Advantages of core–shell particle columns in Sequential Injection Chromatography for determination of phenolic acids

Abstract

Currently, for Sequential Injection Chromatography (SIC), only reversed phase C18 columns have been used for chromatographic separations. This article presents the first use of three different stationary phases: three core–shell particle-packed reversed phase columns in flow systems. The aim of this work was to extend the chromatographic capabilities of the SIC system. Despite the particle-packed columns reaching system pressures of ≤610PSI, their conditions matched those of a commercially produced and optimised SIC system (SIChrom™ (FIAlab®, USA)) with a 8-port high-pressure selection valve and medium-pressure Sapphire™ syringe pump with a 4mL reservoir and maximum system pressure of ≤1000PSI. The selectivity of each of the tested columns, Ascentis® Express RP-Amide, Ascentis® Express Phenyl-Hexyl and Ascentis® Express C18 (30mm×4.6mm, core–shell particle size 2.7μm), was compared by their ability to separate seven phenolic acids that are secondary metabolite substances widely distributed in plants. The separations of all of the components were performed by isocratic elution using binary mobile phases composed of acetonitrile and 0.065% phosphoric acid at pH 2.4 (a specific ratio was used for each column) at a flow-rate of 0.60mL/min. The volume of the mobile phase was 3.8mL for each separation. The injection volume of the sample was 10μL for each separation. The UV detection wavelengths were set to 250, 280 and 325nm. The RP-Amide column provided the highest chromatographic resolution and allowed for complete baseline separation of protocatechuic, syringic, vanillic, ferulic, sinapinic, p-coumaric and o-coumaric acids. The Phenyl–Hexyl and C18 columns were unable to completely separate the tested mixture, syringic and vanillic acid and ferulic and sinapinic acids could not be separated from one another. The analytical parameters were a LOD of 0.3mgL−1, a LOQ of 1.0mgL−1, a calibration range of 1.0–50.0 (100.0)mgL−1 (r>0.997) and a system precision of 10mgL−1 with a RSD ≤1.65%. The high performance of the chromatography process with the RP-Amide column under optimised conditions was highlighted and well documented (HETP values ≤10μm, peak symmetry ≤1.33, resolution ≥1.87 and time for one analysis <8.0min). The results of these experiments confirmed the benefits of extending chromatographic selectivity using core–shell particle column technology in a SIC manifold.