WO2012145522A2 - Deep eutectic solvent systems and methods - Google Patents

Abstract

Disclosed is a Deep Eutectic Solvent (DES) system that comprises betaine monohydrate as one component. The other component can be urea or acids, such as malonic acid or citric acid. Depending on the composition, the melting point of the mixture can be considerably lower than the melting point of either component. The use of betaine monohydrate is advantageous because of its low cost, among other reasons. The DES can be used to dissolve components, such as cellulose, starch, lignin, synthetic polymers, and others, that are not soluble in other media. After treatment of Avicel® (cellulose) with a DES comprising betaine monohydrate and urea, there can be a decrease of crystallinity of at least about 10-15%.

Description

DEEP EUTECTIC SOLVENT SYSTEMS AND METHODS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/477,024 filed 19 April 2011, the entire contents and substance of which is hereby incorporated by reference in its entirety as if fully set forth below.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to deep eutectic solvents, and more particularly, to deep eutectic solvents that comprise betaine monohydrate.

2. Description of Related Art

A eutectic system is a mixture of at least two compounds that solidifies at lower temperature than either one of them at the pure state. A deep eutectic solvent (DES) is a type of ionic solvent with special properties composed of a mixture that forms a eutectic with a melting point much lower than either of the individual components. DES is used herein to include both the singular "solvent" and the plural "solvents", and in some cases, will refer also to deep eutectic system(s). At other times, the term "deep eutectic solvent system" may be used, including "DES system". Those of ordinary skill in the art will recognize difference uses in context. To differentiate eutectic mixtures, the term Deep Eutectic Solvents (DES), also Room Temperature Ionic Liquids (RTIL), has been introduced, which refers to eutectic mixtures that are liquid at or below room temperature.

The principle is illustrated in Fig. 1. Fig. 1 shows that a eutectic occurs at a defined ratio of the compounds, the eutectic composition. At this point all three phases are in a thermal equilibrium: melt(L), a and β. Conventional eutectic solvents are based on mixtures of metals such as alloys.

DES are currently applied in large scale applications such as electro winning of metals or electro polishing of stainless steel, where their advantage of a high solubility for metal oxides is applied. Furthermore, they can be employed as solvents in organic reactions such as Diels-Alder reactions and transesterifications. By mixing a quaternary ammonium salt and a metal salt (or hydrogen bond donor), a eutectic can be observed. Early work focused on eutectic systems comprising the quaternary ammonium salts imidazolium or pyridinium chloride, and the metal salts such as SnCl₂ or ZnCl₂, whereas more recent work involves choline chloride-derived deep eutectic .

There are generally two different methods to form a DES with choline chloride (ChCl) as the quaternary ammonium salt. First the combination of tin(II) chloride or zinc chloride in a molar ratio of 1:2 forms solvents with melting points of 23-25°C and 43-45°C, respectively. These solvents, however, have a disadvantage of high viscosity.

A second method is the combination of various ureas or a carboxylic acid. These solvents, such as urea/choline chloride with molar ratio 2: 1 have a melting point of 12°C with lower viscosity than the metal choline chlorides. However, they too are still viscous at 1100 cP.

Nevertheless, these solvents exhibit good conductivity and moisture stability. Highly ionic and strongly hydrogen-bonding compounds are freely soluble in theses solvents. AgCl, which is insoluble in water, is soluble in the urea/ChCl DES up to 0.66 M. On the other hand, compounds which are not highly ionic or strong hydrogen bonding, are widely immiscible.

The deep eutectic is mainly caused by shielding the charge of the anion by means of complexing it with hydrogen bond donors, as shown in Fig. 2 for the example urea/choline chloride. These interactions led to the significant reduction of the freezing point up to 200°C. A correlation was found between the strength of the hydrogen bond donor and the depression of the freezing point.

DES or Room Temperature Ionic Liquids (RTIL) are mostly formed by use of choline chloride. This, however, is too expensive to be employed in large scale processes. The cost for choline chloride can be as high as $745.00 U.S.

Cellulose

Cellulose is the most abundant renewable biomaterial known and is considered to be a key target for the replacement of fossil energy, as it does not compete with the food market like starch. Cellulose comprises of several hundred D-glucose units linked by a β 1→4 glycosidic linkage. These glucose chains form intermolecular hydrogen bonds which lead to the supramolecular structure of cellulose-like fibers. It is of general interest to hydrolyze cellulose to glucose, which can be further processed with yeast to obtain ethanol.

Due to its crystalline nature, cellulose is not soluble in water or most organic liquids. To apply cellulose in large scale applications, a low cost and environmentally benign solvent with a high availability is needed. Much effort was put into the development of ionic liquids to obtain reasonable dissolution for cellulose. However, ionic liquids too often turned out to be high cost and toxic, which make them inappropriate for large-scale applications.

Eutectic mixtures of salts have been extensively used to decrease the temperature for molten salt applications. They are comprised mostly of a quaternary ammonium salt and an amine, amide, alcohol, or carboxylic acid. They provide a solvent including neutral molecules as well as anions or cations. As they are less toxic, easy to prepare, less water-sensitive and low cost, they offer a good alternative to organic solvents or ionic liquids. As discussed above, DES/RTIL, specifically refer to eutectic mixtures that are liquid at or below room temperature. Due to their unique properties, DES are assumed to dissolve cellulose.

Cellulose comprises of polydisperse linear glucose chains which form a supramolecular structure through inter- and intra-molecular hydrogen bonds, which is illustrated in Fig. 3. For each glucose unit, three hydroxyl groups can be noted. In contrast to starch or glycogen, no linkage of the chains occurs.

In nature, cellulose is produced as continuous crystalline filaments called microfibrils with nanometric lateral dimensions. This is a result of the simultaneous polymerization of the chains. Native cellulose is chemically stable and does not allow water to penetrate under ambient conditions.

The low solubility and turnover rate of cellulose is mainly caused by its high crystallinity. It has been shown that a lower degree of crystallinity (totally amorphous cellulose has a crystallinity of 0%) leads to higher initial hydrolysis rate. The reduction of crystallinity of cellulose is therefore of prime interest.

Cellulose has not yet reached its full potential as a biofuel, as it cannot yet be processed in an economical way. The bottleneck in this context is the insolubility of cellulose for common inexpensive solvents. Nevertheless, in large scale-applications, a homogeneous phase is desired for many reasons such as local effects of overheating or fouling. Therefore, solvents for cellulose are needed which are environmentally benign and enable reactions with cellulose in a homogeneous phase.

What is needed, therefore, is a DES that does not comprise choline chloride, and instead comprises less expensive components. Solvents for cellulose that are environmentally benign and that enable reactions with cellulose in a homogeneous phase are also needed. It is to such systems and methods that the present invention is primarily directed.

BRIEF SUMMARY OF THE INVENTION

Briefly described, in a preferred form, the present invention is a Type III Eutectic comprising an organic salt and a hydrogen bond donor, wherein the organic salt preferably comprises betaine monohydrate. The hydrogen bond donor can comprises urea or acids, such as malonic acid or citric acid. Depending on the composition, the melting point of the mixture can be considerably lower than the melting point of either component, which can be 240°C for urea and 130°C for betaine monohydrate. The melting point for urea/betaine monohydrate with 1:2 molar ratio was found to be approximately 10°C, the melting point for malonic acid/betaine monohydrate with molar ratio 1: 1 was found to be approximately 46°C, and the melting point for citric acid/betaine monohydrate with 3: 1 molar ratio was found to be approximately 42°C, with formation of a gel.

The use of betaine monohydrate is advantageous over that of choline chloride, owing in large measure to the much lower cost of betaine monohydrate, often more than one order of magnitude. The present DES can be used to dissolve components, such as cellulose, starch, lignin, synthetic polymers, and others, that are not soluble in other media. There can be a decrease of crystallinity of cellulose of 10-15 percentage points after treatment of Avicel® with urea/betaine monohydrate.

In embodiments of the present invention, DES are evaluated based on their ability to dissolve cellulose (Avicel®). Currently, no known DES has achieved dissolution using pure DES or DES diluted with buffer. Therefore, the temperature for the incubation of Avicel® in these solvents was increased to gain a better suspension. Furthermore, DES were diluted with a strong base (NaOH) and acid (HC1) along with extension of the incubation time from 1 hour to 10.5 hours. Only the base treated DES showed significant swelling and dissolution of cellulose. From these results, the focus was set on the basic treatment of Avicel®, first using an aqueous sodium hydroxide solution and later an aqueous ammonium hydroxide solution. Considerable swelling and dissolution of Avicel® could only be found for the sodium hydroxide solution.

To check if there is any interaction between Avicel® and the DES, a crystallinity measurement was conducted. The best results were produced with the DES malonic acid/ChCl. The reduction of crystallinity was about 15-20%. It was expected that the hydrolysis rate of Avicel® pretreated with DES and verifiably lower crystallinity, is higher than that for native Avicel®. However, this could not be confirmed.

Knowing that DES are formed between a quaternary ammonium salt and a hydrogen bond donor, further investigation was focused on inventing new DES. To replace the high cost of ChCl, the compound betaine monohydrate was found to be appropriate. DES were formed between betaine monohydrate and citric acid, malonic acid and urea. For the DES betaine monohydrate/urea, the freezing point was plotted as a function of the mol-% urea. It was also found that there is a hysteresis between the melting and freezing points of this system.

The focus was set on the investigation of DES that have not been reported before. Due to the high cost of Choline Chloride (approximately $745.00 U.S. for 100 g), the replacement of this quaternary ammonium salt was of higher priority.

Disregarding the fact that DES are formed between quaternary ammonium salts and hydrogen bond donors, an experiment was conducted using betaine monohydrate to replace ChCl. Betaine monohydrate shows strong hydrogen bonding behavior for itself. It is proposed that, the carboxylated oxygen acts as a hydrogen Bond acceptor, whereas the hydrogen from water acts as a hydrogen bond donor. A DES could be observed for the mixtures shown in Table 1:

Table 1: New DES Formed Between betaine Monohydrate and Hydrogen Bond Donors

This DES show the same properties concerning the viscosity as the DES formed between ChCl and the equivalent compound. No DES could be found for a mixture of betaine monohydrate/acetamide and betaine monohydrate/oxalic acid (molar ratio, each 1:2).

To verify the claim of a new DES, the system betaine monohydrate/urea was investigated. The melting point of mixtures of different ratios in the range of 50 to 75 mol-% urea was determined.

For the compositions 40 and 50 mol-% urea, the melting point is over 100°C which is too high to be of interest in this context. The formation of ammonia could be observed, by its strong smell and ascending bubbles for temperatures about 98°C. For 60, 67 and 70mol-% urea, the freezing point is below 1°C. These samples were therefore set on -20°C for 5 minutes. After that they were removed and a white crystal could be observed. However, these crystals grow fast at room temperature until the whole solution turns solid. The solid remains at room temperature.

Hysteresis could be observed for the freezing point and melting point of the system urea/betaine monohydrate, respectively. The preparing of the DES was conducted for 55, 60, 67, 70 and 75 mol-% urea as described above. Instead of mixing the solids and heating them they were stored at room temperature for 3 hours. In all cases a liquid with crystals could be observed. The samples were heated up and the temperature for which all the solid turned liquid was noted.

To make sure that these samples are not just sub cooled liquids, they were centrifuged for 3 minutes at 505 x g and for 3 minutes at 11,000 x g these samples remained liquid. In another step, they were inoculated with a betaine monohydrate crystal. This also did have an effect on the solvents.

Further investigations concerning the hysteresis were conducted to examine the phase behavior of urea/betaine monohydrate. The melting and freezing points for urea concentrations of 52.6 mol-% and 80 mol-% were determined.

It is evident, that the hysteresis gets insignificant for high and low urea concentrations. On the other side, the stabilizing effect of urea can be seen, by means of a significant higher melting and freezing point for lower concentrations of urea, than the expected eutectic concentration of 67%. The system betaine monohydrate/urea stands out with its extreme low freezing point and low viscosity at room temperature. It is therefore appropriate for further work and application replacing the DES urea/ChCl.

DES have also been used as solvents for starch. In a first step, the dissolution of starch in the DES urea/betaine monohydrate should be determined. In 1 ml of urea/betaine monohydrate DES, 48.2 g starch in a first trial and 46.1 g starch in the second trial was incubated at 90°C mixed at 1400 rpm for 30 minutes. After cooling, a gel can be observed for the urea/choline chloride DES.

In an exemplary embodiment, the present invention is a eutectic solvent comprising a betaine compound and a hydrogen bond donor. The betaine compound can comprise betaine monohydrate. The hydrogen bond donor can comprises urea, malonic acid or citric acid.

In another exemplary embodiment, the present invention is a eutectic solvent comprising a betaine compound and a hydrogen bond donor, wherein the molar ratio of betaine compound:hydrogen bond donor is 2: 1. In other embodiments, the molar ratio of betaine compound:hydrogen bond donor can be 1 : 1 and 3: 1.

In a further exemplary embodiment, the present invention is a composition of matter comprising urea and betaine monohydrate. In various embodiments, at 0 mol-% urea, the freezing point is approximately 241°C, and the melting point is approximately 241°C. At 52.6 mol-% urea, the freezing point is approximately 93°C, and the melting point is approximately 96°C. At 55 mol-% urea, the freezing point is approximately 44°C, and the melting point is approximately 71°C. At 60 mol-% urea, the freezing point is approximately 1°C, and the melting point is approximately 69°C. At 67 mol-% urea, the freezing point is approximately 1°C, and the melting point is approximately 60°C. At 70 mol-% urea, the freezing point is approximately 1°C, and the melting point is approximately 60°C. At 75 mol-% urea, the freezing point is approximately 30°C, and the melting point is approximately 62°C. At 100 mol- % urea, the freezing point is approximately 134°C, and the melting point is approximately 134°C.

In a further exemplary embodiment, the present invention is a deep eutectic solvent comprising betaine monohydrate and a hydrogen bond donor. The hydrogen bond donor preferably comprises urea. In a further exemplary embodiment, the present invention is a method for the dissolution of cellulose comprising mixing cellulose in a eutectic solvent comprising a betaine compound and a hydrogen bond donor.

In a further exemplary embodiment, the present invention is a method for the dissolution of a carbohydrate comprising mixing a carbohydrate in a eutectic solvent comprising a betaine compound and a hydrogen bond donor.

In a further exemplary embodiment, the present invention is a method for the dissolution of starch comprising mixing starch in a eutectic solvent comprising a betaine compound and a hydrogen bond donor.

In a further exemplary embodiment, the present invention is a method of reducing the crystallinity of cellulose comprising mixing cellulose in a eutectic solvent comprising a betaine compound and a hydrogen bond donor, and providing a reduction of crystallinity below 20%. Preferably the invention provides a reduction of crystallinity of approximately 10-15%.

These and other objects, features and advantages of the present invention will become more apparent upon reading the following specification in conjunction with the accompanying drawing figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Various features and advantages of the present invention may be more readily understood with reference to the following detailed description taken in conjunction with the accompanying drawings, wherein like reference numerals designate like structural elements, and in which:

Fig. 1 shows that a eutectic occurs at a defined ratio of compounds.

Fig. 2 shows, for the example urea/choline chloride, that a deep eutectic is mainly caused by shielding the charge of the anion by means of complexing it with hydrogen bond donors.

Fig. 3 shows that cellulose comprises of polydisperse linear glucose chains which form a supramolecular structure through inter- and intra-molecular hydrogen bonds.

Fig. 4 shows a standard calibration curve with pure glucose standards.

Fig. 5 shows the data for a pretreatment with Acetamide/ChCl DES mixed with DL- water. Fig. 6 shows data for hydrolysis of pretreated Avicel® with formamide/ChCl.

Fig. 7 shows a spectrum of crystallinity for Avicel® treated with malonic/ChCl.

Fig. 8 shows another spectrum of crystallinity for Avicel® treated with malonic/ChCl.

Fig. 9 shows a spectrum of crystallinity for Avicel® treated with urea/betaine monohydrate (molar ratio 2: 1).

Fig. 10 shows a freezing point and melting point of urea/betaine monohydrate mixtures as a function of composition.

DETAILED DESCRIPTION OF THE INVENTION

To facilitate an understanding of the principles and features of the various embodiments of the invention, various illustrative embodiments are explained below. Although exemplary embodiments of the invention are explained in detail, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the invention is limited in its scope to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or carried out in various ways. Also, in describing the exemplary embodiments, specific terminology will be resorted to for the sake of clarity.

It must also be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the context clearly dictates otherwise. For example, reference to a component is intended also to include composition of a plurality of components. References to a composition containing "a" constituent is intended to include other constituents in addition to the one named.

Also, in describing the exemplary embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term contemplates its broadest meaning as understood by those skilled in the art and includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.

Ranges may be expressed herein as from "about" or "approximately" or "substantially" one particular value and/or to "about" or "approximately" or "substantially" another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and/or to the other particular value. Similarly, as used herein, "essentially free" or "substantially free" of something, or "substantially pure", and like characterizations, can include both being "at least substantially free" of something, or "at least substantially pure", and being "completely free" of something, or "completely pure".

By "comprising" or "containing" or "including" is meant that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same function as what is named.

It is also to be understood that the mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a composition does not preclude the presence of additional components than those expressly identified.

The materials described as making up the various elements of the invention are intended to be illustrative and not restrictive. Many suitable materials that would perform the same or a similar function as the materials described herein are intended to be embraced within the scope of the invention. Such other materials not described herein can include, but are not limited to, for example, materials that are developed after the time of the development of the invention.

In a preferred form, the present invention is a deep eutectic solvent system that comprises betaine monohydrate as one component. The other component can be urea or acids, such as malonic acid or citric acid. Depending on the composition, the melting point of the mixture can be considerably lower than the melting point of either component, which can be 240°C for urea and 130°C for betaine monohydrate. The melting point for urea/betaine monohydrate with 1:2 molar ratio was found to be 10°C, the melting point for malonic acid/betaine monohydrate with molar ratio 1: 1 was found to be 46°C, and the melting point for citric acid/betaine monohydrate with 3: 1 molar ratio was found to be 42°C, with formation of a gel. The low melting points were not observed with betaine chloride. Embodiments of the present invention comprise a eutectic solvent having a betaine compound and a hydrogen bond donor. In some embodiments, the betaine compound can be betaine monohydrate, and the hydrogen bond donor can be urea malonic acid, or citric acid.

The use of betaine monohydrate is advantageous over that of choline chloride, owing to the much lower cost of betaine monohydrate, often more than one order of magnitude. DES can be used to dissolve components, such as cellulose, starch, lignin, synthetic polymers, and others, that are not soluble in other media. There can be a decrease of crystallinity of cellulose of 10-15 percentage points after treatment of Avicel® with urea/betaine monohydrate.

Experimental Methods

Preparation of Deep Eutectic Solvents

Substances were weighed out in the given ratio, (see Table 2) and mixed together in their solid state in a 24 ml glass vial. For DES with a relatively low freezing point such as urea/ChCl an aggregation during mixing in the solid state at room temperature could be observed. The mixture is heated while stirring constantly to 100°C, until a clear solution can be observed. DES which have melting point below room temperature are stored at room temperature. DES which have a higher melting point are stored at 60°C.

Table 2: Molar Ratio of DES

¹ No DES ² Mass ratio

For the DES malonic acid/ChCl, a decomposition of malonic acid can be observed by heating over 70°C. The decomposition can be verified by bubbles and a strong acetic acid smell, which signifies the formation of acetic acid and carbon dioxide. Therefore, it was decided to heat the system to 70°C only. The same effect was observed for the DES malonic acid/betaine monohydrate.

Dissolution of Avicel® in DES

Twenty gram Avicel® was incubated in the DES at 45°C, with mixing at 505 xg for 1 hour in a Thermomixer®. To lower the viscosity of the system, a 50 mM NaOAc buffer with pH 5 was added. The total concentrations of buffer in the Avicel® solution was 3, 4, 5 and 10%. After the incubation, the solution was centrifuged for 3 minutes at 3000 rpm. The supernatant was removed, and the pellet was washed with Dl-Water. The solution was resuspended and centrifuged again for 3 minutes at 3000 rpm. The pellet of this solution was washed with NaO Ac-buffer and centrifuged again under the same conditions as before. The supernatant was removed and the pellet was freeze dried. For the first experiments, the pellet was dried in an oven at 60°C.

To freeze the pellet, it was stored at -20°C for one hour. The pellet was freeze-dried under vacuum at 0.05 mbar. To indicate the presence of Avicel® which went in solution, the mass of the empty eppendorf tube and the mass of Avicel® was measured for each tube before the incubation. After freeze drying, the total mass of each tube was measured. A delta mass was calculated which is the difference of the mass after freeze drying and the mass of Avicel® plus the empty tube before incubation. Therefore a negative value stands for successful dissolution, while a positive value refers to no dissolution respectively remaining solvent.

The conditions for the incubation were changed later to check for better results. These conditions are described below.

Hydrolysis of Avicel®

To check if there is a better hydrolysis rate due to the treatment of Avicel® with DES, a hydrolysis procedure was applied to the freeze dried Avicel® in a 1.5 ml eppendorf tube. To measure the hydrolysis rate in DES a DNS-Assay was enforced in two DES. Avicel® was incubated in 916 μΐ NaO Ac -buffer at 45 °C, mixed at 1000 rpm for 1 hour. The hydrolysis was started by adding 60 μΐ β-glucosidase and 24 μΐ Cellulase. The reaction was carried out at 45°C, mixed at 1000 rpm for 1 hour. The total concentration of Avicel® was 20 mg/ml.

DNS Assay

To check for the concentration of glucose after hydrolysis an assay for reducing sugars was applied. Therefore, as shown in Fig. 4, a standard calibration curve with pure glucose standards was generated.

The hydrolyzed Avicel® solution was centrifuged for 3 minutes at 3000 rpm. Five hundred μΐ of the supernatant were removed and centrifuged again the same way. 70 μΐ of this supernatant were diluted with 80 μΐ DL- Water.

To detect reducing sugar ends, 150 μΐ DNS reagent (1,4 Dinitrosalicilic acid) was added and the sample was incubated at 90°C for 15 minutes to develop the red/brown color. For every series of measurements, a new blank was generated consisting of 150 μΐ DL- Water and 150 μΐ DNS reagent. After incubation at 90°C, 50 μΐ of a 40% potassium sodium tartrate (Rochelle salt) solution was added to stabilize the color. The mixture was cooled down and diluted with 400 μΐ DL- Water to fit in the linear range. The absorbance was measured at 575 nm with the spectral photometer.

The DNS reagent was comprised of the following substances which are solved in 50 ml DL- Water: 0.5g 1,4-dinitrosalicylic acid, O. lg phenol, 0.025g sodium sulfite, 0.5g sodium hydroxide. The DNS reagent can be used for one month when stored at 4°C.

Due to the influence of the mass of Avicel® used for each sample on the resulting glucose concentration, a normalization is done. The glucose concentration is multiplied by 20 mg Avicel® divided by the actual mass of Avicel®.

Crystallinity Check

The crystallinity (Crl) of cellulose samples incubated in DES was calculated by quantifying the contribution of amorphous cellulose (Phosphoric Acid Swellen Cellulose, PASC) and Avicel® to its normalized X-ray diffraction spectra: Equation 1: Ιβθ) = _ρ(2θ) + (1 - f_j)I_c(29) + e

I_j is the intensity of the ^th sample at diffraction angle 2(9. I_p is the intensity of PASC at diffraction angle 2Θ. Ι₀(2Θ) is the intensity of untreated Avicel® at diffraction angle 2θ, /_ί is the contribution of PASC to the spectrum and e is the random error. To calculate the crystallinity of Avicel® }, the least square estimate of f₇- was used by multiplying the contribution of Avicel® (1-f/) by its crystallinity. The crystallinity of Avicel® was calculated by CP/MAS and ¹³C-NMR to 60%:

Equation 2: Crl_j = (l - f_j) x Crl_c

In this correlation, Crl₇ is the crystallinity in percentage of the ^th sample of Avicel®, and Crl_c is the crystallinity of Avicel® (60%).

Determination of the Freezing and Melting Point

To determine the freezing point, the DES is heated up until a clear, transparable solution can be observed. The solution is cooled down at room temperature. The freezing point is the temperature for that the first crystal can be observed. For the determination of freezing points below room temperature the DES was cooled with ice.

The melting point was determined by mixing the solids together in a 24 ml glass vial. The mixture was set on room temperature for 3 hours, followed by incubation at 60°C for 30 minutes. Mixtures with higher melting points were heated up slowly on a heating plate, while shaking gently. The temperature for all the liquid went transparent is the melting point. Pretesting the Behavior of Avicel® in Urea/Choline Chloride DES and Formamide/ Ammonium Formate Solution

Pretests were carried out for the DES urea/choline chloride. It was found to be miscible with water for 1, 2, 3, 4, 5, 10, 20, 40, 50, 60 and 80 vol-% water at room temperature. The viscosity decreases significantly by adding water, this can be observed starting from 3 vol-% water. A decrease in viscosity can also be observed by increasing the temperature to 45°C and 60°C.

No suspension of Avicel® in the pure urea/choline chloride DES could be observed for 45°C and Avicel® concentrations of 10, 15 and 20 mg/ml. In a first run, Avicel® was incubated at 45°C and mixed at 1000 rpm for 30 minutes. The results are given in the supplementary material in Table 3 for the urea/ChCl DES and in Table 4 for the formamide/ammonium formate mixture. A blank was done containing Avicel® in Dl-water to have a reference for dissoluted Avicel®.

Table 3: Pretest with Urea/Choline Chloride DES¹

¹ incubated at 45°C, mixed at 1000 rpm for 30 minutes

Table 4: Pretest with Formamide/ Ammonium Formate Solution

incubated at 45°C, mixed at 1000 rpm for 30 minutes

The results show no dissolution of Avicel® in either one of the solvents. An increase in mass is noticeable, which could be caused by remaining DES on the surface of Avicel®. In this run, Avicel® was washed once with water. Due to the high mass of remaining DES, it was decided to wash twice for all future experiments. Furthermore, it is evident that additional time for drying does not lead to less residual DES. This is mainly caused by the low volatility of these solvents. Therefore a drying time of about 17 hours can be regarded as sufficient to get comparable results.

Dissolution and Hydrolysis of Avicel® Applying DES Due to their unique solvent properties, the behavior of DES concerning Avicel® was to be investigated. Beside the dissolution of Avicel®, the influence on the hydrolysis of pretreated Avicel® with DES was under investigation.

Hydrolysis of Avicel® in DES

The hydrolysis of Avicel® was checked in the DES urea/ChCl and malonic acid/ChCl.

The hydrolysis in the urea/ChCl DES was carried out in a mixture of 50 vol-% water and 50 vol- % DES. The hydrolysis in the malonic acid/ChCl DES was conducted in a mixture of 11 vol-% NaO Ac-buffer and 89 vol-% DES.

For the urea/ChCl mixture, a glucose concentration of 0.38 mg/ml could be measured. Whereas for the malonic acid/ChCl a glucose concentration of 0.55 + 0.05 mg/ml(double check) was obtained. The glucose concentration for hydrolysis of standard NaOAc -buffer was determined to 6.8 + 0.02 mg/ml(double check). This indicates that the enzyme activity is almost zero in these systems.

Dissolution of Avicel® in DES at 45°C

Avicel® was incubated in the DES urea/ChCl, formamide/ammonium formate and oxalic acid/ChCl at 45 °C, mixed at 1000 rpm for 1 hour. The mixture was centrifuged and washed twice. The pellet was oven dried for 22 hours and the mass of the remaining Avicel® was measured. The data for these experiments is given in Table 5.

Table 5: Incubation of Avicel® in DES at 45 °C

oxalic acid/ChCl 965.2 949.6 981.7 1001.0 985.4 delta mass [mg] urea/ChCl 1.5 5.1 8.0 10.6 0.0 formamide/ammonium format 2.3 1.2 1.0 2.1 -0.3 oxalic acid/ChCl 7.8 11.0 22.7 22.9 -1.1

The resulting data from these experiments shows no dissolution of Avicel® in either of the applied solvents. It is noticeable that for the DES urea/ChCl and oxalic acid/ChCl, a reasonable amount of solvent remains. For the system oxalic acid/ChCl, this effect is even stronger, due to its higher viscosity. Whereas for the solvent system formamide/ammonium formate, this effect is negligible.

Experiments were conducted for the DES phenylacetic acid/ChCl. While washing the pellet after the first centrifugation white precipitation was observed. To give evidence that the white precipitation does not refer to Avicel®, a run was conducted with Avicel® and without Avicel®. The white precipitation was observed in both cases, which lead to the conclusion that it is phenylacetic acid, due to its low solubility in water.

The dissolution of Avicel® in the DES malonic acid/ChCl was checked, results are given in Table 6. The dissolution and the drying procedure was the same as described above. Data shows no dissolution of Avicel® under these conditions in the DES.

Table 6: Dissolution of Avicel® in Malonic Acid/ChCl DES with 10.9% buffer at 45°C

To lower the viscosity without addition of buffer, the temperature was increased. This led to a better suspension of Avicel® in the DES. Additionally, the mixing speed was increased to 1400 rpm.

Dissolution of Avicel® in DES at 80°C Experiments to check for dissolution of Avicel® at 80°C for 1 hour were run for the DES urea/ChCl, oxalic acid/ChCl and malonic acid/ChCl. Additionally, the solvents formamide/ammonium format and glycerol/ChCl were applied. The pellets resulting from the solvents urea/ChCl, oxalic acid/ChCl and glycerol/ChCl were oven dried, while the pellet from the solvents malonic acid/ChCl and formamide/ammonium format were freeze dried. Data is listed in Tables 7-11.

Table 7: Dissolution of Avicel® in Urea/ChCl DES at 80°C

Table 8: Dissolution of Avicel® in Oxalic Acid/ChCl DES at 80°C

Table 9: Dissolution of Avicel® in Malonic Acid/ChCl DES at 80°C

Table 10: Dissolution of Avicel® in Formamide/ Ammonium Formate at 80°C

Table 11: Dissolution of Avicel® in glycerol/ChCl at 80°C

For the DES urea/ChCl and oxalic acid/ChCl, a long term experiment was done to check the influence of time on the dissolution of Avicel®. Avicel® was incubated at 80°C, mixed at 1400 rpm for 10.5 hours. Samples were oven dried for 36 hours, the data is given in the Tables 12-13.

Table 12: Dissolution of Avicel® in Urea/ChCl at 80°C for 10.5 hours

Table 13: Dissolution of Avicel® in Oxalic Acid/ChCl at 80°C for 10.5 hours

mass empty [mg] 993.7 937.2 936.7 941.6

mass Avicel® [mg] 17.1 23.8 24.4 22.7 mass after drying [mg] 1010.3 961.8 961.1 964.3 delta mass [mg] -0.5 0.8 0.0 0.0

Results from these experiments didn't show any significant dissolution for Avicel®. In the case of oxalic acid/ChCl, a black color of Avicel® can be observed for the both times 1 hour and 10.5 hours. In a next step, the dissolution behavior of Avicel® in DES was to be determined in both, acidic and basic milieu. This is described below.

Dissolution of Avicel® in Urea/CaC12 DES at 90°C

The DES Urea/CaC12 is used to dissolute Avicel® at 90°C mixed at 1000 rpm for 1 hour. A good suspension could be observed. The pellet was freeze dried. Data is given in Table 14.

Table 14: Dissolution of Avicel® in Urea/CaC12 at 90°C for 1 hour

Dissolution of Avicel® in Acidic/Basic Buffered DES

To create an acidic milieu for the DES, 100 μΐ of cone. HC1 (12 M) was added to 900 μΐ DES in an eppendorf tube. To create a basic milieu for the DES, 100 μΐ of cone. NaOH (10 M) was added to 900 μΐ DES. The resulting total molarity is therefore 1.2 M (ph = 0) for the acidic and 1.0 M (ph = 14) for the basic milieu, respectively. Experiments were carried out for urea/ChCl and oxalic acid/ChCl DES.

The results for the acidic treatment experiments are given in Tables 15-17. The results for the basic treatment are listed in Tables 18-20. Table 15: Dissolution of Avicel® in Urea/ChCl DES at 80°C, pH = 0

Table 16: Dissolution of Avicel® in Oxalic Acid/ChCl DES at 80°C, pH = 0, 1 h

Table 17: Dissolution of Avicel® in Formamide/ Ammonium Formate at 80°C, pH = 0, 1 h

Table 18: Dissolution of Avicel® in Urea/ChCl DES at 80°C, pH = 14

mass Avicel® [mg] 22.8 22.1 23.0 21.6

mass after drying [mg] 940.3 924.2 955.8 966.1 delta mass [mg] -5.7 -6.1 -3.4 -3.3

Table 19: Dissolution of Avicel® in Oxalic Acid/ChCl DES at 80°C, pH = 14, 1 h

Table 20: Dissolution of Avicel® in Formamide/ Ammonium Formate at 80°C, pH

For the basic treated DES urea/ChCl a significant dissolution of Avicel® of 21+ 15% was noticed, whereas for the acidic treated Avicel® this is not the case. The percentage of dissoluted Avicel® is the ratio of the delta mass for each trial to the mass of Avicel® weighed in for each tube. Therefore, experiments were conducted to examine the behavior of Avicel® in a basic solution to verify the assumption, that the dissolution is caused only by the basic pH.

Dissolution of Avicel® in Aqueous Basic Solution Avicel® was dissoluted in a sodium hydroxide solution. The basicity was lowered starting from pH 15 to pH 13.8-13.6. To reduce basicity, the 10 M sodium hydroxide solution was diluted with the NaOAc-buffer. To get a pH of 13.8 a mixture of 50% buffer and base was mixed, for pH 13.6 the percentage of buffer is 70%. The temperature for these experiments was set to 70°C, with 1 hour incubation time and a mixing speed of 1400rpm. The results are given in Tables 21-23. All samples were freeze dried for 9 hours.

Table 21: Dissolution of Avicel® in Basic Solution at 70°C, 10 M NaOH

Table 22: Dissolution of Avicel® in Basic Solution at 70°C, 5 M NaOH

Table 23: Dissolution of Avicel® in Basic Solution at 70°C, 3M NaOH

For the 10 M sodium hydroxide solution, a yellow color and agglomeration for the Avicel® was observed. For lower sodium hydroxide concentrations, the yellow color cannot be observed, and the Avicel® was suspended well. Surprisingly, a lower sodium hydroxide concentration led to a higher dissolution of Avicel®. Giving the best result thus far, the experiment with the 3 M sodium hydroxide solution was repeated. The resulting data is listed in Table 24.

Table 24: Dissolution of Avicel® in Basic Solution at 70°C, 3M NaOH, Trial 2

The results for the dissoluted Avicel® from the described experiments are shown in Table 25 below. The fraction of dissolved Avicel® was calculated as shown above. For the 3 M NaOH solution, eight trials were done.

Table 25: Dissolution of Avicel® in Basic Solution at Different pH.

;^')lantv of NaOH imol/i! Dissoluted Avicel. |%1 Error \ψ_<

0

6 ^'

The application of sodium hydroxide to dissolute Avicel® appears infeasible, because this would require too much sodium hydroxide. In a further step, it was therefore checked if sodium hydroxide could be replaced with ammonium hydroxide. Avicel® was dissoluted in a solution of ammonium hydroxide comprising of 700 μΐ NH40Ac buffer 50mM at pH 5 and 300 // of cone, ammonia hydroxide (total pH 11.2), at 70°C mixed at 1400 rpm for 1 hour. The results are given in Table 26.

Table 26: Dissolution of Avicel® in basic solution at 45°C, pH 11.2

Resulting data shows no comparable dissolution for Avicel® in ammonium hydroxide as it was found for the sodium hydroxide runs. To check the influence of a higher pH, the volume of cone, ammonium hydroxide was increased. To avoid popping of the eppendorf tube lids, the temperature had to be reduced. Runs were carried out at 45°C at a pH 11.5 and 11.8 for 1 hour. Data is given in Tables 27-28.

Table 27: Dissolution of Avicel® in basic solution at 45°C, pH 11.5

Table 28: Dissolution of Avicel® in basic solution at 45°C, pH 11.8

mass after drying [mg] 968.9 932.2 956.6 94.1

delta mass [mg] -0.4 -1.1 -2.7 -1.4

Dissolution of Avicel® in Acetamide/Choline Chloride DES

The dissolution of Avicel® in acetamide/ChCl was tested in a pure DES solution. The temperature was set to 55°C, for 1 hour at 1400 rpm. After washing in the same manner as shown above, a delta mass of 1.6mg could be obtained, which leads to the conclusion that Avicel® is not soluble in this DES.

To check for an interaction between the solvent and Avicel®, a DNS-Assay was applied. The Avicel® was incubated in a mixture of DL- Water and acetamide/ChCl DES at 55°C mixed at 1400 rpm for 1 hour beginning from 0% water to 50% water in 10% intervals. After freeze- drying, the sample was incubated in NaOAC-buffer and hydrolyzed. The glucose concentration was detected via the DNS-assay.

Fig. 5 shows the data for the pretreatment with Acetamide/ChCl DES mixed with DL- water. The dashed lines signify the upper and lower concentration of glucose for a pretreatment of Avicel® in NaO Ac-buffer at same conditions. From this Fig. 5 no significant increase in glucose concentration can be measured for pretreating Avicel® in acetamide/ChCl DES. The dissolution of Avicel® in a diluted acetamide/ChCl DES diluted with concentration ammonium hydroxide was investigated. The DES was mixed with 50 μΐ and 100 μΐ total volume in tube remained constant 1000 μΐ incubated for 1 hour at 55°C mixed at 1400 rpm. The resulting Avicel® was hydrolyzed afterwards.

Table 29: Dissolution and Hydrolysis of Pretreated Avicel® with Acetamide/ChCl and

Ammonia Hydroxide

Dissolution of Avicel® in a Formamide/Choline Chloride Solution

To reduce the viscosity of the solvent system, it advisable to apply smaller molecules. Therefore, the system formamide/ChCl in a molar ratio of 2: 1 was produced. The melting point of this solution was determined to be 45 °C. Although the system formamide/ChCl cannot be regarded as a DES due to the low melting point of formamide, it is nevertheless important to continue experiments concerning the dissolution of Avicel®. Experiments were conducted in the same way as for acetamide/ChCl with exception of the incubation temperature, which was risen to 70°C instead of to 55°C for the acetamide/ChCl experiments. The data is given in Table 30.

Table 30: Dissolution of Avicel® in Formamide/ChCl Solution with Increasing Buffer

Concentration

Glucose concentration after hydrolysis of Avicel® pretreated with formamide/ChCl solution was measured in duplicate. The experiment procedure is described above. Formamide/ChCl was diluted with the NaO Ac-buffer as necessary. Fig. 6 shows the data for hydrolysis of pretreated Avicel® with formamide/ChCl.

Hydrolysis of Avicel® Pretreated with Formamide/ Ammonium Formate and Glycerol/Choline Chloride

The effect of the solvent system formamide/ Ammonium Formate and glycerol/ChCl on Avicel® was checked by applying hydrolysis on Avicel® treated with these solvents. The incubation in the solvent systems was at 70°C mixed at 1400 rpm for 1 hour. Samples were freeze-dried for 22 hours. The results of the hydrolysis are shown in Table 31. Table 31: Hydrolysis of Pretreated Avicel® via Formamide/ Ammonium format and

Glycerol/ChCl

Hydrolysis with Urea Treated Avicel®

The high crystallinity of cellulose is mainly caused by inter- and intra-molecular hydrogen bonds. Therefore the effect of urea well known for disrupting hydrogen bonds, on Avicel® was also examined. Avicel® was incubated in a 9.1 M aqueous urea solution at 90°C mixed at 1400 rpm for 1 hour, samples were freeze dried for 22 hours. The absorbance of each trial was run in duplicate. Results are shown in Table 32.

Table 32: Hydrolysis of Pretreated Avicel® with Urea

Crystallinity of Avicel® Treated with PES

Crystallinty measurements were carried out for Avicel® incubated for selected DES as described briefly above. The incubation time remained constant at 1 hour. Results are given in the Table 33 below. The temperature listed refers to the incubation temperature and the glucose concentration to the hydrolyzed Avicel® after the pretreatment. For the first three glucose concentrations the Avicel® control is 5.05 mg/ml, compared to 6.54 mg/ml for the last three samples.

Table 33: Crystallinity of DES Pretreated Avicel® and Glucose Concentration

[C] [Vol-%] [ ] Cone. [Mg/Ml]

Urea/ChCl 60 0 46.4 4,45

Phenylacetic/ChCl 45 0 61.6 4,19

Malonic acid/ChCl 1st 45 11 36.7 4,26

Malonic acid/ChCl 2nd 45 11 60.7 n.d.

Malonic acid/ChCl 3rd 45 11 42.7 6.54+0.08

Urea/betaine monohydr. 1st 70 0 48.1 6.27

Urea/betaine monohydr. 2nd 70 0 44.5 6.21

Fig. 7 shows a plot of the intensity as function of the diffraction angle for the 3rd trial plotted. Spectrums are given for the original, reconstructed and Avicel® sample. The reconstructed spectrum was calculated from the original by normalizing the original spectrum with the area underneath it.

In Fig. 7, a significant reduce of the peak from the 220 layer can be observed compared with the Avicel® peak for this layer. The reduce of this peak is the main reason for the less overall crystallinity. The R2-value for this sample is 0.95 crystallinity is 42.7%.

In contrast to this trial the 2nd trial showed no reduction in crystallinity, which is shown in Fig. 8. The spectrum for the pretreated Avicel® overlays with the spectrum for the non- pretreated Avicel®, which signifies no effect of the DES on the crystallinity.

In Fig. 9, the spectrum for the second pretreatment of Avicel® with urea/betaine monohydrate (molar ratio 2: 1) is shown. The reduction of crystallinity was not as high as in the first trial with malonic acid/ChCl. The Present DES

Building on the theory of DES described above, the focus was set on the investigation of DES which have not been reported before. Due to the high cost of Choline Chloride ($745.00 U.S. for 100 g), the replacement of this quaternary ammonium salt was of higher priority.

The compound next similar to ChCl is betaine hydrochloride, therefore experiments were done to form a DES comprising betaine hydrochloride and malonic acid in a molar ratio 1 : 1 and betaine hydrochloride and urea in a molar ratio 1:2. However, no DES could be observed for either of those mixtures.

Disregarding the fact that DES are formed between quaternary ammonium salts and hydrogen bond donors, an experiment was conducted using betaine monohydrate to replace ChCl. Betaine monohydrate shows strong hydrogen bonding behavior for itself. It is proposed that the carboxylated oxygen acts as a hydrogen bond acceptor, whereas the hydrogen from water acts as a hydrogen bond donor. A DES could be observed for the mixtures shown in Table 1.

This DES show the same properties concerning the viscosity as the DES formed between ChCl and the equivalent compound. No DES could be found for a mixture of betaine monohydrate/acetamide and betaine monohydrate/oxalic acid.

Phase Behavior of the System betaine Monohydrate/Urea

To verify the claim of a new DES, the system betaine monohydrate/urea was investigated. The melting point of mixtures of different ratios in the range of 50 to 75 mol-% urea was determined as shown in Fig. 10.

For the compositions 40 and 50 mol-% urea, the melting point is over 100°C, which is too high to be of interest in this context. The formation of ammonia could be observed, by its strong smell and ascending bubbles for temperatures about 98°C. For 60, 67 and 70 mol-% urea, the freezing point is at or below 1°C. These samples were therefore set on -20°C for 5 minutes. After that they were removed and a white crystal could be observed. However, these crystals grow fast at room temperature until the whole solution turns solid. The solid remained at room temperature.

Hysteresis could be observed for the freezing point and melting point of the system urea/betaine monohydrate, respectively. The preparing of the DES was conducted for 55, 60, 67, 70 and 75 mol-% urea as described above. Instead of mixing the solids and heating them they were stored at room temperature for 3 hours. In all cases a liquid with crystals could be observed. The samples were heated up and the temperature for which all the solid turned liquid was noted. This temperature as function of the mol-% urea was plotted also in Fig. 10. To make sure that these samples are not just sub cooled liquids, they were centrifuged for 3 minutes at 505 x g and for 3 minutes at 11,000 x g these samples remained liquid. In another step, they were inoculated with a betaine monohydrate crystal. This also had no affect on the solvents.

Further investigations concerning the hysteresis were conducted to examine the phase behavior of urea/betaine monohydrate. The melting and freezing points for urea concentrations of 52.6 mol-% and 80 mol-% were determined. The results are given in Fig. 10 and Table 34.

Table 34: Freezing and melting point of urea/betaine monohydrate DES for different ratios

It is evident, that the hysteresis gets insignificant for high and low urea concentrations.

On the other side, the stabilizing effect of urea can be seen, by means of a significant higher melting and freezing point for lower concentrations of urea than the expected eutectic concentration of 67%.

Dissolution of Starch in Betaine Monohydrate/Urea DES

DES have been used as solvents for starch. In a first step, the dissolution of starch in the

DES urea/betaine monohydrate should be determined. In 1 ml of urea/betaine monohydrate DES, 48.2 g starch in a first trial and 46.1 g starch in the second trial was incubated at 90°C mixed at 1400 rpm 30 minutes. After cooling, a gel can be observed for the urea/choline chloride DES. General Discussion

No dissolution for Avicel® could be found by use of the conventional DES and other solvents such as described previously. The main problem is still the high viscosity of the DES, which leads to a poor suspension. This disadvantage was overcome by going to higher temperatures, but these experiments did not always yield better results.

The dissolution of Avicel® in a sodium hydroxide solution appears not to be favorable due to the huge amounts of sodium hydroxide needed for a large scale application. However, the swelling and dissolution of cellulose is caused by partially broken intra-molecular hydrogen bonds which leads to a lower crystallinity. The use of ammonia hydroxide instead of sodium hydroxide appears more sensible, due to the better recover of ammonia by means of evaporation.

The measurements to detect the hydrolysis rate turned out to be difficult to compare due to the effect of Avicel® on the glucose concentration. Even slightly higher Avicel® concentrations of about 2-3 mg/ml caused a significantly higher or lower glucose concentration under the same conditions.

DES or Room Temperature Ionic Liquids (RTIL) are mostly formed by use of choline chloride. This however is too expensive to be employed in large scale processes. As reported in above, it can be replaced by betaine monohydrate to form a DES with most of the compounds DES used with choline chloride. The cost for choline chloride is $745.00 U.S. compared to betaine monohydrate $32.10 U.S. (both 99%, Sigma Aldrich for 100 g). It is notable that we report for the first time, DES which are more than 20 times cheaper than the DES used previously.

The system betaine monohydrate/urea stands out with its extreme low freezing point and low viscosity at room temperature. As described above, the reduction of crystallinity of cellulose is of strong interest. We can detect a significant effect of specific DES on the crystallinity of cellulose. For the DES malonic acid/ChCl, the reduction is in the range of 20%. For the new DES urea/betaine monohydrate, the reduction of crystallinity is about 10-15%.

As described previously, the crystallinity of cellulose correlates with the rate of hydrolysis in that way, that a lower crystallinity increases the rate of hydrolysis. For the Avicel® pretreated with DES and therefore reduced its crystallinity a higher glucose concentration should be oberservable. However, this cannot always be reported.

To gain more accurate results, it is recommended to apply a Differential Scanning Calorimetry to detect the melting points of these system. In the case of urea/betaine monohydrate, the phase behavior for 60, 67 and 70 mol- urea below 1°C should be investigated. On the other hand, it has to be checked if there is a DES for urea/betaine.

Furthermore the investigation of new DES has to be advanced to gain more effective, cheaper and less viscous solvents. In general, there are two possibilities to push in that direction. Either the replacement of ChCl with a cheap and as effective quaternary ammonium salt, or combinations of urea with other metal salts as shown briefly with the system urea/CaC12.

In general, the use of DES is reasonable with respect to their ease in preparation compared to currently used ionic liquids like BMIMAc (-N-Butyl-3-methylimidazolium acetate).

Materials Used

1. Chemical Producers

Alfa Aesar (Pelham, NH USA)

Sigma Aldrich (St. Louis, MO USA) VWR (Radnor, PA USA)

Merck KGaA (Darmstadt, Germany)

2. Enzymes Used

3. Equipment Used

Isotemp Hybridization Incubator Fisher Scientific (Pittsburgh, PA USA) pH-meter accumet®basic AB15 Fisher Scientific (Pittsburgh, USA)

Dual Range Balance AG285 Analytical B Mettler Toledo (Columbus, OH USA)

Microfuge®18 Centrifuge Beckman Coulter (Brea, CA USA)

FreeZone 6 Liter Benchtop Freeze Dry System Labconco Corporation (Kansas City, MO USA)

4. Chemicals to be Weighed In

Numerous characteristics and advantages have been set forth in the foregoing description, together with details of structure and function. While the invention has been disclosed in several forms, it will be apparent to those skilled in the art that many modifications, additions, and deletions, especially in matters of shape, size, and arrangement of parts, can be made therein without departing from the spirit and scope of the invention and its equivalents as set forth in the following claims. Therefore, other modifications or embodiments as may be suggested by the teachings herein are particularly reserved as they fall within the breadth and scope of the claims here appended.

Claims

CLAIMS What is claimed is:

1. A eutectic solvent comprising:

a betaine compound; and

a hydrogen bond donor.

2. The eutectic solvent of Claim 1, wherein the betaine compound comprises betaine monohydrate.

3. The eutectic solvent of Claim 1, wherein the hydrogen bond donor comprises urea.

4. The eutectic solvent of Claim 1, wherein the hydrogen bond donor comprises malonic acid.

5. The eutectic solvent of Claim 1, wherein the hydrogen bond donor comprises citric acid.

6. The eutectic solvent of Claim 1, wherein the molar ratio of betaine compound:hydrogen bond donor is 2: 1.

7. The eutectic solvent of Claim 1, wherein the molar ratio of betaine compound:hydrogen bond donor is 1: 1.

8. The eutectic solvent of Claim 1, wherein the molar ratio of betaine compound:hydrogen bond donor is 3: 1.

9. A composition of matter comprising urea and betaine monohydrate.

10. The composition of Claim 9, wherein at 0 mol-% urea, the freezing point is approximately 241°C, and the melting point is approximately 241°C.

11. The composition of Claim 9, wherein at 52.6 mol-% urea, the freezing point is approximately 93°C, and the melting point is approximately 96°C.

12. The composition of Claim 9, wherein at 55 mol-% urea, the freezing point is approximately 44°C, and the melting point is approximately 71 °C.

13. The composition of Claim 9, wherein at 60 mol-% urea, the freezing point is approximately 1°C, and the melting point is approximately 69°C.

14. The composition of Claim 9, wherein at 67 mol-% urea, the freezing point is approximately 1°C, and the melting point is approximately 60°C.

15. The composition of Claim 9, wherein at 70 mol-% urea, the freezing point is approximately 1°C, and the melting point is approximately 60°C.

16. The composition of Claim 9, wherein at 75 mol-% urea, the freezing point is approximately 30°C, and the melting point is approximately 62°C.

17. The composition of Claim 9, wherein at 100 mol-% urea, the freezing point is approximately 134°C, and the melting point is approximately 134°C.

18. A deep eutectic solvent comprising:

betaine monohydrate; and

a hydrogen bond donor.

19. The deep eutectic solvent of Claim 18, wherein the hydrogen bond donor comprises urea.

20. The deep eutectic solvent of Claim 18, wherein the hydrogen bond donor comprises malonic acid.

21. The deep eutectic solvent of Claim 18, wherein the hydrogen bond donor comprises citric acid.

22. The deep eutectic solvent of Claim 18, wherein the molar ratio of betaine monohydrate:hydrogen bond donor is 2: 1.

23. The deep eutectic solvent of Claim 18, wherein the molar ratio of betaine monohydrate:hydrogen bond donor is 1: 1.

24. The deep eutectic solvent of Claim 18, wherein the molar ratio of betaine monohydrate:hydrogen bond donor is 3: 1.

25. A deep eutectic solvent comprising:

betaine monohydrate; and

urea;

wherein the molar ratio of betaine monohydrate:urea is 2: 1.

26. A method for the dissolution of cellulose comprising mixing cellulose in the eutectic solvent of Claim 1.

27. A method for the dissolution of cellulose comprising mixing cellulose in the composition of Claim 9.

28. A method for the dissolution of cellulose comprising mixing cellulose in the deep eutectic solvent of Claim 18.

29. A method for the dissolution of a carbohydrate comprising mixing a carbohydrate in the eutectic solvent of Claim 1.

30. A method for the dissolution of a carbohydrate comprising mixing a carbohydrate in the composition of Claim 9.

31. A method for the dissolution of a carbohydrate comprising mixing a carbohydrate in the deep eutectic solvent of Claim 18.

32. A method for the dissolution of a starch comprising mixing a starch in the eutectic solvent of Claim 1.

33. A method for the dissolution of a starch comprising mixing a starch in the composition of Claim 9.

34. A method for the dissolution of a starch comprising mixing a starch in the deep eutectic solvent of Claim 18.

35. A method of reducing the crystallinity of cellulose comprising mixing cellulose in the eutectic solvent of Claim 1, and providing a reduction of crystallinity below 20%.

36. A method of reducing the crystallinity of cellulose according to Claim 35, providing a reduction of crystallinity of approximately 10-15%.