Ultrafast micromixer for on-line screening of antioxidants

Abstract

Background

The integration of high-performance liquid chromatography (HPLC) with rapid post-column biochemical detection techniques has gained widespread application in the pinpoint of antioxidant components in complex natural matrices. However, the use of PEEK reaction coils in on-line antioxidant analysis often encounters issues with poor mixing of the reaction solution, leading to peak broadening and a decrease in peak capacity. This may adversely affect the sensitivity and precision of the analysis. Microfluidic technology provides a unique opportunity for rapid solution mixing. Despite many continuous flow-based micromixers being reported, developing an ultrafast micromixer remains challenging.

Results

Here, we demonstrated a new microfluidic mixer and used it to create a highly sensitive on-line HPLC-ABTS system for antioxidant screening, replacing the PEEK reaction coil. The mixer utilizes the engulfment flow to achieve complete mixing of two solutions within sub-microseconds (∼0.6 µs). The system demonstrated consistent half-peak widths under 0.63 min for five phenolic compounds, with a 0.1 min delay time. Optimization of the HPLC-ABTS assay included determining the ideal ABTS^•+ concentration and flow rate ratio of ABTS^•+ solution to post-column mobile phase. Validation showed that the system exhibited low detection limits (0.3–1.4 μg/mL), superior linearity (r > 0.9900), acceptable recovery (92.33–109.33 %), high stability (RSD < 4.96 %), and excellent reproducibility (RSD < 2.3 %). These observations highlight the advantages of employing the micromixer in on-line analysis.

Significance

Peak broadening reduces the selectivity and accuracy of on-line HPLC assays for detecting antioxidants in complex matrices. Compared to conventional on-line assays utilizing a PEEK coil for radical scavenging, our newly developed system demonstrates enhanced selectivity and precision. This on-line system can be combined with mass spectrometry or on-line extraction devices, which presents a promising alternative for the rapid screening of antioxidant compounds in plant products.