Periodic Reporting for period 1 - GREENMULTIDIMCO2 (Towards greener and more sustainable analytical approaches: Development of an integrative supercritical CO2-based multidimensional system for bioactive compound study in natural and biological samples)
Período documentado: 2025-01-13 hasta 2027-01-12
Natural and biological samples, such as plants, foods, and biofluids, contain thousands of compounds with diverse chemical properties. Many of these compounds are bioactive, which means they can influence biological functions and be relevant not only to health-related questions, but also to new industrial developments in fields like medicine, cosmetics, and renewable materials.
Due to their chemical complexity, characterizing these samples is challenging and often requires several analytical steps, including sample preparation, extraction, and separation. These workflows not only take a lot of time and resources, but they also use toxic solvents, which are harmful to the environment and human health.
The goal of this project was to develop a more sustainable analytical approach using carbon dioxide-based supercritical fluid technologies and advanced coupling techniques, to integrate both sample preparation and analysis steps into a single, automated workflow. For this, we aimed to develop an online multidimensional system combining supercritical fluid extraction and two-dimensional supercritical fluid chromatography for extensive bioactive compound characterization in a single run.
To achieve this goal, the project focused on the main bottleneck currently limiting integrated CO2-based workflows: interface compatibility.
The work was organized on three main developments: (i) designing multi-step extraction and separation strategies for analyzing polar and non-polar compounds from the same sample, (ii) developing new ways to transfer extracts and fractions without degrading separation performance, and (iii) demonstrating the approach on real-world applications, such as natural products for nutraceutical and traditional medicine use, and lipid-focused biological analyses.
By advancing online multidimensional CO2-based workflows, the project aimed to create more sustainable analytical tools. These tools are expected to help research and innovation by providing detailed compound analysis in fields like food safety, cosmetic formulation, pharmaceutical development, and environmental monitoring, among others.
Due to their chemical complexity, characterizing these samples is challenging and often requires several analytical steps, including sample preparation, extraction, and separation. These workflows not only take a lot of time and resources, but they also use toxic solvents, which are harmful to the environment and human health.
The goal of this project was to develop a more sustainable analytical approach using carbon dioxide-based supercritical fluid technologies and advanced coupling techniques, to integrate both sample preparation and analysis steps into a single, automated workflow. For this, we aimed to develop an online multidimensional system combining supercritical fluid extraction and two-dimensional supercritical fluid chromatography for extensive bioactive compound characterization in a single run.
To achieve this goal, the project focused on the main bottleneck currently limiting integrated CO2-based workflows: interface compatibility.
The work was organized on three main developments: (i) designing multi-step extraction and separation strategies for analyzing polar and non-polar compounds from the same sample, (ii) developing new ways to transfer extracts and fractions without degrading separation performance, and (iii) demonstrating the approach on real-world applications, such as natural products for nutraceutical and traditional medicine use, and lipid-focused biological analyses.
By advancing online multidimensional CO2-based workflows, the project aimed to create more sustainable analytical tools. These tools are expected to help research and innovation by providing detailed compound analysis in fields like food safety, cosmetic formulation, pharmaceutical development, and environmental monitoring, among others.
To achieve the above-mentioned goal, the project was divided into five work packages (WP), to be completed within three years. During the reporting period, the work focused on WP1 and WP2a, using a synthetic mixture of target compounds with diverse polarities for method development and bamboo as a representative natural sample.
The first objective was to set-up an SFC-MS method robust enough to monitor extraction performance. First, the MS ionization conditions were optimized on a triple-quadrupole instrument. This brought to light compound-dependent ionization and solubility effects associated with modifier composition and residence time in the SFC system, which guided later method decisions. Then, the chromatographic separation was optimized. Several stationary phases, co-solvent, and additives compositions were tested. The results showed that the best selectivity and peak capacity was achieved with a diol-bonded stationary phase and that ammonium acetate outperformed other additives for peak quality under the tested conditions. However, some compounds still suffered from poor peak shapes under the tested conditions. To address this issue and further improve peak shapes, a design-of-experiments (DoE) study was conducted on back-pressure and column temperature. The study highlighted that better peak quality could be achieved with a combination of high-pressure and low temperature, with an optimum around 160 bar and 30 °C. To support the green chemistry objective of the project, Methanol was systematically replaced by Ethanol throughout the workflow. The two solvents were also compared in terms of chromatographic performance and detection sensitivity, which showed that Ethanol is in fact a competitive option.
The second objective was to develop an innovative online CO2-based extraction method for the simultaneous extraction of a broad range of compound polarities. One of the major issues encountered, because compounds with very different solubilities were targeted, was contamination and carry-over. To address this, different sample-loading and system-cleaning procedures were evaluated and adapted, which was critical given the wide polarity range of the compounds. Then, a second DoE study explored the effects of extraction back-pressure and cell temperature on compound recovery. This allowed to identify the most favourable conditions within the tested space (i.e. 150 bar, 40 °C in our initial screening). The influence of modifier percentage (0–30%) on extraction efficiency was also investigated. Finally, a first feasibility test was applied on ground bamboo leaves to test the developed approach.
These results provided a working SFC-MS method and a first set of extraction conditions, which formed a basis for subsequent online SFE-SFC transfer optimization.
The first objective was to set-up an SFC-MS method robust enough to monitor extraction performance. First, the MS ionization conditions were optimized on a triple-quadrupole instrument. This brought to light compound-dependent ionization and solubility effects associated with modifier composition and residence time in the SFC system, which guided later method decisions. Then, the chromatographic separation was optimized. Several stationary phases, co-solvent, and additives compositions were tested. The results showed that the best selectivity and peak capacity was achieved with a diol-bonded stationary phase and that ammonium acetate outperformed other additives for peak quality under the tested conditions. However, some compounds still suffered from poor peak shapes under the tested conditions. To address this issue and further improve peak shapes, a design-of-experiments (DoE) study was conducted on back-pressure and column temperature. The study highlighted that better peak quality could be achieved with a combination of high-pressure and low temperature, with an optimum around 160 bar and 30 °C. To support the green chemistry objective of the project, Methanol was systematically replaced by Ethanol throughout the workflow. The two solvents were also compared in terms of chromatographic performance and detection sensitivity, which showed that Ethanol is in fact a competitive option.
The second objective was to develop an innovative online CO2-based extraction method for the simultaneous extraction of a broad range of compound polarities. One of the major issues encountered, because compounds with very different solubilities were targeted, was contamination and carry-over. To address this, different sample-loading and system-cleaning procedures were evaluated and adapted, which was critical given the wide polarity range of the compounds. Then, a second DoE study explored the effects of extraction back-pressure and cell temperature on compound recovery. This allowed to identify the most favourable conditions within the tested space (i.e. 150 bar, 40 °C in our initial screening). The influence of modifier percentage (0–30%) on extraction efficiency was also investigated. Finally, a first feasibility test was applied on ground bamboo leaves to test the developed approach.
These results provided a working SFC-MS method and a first set of extraction conditions, which formed a basis for subsequent online SFE-SFC transfer optimization.
CO2-based analytical methods are attractive for greener analysis, but fully integrated online multidimensional systems remain complex to implement and often require in-house adaptations, as no commercial instruments are currently available. Integrating multiple techniques such as SFE and SFC or SFC and SFC, requires special interfaces that can collect and transfer effluents at the junction between each technique. Connecting two techniques is already a challenge, but the complexity increases further as more techniques are coupled. While such an approach can save time and support an integrated “all-in-one” workflow, it also requires expertise in each technique as well as in coupling technologies. Without doubt, the most challenging part is thus interfacing. These methods are indeed very sensitive to solvent composition, transfer conditions, and injection effects. This can rapidly become problematic when targeting compounds with a broad polarity range, since their analysis require different solvent compositions. This makes it difficult to combine extraction and separation, or even two separation techniques, in one workflow, without sacrificing separation quality. Within the reporting period, the project produced useful results that go beyond a simple trial-and-error approach and help prepare the next stages of development.
First, a reliable SFC-MS method was developed for separating and identifying compounds with a wide polarity range. A major outcome was the detailed optimization of both MS ionization and chromatographic conditions, which helped identify practical pitfalls and key method-development rules for SFC-MS analysis of complex mixtures with a broad polarity range. In particular, the work showed that co-solvent composition and residence time during MRM optimization can strongly affect not only signal intensity but also peak shape, highlighting the importance of using conditions close to real SFC elution conditions.
An important result from the green chemistry side of the project was that ethanol, used as an alternative to methanol, gave comparable SFC-MS performance to methanol. Although methanol remained slightly better overall under the tested conditions, the differences were rather limited and compound-dependent, supporting ethanol as a realistic option in SFC-MS.
Preliminary offline SFE tests, including a first feasibility test on a real bamboo sample, gave a good starting point for the next stages of the project. Additional work is still needed to build a final method, especially because only part of the compound set was optimized at this stage, but this work already highlighted important practical issues for broad-polarity applications, especially contamination/carry-over and the need for appropriate pre- and post-cleaning procedures. Taken together, these results do not yet correspond to the final integrated online SFE-SFCxSFC-MS platform initially planned, but they provide important technical knowledge and a solid basis for future development. In particular, they reduce uncertainty for the next steps, especially for transfer optimization, interface design, and preserving separation quality during coupling. Further progress will require additional work on SFE method development, online transfer/interface optimization, and feasibility testing on the final integrated setup. Wider use of this approach would also benefit from comparison with more conventional step-by-step workflows to better evaluate its performance and added value.
First, a reliable SFC-MS method was developed for separating and identifying compounds with a wide polarity range. A major outcome was the detailed optimization of both MS ionization and chromatographic conditions, which helped identify practical pitfalls and key method-development rules for SFC-MS analysis of complex mixtures with a broad polarity range. In particular, the work showed that co-solvent composition and residence time during MRM optimization can strongly affect not only signal intensity but also peak shape, highlighting the importance of using conditions close to real SFC elution conditions.
An important result from the green chemistry side of the project was that ethanol, used as an alternative to methanol, gave comparable SFC-MS performance to methanol. Although methanol remained slightly better overall under the tested conditions, the differences were rather limited and compound-dependent, supporting ethanol as a realistic option in SFC-MS.
Preliminary offline SFE tests, including a first feasibility test on a real bamboo sample, gave a good starting point for the next stages of the project. Additional work is still needed to build a final method, especially because only part of the compound set was optimized at this stage, but this work already highlighted important practical issues for broad-polarity applications, especially contamination/carry-over and the need for appropriate pre- and post-cleaning procedures. Taken together, these results do not yet correspond to the final integrated online SFE-SFCxSFC-MS platform initially planned, but they provide important technical knowledge and a solid basis for future development. In particular, they reduce uncertainty for the next steps, especially for transfer optimization, interface design, and preserving separation quality during coupling. Further progress will require additional work on SFE method development, online transfer/interface optimization, and feasibility testing on the final integrated setup. Wider use of this approach would also benefit from comparison with more conventional step-by-step workflows to better evaluate its performance and added value.