Fast separation of flavonoids by supercritical fluid chromatography using a column packed with a sub-2 μm particle stationary phase

Abstract

The purpose of this study was to compare the effects of different chromatographic columns for the separation of seven flavonoids. Four different stationary phases are available, including bridged ethyl hybrid, BEH and the same hybrid phase modified with 2-ethylpyridine, CSH fluorophenyl, and HSS C SB. The analytes included calycosin, genistein, medicarpin, calycosin-7-O-β-d-glucoside, formononetin, formononetin-7-O-β-d-glucoside, and liquiritigenin. The CSH fluorophenyl column was determined to be the most suitable and provided the fastest separation within 17 min using gradient elution with carbon dioxide as the mobile phase and methanol as the co-solvent. Good peak shapes were obtained, and the values of the peak asymmetry were close to 1.0 for all of the flavonoids. The resolution was more than 1.41 for all of the separated peaks. Baseline separation on the optimal columns was achieved by changing the co-solvent type and adjusting the temperature and pressure. Quantitative performance was evaluated under optimized conditions, and method validation was accomplished. The validation parameters, such as linearity, sensitivity, precision, and accuracy, were satisfactory. Good repeatability of both peak area (relative standard deviation <1.02%) and retention time (relative standard deviation <0.88%) was observed. The optimized chromatographic methods were successfully used for the determination of seven flavonoids in Radix astragali. The sensitivity was sufficient for the analysis of real samples.

Towards eco‐friendly secondary plant metabolite quantitation: Ultra high performance supercritical fluid chromatography applied to common vervain (Verbena officinalis L.)

“…In SFC, a decrease of the temperature leads typically to an increase of the mobile phase density and thus to faster elution of the compounds. However, in subcritical conditions with high modifier concentrations, prediction of temperature influence is not that simple and, as reported by different studies, the influence of temperature on the retention time can change during the gradient, related to fluid compressibility changes [20,[35][36][37]. As shown in Figure 2, this trend was observed also in the presented study.…”

Sequential design of experiments approach for the multiproduct analysis of cholesterol‐lowering drugs by ultra‐high‐performance supercritical fluid chromatography

“…To model the retention and separation behavior, a CCD was employed to evaluate the pressure, temperature, and amount of modifier. These factors are known to influence the elution strength of CO 2 ‐based MPs [25–27]. Supporting Information Figure S4 presents the chromatograms according to the conditions determined by the CCD.…”

“…Within these ranges, and for temperatures between 25 and 50°C, the retention factors decreased as the pressure and percentage of the modifier increased for all analytes (therefore these factors have negative coefficients in the model) and increased with temperature (temperature has a positive coefficient). This behavior is expected because in a supercritical fluid, the density increases proportionally with an increase in pressure and a temperature decrease, thereby shifting to a more liquid‐like state or subcritical condition [26,27]. Usually, the higher the density, the more soluble the analytes become.…”

Ultra‐performance convergence chromatography method for the determination of four chromones and quality control of Saposhnikovia divaricata (Turcz.) Schischk.

“…We then examined the effect of column temperature between 35 and 55°C to optimize the separation and stability of the stationary phase. As shown in Figure C, the resolutions of the 4GMV and SGH signals decreased as the temperature increased, which corresponds with previously reported results . Thus, based on the various resolutions and retention times observed over this temperature range, 35°C was selected as the optimal temperature for subsequent experiments.…”