Reproducibility of deep eutectic systems found in the literature is difficult when water content has been disregarded. This method provides a standardization protocol for the development of these systems. This technique will allow the reproducibility of deep eutectic system preparation, contributing to coherent results among the scientific community.
Deep eutectic systems have proven applications in many different areas, including therapeutics and biomedical engineering, biocatalysis, extraction, and CO2 capture. Through a visual demonstration, we highlight the variations encountered in the different methods reported in the literature for deep eutectic systems preparation. This is often not reported in conventional manuscripts.
Demonstrating the procedure will be Ana Rita Gameiro, a post-doctoral fellow of my laboratory. To prepare natural DES by freeze-drying, first add two grams of citric acid monohydrate and 0.9530 grams of glucose monohydrate in separate containers. Add 10 milliliters of deionized water to each container and stir until the compounds are completely dissolved.
Mix the two solutions together and ensure the homogenization of the final solution. Place the solution in a round-bottom flask. Freeze the sample using liquid nitrogen.
Place the flask in a freeze-dryer for 48 hours to ensure that all water is removed from the sample. To prepare natural DES by vacuum evaporation, again weigh two grams of citric acid monohydrate and 0.9530 grams of glucose monohydrate in separate containers. Add 10 milliliters of deionized water to each and stir until the compounds are completely dissolved.
Mix the two solutions together and ensure the homogenization of the solution. Place the combined solution in a round-bottom flask. Using a rotary evaporator, dry the sample until a clear, viscous liquid is formed.
The natural DES sample can also be prepared by heating and stirring. To do so, weigh two grams of citric acid monohydrate, 0.9530 grams of glucose monohydrate into separate containers. Place the two solids in the same vial.
Add 278 microliters of water. Place the vial with a magnetic stirring bar in a 50-degrees Celsius water bath. Leave the sample until a clear, viscous liquid is formed.
To characterize natural DES by polarized optical microscopy, place a droplet of natural DES on a microscope glass slide for observation. The natural DES sample can be further characterized by Karl-Fisher titration, differential scanning calorimetry, and nuclear magnetic resonance as described in the text protocol. When using the freeze-drying method to prepare natural DES, the result should be a solid or very dense paste since all the water is removed from the system.
Conversely, the evaporation method should result in a clear and viscous liquid. Using the heating-and-stirring method with the addition of small amounts of water should also result in a clear and very viscous liquid. Polarized optical microscopy images are shown with cross-polarizers.
The natural DES samples were prepared by the heating-and-stirring method, the vacuum-evaporation method, and the freeze-drying method. Polarized optical microscopy images of the natural DES samples are also shown with parallel polarizers. The NMR technique is used to confirm the existence of hydrogen bond formation, which is the main characteristic of natural DES systems.
This can be confirmed by observation of the change and chemical shifts of each signal in the natural DES sample relative to glucose and citric acid alone. Analysis of the NOESY spectra shows spatial and intermolecular correlations, confirming hydrogen bond formation in the natural DES system. When trying to replicate an experiment in which water content is not reported, use the rotary evaporation methodology and determine the water content by Karl-Fisher titration.
When the exact amount of water is known, follow the heating-and-stirring method as this is easier and faster. It is important to always measure the amount of water present in these systems and to report it in manuscripts. This ensures the readers an accurate reproduction of results.
This technique paves the way to a consistent development methodology in the preparation of natural deep eutectic solvents and is not restricted to any particular system. The systems presented here should be seen as examples.
