The Quest for Greener Sample Preparation in Food Analysis

When analytical chemists apply green chemistry approaches, which seek to minimize negative environmental effects, an important area of focus is reducing the consumption of toxic solvents, such as those used in extraction steps in sample preparation. Developing and testing greener extraction processes for food analysis is a major focus of Elena Ibáñez, a research professor at the Institute of Food Science Research (CIAL-CSIC) in Madrid, Spain. She recently spoke to us about this work.

Your group has been involved in developing environmentally friendly approaches for various extraction processes, one of which was the extraction of natural bioactive compounds (phlorotannins) from marine algae (1). What extraction techniques were examined in this study? Which one turned out to be the best choice for this research, and why?

In our research group, we have been working for many years on the use of compressed fluids as an alternative to conventional extraction solvents; therefore, extraction processes such as pressurized liquid extraction (PLE), subcritical water extraction, gas-expanded liquids extraction, and supercritical fluid extraction (SFE) are commonly the techniques we test. The final selection always depends on the target compound or compounds and the matrix. In this particular case, pressurized ethanol at 100 °C was the best choice, although equivalent results could be obtained using other green solvents such as ethyl lactate under pressurized conditions and using gas-expanded liquids formed by mixing ethanol and carbon dioxide (50:50).

You looked at both subcritical and supercritical fluids as extraction solvents. How did you determine the best green solvent or solvent combination to use in this extraction process?

In this work, we employed predictive tools such as Hansen solubility parameters (HSP), which can be considered a good starting point for the initial selection of the solvents. Using this information, we carried out the experiments also considering the effect of temperature, which is not easy to predict using HSP.

What was the final analytical separation method used to characterize the phlorotannins? Was that method a factor in your choice of extraction solvents?

In this study, and considering the complexity of the target analytes (phlorotannins from brown algae), we decided to use comprehensive two-dimensional liquid chromatography coupled to mass spectrometry (LC×LC–MS/MS). These biopolymers are very difficult to separate because of their complexity, diversity of structures, and possible linkages among phloroglucinol units; as the number of phloroglucinol units becomes bigger, the complexity increases. Only by using this technique were we able to separate and tentatively identify 29 phlorotannins with a degree of polymerization (DP) from 3 to 14 phloroglucinol units. Of course, the solvents chosen must be compatible with the separation technique we are using and, therefore, that compatibility is one of the aspects we consider for solvent optimization.

Another study compared four green extraction processes to evaluate their ability to obtain rosemary extracts with in vitro anticancer activity (2). What were the four processes, and why were they selected for this study?

I always say that in our research group, and because of my background as a chemical engineer, we easily move from big to small and vice versa; we also move easily between chemical engineering and analytical chemistry, and between sample preparation techniques and process design at the pilot scale. Therefore, we develop systems, processes, and methodologies following the "Green Chemistry" rules and always consider ideas about sustainability, environmental impact, green solvents, selectivity, efficiency, and so on that can (and should) be applied in all processes and all scales. In this work, we focused on enriching rosemary extracts in the two main bioactive compounds (carnosic acid and carnosol) against colon cancer. With this idea in mind, we developed several processes aimed at increasing the concentration of these compounds in the extract (which is better than purifying the extract using more costly and environmentally aggressive techniques). To do so, we ran the experiments at small scale using four different techniques: single-step supercritical fluid extraction, two-step supercritical fluid extraction, pressurized liquid extraction, and pressurized liquid extraction plus supercritical antisolvent fractionation. The two-step SFE process and the pressurized liquid extraction plus supercritical antisolvent fractionation process were specifically designed for this purpose and therefore were the ones that provided the best results. In this work, we optimized the processes at small scale and then transferred them to the pilot scale.

What results did you obtain in terms of extraction efficiency for the various processes?

As I mentioned earlier, two processes provided the best concentration of target compounds in the extract (more than 45% of carnosic acid plus carnosol) and gave the best antiproliferative results against colon cancer cell lines (reducing viability to 16%). In terms of extraction efficiency, several aspects should be considered, such as extraction yields, working conditions, and cost. The process that used PLE and supercritical antisolvent fractionation at large scale provided the best results and can be considered a suitable process for enrichment of target bioactives from complex mixtures.

Your group used Hansen solubility parameters in selecting bio-based solvents for the selective extraction of the bioactive compound fucoxanthin from a marine diatom microalga (3). Can you please briefly describe the Hansen solubility parameter concept and how it was used in your study?

HSPs are based on the solubility theory approach and on the principle "like dissolves like." To improve the applicability of the total solubility parameter, Hansen divided the Hildebrand parameter into three-dimensional components that quantify, individually, the contributions of the forces of dispersion, polarity, and hydrogen bonding interactions. This approach can be very advantageous as a predictive tool, since it can give a first approximation in extraction processes, indicating the most suitable solvent for a given application, thus reducing or avoiding the selection of impractical experimental conditions. One of the key aspects in this approach is to get the HSP of the target molecule or molecules; this can be obtained from databases or employing the group contribution method (which makes it possible to predict properties and solubility parameters of molecular structures, using additive rules). Once we know the HSP of the molecule, the solvent with the closest HSP will be selected, because it will better dissolve the target analyte.

In a review of the properties and applications of gas-expanded liquids and switchable solvents, you discussed their use in chemical and technological processes (4). What are gas-expanded liquids and switchable solvents?

As defined in the mentioned article, a switchable solvent is a solvent that can be reversibly converted from one form to another differing in one or more physicochemical properties (high dielectric constant–low dielectric constant, hydrophobic–hydrophilic, high ionic strength–low ionic strength) upon application or removal of a trigger. The originality of our contribution in that paper was considering that gas-expanded liquids are similar to switchable solvents, because both use carbon dioxide as a trigger agent (gas-expanded liquids at medium-high pressure and switchable solvents at low pressure).

What are some of their advantages for use as green solvents in chemical processes?

In our research group, we have developed some of the first applications of gas-expanded liquids as green solvents in extraction processes. In gas-expanded liquids, by varying the CO₂ composition, a continuum of liquid media ranging from neat organic solvent to supercritical CO₂ is generated, and their properties are adjustable by tuning the operating pressure. Some of the advantages are the enhanced transport rates; the ability to use lower working pressures (compared to supercritical fluids) and the subsequent reduction in energy consumption and costs; the improved mass transfer by decreasing interfacial tension, reducing viscosity, and increasing diffusivity; and the important replacement of organic solvents with environmentally benign dense-phase CO₂.

Foodomics, the use of -omics technologies in food science, is an important application area for green chemistry, which includes the implementation of alternative solvents and green approaches in metabolomics and proteomics (5). What are the main ways in which green chemistry has been applied in foodomics?

Foodomics is a green discipline that tries to give new answers to important societal challenges (such as sustainability, food safety and quality, the rational design and development of new foods able to improve our health, and so on). One of the approaches toward greener foodomics is the use of green solvents and integrated processes that produce less waste and require less energy consumption; these approaches can be applied to the development of extraction processes to produce functional food ingredients and to the design of greener analytical methods to undertake challenging issues related to food quality, safety, and traceability. The use of miniaturized sample preparation techniques or greener solvents and the development of new alternatives for greener separation techniques are proposed in green foodomics. Moreover, as you mentioned, green foodomics can also influence other -omics technologies by reducing sample preparation steps and solvent consumption, while maintaining or improving data reliability.

Which sample preparation and analytical techniques are most compatible with the application of green chemistry principles in food science, and why?

In green analytical chemistry, we can consider analytical methods as processes in which preliminary information and knowledge, solvents, reagents, samples, energy, and instrument measurements are used as inputs to solve a specific problem. The outputs of those processes are qualitative or quantitative composition of the analytes, or both. But we have to consider that analytical methodologies can also have side effects (such as energy consumption, and wastes that can pose risks for operators and damage the environment, and so on). The key points that sample preparation and analytical techniques should consider for adherence to green chemistry principles are the reduction of the amount and toxicity of solvents in the sample pretreatment step; the reduction of the amount and toxicity of solvents in the measurement step, especially by miniaturization; and the development of alternative direct analytical methodologies not requiring solvents or reagents.

What avenues of green chemistry research do you plan to explore in the near future?

In the near future, I would like to keep exploring new solvents (such as the new deep eutectic solvents, DES) and their possible use under pressurized conditions or in combined processes for extraction and fractionation of bioactives. Moreover, I expect to go more in depth in learning and applying new tools for modeling solvents and processes that can make working in the green chemistry framework easier.

References

(1) A.P. Sánchez-Camargo, L. Montero, A. Cifuentes, M. Herrero, and E. Ibañez, RSC Adv. 6, 94884–94895 (2016). doi: 10.1039/c6ra16862k

(2) A.P. Sánchez-Camargo, V. García-Cañas, M. Herrero, A. Cifuentes, and E. Ibáñez, Int. J. Mol. Sci. 17, 2046 (2016). doi: 10.3390/ijms17122046

(3) A.P. Sánchez-Camargo, N. Pleite, M. Herrero, A. Cifuentes, E. Ibáñez, B. Gilbert-López, J. Supercrit. Fluids 128, 112–120 (2017). doi: 10.1016/j.supflu.2017.05.016

(4) M. Herrero, J.A. Mendiola, and E. Ibáñez, Current Opinion in Green and Sustainable Chemistry 5, 24–30 (2017). doi: 10.1016/j.cogsc.2017.03.008

(5) B. Gilbert-López, J.A. Mendiola, and E. Ibáñez, Trends Anal. Chem. 96, 31–41 (2017). doi: 10.1016/j.trac.2017.06.013

Elena Ibanez

Elena Ibañez is a full research professor at the Institute of Food Science Research (CIAL), which is part of the Spanish National Research Council (CSIC) in Madrid, Spain. She received her PhD in analytical chemistry from the Autonomous University of Madrid (UAM), Spain, and carried out postdoctoral training at Brigham Young University and the University of California at Davis. Her main research focus is the study and development of new green extraction processes based on the use of compressed fluids to isolate bioactive compounds from natural sources and their characterization using advanced analytical techniques. She has co-authored more than 200 publications, 25 book chapters, and 10 patents. Direct correspondence about this article to elena.ibanez@csic.es.