Synthesis, toxicity, biodegradability and physicochemical properties of 4-benzyl-4-methylmorpholinium-based ionic liquids

Article abstract of DOI:10.1039/c1gc15468k

A series of 4-benzyl-4-methylmorpholinium salts have been synthesized producing morpholinium ionic liquids with inorganic (chloride, nitrate, tetrafluoroborate, hydrogen sulphate) and organic (formate, acetate, methoxyacetate, 2-(2-methoxyethoxy)acetate,

Full text of DOI:10.1039/c1gc15468k

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PAPER
Synthesis, toxicity, biodegradability and physicochemical properties of
4-benzyl-4-methylmorpholinium-based ionic liquids†
Juliusz Pernak,*a Nina Borucka,a Filip Walkiewicz,a Bartosz Markiewicz,a Przemysław Fochtman,b
Stefan Stolte,c Stephanie Steudtec and Piotr Stepnowskic
Received 27th April 2011, Accepted 22nd June 2011
DOI: 10.1039/c1gc15468k
A series of 4-benzyl-4-methylmorpholinium salts have been synthesized producing morpholinium
ionic liquids with inorganic (chloride, nitrate, tetrafluoroborate, hydrogen sulphate) and organic
(formate, acetate, methoxyacetate, 2-(2-methoxyethoxy)acetate, hexanoate, octanoate, dodecyl
sulphate, 2-ethylbutyrate, lactate, crotonate, maleate, salicylate, saccharinate) anions. Their
physicochemical properties, cytotoxicity to the promyelocytic leukaemia rat cell line IPC-81, oral
toxicity and biodegradability were determined. The anion significantly determined the state of
aggregation and cytotoxicity. The results enabled 4-benzyl-4-methylmorpholinium ionic liquids to
be classified as being of moderate or low toxicity (EC50 between 0.15 and 14.13 mM). The acute
toxicity for 4-benzyl-4-ethylmorpholinium acetate is between 300–2000 mg kg-1 b.w. in female rats.
The activity of these morpholinium salts against bacteria and fungi was very low. It was found
that new 4-benzyl-4-methylmorpholinium-based ionic liquids with anions commonly used for
dissolving cellulose, such as formate or acetate, can also be used as new biomass solvents.
imidazolium ILs.5 Their acute ecotoxicity has been studied
Introduction
intensively using several different test systems and species.6–10
Ionic liquids (ILs) with nitrogen, sulphur, or phospho-
For IL cations a general trend was found, namely that acute
rus as the central atom of the cations have been exten-
(eco)toxic effects can be related to a compound’s hydropho-
sively investigated. These include imidazolium, pyrrolidinium,
bicity. Substances with greater hydrophilicity exhibit higher
tetraalkylammonium, pyridinium, piperidinium, sulphonium
and phosphonium-based ILs.1,2
ILs are called ‘green’ usually because of their negligible vapour
(eco)toxicities. Thus, compounds with long alkyl substituents
have greater influence on toxicity than cations with shorter alkyl
residues or side chains containing polar functional groups. Be-
pressure. Low volatility, however, does not completely eliminate
sides the cationic head group of ILs, the anion plays a central role
potential environmental hazards and can pose serious threats
in defining physicochemical as well as toxicological properties.
to aquatic and terrestrial ecosystems.3 There are many other
Several anions, especially highly fluorinated and hydrophobic
factors that determine whether ILs are or are not green. Today,
ones, show higher activity compared to, for example, halides in
it is very important that not only ILs themselves should be
different biological test systems.11
green (low toxicity or biodegradability), but also their synthesis.4
Recently, interest in morpholinium-based ILs has in-
In this respect, however, extensive studies on the toxicity and
creased because of their properties. In fact, they are less
biodegradability of ILs are necessary. The vast majority of
toxic than the commonly used imidazolium, pyridinium or
toxicological studies on ILs available to date have focused on
tetraalkylammonium-based ILs.12 Morpholinium ILs are ap-
plied as electrolytes13,14 and gel electrolytes,15 ionic liquid crystals
(the class of liquid-crystalline compounds),16,17 reaction media18
aDepartment of Chemical Technology, Poznan University of Technology,
or corrosion inhibitors.19 In particular, ILs based on the
pl. M. Sklodowskiej-Curie 2, 60-965, Poznan, Poland.
E-mail: juliusz.pernak@put.poznan.pl; Tel: +48 61 665 36 82
morpholinium cation are preferred because of their electro-
chemical stability.20 However, little data on the (eco)toxicity
of morpholinium-based ILs are available. The functionalized
morpholinium compounds tested so far have shown low acute
cytotoxic effects up to concentrations of 20 mM in tests with the
promyelocytic leukaemia rat cell line IPC-81.21
bInstitute of Industrial Organic Chemistry, Pszczyna Branch, ul.
Dos´wiadczalna 27, 43-200, Pszczyna, Poland.
E-mail: fochtman@ipo-pszczyna.pl; Fax: +48 32 210 30 81 ext. 120;
Tel: +48 32 210 30 81 ext. 124
cDepartment of Environmental Analysis, Faculty of Chemistry, University
of Gdan´sk, ul. Sobieskiego 18, 80-952, Gdan´sk, Poland. E-mail: sox@
chem.univ.gda.pl; Fax: +48 58 523 55 72; Tel: +48 58 523 54 48
Apart from a low (eco)toxicity, current environmental legis-
lation makes insistent demands for non-persistent chemicals.
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1gc15468k
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For chemicals to be described as ‘sustainable’ or ‘green’,
complete and rapid biotic degradation is a crucial requirement.
The biodegradability of IL cations is strongly dependent on
structure. Pyridinium compounds with ethyl and octyl side
chains are completely mineralized within 28 days,22,23 whereas
imidazolium-based cations are only partially degradable, with
the core structure itself remaining resistant to biodegradation.24
However, investigations examining the biodegradability of dif-
ferent head groups such as morpholinium are still required.
This study reports on the synthesis of novel 4-benzyl-
4-methylmorpholinium and 4-benzyl-4-ethylmorpholinium
cations combined with 16 different anions: inorganic (chloride,
nitrate, tetrafluoroborate or hydrogen sulphate), organic
aromatic (salicylate or saccharinate) and aliphatic (formate,
acetate, hexanoate, octanoate, maleate, crotonate or lactate)
acids and dodecyl sulphate. Besides typical physicochemical
properties such as melting points, viscosities, and thermal
stabilities, data describing their toxicological properties (acute
toxicity towards bacteria, fungi, in vitro cell line and rat) and
environmental properties (ready biodegradability) are also
presented.
determined with a pH-meter (inoLab pH720) using a combined
electrode (ERH-11S).
Synthesis of 4-benzyl-4-methylmorpholinium salts
4-Benzyl-4-methylmorpholinium and 4-benzyl-4-ethylmor-
pholinium chlorides have recently been described.25,26
Method I: Morpholinium salts were synthesized by treating
equimolar quantities of sodium or potassium salts with 4-
benzyl-4-methylmorpholinium chloride in methanol. The so-
lutions were stirred at room temperature for 1 h, after which
the precipitated sodium or potassium chloride was removed by
filtration. After evaporation of the methanol, the product was
dissolved in acetone and the remaining inorganic salt removed.
The product (1, 2, 10 and 16) was dried under vacuum at 45 ◦C
for 10 h.
Method II: Two salts (3 and 14) were synthesized by the
dropwise addition of sulphuric acid or maleic acid (0.02 mol) dis-
solved in dichloromethane to 4-benzyl-4-methylmorpholinium
chloride (0.02 mol) in dichloromethane, with initial cooling in
ice, followed by warming to room temperature and overnight
stirring. Dry air was passed through the salts obtained to
remove hydrochloric acid. The products were then separated
from dichloromethane and dried under vacuum at 55 ◦C for
8 h.
Experimental
Materials
Method III: The remaining morpholinium salts were synthe-
sized in two stages. In the first stage, a stoichiometric quantity
of potassium hydroxide was added to a portion of 4-benzyl-
4-methylmorpholinium chloride (0.015 mol) or 4-benzyl-4-
ethylmorpholinium chloride (0.015 mol), dissolved in methanol.
The solutions were stirred at room temperature for 0.5 h, after
which the partially precipitated potassium chloride was removed
by filtration. In the second stage, a stoichiometric amount of
an appropriate carboxylic acid was added to the filtrate. The
solutions were stirred again at room temperature for 0.5 h
and, then, after evaporation of the methanol, the product was
dissolved in acetone and the remaining inorganic salt removed.
The products (4–9, 11–13, 15 and 17) were dried under vacuum
at 50 ◦C for 9 h.
4-Methylmorpholine, 4-ethylmorpholine, benzyl chloride,
potassium nitrate, sodium tetrafluoroborate, sulphuric acid,
formic acid, acetic acid, methoxyacetic acid, 2-(2-methoxy-
ethoxy)acetic acid, hexanoic acid, octanoic acid, sodium do-
decyl sulphate, lactic acid, salicylic acid, sodium saccharin,
dimethylsulphoxide, trifluoroacetic acid, potassium phosphate
monobasic, potassium phosphate dibasic, sodium phosphate
dibasic dihydrate, calcium chloride dihydrate, magnesium sul-
phate heptahydrate, iron(III) chloride and microcrystalline cel-
lulose were obtained from Sigma Aldrich and used without
further purification. Crotonic acid, maleic acid, 2-ethylbutyric
acid, acetonitrile, ammonium chloride, potassium hydroxide and
mercury(II) chloride were purchased from Fluka. All solvents
were used as obtained without further purification. Cell culture
media, sera and phosphate buffer were purchased from Invitro-
gen Life Technologies (Frankfurt, Germany), WST-1 reagent
was obtained from Roche Diagnostics GmbH (Mannheim,
Germany).
4-Benzyl-4-methylmorpholinium nitrate (1). 1H NMR
(DMSO-d6) d (ppm) 3.11 (s, 3H); 3.38 (t, J = 4.29 Hz, 2H); 3.58
(qw, J = 2.61 Hz, 2H); 3.99 (t, J = 4.31 Hz, 4H); 4.78 (s, 2H);
7.54 (m, 3H); 7.60 (m, 2H); 13C NMR (DMSO-d6) d (ppm) 45.0;
58.5; 59.9; 67.6; 127.2; 128.9; 130.4; 133.3. Elemental analysis
calc. (%) for C12H18N2O4 (254.28): C 56.68, H 7.13, N 11.02.
Found: C 57.03, H 6.85, N 11.46.
General
1H NMR spectra were recorded on a Mercury Gemini
300 spectrometer operating at 300 MHz with TMS as the
internal standard. 13C NMR spectra were obtained with the same
instrument at 75 MHz. CHN elemental analyses were performed
at the Adam Mickiewicz University, Poznan (Poland). The
water content was determined using an Aquastar volumetric
Karl-Fischer titration with Composite 5 solution as titrant and
anhydrous methanol as solvent. Melting points were determined
by visual observation using a hot-plate apparatus. Viscosity was
determined using a rheometer (Rheotec RC30-CPS) with cone-
shaped geometry (C50-2). The viscosity of the samples, about
1.5◦mL, was measured with respect to temperature, from 20 to
55 C. The pH values of aqueous solutions of IL, 0.1 M, were
4-Benzyl-4-methylmorpholinium tetrafluoroborate (2). solid.
1H NMR (DMSO-d6) d (ppm) 3.06 (s, 3H); 3.32 (t, J = 4.25 Hz,
2H); 3.53 (qw, J = 5.21 Hz, 2H); 3.98 (t, J = 3.29 Hz, 4H); 4.68
(s, 2H); 7.54 (m, 5H); 13C NMR (DMSO-d6) d (ppm) 44.9; 58.6;
59.8; 67.7; 127.1; 129.0; 130.4; 133.2. Elemental analysis calc.
(%) for C12H18NOBF4 (279.08): C 51.64, H 6.50, N 5.02. Found:
C 51.93, H 6.12, N 5.49.
4-Benzyl-4-methylmorpholinium hydrogen sulfate (3). 1H
NMR (DMSO-d6) d (ppm) 3.10 (s, 3H); 3.17 (s,1H); 3.37 (t,
J = 4.29 Hz, 2H); 3.56 (qw, J = 5.24 Hz, 2H); 3.98 (t, J = 1.71
Hz, 4H); 4.77 (s, 2H); 7.54 (m, 3H); 7.60 (m, 2H); 13C NMR
(DMSO-d6) d (ppm) 45.0; 58.5; 59.9; 67.6; 127.3; 129.0; 130.4;
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133.4. Elemental analysis calc. (%) for C12H19NO5S (289.35): C
49.81, H 6.62, N 4.84. Found: C 49.37, H 7.03, N 4.42.
3H); 7.60 (m, 2H); 13C NMR (DMSO-d6) d (ppm) 13.9; 22.1;
25.5; 28.71; 28.77; 29.0; 31.3; 44.8; 58.4; 59.8; 65.5; 67.4; 127.2;
128.8; 130.3; 133.2. Elemental analysis calc. (%) for C24H43NO5S
(457.66): C 62.98, H 9.47, N 3.06. Found: C 62.46, H 9.93, N
2.67.
4-Benzyl-4-methylmorpholinium formate (4). 1H NMR
(DMSO-d6) d (ppm) 3.13 (s, 3H); 3.41 (t, J = 4.27 Hz, 2H); 3.60
(qw, J = 5.13 Hz, 2H); 3.99 (t, J = 6.35 Hz, 4H); 4.84 (s, 2H);
7.53 (m, 3H); 7.62 (m, 2H); 8.62 (s, 1H); 13C NMR (DMSO-d6)
d (ppm) 44.8; 58.4; 59.9; 67.4; 127.4; 128.9; 130.3; 133.4; 165.7.
Elemental analysis calc. (%) for C13H19NO3 (237.29): C 65.80, H
8.07, N 5.90. Found: C 65.21, H 8.84, N 5.39.
4-Benzyl-4-methylmorpholinium 2-ethylbutyrate (11). 1H
NMR (DMSO-d6) d (ppm) 0.78 (t, J = 4.90 Hz, 6H); 1.23 (qw,
J = 4.04 Hz, 2H); 1.40 (qw, J = 4.28 Hz, 2H); 1.73 (qw, J = 3.38
Hz, 1H); 3.14 (s, 3H); 3.43 (t, J = 4.30 Hz, 2H); 3.61 (qw, J =
5.19 Hz, 2H); 3.98 (t, J = 2.42 Hz, 4H); 4.85 (s, 2H); 7.52 (m,
3H); 7.63 (m, 2H); 13C NMR (DMSO-d6) d (ppm) 12.6; 25.7;
44.8; 52.1; 58.3; 59.9; 67.3; 127.5; 128.8; 130.2; 133.4; 178.1.
Elemental analysis calc. (%) for C18H29NO3 (307.43): C 70.32, H
9.51, N 4.56. Found: C 70.02, H 10.11, N 3.95.
4-Benzyl-4-methylmorpholinium acetate (5). 1H NMR
(DMSO-d6) d (ppm) 1.61 (s, 3H); 3.15 (s, 3H); 3.43 (t, J = 4.27
Hz, 2H); 3.60 (qw, J = 5.24 Hz, 2H); 3.98 (t, J = 5.86 Hz, 4H);
4.88 (s, 2H); 7.52 (m, 3H); 7.64 (m, 2H); 13C NMR (DMSO-d6)
d (ppm) 26.1; 44.7; 58.3; 59.9; 67.2; 127.6; 128.8; 130.2; 133.4;
173.2. Elemental analysis calc. (%) for C14H21NO3 (251.32): C
66.91, H 8.42, N 5.57. Found: C 67.47, H 8.93, N 4.96.
4-Benzyl-4-methylmorpholinium lactate (12). 1H NMR
(DMSO-d6) d (ppm) 1.32 (d, J = 3.36 Hz, 3H); 3.29 (s, 3H);
3.56 (t, J = 4.26 Hz, 2H); 3.68 (qw, J = 5.27 Hz, 2H); 3.92 (d, J =
3.36 Hz, 1H); 3.97 (t, J = 3.89 Hz, 4H); 4.46 (m, 1H); 4.94 (s,
2H); 7.43 (m, 3H); 7.56 (m, 2H); 13C NMR (DMSO-d6) d (ppm)
21.3; 45.1; 58.2; 60.3; 68.0; 68.9; 126.2; 129.0; 130.5; 133.1; 179.6.
Elemental analysis calc. (%) for C15H23NO4 (281.35): C 64.03, H
8.24, N 4.98. Found: C 63.55, H 8.79, N 4.31.
4-Benzyl-4-methylmorpholinium methoxyacetate (6). 1H
NMR (DMSO-d6) d (ppm) 3.12 (s, 3H); 3.21 (s, 3H); 3.40 (t,
J = 4.27 Hz, 2H); 3.59 (qw, J = 4.06 Hz, 2H); 3.98 (t, J = 2.50
Hz, 4H); 4.11 (s, 2H); 4.82 (s, 2H); 7.53 (m, 3H); 7.62 (m, 2H);
13C NMR (DMSO-d6) d (ppm) 44.8; 57.5; 58.4; 59.9; 67.4; 72.0;
127.4; 128.9; 130.3; 133.3; 172.1. Elemental analysis calc. (%) for
C15H23NO4 (281.35): C 64.03, H 8.24, N 4.98. Found: C 64.48,
H 7.84, N 4.47.
4-Benzyl-4-methylmorpholinium crotonate (13). 1H NMR
(DMSO-d6) d (ppm) 1.68 (d, J = 4.20 Hz, 3H); 3.26 (s, 3H);
3.52 (t, J = 4.20 Hz, 2H); 3.59 (qw, J = 5.28 Hz, 2H); 3.93 (t, J =
5.93 Hz, 4H); 4.83 (s, 2H); 5.88 (d, J = 7.70 Hz, 1H); 6.53 (qw, J =
4.44 Hz, 1H); 7.37 (m, 3H); 7.54 (m, 2H); 13C NMR (DMSO-d6)
d (ppm) 17.2; 45.4; 58.0; 60.4; 69.1; 126.6; 128.9; 130.2; 130.4;
133.4; 136.8; 172.3. Elemental analysis calc. (%) for C16H23NO3
(277.36): C 69.29, H 8.36, N 5.05. Found: C 69.86, H 8.78, N
4.64.
4-Benzyl-4-methylmorpholinium 2-(2-methoxyethoxy)acetate
(7). 1H NMR (DMSO-d6) d (ppm) 3.11 (s, 3H); 3.23 (s, 3H);
3.41 (t, J = 1.83 Hz, 2H); 3.49 (qw, J = 1.98 Hz, 2H); 3.57 (t, J =
3.66 Hz, 4H); 3.76 (s, 2H); 3.98 (t, J = 6.23 Hz, 4H); 4.81 (s, 2H);
7.53 (m, 3H); 7.61 (m, 2H); 13C NMR (DMSO-d6) d (ppm) 44.8;
58.0; 58.4; 59.9; 67.5; 68.7; 71.0; 71.4; 127.4; 128.9; 130.3; 133.4;
172.2. Elemental analysis calc. (%) for C17H27NO5 (325.40): C
62.75, H 8.36, N 4.30. Found: C 63.18, H 7.84, N 4.84.
4-Benzyl-4-methylmorpholinium maleate (14). 1H NMR
(DMSO-d6) d (ppm) 3.11 (s, 3H); 3.38 (t, J = 4.10 Hz, 2H);
3.58 (qw, 2H); 3.98 (t, 4H); 4.82 (s, 2H); 6.28 (d, 2H); 7.53 (m,
3H); 7.61 (m, 2H); 13C NMR (DMSO-d6) d (ppm) 44.9; 58.5;
59.9; 67.4; 127.3; 128.9; 130.3; 130.4; 133.3; 166.7. Elemental
analysis calc. (%) for C16H21NO5 (307.34): C 62.53, H 6.89, N
4.56. Found: C 62.98, H 7.47, N 3.94.
4-Benzyl-4-methylmorpholinium hexanoate (8). 1H NMR
(DMSO-d6) d (ppm) 0.83 (t, J = 4.66 Hz, 3H); 1.21 (m, 4H);
1.42 (qw, J = 5.92 Hz, 2H); 1.89 (t, J = 4.93 Hz, 2H); 3.15 (s,
3H); 3.43 (t, J = 4.26 Hz, 2H); 3.62 (qw, J = 5.26 Hz, 2H);
3.98 (t, J = 2.36 Hz, 4H); 4.88 (s, 2H); 7.51 (m, 3H); 7.64 (m,
2H); 13C NMR (DMSO-d6) d (ppm) 14.0; 22.2; 26.1; 31.7; 38.3;
44.6; 58.3; 59.9; 67.2; 127.5; 128.7; 130.1; 133.4; 175.6. Elemental
analysis calc. (%) for C18H23NO3 (307.43): C 70.32, H 9.51, N
4.56. Found: C 69.84, H 9.02, N 4.97.
4-Benzyl-4-methylmorpholinium salicylate (15). 1H NMR
(DMSO-d6) d (ppm) 3.08 (s, 3H); 3.35 (t, J = 4.21 Hz, 2H);
3.56 (qw, J = 5.22 Hz, 2H); 3.98 (t, J = 2.70 Hz, 4H); 4.74 (s,
2H); 6.66 (m, 2H); 7.18 (t, J = 5.72 Hz, 1H); 7.53 (m, 3H); 7.57
(m, 2H); 7.69 (d, J = 4.67 Hz, 1H); 13C NMR (DMSO-d6) d
(ppm) 44.9; 58.5; 59.8; 67.6; 116.0; 116.3; 119.6; 127.2; 128.9;
130.0; 130.3; 131.9; 133.2; 162.7; 171.5. Elemental analysis calc.
(%) for C19H23NO4 (329.39): C 69.28, H 7.04, N 4.25. Found: C
68.74, H 7.55, N 4.82.
4-Benzyl-4-methylmorpholinium octanoate (9). 1H NMR
(DMSO-d6) d (ppm) 0.84 (t, J = 4.52 Hz, 3H); 1.21 (qw, J =
8.42 Hz, 8H); 1.40 (qw, J = 5.64 Hz, 2H); 1.86 (t, J = 5.01 Hz,
2H); 3.12 (s, 3H); 3.41 (t, J = 4.27 Hz, 2H); 3.59 (qw, J = 5.20 Hz,
2H); 3.95 (t, J = 2.20 Hz, 4H); 4.83 (s, 2H); 7.52 (m, 3H); 7.62
(m, 2H); 13C NMR (DMSO-d6) d (ppm) 14.0; 22.1; 26.6; 28.8;
29.5; 31.4; 38.6; 44.7; 58.3; 60.0; 67.4; 127.4; 128.8; 130.2; 133.3;
175.6. Elemental analysis calc. (%) for C20H33NO3 (335.48): C
71.60, H 9.91, N 4.17. Found: C 71.04, H 10.37, N 4.59.
4-Benzyl-4-methylmorpholinium saccharinate (16). 1H NMR
(DMSO-d6) d (ppm) 3.28 (s, 3H); 3.55 (t, J = 4.26 Hz, 2H); 3.65
(qw, J = 5.30 Hz, 2H); 3.92 (t, J = 6.13 Hz, 4H); 4.91 (s, 2H);
7.35 (m, 3H); 7.56 (m, 4H); 7.75 (m, 2H); 13C NMR (DMSO-d6)
d (ppm) 45.3; 58.2; 60.4; 69.3; 119.5; 123.2; 126.1; 129.1; 130.6;
131.4; 132.0; 133.4; 134.6; 144.4; 170.1. Elemental analysis calc.
(%) for C19H22N2O4S (374.45): C 60.94, H 5.92, N 7.48. Found:
C 60.86, H 5.84, N 7.59.
4-Benzyl-4-methylmorpholinium dodecyl sulfate (10). solid.
1H NMR (DMSO-d6) d (ppm) 0.85 (t, J = 4.56 Hz, 3H); 1.23
(qw, J = 4.32 Hz, 18H); 1.47 (qw, J = 4.00 Hz, 2H); 3.10 (s, 3H);
3.36 (t, J = 4.33 Hz, 2H); 3.57 (qw, J = 4.22 Hz, 2H); 3.69 (t, J =
4.76 Hz, 2H); 3.98 (t, J = 2.90 Hz, 4H); 4.78 (s, 2H); 7.53 (m,
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4-Benzyl-4-ethylmorpholinium acetate (17). 1H NMR
(DMSO-d6) d (ppm) 1.36 (t, J = 4.78 Hz, 3H); 1.64 (s, 3H); 3.44
(m, 6H); 3.97 (t, J = 2.83 Hz, 4H); 4.74 (s, 2H); 7.53 (m, 3H);
7.56 (m, 2H); 13C NMR (DMSO-d6) d (ppm) 7.2; 25.1; 51.2;
55.9; 59.7; 62.3; 127.3; 129.0; 130.3; 133.1; 173.5. Elemental
analysis calc. (%) for C15H23NO3 (265.35): C 67.90, H 8.74, N
5.28. Found: C 67.42, H 9.25, N 4.76.
DMSO concentration has been proven not to be cytotoxic.
For the test, IPC-81 cells in a concentration of 15 ¥ 105 cells
mL-1 (in RPMI with 8% foetal calf serum) were incubated for
44 h in 96-well plates in the presence of a morpholinium salt
and for an additional 4 h in the presence of WST-1 reagent.
Cell viability as the ability to reduce WST-1 was observed
photometrically at 450 nm in a microplate reader (MRX,
Dynatech Laboratories, Chantilly, USA). The cytotoxicity of
the compounds was expressed as the percentage of cell viability
measured as WST-1 reduction compared to the controls. Each
dose–response curve was recorded for at least 9 parallel dilution
series on three different 96 well plates. Dose–response curve
parameters and plots were obtained using the drift package
(version 0.05–63) for R.30 Positive controls with carbendazim
were checked at regular intervals.
Thermal analysis
Thermal transition temperatures of the morpholinium salts
were determined by DSC, with a Mettler Toledo Stare DSC1
(Leicester, UK) unit, under nitrogen. Samples between 5 and
15 mg were placed in aluminium pans and heated from 25
◦
to 120 ◦C at a heating rate of 10 C min-1 and cooled with
◦
an intracooler at a cooling rate of 10 C min-1 to -100 ◦C
and then heated again to 120 ◦C. Thermogravimetric analysis
was performed using a Mettler Toledo Stare TGA/DSC1 unit
(Leicester, UK) under nitrogen. Samples between 2 and 10 mg
were placed in aluminium pans and heated from 30 to 450 ◦C at
a heating rate of 10 ◦C min-1.
Biodegradation tests
The primary biodegradation test was carried out using a
modified version of OECD guideline 301 D.31 The primary
biodegradation of the compound was monitored via HPLC-UV
for 28 days. The inoculum was obtained from the wastewater
treatment plant at Gdan´sk (Poland) and aerated for 48 h. A
mineral medium containing final concentrations of 8.5 g L-1
KH2PO4, 21.75 mg L-1 K2HPO4, 22.13 mg L-1 Na2HPO4·2H2O,
1.7 mg L-1 NH4Cl, 36.4 mg L-1 CaCl2·2H2O, 22.5 mg L-1
MgSO4·7H2O and 0.25 mg L-1 FeCl3 (pH 7.2) was added to
the suspension. The dry weight of the sludge was determined
gravimetrically (10 g L-1). Samples containing 200 mM (ca. 45 mg
L-1) 4-benzyl-4-methylmorpholinium chloride were prepared, as
well as blank samples (inoculated media without test substance)
and abiotic control samples (inoculated media containing
200 mM 4-benzyl-4-methylmorpholinium chloride poisoned
with 50 mg L-1 HgCl2), each in replicates. All samples were
aerated at 20 ◦C in the dark during the test. Losses due to
evaporation were checked regularly by weighing and made good
by the addition of water. The oxygen content was checked as
well. For analysis of the biodegradation 1000 mL of all samples
were taken at regular intervals and centrifuged (5000 rpm,
15 min, MiniSpin, Eppendorf, Hamburg, Germany). 500 mL
of the supernatant were analysed via HPLC-UV (212 nm)
Antimicrobial activity
The antimicrobial activity was determined by the tube dilution
method and described earlier.27 The lowest concentration of
compound at which there was no visible growth (turbidity) was
taken as representing the MIC (minimum inhibitory concen-
tration). The lowest concentration of compound supporting
no colony formation was defined as the MBC for bacteria
and MFC for fungi. The following microorganisms were used:
Micrococcus luteus NCTC 7743, Staphylococcus aureus NCTC
4163, Staphylococcus epidermidis ATCC 49134, Enterococcus
faecium ATCC 49474, Moraxella catarrhalis ATCC 25238,
Escherichia coli ATCC 25922, Serratia marcescens ATCC 8100,
Proteus vulgaris NCTC 4635, Pseudomonas aeruginosa NCTC
6749, Bacillus subtilis ATCC 6633, Candida albicans ATCC
10231, and Rhodothorula rubra (Demml 1889, Lodder 1934).
Standard strains were supplied by the National Collection of
Type Cultures (NCTC) London and the American Type Culture
Collection (ATCC). Rhodothorula rubra was obtained from
the Department of Pharmaceutical Bacteriology, University of
Medical Sciences, Poznan.
˚
on a C6-Phenyl column (Gemini 5u C6-Phenyl 110 A 150 ¥
4.60 mm). An isocratic method using 7% acetonitrile and 93%
0.1% trifluoroacetic acid and a flow rate of 0.7 mL min-1
was applied (retention time for 4-benzyl-4-methylmorpholinium
8.05 min, limit of detection 1 mM, limit of quantification 3 mM).
The percentage of biodegradation was determined by integrating
the peak area in comparison to the initial concentration (day 0).
The manometric respirometry test was performed according
to OECD guideline 301 F.32 The biological oxygen demand of
the substance was determined for 28 days using BOD TRAK
(Hach Lange GmbH). The inoculum used was obtained from
the wastewater treatment plant at Delmenhorst (Germany).
The inoculum was filtered and aerated for 24 h before use. A
mineral medium containing final concentrations of 85 mg L-1
KH2PO4, 217.5 mg L-1 K2HPO4, 221.3 mg L-1 Na2HPO4·2H2O,
17 mg L-1 NH4Cl, 36.4 mg L-1 CaCl2·2H2O, 22.5 mg L-1,
MgSO4·7H2O and 0.25 mg L-1 FeCl3 (pH 7.2) was added
to the filtrate. Samples containing 20 mg L-1 and 100 mg
Cell viability assay
The cytotoxicity of the morpholinium salts was determined for
the promyelocytic leukaemia rat cell line IPC-81.28 Cultures of
IPC-81 were grown in RPMI medium (with L-glutamine, with-
out NaHCO3, supplemented with 1% penicillin–streptomycin
and 1% glutamine, pH 7) with 10% horse serum at 37 ◦C
(5% CO2).
The cytotoxicity assay was carried out according to Ranke
et al.29 Cell viability was measured using a colorimetric assay
for 96 well plates with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-
(2,4-disulphophenyl)-2H-tetrazolium monosodium salt (WST-
1) reagent. Each plate contained blanks (no cells), controls (no
toxicants), and substance in 1 : 1 dilution series. Stocks of ILs
were prepared in a culture medium with 0.5% dimethylsulphox-
ide (DMSO) to improve the solubility of the substances. This
2904 | Green Chem., 2011, 13, 2901–2910
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L-1 4-benzyl-4-methylmorpholinium chloride respectively were
prepared, as well as blank samples (inoculated media without
test substance). The bottles containing vessels with potassium
hydroxide to ensure absorption of the carbon dioxide produced
were hermetically sealed and kept in the dark at 20 ◦C.
The oxygen consumption was determined manometrically. The
biodegradation of the test substance was calculated by the
oxygen uptake for the test substance (corrected by the oxygen
demand of blank samples) related to the theoretical oxygen
demand (ThOD) of the substance and the amount of substance
present in the sample:
Dissolution of cellulose in the morpholinium ILs
Microcrystalline cellulose (MCC) was added to a 20 mL vial
containing 1.0 g of the dried IL. The vial, sealed with parafilm,
was then immersed in an oil bath; the instability of the bath
temperature was estimated to be
1
◦C. The mixture was
heated at a given temperature (95 ◦C) and stirred. Additional
cellulose was added until the solution became optically clear
under a microscope (Bresser Biolux LCD Microscope). When
the cellulose became saturated, this was judged to be the point
where cellulose could not be dissolved further within 1 h.
O2(s) −O2(z)
Results and discussion
B =
×100%
C(s) ×ThOD(s)
Seventeen new 4-benzyl-4-methylmorpholinium salts (structure
of cations in Scheme 1 and of organic anions in Scheme 2) were
synthesized via anion-exchange reaction but in three different
ways (Scheme 3) with high yields (Table 1, only two salts
7 and 9 gave a yield of 70%). Although it was possible to
carry out the reactions in aqueous solution, better yields were
obtained in an organic solvent. The yield of anion-exchange
reaction and total yield for the reaction of quaternization and
anion-exchange are showed in Table 1. Potassium or sodium
chloride were a by-product of the exchange reaction according
to methods I and III, and were removed from anhydrous
acetone solution. In method II hydrochloric acid was the by-
product; it was removed by passing dry air, which was then
where:
B is percentage of biodegradation of the test substance, %;
O2(s) is uptake of oxygen in the sample, mg(O L-1;
)
2
O2(z) is the oxygen demand of blank samples, mg(O L-1;
)
2
C(s) is the concentration of substance present in the sample,
mg(s) L-1;
ThOD(s) is the theoretical oxygen demand (ThOD) of the
substance, mg(O mg(s)
-1
.
)
2
Acute oral toxicity test
The studies were conducted at the Institute of Industrial
Organic Chemistry, Pszczyna Branch, Poland. The toxicity was
tested according to the acute toxic class method (OECD No.
420: Method UE B.1.BIS). The studies were started with a
preliminary experiment in which 17 was administered once to a
female Wistar rat, 171 g body weight (b.w.), at a dose of 2000 mg
kg-1 b.w. and to another female rat, b.w. of 167 g, at a dose of
300 mg kg-1 b.w. 17 was first suspended in distilled water and then
administered intragastrically at doses of 300 and 2000 mg kg-1
b.w. In the experiment proper, four 9-week-old rats were used
(mean body weight 172 g). After the dose was administered, the
rats were observed for 14 days.
Scheme 1 Structure of cations.
Table 1 4-Benzyl-4-methylmorpholinium [BMmorf] (1–16) and 4-benzyl-4-ethylmorpholinium [BEmorf] (17) salts
Yield/%
Salt
Anion
Metathesis
Total
mpa/◦C
Viscosityc/Pa s
pHd
1
Nitrate, [NO3]
88.0
83.0
94.0
83.0
90.5
85.5
70.0
86.0
70.5
98.5
83.0
89.0
98.0
93.0
88.0
88.0
84.5
83.0
78.5
89.0
78.5
85.5
81.0
66.0
81.5
66.5
93.0
78.5
84.0
92.5
88.0
83.0
83.0
80.0
Liquid
88–92b
Liquid
Wax
Liquid
Wax
143.64
—
8.31
—
5.81
—
—
5.41
0.49
—
6.12
4.16
—
11.54
80.51
—
7.18
2.81
0.87
6.84
8.33
3.98
4.43
5.48
6.28
7.62
5.29
7.33
5.22
2.48
3.15
4.48
5.25
2
Tetrafluoroborate, [BF4]
Hydrogen sulfate, [HSO4]
Formate, [Fmt]
3
4
5
Acetate, [Ac]
6
Methoxyacetate, [MAc]
2-(2-Methoxyethoxy)acetate, [MEAc]
Hexanoate, [Hex]
7
Wax
8
Liquid
Liquid
65–68
Liquid
Liquid
Wax
Liquid
Liquid
Wax
9
Octanoate, [Oct]
10
11
12
13
14
15
16
17
Dodecyl sulfate, [C12OSO3]
2-Ethylbutyrate, [2-C2Bu]
Lactate, [Lac]
Crotonate, [Cro]
Maleate, [Mal]
Salicylate, [Sal]
Saccharinate, [Sac]
Acetate, [Ac]
Liquid
0.33
a Melting point determined by hot-plate apparatus. b Crystallized from water. c Measured at 25 ◦C. d For concentration 100 mM.
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Table 2 Chemical shifts
1H NMR
13C NMR
IL
+N-CH3
+N-CH2-Ph
+N-CH2-Ph
[BMmorf][Cl]
3.10
3.06
3.10
3.15
3.15
3.14
3.29
3.26
3.28
4.81
4.68
4.77
4.88
4.88
4.85
4.94
4.83
4.91
67.3
67.7
67.6
67.2
67.2
67.3
68.9
69.1
69.3
2
3
5
8
11
12
13
16
Of the seventeen synthesized salts, ten were liquids, two
were crystalline and the other five were a wax. The liquid
products were dried under vacuum and stored over P4O10.
The synthesized salts were air and moisture stable and soluble
in water, methanol, dichloromethane (except 3 and 14) and
acetone. They were immiscible with hexane and toluene. The
water content, determined by Karl-Fischer measurements, was
found to be less than 500 ppm. All the morpholinium salts
◦
obtained were ILs with melting points below 100 C and were
characterized by 1H and 13C NMR spectroscopy and elemental
CHN analysis. In the 1H NMR spectra, proton chemical shifts as
large as 0.25 ppm were observed for the protons located around
the quaternary nitrogen atom (see Table 2). On the other hand,
the 13C NMR spectra indicated weak variation in the carbon
signal shifts at the level of 2 ppm (Table 2).
Scheme 2 Structure of organic anions.
The anions significantly determined the state of aggregation
on morpholinium ILs. By selecting an appropriate anion, it was
possible to obtain an IL resembling water (low viscosity), a
viscous liquid, wax or a solid substance with a high melting
point. Correspondingly, the viscosity of the liquid ILs at room
temperature (Table 1) varied widely over a wide range from
0.33 for 17 to 143.64 Pa s for 1. The value was strongly
temperature-dependent, as evident from Fig. 1. The results
show how strongly the viscosity, measured at room temperature,
depended on the type of anion. With increasing temperature
the effect of the anion on viscosities was overridden and similar
for each counterion (Fig. 1). The dependence of shear stress
versus shear rate at 25 ◦C was rectilinear, so morpholinium ILs
exhibited the character of a Newtonian liquid.
Scheme 3 Scheme of the synthesis methods.
reacted with potassium hydroxide in a trap until the colour of
phenolphthalein disappeared. This method was used for acids,
whose pKa did not exceed 2 (sulphuric acid pKa = 1.99, maleic
acid pKa1 = 1.9333,34).
Fig. 1 Viscosity of the liquid compounds.
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Table 3 Thermal stability and phase transition of the ILs
IL Tga/◦C Tmb/◦C Tonset(5%)c/◦C Tonset(50%)d/◦C T1e/◦C
used the concept of “anion effect ratio” to classify relative
toxicities of anions in ILs.11,39 It is defined the ratio (AR) of
the EC50 value of the chloride-containing reference ionic liquid
(here 4-benzyl-4-methylmorpholinium chloride) and the EC50
value measured for the same cation combined with a different
anion. Anions in ionic liquids with AR values smaller than 5
are considered as non-cytotoxic or only marginal cytotoxic.
In contrast, anions exhibiting AR values greater than 5 are
considered as significantly influencing the cytotoxicity of the
corresponding IL. In general, all the compounds showed a
cytotoxicity level from 0.25 to 14.13 mM and AR vaules from 1
to 43. Comparison of 4-benzyl-4-methylmorpholinium chloride
(EC50 11 mM) with 1-methyl-3-octylimidazolium chloride (EC50
0.10 mM28) shows that the cytotoxicity of the morpholinium
cation itself is low.
The cytotoxicity effect is dramatically changed by the anion to
lower EC50 and higher AR values. When combining the 4-benzyl-
4-methylmorpholinium cation with nitrate [NO3], hydrogen
sulphate [HSO4], formate [Fmt], acetate [Ac], methoxyacetate
[Mac], 2-(2-methoxyethoxy)acetate [MEAc], hexanoate [Hex],
2-ethylbutyrate [2-C2Bu], lactate [Lac], crotonate [Cro] or
saccharinate [Sac]] anion, the cytotoxicity of the IL is not
considerably increased (AR values < 5).
Whereas the combination with, tetrafluoroborate [BF4], oc-
tanoate [Oct], dodecyl sulphate [C12OSO3], maleate [Mal], and
salicylate [Sal], gives a lower EC50 values and AR values ranging
from 5 to 43. The low EC50 value of the dodecyl sulphate anion
can be related to the long alkyl side chain, which is responsible
for the high lipophilicity. The cytotoxicity of sodium dodecyl
sulphate was already known to be 0.5 mM;24 the results obtained
in this study verified this value. Surprisingly, the IL containing
[BF4] anion also had a low EC50 value (AR 26). Toxicity studies
of this anion combined with an inorganic cation have indicated
its much lower toxicity.11
The acute toxicity values listed in Fig. 2 indicate that
substitution of chloride by an organic anion containing two
oxygen atoms results in decreased toxicity, whereas three or
more oxygen atoms in the anion or the presence of a linear alkyl
group containing five or more carbon atoms, or the presence of
an aromatic ring is associated with an increase in toxicity.
1
2
4
5
6
7
8
9
-19.4
-1.3
—
88–92
—
—
—
—
—
—
74–77
—
—
—
—
—
—
—
196
240
114
107
95
116
100
112
226
120
146
113
115
145
182
120
250
312
178
175
189
247
180
210
276
193
198
184
236
223
280
172
—
—
—
—
-52.4
-56.7
-57.6
-57.3
-55.7
-56.2
—
247 (50.0)
189 (47.3)
191 (55.8)
—
185 (45.5)
—
10 -42.0
11 -44.8
12 -34.8
13 -29.8
14 -43.4
15 -12.4
16 8.6
—
—
220 (49.8)
255 (57.5)
—
17 -56.6
a Glass transition temp. determined by DSC. b Melting point determined
by DSC. c Decomposition temp. determined from onset to 5 wt%
mass loss. d Decomposition temp. determined from onset to 50%
mass loss. e Second step decomposition (percentage decomposition in
parentheses).
Since the ILs synthesized were soluble in water, it was decided
to measure the pH of their 100 mM solutions in water. The
results are listed in Table 1. Following dissolution in water,
morpholinium ILs with the anions presented in Scheme 2 yielded
solutions with pH values ranging from 8.33 to 0.87. The pH
values for 4-benzyl-4-methylmorpholinium salts changed in the
following sequence:
[Ac] > [C12OSO3] > [Lac] > [NO3] > [Fmt] > [Oct] > [Cl] (pH
= 5.76) > [Hex] > [2-C2Bu] > [Cro] > [Sac] > [MEAc] > [MAc]
> [Sal] > [BF4] > [Mal] > [HSO4].
To a large extent the pH of the aqueous solution was determined
by the anion.
As shown in Table 3, the thermal stability of morpholinium
ILs also depended on the anion. The most stable ILs were those
with [BF4] (2) and [C12OSO3] (10) anions, where decomposition
started above 200 ◦C. In six morpholinium ILs, a second
◦
decomposition step was observed – in 7 at 247 C (50% mass
loss), in 8 at 189 ◦C (47% mass loss), in 9 at 191 ◦C (56% mass
loss), in 11 at 185 ◦C (46% mass loss), in 15 at 220 ◦C (50% mass
loss), and in 16 at 255 ◦C (58% mass loss). A glass transition was
observed with almost every salt. The melting point was observed
in two morpholinium ILs.
For a first toxicity evaluation we investigated the antimicrobial
and in vitro toxicity with IPC-81 cells isolated from rats. These
test systems provide reproducible results for measuring the acute
toxicity of ILs, and many data are available for comparison.35–37
For four ILs (6, 10, 12, 17) and 4-benzyl-4-methy-
lmorpholinium chloride the determined values of MIC, MBC
and MFC are listed in Table 4. The IL with the dodecyl sulphate
anion (10) was slightly more effective than IL with lactate (12)
because of its long alkyl chain. Summing up, the activity of
morpholinium salts against bacteria and fungi was very low.
This was confirmed by tests on the antimicrobial activity of
4-carbalkoxymethyl-4-methylmorpholinium chlorides.38
The cytotoxicity of the compounds synthesized to the IPC
81 rat leukaemia cell line is summarized in Table 5. We
Fig.
2 Acute in vitro cytotoxicity of the ILs and 4-benzyl-4-
methylmorpholinium chloride (EC50 values in mM).
The acute oral toxicities of 4-benzyl-4-ethylmorpholinium
acetate (17) was determined in six female Wistar rats, which
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Table 4 MICa, MBCb and MFCc valuesd for the ILs and [BMmorf][Cl]e
ILs
Strain
6
10
12
17
[BMmorf][Cl]e
M. luteus
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MFC
MIC
MFC
>1.8
>1.8
>1.8
>1.8
>1.8
>1.8
0.9
>1.8
0.9
0.9
>1.8
>1.8
>1.8
>1.8
>1.8
>1.8
>1.8
>1.8
0.9
0.5
0.5
0.3
0.3
0.3
>1.1
0.5
>1.1
0.1
0.3
>1.8
>1.8
0.9
>1.8
0.9
>1.8
0.9
0.9
>1.9
>1.9
>1.9
>1.9
>1.9
>1.9
>1.9
>1.9
0.9
>2.2
>2.2
>2.2
>2.2
>2.2
>2.2
>2.2
>2.2
1.1
S. aureus
S. epidermidis
E. faecium
M. catarrhalis
E. coli
0.9
0.9
0.9
0.9
0.9
1.1
1.1
0.3
0.3
>1.8
>1.8
>1.8
0.9
>1.8
>1.8
>1.8
0.9
>1.9
>1.9
>1.9
>1.9
>1.9
>1.9
>1.9
0.9
>2.2
>2.2
>2.2
>2.2
>2.2
>2.2
>2.2
1.1
S. marcescens
P. vulgaris
P. aeruginosa
B. subtilis
C. albicans
R. rubra
>1.1
>1.1
>1.1
>1.1
>1.1
>1.1
0.3
0.9
0.3
0.9
0.9
1.1
>1.8
>1.8
>1.8
>1.8
>1.1
>1.1
0.5
>1.8
>1.8
>1.8
>1.8
>1.9
>1.9
>1.9
>1.9
>2.2
>2.2
>2.2
>2.2
>1.1
a minimum inhibitory concentration; b minimum bactericidal concentration; c minimum fungicidal concentration; d in mM; e 4-benzyl-4-
methylmorpholinium chloride.
Table 5 EC50 values for the acute toxicity of IL towards IPC-81 cells
thus be classified as a category 4 (R22 Harmful if swallowed)
toxin, according to standard GHS grading.
IL
anion
EC50/mM
Anion effect ratio (AR)
Compared to the imidazolium and pyridinium ILs5 described,
the low biological activity of morpholinium ILs and the
moderate oral acute toxicity towards rats deserves attention: this
trait significantly influences the application of morpholinium-
based ILs.
[Cl]
[NO3]
[BF4]
[HSO4]
[Fmt]
[Ac]
[MAc]
[MEAc]
[Hex]
[Oct]
[C12OSO3]
[2-C2Bu]
[Lac]
[Cro]
[Mal]
[Sal]
10.72
10.00
0.41
1
1
26
3
1
1
1
1
3
7
43
1
<1
1
5
1
2
3
4.37
4
12.59
12.59
7.94
7.76
4.27
1.66
0.25
13.49
>10.00
14.13
2.24
The biodegradability of 4-benzyl-4-methylmorpholinium
cation combined with chloride has been tested in primary
biodegradation and oxygen consumption tests. As neither test
showed any biodegradation of the cation, the cation cannot
be classified as readily biodegradable. It does not necessarily
follow that it will not degrade in the environment, but further
testing (e.g. for inherent biodegradation) will be necessary to
clear this initial suspicion of persistence. Also, the introduction
of functionalized side chains instead of the phenyl group could
help to improve biodegradability.
5
6
7
8
9
10
11
12
13
14
15
16
0.79
3.16
14
3
[Sac]
Dissolution of cellulose was tested for six morpholinium ILs
with carboxylate anions – 4, 5, 7, 8, 9 and 17. Because of the
low stability of ILs, the dissolution was carried out at 95 ◦C.
All compounds showed the ability to dissolve microcrystalline
cellulose but needed different times to dissolve 1% of cellulose:
for example, 7 mixed with cellulose for 3 h showed a significant
fragmentation of cellulose pulp and partial dissolution of
cellulose. Morpholinium acetate dissolved cellulose best, and
elongation of the alkyl chain in the cation shortened the
dissolution time. As shown in Fig. 3, 1% cellulose dissolved
completely after just 0.5 h of heating the cellulose with 4-
benzyl-4-ethylmorpholinium acetate, 17. This means that the
morpholinium ILs have the ability to dissolve cellulose, thus
forming a new class of compounds able to dissolve cellulose.40
received a dose of 300 mg kg-1 b.w. (mg of substance per kg of
body weight) and 2000 mg kg-1 b.w. of 17. In the preliminary
experiment, a single administration of 17 to a rat at a dose of
2000 mg kg-1 b. w. was followed within 30 min by development of
clinical signs, which persisted until the rat died two hours later.
In the experiment proper, following a single administration of 17
at a dose of 300 mg kg-1 b.w. to four other rats no signs of toxicity
were observed. In the second week of the experiment two of the
animals demonstrated a slight decrease in body weight, while
the remaining animals gained weight. The above results indicate
that the acute toxicity range for 17 is between 300–2000 mg kg-1
b.w. in female rats (Imp: WIST – outbred strain). This IL would
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Fig. 3 Micrographs of 1% of microcrystalline cellulose dissolved in 4-benzyl-4-ethylmorpholinium acetate - 17 (a) after adding cellulose, (b) after
0.5 h of stirring (temperature: 95 ◦C; fourfold enlargement).
5 M. Petkovic, K. R. Seddon, L. P. N. Rebelo and C. S. Pereira, Chem.
Soc. Rev., 2011, 40, 1383.
Conclusions
6 J. Arning, S. Stolte, A. Bo¨schen, F. Stock, W. R. Pitner, U. Welz-
Biermann, B. Jastorff and J. Ranke, Green Chem., 2008, 10, 47.
7 S. Stolte, M. Matzke, J. Arning, A. Bo¨schen, W. R. Pitner, U.
Welz-Biermann, B. Jastorff and J. Ranke, Green Chem., 2007, 9,
1170.
8 J. Pernak, K. Sobaszkiewicz and I. Mirska, Green Chem., 2003, 5, 52.
9 D. J. Couling, R. J. Bernot, K. M. Docherty, J. K. Dixon and E. J.
Maginn, Green Chem., 2006, 8, 82.
10 A. Latała, M. Ne˛dzi and P. Stepnowski, Green Chem., 2009, 11, 1371.
11 S. Stolte, J. Arning, U. Bottin-Weber, M. Matzke, F. Stock, K. Thiele,
M. Uerdingen, U. Welz-Biermann, B. Jastorff and J. Ranke, Green
Chem., 2006, 8, 621.
By using an anion-exchange reaction, a number of new mor-
pholinium ILs were synthesized. Optimal methods of synthesiz-
ing various salts were developed to obtain the highest possible
yield. All the salts obtained are stable in air, water and commonly
used organic solvents. The anions determined the state of
aggregation on morpholinium ILs to a significant extent. By
selecting an appropriate anion it was possible to obtain an IL
resembling water, a viscous liquid, wax or a solid salt, and also
to regulate thermal stability.
The activity of 4-benzyl-4-methylmorpholinium ILs against
bacteria and fungi was very low. The cytotoxicity of these
compounds can be classified as moderate or low, but may vary
dramatically – from 0.25 to 14.13 mM – depending on the anion.
The acute toxicity of 4-benzyl-4-ethylmorpholinium acetate is
between 300–2000 mg kg-1 b.w. in female rats. This IL would
thus be classified as a category 4 (R22 Harmful if swallowed)
toxin, according to standard GHS grading. The biodegradation
tests cannot classify the 4-benzyl-4-methylmorpholinium cation
as readily biodegradable. However, this does not mean that these
compounds will not decompose in the environment, only that
to determine their behaviour in the environment other, more
comprehensive tests should be carried out.
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In addition, 4-benzyl-4-ethylmorpholinium acetate, because
it has the lowest viscosity, can be effectively used as a solvent for
biomass.
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Acknowledgements
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This work was supported by the Foundation for Polish Science,
academic grants for professors, MISTRZ Programme, 2009
edition. This research was also supported by the Polish Ministry
of Research and Higher Education under grants DS 8200-4-
0085-11 and SPB/260/7PR/2010.
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