy]ethanol. Initially, a reduction of the intensity at 1055 cm-1 occurred.
Fig. 1. Chemical structures of the cations.
Table 1. Prepared precursors of ILs.
Tm
(°C)
Time
(h)
Compounds
R1
R2
Yield(%)
[C10DMEA][Br]
[C10MMorf][Br]
[C12DMEEA][Br]
[C12Bet]
C10H21
C10H21
C12H25 (CH2CH2O)2H
C12H25
CH2CH2OH
-
2.75
23.00
2.50
94
95
98
96
156
188
56
-
-
2.00
The reaction also results in the appearance of specific bands which can be assigned to the
complexes with the quaternary ammonium ion.22 The new bands appeared in the region
ranging from 1100 to 700 cm−1 (1089, 970, 921 cm-1 for [C10DMEA][Br]; 1044, 917, 899 cm-
1
for [C10MMorf]; and 1070, 977, 921 cm-1 for [C12DMEEA][Br]). The quaternization
reaction between a fatty amine and the potassium salt of chloroacetic acid was applied for the
preparation of alkyl betaines. The characteristic absorption peaks of C=O stretching vibrations
of the carbonyl group appeared at 1648 cm-1. The longest reaction time necessary to obtain
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the maximum concentration of the product was observed in the case of [C10MMorf][Br] and
was 23 h (Table 1). The other precursors of ILs could be synthesized more rapidly (in the
range of 2-2.75 h). No major side products were observed. After the purification of products
from the starting reactants, the compounds were obtained in yields ranging from 94 to 98%.
The resulting bromides were white or yellow solids with a melting point in the range of 56-
188 °C and the amphoteric surfactant had a greasy consistency.
2.2. Synthesis of ILs
Antioxidant ILs were prepared using the two methods presented in Scheme 1. The first
method, based on a neutralization reaction, consists of two stages and can be used to
synthesize various types of ILs.23
Scheme 1. Synthesis of gallate ILs.
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Table 2. Prepared ILs and their physicochemical properties.
a
b
c
Yield
(%)
98
97
98
State at
25˚C
liquid
wax
liquid
wax
Tg
Tonset5
(oC)
174
193
185
Tonset50
(oC)
241
254
260
ILs
Method
(°C)
6.7
35.8
-3.6
15.3
[C10DMEA][Gal]
[C10MMorf][Gal]
[C12DMEEA][Gal]
[C12Bet][Gal]
I
I
I
II
99
194
275
aTg – glass transition temperature; Tonset5 – decomposition temperature of 5% sample;
cTonset50 – decomposition temperature of 50% sample.
b
The synthesis started by preparation of a solution of quaternary ammonium hydroxide in
methanol using an anion exchange resin to replace the bromide ion. Next, a neutralization
reaction with GA was conducted at low temperatures. In addition, a hydroxide solution was
progressively introduced to the acid solution due to the low stability of GA at high pH.24 The
IL with [C12Bet] cation was obtained using the second method, which involved a one-step
protonation of the carboxylate group of betaine25 in methanol with GA. An overview of the
prepared ILs is shown in Table 2. In both methods the synthesized ILs do not require further
purification after evaporation of the solvent. In addition, the products were dried under
reduced pressure (5 mbar) for 24 h at ambient temperature and stored at a low temperature in
the dark. The described methods gave the ILs with a yield exceeding 97%. Water content was
determined by Karl-Fischer titration and amounted to 500-800 ppm. Two of the obtained ILs
containing hydroxyl groups were liquids at room temperature. Other salts were waxes. The
1
13
structure of the synthesized compounds was confirmed by H and C NMR spectroscopic
analysis. The obtained spectra are presented in Fig. S1-S4, ESI. The synthesized ILs showed
only one signal derived from the anion in the form of a singlet in the range of 6.84-6.93 ppm,
which is generated by the protons of the gallic acid aromatic ring.26 In the case of the cations,
the protons of the long alkyl chain are located at 0.86 ppm for the methyl group and
approximately 1.24 and 1.63 ppm for the methylene groups.27
2.3. Thermal properties
Short-term thermal stabilities of the salts were determined by temperature-ramped TGA
experiments.28 As shown in Table 2, all the prepared ILs exhibited high thermal stability and
manifested Tonset50 in the range of 241-274 °C. Nonetheless, obtained compounds were less
stable than GA.18 According to the literature, ILs containing short ether groups are less
thermally stable compared to alkyl-substituted analogues.29 However, the least stable salt
among the antioxidant ILs was [C10DMEA][Gal], without an ether linkage, which also
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exhibited the lowest value of Tonset5 (174 °C). The obtained results indicate that the presence
of long alkyl chains and their elongation does not affect the stability of ILs. The prepared
antioxidant ILs with long alkyl chains mainly underwent a three-step decomposition (Fig.
S20. ESI). [C12Bet][Gal] was an exception, with a clear two-step thermal degradation process.
No prepared ILs demonstrated melting points and their crystallization temperatures were in
the range of -100 to 120 °C. Based on the results of DSC analyses, it could be concluded that
gallate ILs have no tendency to crystallize upon cooling and mainly remain in the glass state.
Additionally, they can exhibit high glass transition temperature e.g. Tg = 35.8 ˚C in the case of
a compound with a morpholinium cation. The lowest values (Tg = -3.6 °C and 6.7 °C) were
observed for ILs [C12DMEEA][Gal] and [C10DMEA][Gal], which comprised the ethoxyl or
the hydroxyl substituent in the structure of the cation, respectively.
2.4. Octanol–water partition coefficient
The octanol–water partition coefficients (Kow) were determined in order to describe the
hydrophobicity of the gallate ILs. In addition, this parameter allows definition of the
environmental risk as associated with bioaccumulation or toxicity, because it reflects the
partition of the compounds in biotic systems. The Kow values calculated for gallate ILs are
shown in Table 3.
Table 3. Solubility in water and octanol-water partition coefficients of gallate ILs and GA
(2 mmol L-1) at 25 °C .
Solubility ± sd
Compound
Log Kow
(g L-1)
[C10DMEA][Gal]
[C10MMorf][Gal]
[C12DMEEA][Gal]
[C12Bet][Gal]
GA
49.16 ± 1.79
33.92 ± 2.17
totally miscible
20.77 ± 0.71
11.00 ± 0.06
-0.35 ± 0.02
-0.42 ± 0.03
0.07 ± 0.01
0.31 ± 0.01
0.32 ± 0.01
Based on the obtained data, it was established that ILs containing the [C10MMorf] and
[C10DMEA] cations were the least hydrophobic compounds. Elongation of the alkyl chain in
the cation increased the Kow, due to their higher solubility in n-octanol. Similar relationships
were found for imidazolium ILs.30 Only [C12Bet][Gal] exhibited a similar hydrophobicity to
gallic acid. The advantages of synthesized ILs can be observed in comparison to other forms
of this antioxidant acid. The most commonly used esters of gallic acid with a long alkyl chain
(e.g. dodecyl gallate) exhibit significant hydrophobicity (log Kow = 5.3), which can
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theoretically facilitate their interaction with the lipid membrane.31 Based on the measured Kow
values, it can be concluded that obtained ILs have a very promising properties. Basically,
compounds with low hydrophobicity and Kow values may exhibit a less risk of
bioaccumulation.32 However, the Kow of different ILs was not constant and depended on the
concentration of the compound in the aqueous phase due to the different dissociation of the
ILs in the water and the octanol phases.33 The Kow were measured in the concentration range
of 0.25-2 mmol L-1 in order to evaluate this relationship in case of gallate ILs (Fig. S14, ESI).
A slight increase in the partition coefficient with increasing concentrations of obtained ILs
was observed, except for [C10MMorf][Gal]. For this IL the greatest range of log Kow was
measured between -0.93 and -0.42.
2.5. Solubility
All the prepared ILs exhibited higher solubility in water than GA (11.00 ± 0.06 g L-1) which is
shown in Table 3. However, the properties of gallate ILs strongly depended on the structure of
the cation. The highest solubility in water (beyond 1000 g L-1) at 25 °C was observed for
[C12DMEEA][Gal]. The presence of alkoxy chains in the cation and the hydroxyl group can
significantly improve the hydrophilicity and water miscibility of salts.29 In addition, the
increase of hydrophobicity due to the longer alkyl chain does not significantly affect the
solubility in the presence of strongly hydrophilic groups. The lowest water solubility was
determined for [C12Bet][Gal] and amounted to 20.77 ± 0.71 g L-1. Thus, the selection of an
appropriate cation can successfully change the solubility of ILs with the gallate anion to a
different degree, until complete dissolution is achieved. The solubility of the synthesized ILs
in other solvents is presented in Table 4. Due to their polar character, all the obtained ILs
were also highly soluble in polar protic and aprotic solvents such as methanol and DMSO.
However, not all gallate ILs exhibited solubility in acetonitrile. Likewise, the prepared ILs
were characterized by low solubility in the case of other, less polar solvents. ILs containing an
alkyl group with 12 carbon atoms exhibited solubility in isopropanol, but only [C12Bet][Gal]
was easy dissolved in acetone and showed limited solubility in ethyl acetate. Like most of the
ILs, the antioxidant salts are not soluble in alkanes34 (e.g. hexane) and other non-polar
solvents due to the significant difference in polarity.
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Table 4. Solubility of the prepared ILs at 25 °Ca.
Cation
Solvent
Methanol
DMSO
[C10DMEA] [C10MMorf] [C12DMEEA]
[C12Bet]
Acetonitryle
Acetone
2-Propanol
Ethylacetate
Chloroform
Toluene
Hexane
a “green”, complete solubility; “yellow”, limited solubility; “red”, insoluble.
2.6. Antioxidant properties
Antioxidant activity of gallate ILs was measured using the DPPH assay. This method involves
the reduction of a stable free radical located on the nitrogen atom of the DPPH molecule by
the tested antioxidants and is widely used to evaluate the scavenging properties of a single
compound or plant extracts containing various antioxidant substances35 due to its simplicity
and high repeatability.
Fig. 2. Measured IC50 values of tested ILs.
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The scavenging efficacy of ILs against DPPH radicals in methanol as a solvent is shown in
Fig. 2. Our study indicates that all gallate ILs are very good antioxidants and exhibit better
activity than commonly used compounds, such as Trolox.36 GA was used as a positive control
and the results were expressed as the IC50 parameter. Generally, GA derivatives (such as
esters) exhibit lower antiradical activity in comparison to the free acid.37 The presence of a
linear chain and the steric effect or the intramolecular and intermolecular hydrogen bonding
of such chains could affect the activity of antioxidants.38 The obtained results indicate that all
the synthesized ILs exhibited slightly higher or statistically insignificant differences in radical
scavenging capacity compared to GA (IC50 = 5.53 ± 0.15). Additionally, the negative impact
of a long alkyl chain in the cation on the antioxidant properties was not observed in case of
the gallate ILs. [C12Bet][Gal] and [C12DMEEA][Gal] were the most effective in the inhibition
of DPPH radicals in a methanol solution, with IC50 values equal to 4.69 ± 0.15 and 4.82 ±
0.19, respectively. In general, betaine derivatives may exhibit a different activity e.g. glycine
betaine does not have antioxidant activity.39
2.7. Anti-microbial activity
Phenolic acids, such as gallic acid, exhibit antibacterial activity against both Gram-positive (S.
aureus and L. monocytogenes) and Gram-negative bacteria (E. coli , MIC = 1.5 mmol L-1 and
P. aeruginosa, MIC = 5.9 mmol L-1).40 The determined minimal inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC) values are shown in Table 5. The
results indicate that all the synthesized ILs exhibit antimicrobial properties and that their
activity is selective. Generally, the synthesized gallate ILs are moderate bactericides. All salts
exhibit weaker activity in comparison to e.g. benzalkonium chloride or
didecydimethylammonium chloride.41 The lowest antimicrobial activity was observed for
[C10MMorf][Gal] with MBC values of 9.5 mmol L-1 against M. catarhallis and E. faecium,
and 19.0 mmol L-1 against S. marescens. The most effective ILs were [C12Bet][Gal] and
[C12DMEEA][Gal], followed by [C10DMEA][Gal] and [C10MMorf][Gal]. [C12Bet][Gal]
exhibited anti-microbial activity against all tested bacterial strains at a concentration of 2.2
mmol L-1 and lower, except for P. aeruginosa (activity at 8.8 mmol L-1 and higher).
Generally, the antimicrobial activity of ILs increased with the extension of the alkyl chain and
the optimal number of carbon atoms was usually between 10 and 16.42 It was also observed
that the differences between the MIC and MBC values obtained for the studied ILs were
marginal.
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Table 5. MIC and MBC valuesa for gallate ILs.
Strains
M. luteus
[C10DMEA]
4.9
[C10MMorf] [C12DMEEA]
[C12Bet]
2.2
2.2
1.1
1.1
2.2
2.2
1.1
1.1
1.1
1.1
1.1
1.1
8.8
8.8
1.1
1.1
1.1
1.1
1.1
1.1
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
9.5
9.5
4.7
9.5
2.4
2.4
9.5
9.5
9.5
9.5
9.5
9.5
9.5
9.5
19.0
19.0
4.7
9.5
2.4
2.4
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
1.0
1.0
2.1
4.1
4.1
4.1
2.1
2.1
1.0
1.0
4.9
S. epidermidis
S. aureus
2.5
2.5
2.5
4.9
M. catarrhalis
E. faecium
4.9
4.9
2.5
2.5
E. coli
2.5
4.9
P. aeruginosa
S. marescens
P. vulgaris
B. subtilis
4.9
9.8
9.8
9.8
4.9
9.8
1.2
1.2
a in mmol L-1.
2.8. Surface activity
Critical micelle concentration (CMC), surface tension at CMC (γCMC), maximum surface
excess (Γmax), minimum area per molecule (Amin), adsorption efficiency (pC20) and contact
angle (CA) were characterized and are shown in Table 6. The precursors of the obtained ILs
demonstrated good surface activity. In the case of [C10Morf][Br] and [C10DMEA][Br] the
critical micelle concentrations were at 61 mmol L-1 and 63 mmol L-1, respectively.43 In
contrast, pure GA did not reduce the surface tension of water.
Table 6. Surface properties of synthesized ILs.
γcmc
CMC
106Γmax
(mol m-2) (nm2)
Amin
pC20
CA
(˚)
ILs
(mN m-
(mmol L-1)
1
)
[C10DMEA][Gal]
[C10MMorf][Gal]
[C12DMEEA][Gal]
[C12Bet][Gal]
27.54
21.87
8.71
35.12
35.63
30.34
34.61
2.55
2.49
2.39
2.32
6.51
6.65
6.96
7.15
2.16
2.25
2.88
3.82
75.9
65.2
72.4
53.8
0.71
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However, the aqueous solutions of the obtained antioxidant ILs efficiently reduced the surface
tension of water. Significantly lower values of CMC are characteristic for ILs containing the
dodecyl alkyl chain in the cation. The lowest value of CMC was observed for [C12Bet][Gal]
(0.71 mmol L-1) and for [C12DMEEA][Gal] (8.71 mmol L-1). Thanks to low CMC values,
these salts can be described as good surfactants with surface activity similar to conventional
compounds such as cetyltrimethylammonium bromide (1.0 mmol L-1)44 or sodium dodecyl
sulfate (8.4 mmol L-1).45 Antioxidant ILs have good wettability properties. Contact angle
(CA) values of gallate ILs were determined based on drop shape analysis on the examined
surface (paraffin) and are also presented in Table 6. The value of CA of water as a pure
solvent on paraffin is approximately 111°. In turn, the lowest value of contact angle on the
same tested surface was observed for [C12Bet][Gal] – 53.8°. Fig. S21. in Supplementary data
shows the surface tension as a function of the logarithm of concentration. As can be seen, the
surface tension values of the aqueous solutions showed a progressive decrease with increasing
concentration and remained constant above the CMC.
3. Conclusions
The conducted studies introduce a new direction in the synthesis of antioxidants by
preparation of ILs, which may exhibit strong radical scavenging activity and also act as
efficient bactericidal and surface active agents. Different ILs derived from naturally occurring
gallic acid can be easily prepared with high efficiency using solvents with low toxicity
(methanol, acetone). The selection of an appropriate cation allows to design the properties of
the gallate ILs, such as solubility in water, octanol-water partition coefficient or surface
activity.
Introduction of an antioxidant anion into the structure of ILs allows to obtain salts, which may
be an alternative to commonly applied compounds, e.g. gallic acid esters commonly applied
in the cosmetics industry. In contrast to such compounds, the gallate ILs exhibit several
additional advantages (low CMC, beneficial octanol-water partition coefficient, low
MIC/MBC values against Gram-positive and Gram-negative bacteria).
4. Experimental
4.1. Materials
The following reagents used throughout the study were purchased from Sigma-Aldrich and
used without further purification: 1-Bromodecane (98%, CAS 112-29-8), 1-bromododecane
(97%,
CAS
143-15-7),
dimethyldodecylamine
(97%,
CAS
112-18-5),
2-
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dimethylaminoethanol (99,5%, CAS 108-01-0), 2-[2-(dimethylamino)ethoxy]ethanol (98%,
CAS 1704-62-7), 4-methylmorpholine (≥98%, CAS 109-02-4), chloroacetic acid (≥99%, CAS
79-11-8), gallic acid (97.5-102.5%, CAS 149-91-7) and 2,2-diphenyl-1-picrylhydrazyl
(DPPH, 95%, CAS 1898-66-4). Other chemicals and all solvents were obtained from Avantor
Performance Materials Poland S.A. and used as received from the supplier.
4.2. General
1H and 13C NMR spectroscopic analyses were carried out on a Varian Mercury 300
spectrometer operating at 300 and 75 MHz. Elemental analyses were performed at the Adam
Mickiewicz University, Poznan (Poland). The water content measurements were conducted
using Aquastar volumetric Karl Fischer titration with composite 5 solution as the titrant and
anhydrous methanol as the solvent.
4.3. Thermal analysis
Thermogravimetric analysis was performed using a Mettler Toledo Stare TGA/DSC1 unit.
o
ILs (between 2 and 10 mg) were placed in aluminum pans and heated from 30 to 450 C at a
heating rate of 10 oC min-1 under the flow of nitrogen.
Thermal transition temperatures of the prepared salts were determined using a Mettler Toledo
Stare DSC1 apparatus under nitrogen. ILs (between 5 and 15 mg) were placed in aluminum
pans and heated from 25 to 120 oC at a heating rate of 10 oC min-1, cooled with an intracooler
at a cooling rate of 10 oC min-1 to -100 oC and then heated again to 120 oC.
4.4. Antioxidant properties
The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay was carried out
according to the method described by Brand-Williams et al.46 Methanol solutions of ILs at
different known concentrations (0.1 mL) was added to the DPPH solution (3.9 mL, 0.06
mmol L-1). The mixture was shaken vigorously and left in the dark. After 30 min of
incubation at room temperature, the absorbance was measured at 515 nm with a Rayleigh UV-
1800 spectrophotometer. Gallic acid was used as the reference standard. All determinations
were performed in triplicate. DPPH antioxidant activity was calculated on the basis of the
following equation:
% radical scavenging activity = ꢀꢁꢂꢃ
ꢄ
∙ 100%
ꢁ
where A is the absorbance of the control and B is the absorbance of the samples.
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The concentrations of ILs which reduced 50% of the free-radical concentration (IC50 values,
Table S1, ESI) were processed from the sigmoidal dose response curves (Fig. S15-S19, ESI)
using the OriginPro 8.5 program.47
4.5. Solubility
The water solubility was determined according to a modified method described by Marrucho
et al.48 Saturated solutions of ILs were prepared in 10 mL glass vials by addition of an excess
amount of salts to DI water (1 mL). The vials were mixed at a temperature of 25 ± 0.2 °C
using a Heidolph MR Hei-End stirrer equipped with a Heat-On anodized block. After 48 h of
stirring, equilibrium was reached and the samples were centrifuged in order to enhance the
complete phase separation. The concentrations of ILs in the saturated solution were quantified
via UV/Vis spectrophotometry. All measurements were performed in triplicate. The
calibration curves of ILs were prepared in DI water and the maximum absorbance values of
salts at different known concentrations were collected at the characteristic wavelength of 265
nm for [C12Bet][Gal] and gallic acid or 259 nm for the other ILs. All obtained calibration
curves are presented in the ESI (Table S2, ESI).
The solubility of the ILs (0.1 g) in other solvents was measured using the method described in
Vogel's Textbook of Practical Organic Chemistry.49 ‘Complete solubility’ applies to ILs,
which were dissolved in 1 mL of the solvent, ‘limited solubility’ means that ILs were
dissolved in 3 mL of the solvent, ‘insoluble’ applies to ILs, that did not dissolve in 3 mL of
the solvent. All ILs were thermostated at 25 °C in a water bath MEMMERT WNB 7.
4.6. Octanol–water partition coefficients
Octanol-water partition coefficients (Kow) of gallic acid and gallate ILs were estimated by the
shake-flask method according to OECD guidelines.50 Measurements of Kow values were
conducted using mutually saturated solvents (water and n-octanol) and in a 15 mL glass vial
containing a magnetic stir bar. Firstly, 5 mL of ILs or free acid solution in saturated water
(concentrations in the range of 0.25 to 2 mmol L-1) were added to 5 mL of saturated n-octanol.
All vials were shaken at a constant temperature of 25 ˚C. After 24 h, all the samples were
centrifuged and the water phase was collected by a syringe. The concentrations of ILs in the
water solution were determined spectrophotometrically using a UV/Vis spectrophotometer.
Three repetitions of each measurement were performed. The calibration curves are presented
in Table S3, ESI.
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4.7. Anti-microbial activity test procedure
Anti-microbial activity was determined by the broth microdilution method, according to the
approved standard for aerobic bacteria (CLSI document M07-A9). Minimal Inhibitory
Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values were
determined. All antimicrobial agents were tested in a series of twofold dilutions from 0.49 to
500 mg mL-1. Cultures of standard tested strains (24 hrs) were suspended in Mueller-Hinton
Broth (MHB) to a concentration of 105 –106 CFU mL-1.
The following microorganisms were used during the tests: Micrococcus luteus NCTC 7743,
Staphylococcus aureus NCTC 4163, Staphylococcus epidermidis ATCC 49134, Moraxella
catarrhalis ATCC 25238, Enterococcus faecium ATCC 49474, Pseudomonas aeruginosa
NCTC 6749, Escherichia coli ATCC 25922, Serratia marescens ATCC 8100, Proteus
vulgaris NCTC 4635, and Bacillus subtilis ATCC 6633. Reference strains were supplied by
the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures.
4.8. Surface activity
Surface tension measurements were carried out using a DSA 100 analyzer (Krüss, Germany,
accuracy ±0.01 mN m-1), at 25 oC. The surface tension was determined using the pendant drop
method. Basically, the principle of this method is to form an axisymmetric drop at the tip of a
needle of a syringe. The image of the drop (3 mL) is taken from a CCD camera and digitized.
The surface tension (γ in mN m-1) is calculated by analyzing the profile of the drop according
to the Laplace equation. Temperature was controlled using a Fisherbrand FBH604
thermostatic bath (Fisher, Germany, accuracy 0.1 ±˚C. The values of the critical micelle
concentration (CMC) and the surface tension at the CMC (γCMC) were determined from the
intersection of the two straight lines drawn in low and high concentration regions in surface
tension curves (γ vs log C curves) using a linear regression analysis method. The image of the
drop on the examined surface (paraffin) is the basis for the determination of the contact angle.
After determination of the actual drop shape and the contact line, the drop shape is adapted to
fit a mathematical model used to calculate the contact angle. The most exact method to
calculate this value is Young–Laplace fitting (sessile drop fitting). Complete drop contour is
evaluated. After successful fitting of the Young–Laplace equation, the contact angle is
determined as the slope of the contour line at the 3-phase contact point (solid–liquid and
liquid–air).
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4.9. Synthesis of ILs precursors
An acetonitrile mixture (15 ml) containing the long alkyl bromoalkane (15 mmol) or the
potassium salt of chloroacetic acid (11.25 mmol) was placed in a semi-automated system
EasyMax 102 (Mettler Toledo) equipped with a 50 mL glass reactor, magnetic stirring bar and
ReactIR probe. After 5 min of stirring at 80 °C, the appropriate tertiary amine (15 mmol) or
dimethyldodecylamine (11.25 mmol) was quickly added. The reaction was carried out at 80
°C and the optimal time of the reaction was determined by ReactIR iC15 (Mettler Toledo)
equipped with a MCT detector and a 9.5 mm AgX probe with a diamond tip. Data were
sampled from 2500 to 650 cm-1 with 8 cm-1 resolution and processed by iCIR 4.3 software.
Upon completion, the mixture was cooled to 20 °C and the solvent was removed by rotary
evaporator under vacuum. Next, the obtained precursors were purified by washing repeatedly
with acetone and dried under reduced pressure (5 mbar) at 60 °C for 24 h.
4.10. Synthesis of ILs
Preparation of ILs was carried out in the semi-automated reactor system EasyMax 102
(Mettler Toledo).
Method I: the appropriate quaternary ammonium bromide (0.01 mol) was dissolved with 50
mL of methanol in a 100 mL reaction glass equipped with and mechanical stirrer. Next, 40
mL of anionic resin Dowex Monosphere 550A in the form of methanolic suspension were
added and the mixture was stirred for 5 h at 25 °C. After the anion exchange reaction, the
resin was filtered off and rinsed repeatedly with methanol. Afterwards, the obtained solution
of quaternary ammonium hydroxides was slowly introduced into 50 mL of a methanolic
solution of GA (0.01 mol). The neutralization reaction was conducted for 1 h at 0 °C. At the
end, the solvent was evaporated by rotary evaporator under vacuum and the product was dried
under reduced pressure (5 mbar) at 25 °C for 24 h.
Method II: the amphoteric surfactant (0.01 mol) and 20 mL of methanol were poured into a 50
mL glass reactor containing a magnetic stirring bar. Next, equimolar amount of GA dissolved
in 20 mL of methanol was added and the reaction was conducted for 1 h at a temperature of
25 °C. Afterwards, methanol was evaporated by a rotary evaporator under vacuum and the
product was dried under vacuum for 24 h at a temperature of 25 °C.
[C10DMEA][Gal] - decyl(2-hydroxyethyl)dimethylammonium gallate:
1H NMR (DMSO-d6, 300 MHz) δ (ppm) = 0.83-0.87 (m, 3H, (CH2)7CH3), 1.18-1.31 (m, 14H,
CH2(CH2)7CH3), 1.63 (br.s, 2H, N+CH2CH2(CH2)7), 3.04 (s, 6H, N+(CH3)2), 3.26-3.31 (m,
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2H, HOCH2CH2N+), 3.35-3.38 (m, 2H, N+CH2CH2(CH2)7), 3.81 (br.s, 2H, HOCH2CH2N+),
6.85 (s, 2H, Ar); 13C NMR (DMSO-d6, 75 MHz) δ (ppm) = 14.0, 21.8, 22.1, 28.5, 28.7, 28.8,
28.9, 31.3, 50.7, 54.9, 64.2, 64.7, 109.0, 127.0, 136.8, 145.9, 171.4; elemental analysis calcd
(%) for C21H37NO6 (M = 399.53 g mol-1): C = 63.13, H = 9.34, N = 3.51, found: C = 63.29, H
= 9.21, N = 3.37; IR (neat) 3600-3100 (br), 2927, 2857, 1681, 1546, 1467, 1348, 1220, 1180,
1091, 1041, 965, 883, 796, 733, 679 cm-1.
[C10MMorf][Gal] - 4-decyl-4-methylmorpholinium gallate:
1H NMR (DMSO-d6, 300 MHz) δ (ppm) = 0.83-0.88 (m, 3H, (CH2)7CH3), 1.18-1.31 (m, 14H,
CH2(CH2)7CH3), 1.63 (br.s., 2H, N+CH2CH2(CH2)7), 3.10 (s, 3H, N+CH3), 3.37-3.42 (m, 6H,
N+CH2CH2(CH2)7, (N+CH2CH2O)2), 3.82-3.93 (m, 4H, (N+CH2CH2O)2), 6.84 (s, 2H, Ar); 13C
NMR (DMSO-d6, 75 MHz) δ (ppm) = 14.0, 22.1, 25.8, 28.5, 28.7, 28.8, 28.9 (2x), 29.0, 29.1,
31.3, 45.8, 58.9, 59.8, 64.0, 109.1, 127.3, 136.9, 146.1, 171.8; elemental analysis calcd (%)
for C22H37NO6 (M = 411.54 g mol-1): C = 64.21, H = 9.06, N = 3.40, found: C = 64.53, H =
9.22, N = 3.21; IR (neat) 3600-3100 (br), 2931, 2857, 1685, 1550, 1525, 1469, 1346, 1217,
1180, 1120, 1092, 1041, 968, 891, 856, 794, 727 cm-1.
[C12DMEEA][Gal] - dodecyl[2-(2-hydroxyethoxy)ethyl]dimethylammonium gallate:
1H NMR (DMSO-d6, 300 MHz) δ (ppm) = 0.83-0.88 (m, 3H, (CH2)9CH3), 1.18-1.31 (m, 18H,
CH2(CH2)9CH3), 1.62 (br.s., 2H, N+CH2CH2(CH2)9), 3.03 (s, 6H, N+(CH3)2), 3.24-3.29 (m,
2H, OCH2CH2N+), 3.44-3.53 (m, 6H, N+CH2CH2(CH2)9, CH2OCH2), 3.79 (s, 2H,
13
HOCH2CH2O), 6.85 (s, 2H, Ar); C NMR (DMSO-d6, 75 MHz) δ (ppm) = 14.0, 22.1, 25.8,
28.5, 28.8, 28.9, 29.0, 29.1, 31.3, 50.7, 60.0, 62.3, 63.9, 64.3, 72.2, 109.1, 126.5, 137.1, 146.0,
171.4; elemental analysis calcd (%) for C23H41NO7 (M = 443.58 g mol-1): C = 62.21, H =
9.32, N = 3.16, found: C = 62.02, H = 9.17, N = 3. 25, IR (neat) 3600-3100 (br), 2927, 2857,
1697, 1538, 1467, 1350, 1218, 1182, 1124, 1041, 974, 881, 794, 729, 659 cm-1.
[C12Bet][Gal] - dodecyldimethylglycine gallate:
1H NMR (DMSO-d6, 300 MHz) δ (ppm) = 0.83-0.88 (m, 3H, (CH2)9CH3), 1.17-1.30 (m, 18H,
CH2(CH2)9CH3), 1.62 (br.s., 2H, N+CH2CH2(CH2)9), 3.14 (s, 6H, N+(CH3)2), 3.45-3.51 (m,
13
2H, N+CH2CH2(CH2)9), 3.69 (s, 2H, HOOCCH2N+), 6.93 (s, 2H, Ar); C NMR (DMSO-d6,
75 MHz) δ (ppm) = 14.0, 22.2, 26.0, 28.7, 28.8, 28.9, 29.1 (2x), 29.2, 31.4, 50.1, 63.0, 63.9,
109.0, 121.4, 138.0, 145.8, 165.6, 168.1; elemental analysis calcd (%) for C23H39NO7 (M =
441.57 g mol-1): C = 62.56, H = 8.90, N = 3.17, found: C = 62.73, H = 8.79, N = 3.25, IR
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(neat) 3600-3100 (br), 2927, 2855, 1689, 1614, 1594, 1554, 1454, 1394, 1332, 1270, 1242,
1188, 1047, 1024, 900, 879, 773, 727, 692 cm-1.
4.11. Statistical analysis
Comparisons of antioxidant measurements were carried out in STATISTICA 12 (StatSoft)
using one-way analysis of variance (ANOVA) with Tukey’s test for post hoc analysis (p <
0.05).
Acknowledgements
This work was supported by 03/32/DSPB/0608, Poznan University of Technology,
Department of Chemical Technology.
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