This project has received funding from the European Research Council (ERC) under the European Union&#8217;s Horizon 2020 research and innovation program, under grant agreement No ERC-2016-CoG 725034. This work was also supported by the Associate Laboratory for Green Chemistry-LAQV which is financed by national funds from FCT/MCTES (UID/QUI/50006/2019) and by FCT/MCTES through the project CryoDES (PTDC/EQU-EQU/29851/2017).

NOTE: The NADES studied were betaine:L-(+)-tartaric acid (2:1), &#946;-alanine:DL-malic acid (3:2), glucose:sucrose (1:1) and citric acid:glucose (2:1). These systems were prepared by different methods: freeze-drying (FD), vacuum evaporation (VE), and heat and stirring (HS) with and without water. As an example, the protocol for the system citric acid:glucose (2:1) is given. The NADES were characterized by differential scanning calorimetry (DSC), polarized optical microscopy (POM), water content and nuclear magnetic resonance (NMR) spectroscopy.
1. NADES preparation
<ol>
	<li>Freeze drying
	<ol>
		<li>In separate containers, add 2 g of citric acid monohydrate and 0.9530 g of glucose monohydrate. Add 10 mL of deionized water to each and stir until the compounds are completely dissolved.</li>
		<li>Mix the two solutions together and ensure the homogenization of the final solution. Place the solution in a round bottom flask.</li>
		<li>Freeze it using liquid nitrogen. Place the flask in a freeze-dryer for 48 h to ensure that all the water is removed from the sample.</li>
	</ol>
	</li>
	<li>Vacuum evaporation
	<ol>
		<li>Weigh 2 g of citric acid monohydrate and 0.9530 g of glucose monohydrate in separate containers. Add 10 mL of deionized water to each and stir until the compounds are completely dissolved.</li>
		<li>Mix the two solutions together and ensure the homogenization of the solution. Place the solution in a round bottom flask.</li>
		<li>Using a rotary evaporator, dry the sample until a clear, viscous liquid is formed.</li>
	</ol>
	</li>
	<li>Heating and stirring
	<ol>
		<li>Weigh 2 g of citric acid monohydrate and 0.9530 g of glucose monohydrate into the same vial. Add 278 &#956;L of water.</li>
		<li>Place the vial with a magnetic stirring bar in a 50 &#176;C water bath.</li>
		<li>Leave the sample until a clear, viscous liquid is formed.</li>
	</ol>
	</li>
</ol>
2. NADES characterization
<ol>
	<li>Polarized optical microscopy (POM)
	<ol>
		<li>Place a droplet of NADES on a microscope glass slide for observation.</li>
		<li>Using the transmission mode of a microscope, perform the optical characterization of the sample at room temperature.</li>
	</ol>
	</li>
	<li>Karl-Fisher titration
	<ol>
		<li>Collect 100 &#956;L of NADES in a syringe, and then clean the excess liquid on the outside.</li>
		<li>Place the syringe on a scale and tare it.</li>
		<li>Press START on the KF equipment and add a small drop of the sample to the vessel.</li>
		<li>Weigh the syringe, enter the mass on the KF equipment and press ENTER. The result will appear on the screen in ppm of water.</li>
	</ol>
	</li>
	<li>Differential scanning calorimetry (DSC)
	<ol>
		<li>Place 3-10 mg of each sample in a hermetic aluminum pan with a covering lid. Close the pan with a sample press.</li>
		<li>Analyze the samples using a DSC with a temperature range of -90 &#176;C up to the degradation temperature, with a heating rate of 10 &#176;C/min. Perform two cycles with an isothermal hold of 2 min and analyze under a nitrogen atmosphere (50 mL/min).</li>
	</ol>
	</li>
	<li>Nuclear magnetic resonance (NMR)
	<ol>
		<li>Prepare a 5 mm NMR tube by dissolving 250 &#181;L of NADES with 250 &#181;L of dimethyl sulfoxide-d6 (DMSO-d6).</li>
		<li>Acquire the 1H and NOESY spectra at 25 &#176;C on a 400 MHz spectrometer.</li>
		<li>Use an appropriate software to analyze the spectra, and use the chemical shift of DMSO-d6 (&#948; 2.50 ppm) to assign all the signals of each component.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Paiva, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+deep+eutectic+solvents+-+solvents+for+the+21st+century.">Natural deep eutectic solvents - solvents for the 21st century.</a> ACS Sustainable Chemistry &amp; Engineering. 2, 1063-1071 (2014).</li><li>Abbott, A. P., Capper, G., Davies, D. L., Rasheed, R. K., Tambyrajah, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+solvent+properties+of+choline+chloride/urea+mixtures.">Novel solvent properties of choline chloride/urea mixtures.</a> Chemical Communications. , 70-71 (2003).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+deep+eutectic+solvents:+properties,+applications,+and+perspectives.">Natural deep eutectic solvents: properties, applications, and perspectives.</a> Journal of Natural Products. 81, 679-690 (2018).</li><li>Choi, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+natural+deep+eutectic+solvents+the+missing+link+in+understanding+cellular+metabolism+and+physiology.">Are natural deep eutectic solvents the missing link in understanding cellular metabolism and physiology.</a> Plant Physiology. 156, 1701-1705 (2011).</li><li>Dai, Y., Spromsen, J. V., Witkamp, G. -. J., Verpoorte, R., Choi, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+deep+eutectic+solvents+as+new+potential+media+for+green+technology.">Natural deep eutectic solvents as new potential media for green technology.</a> Analytica Chimica Acta. 766, 61-68 (2013).</li><li>Espino, M., Fern&#225;ndez, M. A., Gomez, F. J. V., Silva, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+designer+solvents+for+greening+analytical+chemistry.">Natural designer solvents for greening analytical chemistry.</a> Trends in Analytical Chemistry. 76, 126-136 (2016).</li><li>Hayyan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+deep+eutectic+solvents:+cytotoxic+profile.">Natural deep eutectic solvents: cytotoxic profile.</a> Springer Plus. 5, 913 (2016).</li><li>Dai, Y., Witkamp, G. -. J., Verpoorte, R., Choi, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+properties+of+natural+deep+eutectic+solvents+with+water+to+facilitate+their+applications.">Tailoring properties of natural deep eutectic solvents with water to facilitate their applications.</a> Food Chemistry. 187, 14-19 (2015).</li><li>Choi, Y. H., Verpoorte, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+solvents+for+the+extraction+of+bioactive+compounds+from+natural+products+using+ionic+liquids+and+deep+eutectic+solvents.">Green solvents for the extraction of bioactive compounds from natural products using ionic liquids and deep eutectic solvents.</a> Current Opinion in Food Science. 26, 87-93 (2019).</li><li>Guit&#233;rrez, M. C., Ferrer, M. L., Mateo, C. R., Del Monte, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-drying+of+aqueous+solutions+of+deep+eutectic+solvents:+a+suitable+approach+to+deep+eutectic+suspensions+of+self-assembled+structures.">Freeze-drying of aqueous solutions of deep eutectic solvents: a suitable approach to deep eutectic suspensions of self-assembled structures.</a> Langmuir. 25, 5509-5515 (2009).</li><li>Gomez, F. J. V., Espino, M., Fern&#225;ndez, M. A., Silva, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+greener+approach+to+prepare+natural+deep+eutectic+solvents.">A greener approach to prepare natural deep eutectic solvents.</a> Chemistry Select. 3, 6122-6125 (2018).</li></ol>

The authors have nothing to disclose.

The different methodologies reported in the literature for the preparation of NADES are a heating and stirring method (HS), vacuum evaporation (VE), and freeze-drying (FD). The systems we have prepared in this work are described by different authors in the literature4,5,6,10,11. Table 1 lists the components of each mixture, as reported in the original manuscript as well as their preparation method.
Upon our investigations to reproduce the systems described, we realized that in some cases it was not possible to achieve a similar NADES, as a clear, viscous, liquid sample at room temperature. Preparing a NADES relies on many factors. Some can be easily controlled, but others are more difficult to standardize. The most important thing to consider is that the final product cannot rely on external factors such as the equipment used.
The systems prepared by different methods were then characterized. With polarized optical microscopy (POM), it was observed that with the HS method without water, even at different temperatures, the NADES did not form a clear and viscous liquid. However, a homogeneous and clear, viscous liquid was observed as represented in Figure 1 when applying the HS method with small amounts of water and the VE method for the preparation of the NADES.
DSC was used to determine the thermal events of the mixture. The results showed that the system is liquid at room temperature and up to 130 &#176;C, since the thermogram shows no thermal events. The water content of each sample was measured by Karl-Fischer titration, and the results are represented in Table 2. The water content of the systems must be reported, since it is the parameter that most influences the properties of the obtained liquid, such as viscosity and polarity. These changes have great impact on the outcome of the application for which the NADES is designed.
NMR was also used to confirm the formation the mentioned NADES systems, through the formation of hydrogen bonds between the molecules of each system. One example is given in Figure 2 for the NADES system citric acid:glucose (2:1) with 17% water obtained by HS where the proton spectrum of this NADES and the starting materials (citric acid and glucose) are overlaid (Figure 2a). From this, it is possible to observe changes in the chemical shifts of some protons from each molecule. The major change is the shifting of the OH proton from citric acid. Originally, this signal appears at 5.16 ppm, but this signal shifts to 6.22 ppm because of the formation of hydrogen bonds. This is confirmed by the NOESY spectrum (Figure 2b), where the strong interaction between the OH from citric acid and the remaining protons is visible. A similar interaction was observed for the other NADES systems.
In this study we observed that the description of the preparation method for eutectic systems reported in literature sometimes are incomplete, due to the lack of information regarding the water content of most systems. In the VE method, the water is added by preparing solutions of different components and mixing at a temperature that leads to the formation of eutectic systems; however, we cannot be sure of the minimum required water content. The knowledge of percentage of water needed to form the systems is considered hence, a crucial point that should always be reported, for others to be able to reproduce the preparation of the different eutectic mixtures.
The best method to use is the HS method with water added as it takes less time to prepare, for cases where the water content is already described. However, if this information is not available, the easiest method is the VE method, where all the available water is removed and only the water interacting with the NADES components remains in the system. In any case, researchers should let the systems evaporate for enough time to ensure that free water is removed from the system. This timing is dependent on the equipment and therefore it is not enough to describe in the materials section the duration of the VE method, but the water content has always to be reported.

Deep eutectic solvents have been named the solvents for the 21st century and are considered a new generation of solvents. They are defined as a mixture of two or more chemical compounds at a particular molar ratio to result in a significant decrease in the melting temperature of the individual components, becoming liquid at room temperature1,2,3. In this sense, the preparation of the solvents does not require any chemical reaction and hence the production yield is 100%. In 2011, Choi and co-workers reported the possibility of naturally occurring DES and named them, natural deep eutectic solvents (NADES)3,4,5. NADES can be prepared from different combinations of sugars, amino acids, organic acids and choline derivatives; and these systems prepared from natural components are inherently biocompatible and biodegradable, presenting considerably less toxicity compared to other alternative solvents (e.g., ionic liquids)5,6,7,8. Since 2015, the number of publications in the field has risen exponentially and the possible applications of NADES are very broad3. Even though many manuscripts and reviews have been published, there are fundamental questions that persist, and scientists have not yet found the answer to intriguing questions such as the mechanisms underlying DES formation. Understanding the DES formation mechanism would lead to a consolidated approach towards the development of new systems, rather than the current trial and error approach. Furthermore, the opportunities in the field are growing each day, as consumers become more aware of the sustainability of their products, not only in terms of their end-life but also in terms of processing itself8,9,10. To drive major innovations in the field of deep eutectic solvents, the standardization of the production and characterization methods is first required. The lack of reproducibility of some of the systems reported in the literature was the motivation to develop this work as we faced this issue several times. Herein, we demonstrate the need and crucial importance to accurately describe the materials and methods and show that although the preparation of DES is a simple and straightforward procedure, there are some key aspects (e.g., the presence/amount of water) that must always be discussed.

<table>
	<tbody>
		<tr>
			<td>5 mm NMR tube</td>
			<td>Norell</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acid citric monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Advance III spectrometer</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide-d6</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DSC Q200</td>
			<td>TA Instruments, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze-dryer CHRIST ALPHA 1-4</td>
			<td>Braun Biotec International</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose monohydrate</td>
			<td>Cmd chemicals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Karl Fisher Coulometer</td>
			<td>Metrohm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX-51 polarized optical microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of binary and ternary deep eutectic systems

The preparation of deep eutectic systems (DES) is a priori a simple procedure. By definition, two or more components are mixed together at a given molar ratio to form a DES. However, from our experience in the laboratory, there is a need to standardize the procedure to prepare, characterize and report the methodologies followed by different researchers, so that the results published can be reproduced. In this work, we test different approaches reported in the literature to prepare eutectic systems and evaluated the importance of water in the successful preparation of liquid systems at room temperature. These published eutectic systems were composed of citric acid, glucose, sucrose, malic acid, &#946;-alanine, L-tartaric acid and betaine and not all of preparation methods described could be reproduced. However, in some cases, it was possible to reproduce the systems described, with the inclusion of water as a third component of the eutectic mixture.

From the preparation of NADES, the results we expect to obtain are shown on Figure 1. A description of each system is made below. Using the freeze-drying method, the result should be a solid or a very dense paste since all the water is removed from the system. Using the evaporation method, the result should be a clear and viscous liquid. Using the heating and stirring method with the addition of small amounts of water, the result should be a clear and very viscous liquid.
The results obtained from POM can be seen on Figure 1. When a NADES is completely formed, we expect to see a black image, indicating that the sample is completely amorphous and that there are no crystals remaining in the system. The results obtained from KF titration are described in Table 2. Besides the amount of water that is added to the systems, the percentage of water of the final mixture also depends on the water content of the reagents.
Concerning the DSC, the goal of this technique is also to confirm that the system is liquid in the temperature range that it will be applied, so the expected result is to have a thermogram that shows no thermal events on the temperature range of interest (Table 2). The NMR technique is used to confirm the existence of hydrogen bond formation, which is the main characteristic of NADES systems. This can be confirmed by observation of the change in chemical shifts of each signal, and by analysis of the NOESY spectra, that shows spatial and intermolecular correlations (Figure 2).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component 1</td>
			<td>Component 2</td>
			<td>Preparation Method</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>Betaine (Bet)</td>
			<td>L-(+)-Tartaric Acid (LTA)</td>
			<td>Vacuum evaporating (VE)</td>
			<td>Dai et al. (2013)5 and Espino et al. (2016)6</td>
		</tr>
		<tr>
			<td>&#946;-Alanine (&#946;-A)</td>
			<td>DL-Malic Acid (MA)</td>
			<td>Vacuum evaporating (VE)</td>
			<td>Dai et al. (2013)5 and Espino et al. (2016)6</td>
		</tr>
		<tr>
			<td>Glucose (Gluc)</td>
			<td>Sucrose (Suc)</td>
			<td>Freeze-dried (FD)</td>
			<td>Choi et al. (2011)4 and Espino et al. (2016)6</td>
		</tr>
		<tr>
			<td>Citric Acid (CA)</td>
			<td>Glucose (Gluc)</td>
			<td>Freeze-dried (FD)</td>
			<td>Choi et al. (2011)4 and Espino et al. (2016)6</td>
		</tr>
	</tbody>
</table>
Table 1: Systems reported in literature and their preparation method.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">NADES</td>
			<td rowspan="2">Preparation Method</td>
			<td>Water Content (%)</td>
		</tr>
		<tr>
			<td>Karl Fischer measure</td>
		</tr>
		<tr>
			<td>Bet:LTA (2:1 + 20% water)</td>
			<td>Heating and stirring, adding water</td>
			<td>19.94 &#177; 1.28</td>
		</tr>
		<tr>
			<td>Bet:LTA (2:1)</td>
			<td>Vacuum evaporating</td>
			<td>11.36 &#177; 0.78</td>
		</tr>
		<tr>
			<td>&#946;-A:MA (3:2 + 11% water)</td>
			<td>Heating and stirring, adding water</td>
			<td>11.45 &#177; 0.25</td>
		</tr>
		<tr>
			<td>&#946;-A:MA (3:2)</td>
			<td>Vacuum evaporating</td>
			<td>18.84 &#177; 1.78</td>
		</tr>
		<tr>
			<td>Gluc:Suc (1:1 + 21% water)</td>
			<td>Heating and stirring, adding water</td>
			<td>20.88 &#177; 0.13</td>
		</tr>
		<tr>
			<td>Gluc:Suc (1:1)</td>
			<td>Vacuum evaporating</td>
			<td>22.56 &#177; 0.48</td>
		</tr>
		<tr>
			<td>CA:Gluc (2:1 + 17% water)</td>
			<td>Heating and stirring, adding water</td>
			<td>17.33 &#177; 0.68</td>
		</tr>
		<tr>
			<td>CA:Gluc (2:1)</td>
			<td>Vacuum evaporating</td>
			<td>20.04 &#177; 0.26</td>
		</tr>
	</tbody>
</table>
Table 2: Water content (%) of the systems prepared by different methods.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60326/60326fig01.jpg" /> 
Figure 1: Representative results of the NADES when prepared by a) freeze-drying, b) vacuum evaporation and c) heating and stirring with addition of water.&#160;The picture shows that when the system is freeze-dried, the result obtained is a crystal since all the water is removed from the mixture whereas when VE and HS methods are used, the amount of water needed for the NADES to form is present and the obtained result is a homogenous liquid at room temperature. <a href="https://www.jove.com/files/ftp_upload/60326/60326fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60326/60326fig02.jpg" /> 
Figure 2: Polarized optical microscopy of CA:Glu (2:1) prepared by different methods, with cross polarizers (left image) and parallel polarizers (right image) &#8211; 100 &#956;m (10x amplification).&#160;The black images show that the sample is a liquid at room temperature. The FD sample is completely crystalized since the result obtained from this technique was not a liquid. <a href="https://www.jove.com/files/ftp_upload/60326/60326fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60326/60326fig03.jpg" /> 
Figure 3: a) Overlay of 1H NMR spectra of the (A) NADES system citric acid:glucose:water (2:1:4), (B) glucose, and (C) citric acid; b) NOESY spectrum of the NADES system citric acid:glucose:water (2:1:4).&#160;The overlaid spectra show the difference in chemical shifts of each component upon DES formation, originated by the establishment of hydrogen bonds between them. The NOESY spectrum shows the interaction between the OH proton from citric acid with the remaining protons from both components. <a href="https://www.jove.com/files/ftp_upload/60326/60326fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation of Binary and Ternary Deep Eutectic Systems at JoVE.com

Preparation of Binary and Ternary Deep Eutectic Systems

This protocol aims to standardize the preparation of deep eutectic systems throughout the scientific community so that these systems can be reproduced.

The preparation of deep eutectic systems (DES) is a priori a simple procedure. By definition, two or more components are mixed together at a given molar ...

preparation-of-binary-and-ternary-deep-eutectic-systems

Research

JoVE Journal

Chemistry

11.6K Views.  Universidade Nova de Lisboa. This protocol aims to standardize the preparation of deep eutectic systems throughout the scientific community so that these systems can be reproduced.

Video: Preparation of Binary and Ternary Deep Eutectic Systems

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. Specifically, the sapphire cell enables visual data collection from multiple loadings to solve a set of material balances to precisely determine phase composition. Ternary phase diagrams can then be established to determine the proportion of each component in each phase at a given condition. In principle, any ternary system can be studied although ternary systems (gas-liquid-liquid) are the specific examples discussed herein. For instance, the ternary THF-Water-CO2 system was studied at 25 and 40&#160;&#176;C and is described herein. Of key importance, this technique does not require sampling. Circumventing the possible disturbance of the system equilibrium upon sampling, inherent measurement errors, and technical difficulties of physically sampling under pressure is a significant benefit of this technique. Perhaps as important, the sapphire cell also enables the direct visual observation of the phase behavior. In fact, as the CO2 pressure is increased, the homogeneous THF-Water solution phase splits at about 2 MPa. With this technique, it was possible to easily and clearly observe the cloud point and determine the composition of the newly formed phases as a function of pressure.
The data acquired with the sapphire cell technique can be used for many applications. In our case, we measured swelling and composition for tunable solvents, like gas-expanded liquids, gas-expanded ionic liquids and Organic Aqueous Tunable Systems (OATS)1-4. For the latest system, OATS, the high-pressure sapphire cell enabled the study of (1) phase behavior as a function of pressure and temperature, (2) composition of each phase (gas-liquid-liquid) as a function of pressure and temperature and (3) catalyst partitioning in the two liquid phases as a function of pressure and composition. Finally, the sapphire cell is an especially effective tool to gather accurate and reproducible measurements in a timely fashion.

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus is a unique tool to study, without sampling, phase behavior under a wide range of pressures. Using a cathetometer, very precise volume measurements can be recorded to measure liquid expansion and phase composition. Thus, this synthetic method enables the study of (1) phase equilibria of multi-component mixtures and (2) the partition behavior of catalyst or model compounds as a function of pressure.

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. ...

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

Preparation and Characterization of SDF-1&amp;#945;-Chitosan-Dextran Sulfate Nanoparticles

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Chemical Gardens as Flow-through Reactors Simulating Natural Hydrothermal Systems

Here we report experimental simulations of hydrothermal chimney growth using injection chemical garden methods. The versatility of this type of experiment allows for testing of various proposed ocean / hydrothermal fluid chemistries that could have driven reactions toward the origin of life in environments on the early Earth, early Mars, or even other worlds such as the icy moons of the outer planets. We show experiments that include growth of chemical garden structures under anoxic conditions simulating the early Earth, inclusion of trace components of phosphates / organics in the injection solution to incorporate them into the structure, a switch of the injection solution to introduce a secondary precipitating anion, and the measurement of membrane potentials generated by chemical gardens. Using this method, self-assembling chemical garden structures were formed that mimic the natural chimneys precipitated at submarine hydrothermal springs, and these precipitates can be used successfully as flow-through reactors by feeding through multiple successive &#8220;hydrothermal&#8221; injections.

We describe chemical garden formation via injection experiments that allow for laboratory simulations of natural chemical garden systems that form at submarine hydrothermal vents.

Here we report experimental simulations of hydrothermal chimney growth using injection chemical garden methods. The versatility of this type of experiment ...

Construction and Systematical Symmetric Studies of a Series of Supramolecular Clusters with Binary or Ternary Ammonium Triphenylacetates

Functions of clusters in nano or sub-nano scale significantly depend on not only kinds of their components but also arrangements, or symmetry, of their components. Therefore, the arrangements in the clusters have been precisely characterized, especially for metal complexes. Contrary to this, characterizations of molecular arrangements in supramolecular clusters composed of organic molecules are limited to a few cases. This is because construction of the supramolecular clusters, especially obtaining a series of the supramolecular clusters, is difficult due to low stability of non-covalent bonds compare to covalent bonds. From this viewpoint, utilization of organic salts is one of the most useful strategies. A series of the supramolecules could be constructed by combinations of a specific organic molecule with various counter ions. Especially, primary ammonium carboxylates are suitable as typical examples of supramolecules because various kinds of carboxylic acids and primary amines are commercially available, and it is easy to change their combinations. Previously, it was demonstrated that primary ammonium triphenylacetates using various kinds of primary amines specifically construct supramolecular clusters, which are composed of four ammoniums and four triphenylacetates assembled by charge-assisted hydrogen bonds, in crystals obtained from non-polar solvents. This study demonstrates an application of the specific construction of the supramolecular clusters as a strategy to conduct systematical symmetric study for clarification of correlations between molecular arrangements in supramolecules and kinds and numbers of their components. In the same way with binary salts composed of triphenylacetates and one kind of primary ammoniums, ternary organic salts composed of triphenylacetates and two kinds of ammoniums construct the supramolecular clusters, affording a series of the supramolecular clusters with various kinds and numbers of the components.

This article describes construction of a series of hydrogen-bonding supramolecular clusters in crystals using primary ammonium triphenylacetates, which are recrystallized from non-polar solvents. This selective construction of the supramolecular clusters leads to effective systematical symmetric studies about a correlation between the supramolecular clusters and their components.

Functions of clusters in nano or sub-nano scale significantly depend on not only kinds of their components but also arrangements, or symmetry, of their ...

Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is a major effort to target PPIs in basic research and in the pharmaceutical industry. Protein-protein interfaces are usually large, flat, and often lack pockets, complicating the discovery of small molecules that target such sites. Alternative targeting approaches using antibodies have limitations due to poor oral bioavailability, low cell-permeability, and production inefficiency.
Using peptides to target PPI interfaces has several advantages. Peptides have higher conformational flexibility, increased selectivity, and are generally inexpensive. However, peptides have their own limitations including poor stability and inefficiency crossing cell membranes. To overcome such limitations, peptide cyclization can be performed. Cyclization has been demonstrated to improve peptide selectivity, metabolic stability, and bioavailability. However, predicting the bioactive conformation of a cyclic peptide is not trivial. To overcome this challenge, one attractive approach it to screen a focused library to screen in which all backbone cyclic peptides have the same primary sequence, but differ in parameters that influence their conformation, such as ring size and position.
We describe a detailed protocol for synthesizing a library of backbone cyclic peptides targeting specific parasite PPIs. Using a rational design approach, we developed peptides derived from the scaffold protein Leishmania receptor for activated C-kinase (LACK). We hypothesized that sequences in LACK that are conserved in parasites, but not in the mammalian host homolog, may represent interaction sites for proteins that are critical for the parasites&#39; viability. The cyclic peptides were synthesized using microwave irradiation to reduce reaction times and increase efficiency. Developing a library of backbone cyclic peptides with different ring sizes facilitates a systematic screen for the most biological active conformation. This method provides a general, fast, and facile way to synthesize cyclic peptides.

A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Solution-Processed "Silver-Bismuth-Iodine" Ternary Thin Films for Lead-Free Photovoltaic Absorbers

Bismuth-based hybrid perovskites are regarded as promising photo-active semiconductors for environment-friendly and air-stable solar cell applications. However, poor surface morphologies and relatively high bandgap energies have limited their potential. Silver-bismuth-iodine (Ag-Bi-I) is a promising semiconductor for optoelectronic devices. Therefore, we demonstrate the fabrication of Ag-Bi-I ternary thin films using material solution processing. The resulting thin films exhibit controlled surface morphologies and optical bandgaps according to their thermal annealing temperatures. In addition, it has been reported that Ag-Bi-I ternary systems crystallize to AgBi2I7, Ag2BiI5, etc. according to the ratio of the precursor chemicals. The solution-processed AgBi2I7 thin films exhibit a cubic-phase crystal structure, dense, pinhole-free surface morphologies with grains ranging in size from 200 to 800 nm, and an indirect bandgap of 1.87 eV. The resultant AgBi2I7 thin films show good air stability and energy band diagrams, as well as surface morphologies and optical bandgaps suitable for lead-free and air-stable single-junction solar cells. Very recently, a solar cell with 4.3% power conversion efficiency was obtained by optimizing the Ag-Bi-I crystal compositions and solar cell device architectures.

Solution-Processed &quot;Silver-Bismuth-Iodine&quot; Ternary Thin Films for Lead-Free Photovoltaic Absorbers

Herein, we present detailed protocols for solution-processed silver-bismuth-iodine (Ag-Bi-I) ternary semiconductor thin films fabricated on TiO2-coated transparent electrodes and their potential application as air-stable and lead-free optoelectronic devices.

Bismuth-based hybrid perovskites are regarded as promising photo-active semiconductors for environment-friendly and air-stable solar cell applications. ...

Development and Validation of Chromium Getters for Solid Oxide Fuel Cell Power Systems

Degradation of cathode in solid oxide fuel cells (SOFC) remains a major concern for the long-term performance stability and operational reliability. The presence of gas phase chromium species in air has demonstrated significant cathode performance degradation during long-term exposure due to unwanted compound formation at the cathode and electrolyte interface which retards the oxygen reduction reaction (ORR). We have demonstrated a novel method to mitigate the cathode degradation using chromium getters which capture the gas phase chromium species before it is ingested in the cathode chamber. Low-cost getter materials, synthesized from alkaline earth and transition metal oxides, are coated on the cordierite honeycomb substrate for application in the SOFC power systems. As-fabricated getters have been screened by chromium transpiration tests for 500 h in humidified air atmosphere in presence of chromium vapor. Selected getters have been further validated utilizing electrochemical tests. Typically, electrochemical performance of SOFCs (lanthanum strontium manganite (LSM) &#449; yttria stabilized zirconia (YSZ) &#449; Pt) was measured at 850 &#176;C in the presence and absence of Cr getter. For the 100 h cell tests containing getters, stable electrochemical performance was maintained, whereas the cell performance in the absence of Cr getters rapidly decreased in 10 h. Analyses of Nyquist plots indicated significant increase in the polarization resistance within the first 10 h of the cell operation. Characterization results from posttest SOFCs and getters have demonstrated the high efficiency of chromium capture for the mitigation of cell degradation.

Cathode poisoning from airborne contaminants in trace levels remains a major concern for long-term stability of high-temperature electrochemical systems. We provide a novel method to mitigate the cathode degradations using getters, which capture airborne contaminants at high temperature before entering electrochemically active stack area.

Degradation of cathode in solid oxide fuel cells (SOFC) remains a major concern for the long-term performance stability and operational reliability. The ...

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. The versatility of the GSMLCs is demonstrated in the context of a study about etching and growth dynamics of gold nanostructures from a HAuCl4 precursor solution. GSMLCs combine the advantages of conventional silicon- and graphene-based liquid cells by offering reproducible well depths together with facile cell manufacturing and handling of the specimen under investigation. The GSMLCs are fabricated on a single silicon substrate which drastically reduces the complexity of the manufacturing process compared to two-wafer-based liquid cell designs. Here, no bonding or alignment process steps are required. Furthermore, the enclosed liquid volume can be tailored to the respective experimental requirements by simply adjusting the thickness of a silicon nitride layer. This enables a significant reduction of window bulging in the electron microscope vacuum. Finally, a state-of-the-art quantitative evaluation of single particle tracking and dendrite formation in liquid cell experiments using only open source software is presented.

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

A protocol for preparation of graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals from HAuCl4 precursor solution is presented. Furthermore, an analysis routine is presented to quantify observed etching and growth dynamics.

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. ...