Fabio Cardellinia,
Matteo Tieccoa,
Raimondo Germani*a,
Gianluigi Cardinaliab,
Laura Corteb,
Luca Roscinib and
Nicoletta Spretic
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
First published on 23rd October 2014
We report the preparation and the study of the properties of novel deep eutectic solvents (DESs) formed by zwitterionic trimethylglicine and high melting point carboxylic acids. These zwitterionic DESs are liquid at room temperature or at temperatures lower than 70 °C and do not have chloride or any metal ions in their composition. These media were characterized in terms of their viscosity, conductivity, density, ionicity (via Walden plots), surface tension and thermal stability. The values observed are similar to other typical DESs, and the mixtures resulted in “poor ionic liquids” in the Walden plot. The values obtained could be correlated to the carboxylic acids' structures. The toxicity of the pure mixtures was evaluated via an FTIR-bioassay on Saccharomyces cerevisiae cells. This method allowed us to define the action of these media as dehydrating agents on eukaryotic model cells, with a mechanism highly correlated with CaCl2, a well-known dehydrating agent. The Glycolic acid/Trimethylglicine eutectic system can be considered a NADES (Natural Deep Eutectic Solvent) and resulted as the best in our set for many reasons. It is formed by natural, renewable, eco-compatible and cheap compounds (both the molecules can be derived from sugar beet) and it can be used in many applications because it has a low melting point (−36 °C) and it is colorless, and therefore suitable for spectroscopic measurements. For these reasons, the solubility of some α-L-amino acids was investigated in this DES, showing a good solubility for aromatic amino acids, which are normally scarcely soluble in water.
For these reasons, more biocompatible novel systems have gained relevance in recent years, like bio-based solvents.13 In this topic important media have been realized, such as bio-ILs14 and biomass derived systems e.g. lactic acid15 and GAAS–meglumine mixture.16
Among these systems, deep eutectic solvents (DESs) represent an interesting class of solvents.17 DESs are a novel family of media generally liquid at temperatures lower than 100 °C.18 DESs show chemico-physical properties similar to the traditionally used ionic liquids; however they are less toxic and more biodegradable.19–22 DESs can be prepared by mere mixing high-melting-point quaternary ammonium or phosphonium salts23 with neutral compounds, which are able to form hydrogen-bond interactions, as alcohols,24 amides,25 carboxylic acids, phenols,26 polyols or carbohydrates.27–30 The strong interaction between the hydrogen-bond donor (HBD) compound and the anion, provided by the salt, leads to a considerable reduction in the melting point of the mixture.18
Due to their properties, DESs are promising green media for several applications. For the simplicity of preparation, atom economy and the low cost of many starting products, DESs have gained particular attention in recent years as substitutes to conventional volatile organic solvents in many chemical processes. DESs have found multiple applications in organic synthesis as solvents and/or catalysts,31–34 in liquid–liquid extraction processes,35,36 in electrochemistry as electrolytes,37–40 in biocatalysys,41–44 in metal oxides solubilization,45,46 and in nanomaterials preparation.47
Among deep eutectic solvents, the binary system choline chloride (ChCl)/urea was the first to be developed25 and it is the most widely studied and used. Recently Choi and co-workers27 developed the family of NADES (Natural Deep Eutectic Solvents) eutectic binary systems formed by mixing natural vegetal compounds. A noteworthy example of these media is represented by sugar-based solvents, which are a chiral media formed by bio renewable compounds.27,48 The NADES showed a good capability of solubilization of molecules as DNA and albumin and other metabolic compounds, endorsing the hypothesis of the use of these molecules for metabolism of plants in absence of water.49
Other binary systems able to form DESs have been studied since 2003.50 Most of them are formed by ammonium or phosphonium salts with halogens counterions, mainly bromide or chloride. However these counterions can have nucleophilic, alkaline, complexing or oxidant properties. These behaviors can lead to side-products using this family of DESs as reaction media. In a previous paper we showed how quaternary ammonium salts, with methanesulfonate or tosylate as counterions, form DESs when mixed with p-toluenesulfonic acid monohydrate as HBD molecule. These DESs were used as the dual solvent–catalyst in the Fisher esterification of carboxylic acid with alcohols.51
This work is a continuation of our investigations to find novel deep eutectic solvents, without chloride or any metal atoms, with acid properties. The focus of the present study is the developing of a new class of DESs using carboxylic acids as HBD compounds and trimethylglicine as HBA. Trimethylglicine (TMG) is a natural bio-renewable and sustainable zwitterionic molecule,52–55 with m.p. 300 °C.27
The novel DESs were characterized in terms of viscosity, conductivity and density to obtain Walden plots of their ionicity. Surface tension and thermogravimetric analyses were carried out as a characterization of the properties of these novel mixtures. The solubilizing capability was tested with α-L-amino acids comparing the results with the solubility of the same compounds in water. A FTIR-based bioassay, developed in our laboratory,56 was performed on Saccharomices cerevisiae yeast cells to determine the presence and the extent of cell stress caused by the DESs, therefore their toxicity. The yeast Saccharomyces cerevisiae is the typical model species to represent eukaryotic cells keeping all advantages of microorganism manipulation.
Entry | M.p. (°C) | pKa | Mixture with TMG at 90 °Ca | |
---|---|---|---|---|
a Molar ratio carboxylic acids–betaine 2![]() ![]() |
||||
Aromatic acids | ||||
1 | Benzoic | 122 | 4.2 | Liquid |
2 | 4-Hydroxy-benzoic | 214 | 4.5 | Solid |
3 | 3-Hydroxy-benzoic | 200 | 4.5 | Solid |
4 | 3,5-Hydroxy-benzoic | 236 | 4.0 | Solid |
5 | 2-Hydroxy-benzoic (salicylic) | 159 | 3.0 | Liquid |
6 | 4-Methoxy-benzoic | 182 | 4.5 | Solid |
7 | 3-Iodo-benzoic | 185 | 3.9 | Solid |
8 | 4-Chloro-benzoic | 140 | 4.0 | Liquid |
9 | 2-Chloro-benzoic | 140 | 2.9 | Liquid |
10 | 3-Chloro-benzoic | 154 | 3.8 | Liquid |
11 | 2 -Bromo-benzoic | 150 | 2.9 | Liquidb |
12 | 4-Bromo-benzoic | 252 | 4.0 | Solid |
13 | 2-Nitro-benzoic | 159 | 2.2 | Liquidb |
14 | 4-Nitro-benzoic | 237 | 3.4 | Solid |
15 | 3-Nitro-benzoic | 139 | 3.5 | Solid |
16 | 3,5-Dinitro-benzoic | 204 | 2.8 | Solid |
17 | 2-Chloro-3-nitro-benzoic | 183 | — | Solid |
18 | 4-Amino-benzoic | 187 | 4.6 | Solid |
19 | Terephtalic | 210 | 3.5–4.8 | Solid |
20 | 1,3,5-Benzentricarboxylic | 300 | 3.1–3.8–4.7 | Solid |
21 | Isonicotinic | 300 | 1.9 | Solid |
22 | Nicotinic | 236 | 2.2 | Solid |
23 | 2-Furoic | 128 | 3.1 | Liquid |
![]() |
||||
Aliphatic acids | ||||
24 | Phenylacetic | 76 | 4.3 | Liquid |
25 | 3-(2-Hydroxy-phenyl)-propionic | 86 | 4.2 | Liquidb |
26 | D-(+)-Mandelic | 132 | 3.4 | Liquid |
27 | Trans-Cinnammic | 132 | 4.4 | Solid |
28 | Glycolic | 75 | 3.8 | Liquid |
29 | Oxalic·2H2O | 101 | 1.2–4.4 | Liquid |
30 | Malonic | 135 | 2.8–5.7 | Liquidb |
31 | Maleic | 137 | 1.9–6.0 | Liquidb |
32 | Adipic | 152 | 4.4–5.4 | Solid |
33 | Sebacic | 131 | 4.7–5.4 | Solid |
34 | Ascorbic | 190 | 4.1–11.6 | Solid |
35 | Citric·H2O | 153 | 3.1–4.8–6.3 | Liquid |
36 | L-Tartaric | 168 | 2.4–4.2 | Solid |
We observed that the melting point of the acid has generally an impact on DES formation, in fact, melting points lower than 160 °C led to liquid mixtures at 90 °C. In the family of benzoic acids (entry 1–20) DESs were formed at 90 °C both in presence of EWG and EDG substituents in the aromatic ring. The –OH, –NO2 and –Br substituted benzoic acids were liquid in mixture with TMG only if the substituent is in ortho-position (entry 5, 11, 13). This might be due to their lower melting points and to their lower pKa, therefore to the capability of H-bond formation. Consequently all the three Cl-benzoic acids (entry 8–10) formed liquid mixtures with TMG at 90 °C for their low melting points. In the aromatic heterocyclic acids class (entry 21–23), 2-furoic acid led to a liquid mixture, while the nicotinic and isonicotinic ones have too high melting points. These two acids moreover are zwitterions with the acid hydrogen bound to pyridine nitrogen and not to carboxyl. The aliphatic monocarboxylic acids (entry 24–28) led to DESs as their melting points are lower than 160 °C. Trans-cinnamic acid (entry 27) did not lead to a liquid mixture, in spite of the fact that its pKa and m.p. are comparable to the other acids generating DESs. This might be due to molecular conformations of the acid.60 In the bi-carboxylic acids class (entry 29–33) the number of methylene groups between the two carboxyls seemed to have more impact on liquid mixture formation rather than their melting points. Sebacic and adipic acids mixtures with TMG, in fact, did not lead to DESs, while malonic and oxalic acids with TMG formed liquid mixtures. A liquid mixture was observed in the citric acid/TMG case, in spite of the fact that the citric acid has an high melting point, but in this case the water hydration of the acid could favor the DES formation lowering the melting point of the mixture. Ascorbic acid (entry 34) has a high m.p. and its acid behavior depends on enolic –OH groups with less capability of H-bond formation; so its mixture with TMG was not liquid.
The eutectic ratio was determined for those mixtures liquid at 90 °C. The melting point of the mixtures acid/betaine were determined by varying the molar fraction (x) of the carboxylic acid. In Fig. 1 is reported a profile of melting point/molar fraction x of two HBD molecules, benzoic and glycolic acids. (See ESI section for all the profiles, Fig. S1†).
![]() | ||
Fig. 1 Melting points of TMG with benzoic and glycolic acids as a function of acid molar fraction x. (♦) Benzoic acid/TMG DES; (□) glycolic acid/TMG DES. |
The liquid mixtures studied in depth in this paper are the ones that showed the most suitable properties (Table 2), the other liquid DESs showed unstable behavior during time. In Table 2 are reported the melting temperature values at the eutectic point, and the decrease of the melting points compared to the ones of the pure carboxylic acid (ΔT).
Entry | Acid | Acid m.p. (°C) | DES m.p (°C) | ΔT (°C) | Molar ratio acid/betaine |
---|---|---|---|---|---|
a Betaine m.p. 300 °C. | |||||
1 | Benzoic | 122 | 53 | 69 | 1.5 |
5 | 2-Hydroxy-benzoic (salicylic) | 159 | 63 | 96 | 1.5 |
8 | 4-Chloro-benzoic | 140 | 28 | 112 | 1.5 |
9 | 2-Chloro-benzoic | 140 | 39 | 101 | 1.5 |
10 | 3-Chloro-benzoic | 154 | 43 | 111 | 1.5 |
23 | 2-Furoic | 130 | 11 | 119 | 2 |
24 | Phenylacetic | 76 | −7 | 83 | 2 |
26 | D-(+)-Mandelic | 132 | 13 | 119 | 1 |
28 | Glycolic | 75 | −36 | 111 | 2 |
29 | Oxalic·2H2O | 101 | 33 | 68 | 2 |
35 | Citric·H2O | 153 | 48 | 105 | 1.5 |
The optimal eutectic ratio of 1.5:
1 was observed for benzoic acid and derivatives (entry 1, 5, 8, 9, 10) and for citric acid (entry 35). The eutectic ratio of 1
:
1 was observed for D-(+)-mandelic acid (entry 26). This could be due to the –OH group in the structure that can interact with betaine carboxylate via H-bonding. The optimal eutectic ratio of 2
:
1 was observed for all the other acid/TMG systems. DESs derived from phenylacetic (entry 24), 2-fuoroic (entry 23), D-(+)-mandelic (entry 26) and glycolic acids (entry 28), are relevant because they are liquid at room temperature. The glycolic acid/TMG system is particularly significant, because it's formed by natural substances extractable from sugar beet, therefore it can be considered a NADES (Natural Deep Eutectic Solvent).
![]() | (1) |
Profiles of viscosity/T of the novel DESs are reported in Fig. 2 (temperature range 35–100 °C).
The viscosity of these liquids resulted quite high (>100 cP) at room temperature, probably due to a network of intramolecular hydrogen bonds and to molecular packing formed in these systems that leads to a reduced mobility of free species. The lowest viscosity value observed in our DESs was η = 93 cP at 40 °C of oxalic acid/TMG, it could be due to hydration water that decreases viscosity increasing mobility. Viscosity value of phenylacetic acid/betaine was η = 523 cP and glycolic acid/TMG showed η = 937 cP (40 °C). This value might be determined by intramolecular hydrogen bonds formed by alcoholic –OH in glycolic acid. Homologous deep eutectic solvents reported in literature show similar viscosity values, so the hydrogen bond network and therefore the lower mobility of free species could determine the values of η in the novel DESs presented in this work.22,29,58
The systems with 2-chlorobenzoic, 4-chlorobenzoic, D-(+)-mandelic acids showed non-Newtonian behaviors after a definite temperature. As showed in the chart in the inset in Fig. 2, the viscosities go through an increase after a specific temperature. A non-Newtonian similar behavior is reported in literature with imidazolinic DESs.59 We can hypothesize a differently structured system, in terms of higher viscosity, that determined discontinuities in the profiles.
Activation energy Eη was determined for those DESs that followed the Arrhenius law (eqn (1)). The values determined are 42.6 kJ mol−1 for glycolic acid DES, 34.2 kJ mol−1 for oxalic acid DES, 45.9 kJ mol−1 for benzoic acid DES, 41.6 kJ mol−1 for phenylacetic acid DES, 61.3 kJ mol−1 for 2-furoic acid DES. The Eη is dependent on the ratio R±/Rh, where R± is the ion radius and Rh is the radius of the vacancies in the liquid. High Eη values mean difficult movement of the molecules in the liquid due to the small dimensions of the vacancies.61,62 The molecular structures of the acids could determine the values we observed in our systems. Planar, symmetrical and rigid molecules (such as benzoic acid) could provoke interactions that can get the molecules closer in the liquid structure, therefore provoke smaller dimensions of the vacancies and higher Eη values. 2-Furoic acid has an oxygen that can play a HBA role in hydrogen bond interactions that can increase the Eη value. The Eη value of oxalic acid/TMG DES is the lowest of our set, and it can be determined by hydration water in the mixture.
The conductivity of these systems resulted low, as expected because of the non-ionicity of the molecules. The hydrogen bonding in fact is not a complete charge transfer, so it does not provoke a formation of two discrete ionic species. Oxalic acid/TMG was the DES that showed the highest conductivity value (5.1 mS cm−1, 30 °C). Glycolic acid mixture showed 0.179 mS cm−1 at the same temperature, the other systems showed values one order of magnitude lower. The conductivity of oxalic acid system is due to the small dimension of the acid molecule (therefore higher mobility) and to the hydration water that enhances the ionicity of the DES. The small dimension of the acid is responsible also of the conductivity value of glycolic acid system. A linear behavior, closer to the Arrhenius equation, was observed for aromatic acids mixtures only at high temperatures (80–100 °C).
![]() | (2) |
The 2-furoic acid showed a linear trend. In the aliphatic family only D-(+)-mandelic acid showed a non-linear trend. The systems following the Arrhenius law are: oxalic (EΛ = −24.4 kJ mol−1), glycolic (EΛ = −31.1 kJ mol−1), citric (EΛ = −48.2 kJ mol−1), phenylacetic (EΛ = −27.1 kJ mol−1) and 2-furoic (EΛ = −47.8 kJ mol−1). These values are comparable to other analogues,29,57,61 so our DESs can be considered similar to the ones in literature in terms of conductivity. The values of EΛ (similarly to Eη) can be related to the energy required to the formation and the expansion of vacancies, responsible of mass transport.61 Oxalic and phenylacetic DESs showed the weaker intramolecular interactions because of their EΛ values. Glycolic and citric acids systems displayed high values for their higher hydrogen bonding capacity. Oxalic acid/TMG mixture has a similar behavior but weaker, due to the hydration water that can weaken the H-bonding interaction, and therefore leading to higher mobility of the molecules. The 2-furoic acid showed a high value; this seems due to further H-bonding interactions as observed also in viscosity measures.
Densities in the temperature range 25–100 °C are reported in Table 3.
Glycolic | Oxalic | D-(+)-Mandelic | Phenylacetic | ||||
---|---|---|---|---|---|---|---|
T, °C | ρ, g cm−1 | T, °C | ρ, g cm−1 | T, °C | ρ, g cm−1 | T, °C | ρ, g cm−1 |
25 | 1.27 | — | — | 25 | 1.22 | 25 | 1.16 |
50 | 1.25 | 50 | 1.27 | 50 | 1.21 | 50 | 1.15 |
60 | 1.24 | 60 | 1.26 | 60 | 1.2 | 60 | 1.13 |
75 | 1.23 | 75 | 1.24 | 75 | 1.19 | 75 | 1.12 |
90 | 1.22 | 90 | 1.21 | 90 | 1.18 | 90 | 1.11 |
2-Furoic | 2-Chloro-benzoic | 4-Chloro-benzoic | Benzoic | ||||
---|---|---|---|---|---|---|---|
T, °C | ρ, g cm−1 | T, °C | ρ, g cm−1 | T, °C | ρ, g cm−1 | T, °C | ρ, g cm−1 |
30 | 1.27 | — | — | — | — | — | — |
55 | 1.25 | 65 | 1.29 | 65 | 1.27 | — | — |
65 | 1.23 | 75 | 1.27 | 75 | 1.25 | 80 | 1.18 |
75 | 1.22 | 90 | 1.25 | 90 | 1.23 | 90 | 1.17 |
90 | 1.21 | 100 | 1.24 | 100 | 1.22 | 100 | 1.15 |
At room temperature the mixtures showed behaviors similar to the ones observed with the other techniques. The lower density was observed in fact for phenylacetic DES, followed by D-(+)-mandelic, glycolic and 2-furoic acids mixtures. The differences of density between the DESs were even at 90 °C, due to weaker interactions between the molecules. All the DESs showed density values higher than water and similar to other DESs in literature.58,61,63
Λη = Constant | (3) |
Angell and co-workers68 proposed to use Walden plot as a qualitative estimation of the ionicity of the media. According to this we made Walden plots of lnΛ/ln
η−1 of our DESs. In this chart the diagonal represent the theoretical behavior of KCl 0.01 M in water, assuming it is fully dissociated and has ions of equal mobility. The reaction media that have ln
Λ/ln
η−1 values next to that diagonal are considered “good ionic liquids”, and the ones far from that diagonal in the lower portion of the chart are considered “poor ionic liquids” in terms of ionicity. Non-ionic liquids are in the lower portion, at a great distance from the diagonal. On the upper side of the Walden plot, super-ionic liquids can be found. Aprotic IL are considered “good ionic liquids” in the Walden plot, while protic ILs are considered “poor ionic liquids” due to not complete charge transfer in HBA–HBD interaction.70–72
The acid/TMG DESs Walden plots are reported in Fig. 5, only the ones with viscosity Newtonian behavior were analyzed.
![]() | ||
Fig. 5 Walden plot of DESs. (◊) glycolic acid/TMG; (■) oxalic acid/TMG; (●) phenylacetic acid/TMG; (▲) benzoic acid/TMG; (○) 2-furoic acid/TMG. |
The acid/TMG DESs analyzed showed a “poor ionic liquid” behavior, except for the oxalic acid/TMG mixture which has high ionicity behavior. This could be due to the hydration water that facilitates the dissociation and has an impact on the conductivity and the viscosity of the DES, therefore on the position on the plot. The values of pKa of the species (Table 1) seem to have relevance on the position in the Walden plot for the DESs analyzed, even if these values of the acids are measured in water solution. Oxalic acid and TMG have similar pKa values in water (acid = 1.2, betaine = 1.8), so the proton results much more delocalized between the two species. 2-Furoic (pKa = 3.1) and glycolic (pKa = 3.8) acids are close in the plot; glycolic acid DES has a higher ionicity. Phenylacetic (pKa = 4.3) and benzoic (pKa = 4.2) acids DESs are far from the diagonal, in the lower portion, therefore they have low ionicity. Our Acid/TMG DESs showed good linear trends, except for benzoic acid/TMG mixture, which has a non-linear behavior at high temperatures. This could be due to a weakening of the H-bond between the two species at high temperatures.
All the DESs analyzed showed a good stability until 200 °C. This temperature was observed for all the systems studied, so the decomposition can be considered independent on the acid structure. Oxalic acid DES showed a variation starting from 100 °C, due to hydration water loss. The best DES in our set resulted glycolic/TMG system, in terms of its liquid range (Δt = 236 °C). Dai and co-workers27 reported decomposition temperatures of various-structured NADES at about 135 °C for sugar-based ones, and over 200 °C for the others. Our DESs showed similar behavior in terms of thermal stability.
Amino acid | SAA (g/100 g solvent) DES | SAA (g/100 g solvent) water70 |
---|---|---|
Phenylalanine | 4.37 | 2.20 |
Tryptophan | 2.49 | 1.00 |
Tyrosine | 1.05 | 0.01 |
Leucine | 0.54 | 1.76 |
Glutamic acid | 0.205 | 2.20 |
Aspartic acid | 0.150 | 2.10 |
The amino acids with an aromatic portion in their structures (Phenylalanine, Tryptophan and Tyrosine) showed higher solubility in the DES than in water. The solubility of apolar amino acids (such as Leucine) in the DES was little lower than in water, whereas polar amino acids (Glutamic acid and Aspartic acid) showed much lower solubility in DES than in water. The Glycolic acid/TMG DES showed good solubilizing capability of amino acids compared with the ones observed in literature for typical ionic liquids.75,76 This could be due to the presence of a zwitterion in its structure. Ionic liquids are able to solubilize amino acids only in presence of water75,76 or in presence of crown-ethers.77
The low cost in consumables, the reproducibility and the rapidity were considered key factors in this application.86 FTIR analysis could discriminate among the physiological states of microbial cells throughout their growth and differentiation, irrespectively of the type of cells considered.87–89
From a previous analysis, it has emerged that these compounds brought 100% of cell mortality after little exposure times (data not shown). Preliminary microbiological tests showed a very rapid decrease of cell viability after few minutes of exposition to the tested DES. Since such a rapid effect was not previously observed,56,89–91 it was hypothesized that the high concentration of these compounds caused a very rapid exit of the cell water and thus their immediate inactivation. In order to test this hypothesis, CaCl2 was chosen as a dehydrating known not to be toxic, in order to avoid the simultaneous presence of cyto-toxic and dehydration effect.92,93 Normalized spectra from cells treated with DES and CaCl2 were almost identical, with the CaCl2 showing some little differences in the region between 1500 cm−1 and 1200 cm−1 (Fig. 7a, dotted line).
The correlation matrix obtained from normalized spectra (Fig. 7b) indicates lower correlation between control and challenged cells than among the various treatment carried out with DESs and CaCl2, in fact the average correlation of control vs. treated cells is 0.9594, whereas, the average correlation between the cells treated with various compounds ranged from 0.9877 (CaCl2) to 0.9932 (4-chlorobenzoic acid/TMG DES). The RSs showed an overall parallel trend throughout the entire spectra range, with CaCl2 differing from the other compounds in the amide 1 region (1700 cm−1 ca.) and within the mixed region (1250 cm−1 ca.) (Fig. 8).
No differences could be found in the fatty acids and in the carbohydrates regions, confirming that the action exerted by CaCl2 is substantially identical to that of the tested DESs. Furthermore, these results indicate the all tested DESs challenge the cells in the same way, indicating that their biological action is rather independent of the compound structure for this class of mixtures. The correlation analysis between the RSs of the cells treated with the studied DESs confirmed the overall results previously described for the spectra analysis.
Pre-culture was inoculated at OD600 = 0.2 in 200 mL of YEPD + chloramphenicol medium (yeast extract 1%, peptone 1%, dextrose 2% – chloramphenicol 0.5 g L−1 – 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 pelleted. 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 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 μL volume was sampled for three independent FTIR readings (35 μL each, according to the technique suggested by Manfait and co-workers95), while 100 μL 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 = [(1 − Cv)/Ct] × 100.
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).
FTIR measurements were performed in transmission mode. All spectra were recorded in the range between 4000 and 400 cm−1. Spectral resolution was set at 4 cm−1, sampling 256 scans per sample. The software 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 first and second derivatives of spectral values.
Glycolic acid/TMG mixture resulted the best DES in our set. This is due to its properties (very low melting point, wide liquid range, absence of color, low cost) and for its “greennes” (the molecules composing it are derived from sugar beet, the effect on eukaryotic cells is simple dehydration). For these reasons this DES has been applied in solubility measures of α-L-amino acids. These showed that this media could solubilize aromatic amino acids, which are usually very little soluble in water.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra10628h |
This journal is © The Royal Society of Chemistry 2014 |