Two-Dimensional Supercritical Fluid Chromatography System Created with Multiple Heart-Cutting Modes

Scientists from the Université d’Orleans in Orléans, France recently created a two-dimensional supercritical fluid chromatography (SFC) system with multiple heart-cutting (MHC) modes. Their research and findings were published in Analytical Chemistry (1).

How work in laboratory really looks like. chromatography with a column and fraction collection in reagent glasses | Image Credit: © Nicole - stock.adobe.com

Multiple heart-cutting (MHC) is an intermediate mode of two-dimensional liquid chromatography (2D-LC), which enhances the separation power compared to conventional LC, particularly in complex samples (2). In MHC, a predefined number of fractions from a first-dimension chromatogram are subjected to the second dimension. This allows several analytes to be subjected to two different separation mechanisms so that sample purification is achieved while increasing chromatographic resolution between analytes and interfering matrix compounds. Meanwhile, supercritical fluid chromatography (SFC) is similar to gas and liquid chromatography, though this uses a supercritical fluid (a liquid or gas that is used above its critical temperature and pressure) as a mobile phase (3).

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In this study, the scientists developed a loop-based two-dimensional supercritical fluid system in multiple heart-cutting modes (mSFC-SFC), with diode-array and mass spectrometric detection. Based on a single SFC instrument, the device was developed to be as simple as possible, with the only addition being three external 2-port 6-position valves. With this system, they hoped to achieve the most complete transfer of a peak from the first dimension to the second, regardless of the mobile phase’s composition. Due to fine adjustment of valve switching times, the first-dimension peaks were parked in 50 or 100 µL loops before later being discharged to the second dimension.

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To test this and demonstrate interest of this instrument, a sample application was performed on a natural product; namely, a Citrus aurantium L. bark extract was analyzed, with focus being placed on chiral flavonoids, neohesperidin, and naringin. The first dimension in this system was an achiral flavonoid separation, based on a polar diethylamine-bonded silica stationary phase, specifically ACQUITY Torus DEA. Meanwhile, the second dimension used CHIRALPAK IB-3, a stereoselective polysaccharide stationary phase, to resolve flavonoid diastereomers. The system demonstrated excellent repeatability, with relative standard deviation values on retention times and peak areas all below 2%, together with excellent peak capacity and peak shapes (all while no distortion was observed), making it possible to quantify diastereomers in the second-dimension separation. Finally, the scientists demonstrated the system’s ability to yield excellent performance by transferring a compressible sample from one dimension to the next, regardless of the large loop volume involved.

With these findings, this system shows promise for improving our capabilities for 2D-SFC systems. More tests will need to be conducted, but the results of this study do foreshadow a promising future for this system.

References

(1) Réset, L.; De Saint Jores, C.; François, I.; West, C. Development of a Two-Dimensional Supercritical Fluid Chromatography System in Multiple Heart-Cutting Modes. Anal. Chem. 2024, 96 (29), 11969–11976. DOI: 10.1021/acs.analchem.4c01795

(2) Fernánded, A. S.; Rodríguez-González, P.; Álvarez, L.; et al. Multiple Heart-Cutting Two-Dimensional Liquid Chromatography and Isotope Dilution Tandem Mass Spectrometry for the Absolute Quantification of Proteins in Human Serum. Anal. Chim. Acta 2021, 1184, 339022. DOI: CVVV FFVFV10.1016/j.aca.2021.339022

(3) Supercritical Fluid Chromatography. Elsevier B.V., 2024. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/supercritical-fluid-chromatography (accessed 2024-09-09)