Development of a Two-Dimensional Supercritical Fluid Chromatography System in Multiple Heart-Cutting Modes.

In this study, we present the development of a loop-based two-dimensional supercritical fluid system in multiple heart-cutting modes (mSFC-SFC), with diode-array and mass spectrometric detection. The instrument design was developed to be as simple as possible, based on a single SFC instrument, with the sole addition of three external 2-port 6-position valves. The objective was to achieve the most complete transfer of a peak from the first to the second dimension, whatever the composition of the mobile phase, i.e., whatever the proportion of carbon dioxide and methanol cosolvent along a wide gradient elution. Thanks to fine adjustment of the valve switching times, the first-dimension peaks were parked in 50 μL or 100 μL loops and later discharged to the second dimension. The interest of this instrument was then demonstrated with a sample application on a natural product: an extract of Citrus aurantium L. bark was analyzed, with a particular focus on chiral flavonoids, neohesperidin, and naringin. In this system, the first dimension was an achiral separation of the flavonoids, based on a polar diethylamine-bonded silica stationary phase (ACQUITY Torus DEA), while the second dimension used a stereoselective polysaccharide stationary phase (CHIRALPAK IB-3) to resolve flavonoid diastereomers. Excellent repeatability was demonstrated, with relative standard deviation values on retention times and peak areas all below 2%, together with excellent peak capacity and peak shapes (no distortion observed), making it possible to quantify diastereomers in the second-dimension separation. This good repeatability was also shown for the transfer rate between the two dimensions, which reached a value of 83%. Finally, transferring a compressible sample from the first to the second dimension is demonstrated to yield excellent performance, despite the large loop volume.