Improvement of an Automated Sample Injection System for Pillar Array Columns to Increase Analytical Reproducibility

Abstract

In our previous study, we developed an automatic sample injection system for pillar array columns for quantitative analysis. An autosampler was used to maintain a constant sample injection volume. However, the sample was diluted during injection using the autosampler, thus deteriorating the analytical reproducibility. In this study, we have substituted the autosampler with a syringe pump to overcome the abovementioned problem and improve the system. Sample dilution was avoided by filling the entire capillary with the sample at a constant rate. This improved system also increased the analytical reproducibility. In the previous system, the relative standard deviation (RSD) exceeded 17% of the peak height for coumarin dyes. In contrast, the improved system decreased the RSD to the range 1.2–1.8%. The analytical reproducibility was evaluated by using five types of amino acids. The RSD of each peak height was within 3.0%, confirming good reproducibility. These results indicate that the sample injection method developed in this study can be applied to biological sample analyses as a simple quantitative analysis method for pillar array columns.

Pillar Array Mixer for Postcolumn Derivatization Integrated into Liquid Chromatography-Based Microfluidic Device

“…While the fluorescein peak broadened by 1.8 times upon passing through the hollow-channel mixer, the peak width hardly changed when using the 5- and 10-μm PA mixers (Figure B). This result confirms the capability of the PA structure to decrease longitudinal dispersion compared to a hollow-channel structure, as previously demonstrated for LC unit with PA structures. − Although the peak width detected at the rear end of the 10-μm PA mixer appeared narrower than expected from the CFD analysis (Figure B), this can be explained by differences in the peak detection conditions. In the CFD analysis, the peak detection was conducted in areas without pillars, resulting in similar linear velocities at the detection points for the 5- and 10-μm PA mixers.…”

“…To achieve these contradictory mixing properties, a PA structure was considered promising. Perfectly ordered PA structures have been used in microfluidic LC units because they reduce eddy diffusion compared to traditional LC column supports. − This, in turn, leads to lower dispersion of the analytes in the flow direction. Additionally, compared to hollow-channel mixers, in which Taylor–Aris dispersion is the primary driver of transverse mixing, PA structures are expected to enhance mixing through additional convection phenomena.…”

“…The microfluidic devices were fabricated following previously reported procedure, with some modifications. − The devices comprised an injection unit, an LC unit (PA column) with a low dispersion turn (Table S1), and a postcolumn derivatization unit (hollow-channel, 5-μm PA, or 10-μm PA mixers). The devices were fabricated on 20 × 20 mm silicon chips via multistep ultraviolet photolithography (MA-6, SUSS MicroTec, Germany) and deep reactive ion etching (RIE-400iPH, Samco Inc., Kyoto, Japan) with anodic bonding (Techno System, Kanagawa, Japan) to a glass plate.…”

“…On-chip injection of the samples was conducted by switching the four-port valve. The inlets of the microfluidic devices were connected to the capillaries of the pump and valve using a custom-made setup, as described previously. − Fluorescence images of the microfluidic devices were obtained using an IX70 inverted fluorescence microscope (Olympus) and an electron-multiplying charge-coupled device (EMCCD) camera, iXon3 (Andor Technologies, South Windsor, CT, USA).…”

“…However, compared to DNA/RNA and protein analyses, which employ complementary strands or antibodies for selective analysis, metabolite analyses via μTAS necessitate a separation step owing to the difficulty in detecting target analytes among similar molecules with high specificity. Therefore, separation-based microfluidic devices have been developed for the analysis of metabolites by LC, − gas chromatography, or capillary electrophoresis. , Additional steps such as sample enrichment, eluent mixing, and detection have also been developed and integrated into separation-based devices. , …”