Enhanced selectivity and sensitivity of sequential injection chromatography: determination of bioflavonoids in...

The gradual development of sequential injection chromatography (SIC) has aimed to increase separation selectivity and sensitivity. In this work, an HPLC column, a sub-2 μL flow cell detector, and advanced processing of UV-vis spectral data were used to determine the concentration of closely related analytes, such as diosmin and hesperidin, in nutraceuticals. The improvements resulted in narrower peaks, higher resolution, and more raw data acquired for effective noise reduction with the Savitzky-Golay filter. This was followed by an automated calculation of peak characteristics, which facilitated easy data evaluation during the method development and sample analysis. The developed SIC used a standard 100 × 4.6 mm, 5 μm multilayer particle-packed column and isocratic separation with a mixture of acetonitrile and aqueous 0.01 mol L^-1 phosphoric acid (pH 2.0) 25:75 (v/v) as the mobile phase and spectrophotometric detection in the range of 200-350 nm with a resolution of 1 nm. The analysis of a 5 μL sample resulted in LODs of 0.6 mg L^-1, LOQs of 2.0 mg L^-1, linearity of 2.0-250.0 mg L^-1 (r > 0.999) for diosmin and of 2.0-150.0 mg L^-1 (r > 0.999) for hesperidin and rutin, and a good repeatability of the peak area at 10 mg L^-1 (RSD ≤2.31 %). The performance of the chromatographic process was highlighted and well documented by HETP values ≤ 25 μm, peak symmetry ≤1.26, resolution ≥2.21, and time for one analysis 8.0 min. The study proved the updates in the capability and applicability of SIC, which aid in solving advanced chromatographic tasks.