RESUMO
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.
RESUMO
When starting a method development in supercritical fluid chromatography (SFC), the first step is usually to screen several stationary phases based on previous experience or simply based on what is available in the laboratory. However, as there are now a large number of stationary phases available for SFC, the choice of an adequate set of columns to rapidly achieve a satisfying result can be difficult. In this project, 16 columns comprising a wide diversity of stationary phases and polarities ranging from the most polar (like bare silica gel) to the least polar (like octadecylbonded-silica) were compared, based on the gradient analysis of 129 probe compounds. The set mostly comprised active pharmaceutical ingredients, natural products and a few metabolites. The columns were ranked with the help of Derringer desirability functions taking account of (i) the number of compounds eluted from the column, (ii) the elution time in a suitable time frame, (iii) the average peak width, (iv) the average peak symmetry and (v) the spreading of retention along the gradient time. The five criteria selected showed no correlation. Overall, it appeared that those columns that had a high overall score were good for several reasons, like bare silica gel, propanediol-bonded silica or pentabromobenzyloxy-bonded silica. Initially, the columns had been screened with a gradient elution starting from 5% co-solvent and ending with 50% co-solvent in CO2. However, for some most retentive columns like amide-bonded silica, too many compounds remained non-eluted from the column. To examine this column more fairly, a second elution gradient was applied that ended with 100% co-solvent. This proved effective in restoring good overall performance through the elution of the most polar compounds.
