A gradient elution system for pressure-driven liquid chromatography (LC) on a chip was developed for carrying out faster and more efficient chemical analyses. Through computational fluid dynamics simulations and an experimental study, we found that the use of a cross-Tesla structure with a 3 mm mixing length was effective for mixing two liquids. A gradient elution system using a cross-Tesla mixer was fabricated on a 20 mm × 20 mm silicon chip with a separation channel of pillar array columns and a sample injection channel. A mixed solution of water and fluorescein in methanol was delivered to the separation channel 7 s after the gradient program had been started. Then, the fluorescence intensity increased gradually with the increasing ratio of fluorescein, which showed that the gradient elution worked well. Under the gradient elution condition, the retention times of two coumarin dyes decreased with the gradient time. When the gradient time was 30 s, the analysis could be completed in 30 s, which was only half the time required compared to that required for an isocratic elution. Fluorescent derivatives of aliphatic amines were successfully separated within 110 s. The results show that the proposed system is promising for the analyses of complex biological samples.
In this study, a fast and quantitative determination method for branched-chain amino acids (BCAAs), namely leucine, isoleucine, and valine, was developed using a pillar array column. A pillar array column with low-dispersion turns was fabricated on a 20 × 20-mm(2) microchip using multistep ultraviolet photolithography and deep reactive ion etching. The BCAAs were fluorescently labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), followed by reversed-phase separation on the pillar array column. The NBD derivatives of the three BCAAs and an internal standard (6-aminocaproic acid) were separated in 100 s. The calibration curves for the NBD-BCAAs had good linearity in the range of 0.4-20 μM, using an internal standard. The intra- and interday precisions were found to be in the ranges of 1.42-3.80 and 2.74-6.97%, respectively. The accuracies for the NBD-BCAA were from 90.2 to 99.1%. The method was used for the analysis of sports drink and human plasma samples. The concentrations of BCAAs determined by the developed method showed good agreements with those determined using a conventional high-performance liquid chromatography system. As BCAAs are important biomarkers of some diseases, these results showed that the developed method could be a potential diagnostic tool in clinical research.
To realize efficient, fast separations on pillar array columns with turns, a novel turn with low-dispersion and low-pressure-drop properties was developed. This "pillar-distribution-controlled" (PDC) turn was designed as a constant-radius turn filled with octagonal pillars that were arranged to control the linear velocity of the mobile phase in the radial direction. After the pillar positions were adjusted by computational fluid dynamics analysis, 27 mm long pillar array columns with two turns were fabricated on a 20 × 20 mm(2) silicon glass plate. The PDC turns suppressed the sample dispersion to a similar extent as the previously developed tapered turn, and the pressure drop of the newly designed turn was reduced to ∼1/6 that of the tapered turn. Moreover, the C18-modified pillar array column with the PDC turns showed good bioanalytical applicability; five fluorescently labeled amino acids were separated in only 24 s at a linear velocity of 7.5 mm/s. The developed turn structure offers the advantages of longer pillar array columns with more turns for the fast analysis of complex samples.
This study reports a fast and quantitative determination method for phenylalanine (Phe) and tyrosine (Tyr) in human plasma using on-chip pressure-driven liquid chromatography. A pillar array column with low-dispersion turns and a gradient elution system was used. The separation of fluorescent derivatives of Phe, Tyr, and other hydrophobic amino acids was successfully performed within 140 s. Under the optimized conditions, Phe and Tyr in human plasma were quantified. The developed method is promising for rapid diagnosis in the clinical field.
Previously, we have developed a gradient elution system for pillar array columns, which achieved faster separation than isocratic elution. In this study, we validated gradient elution in microchip liquid chromatography (LC) and investigated the retention and bandwidth predictions of fluorescently labeled aliphatic amines in fast gradient elution chromatography using semi-empirical retention models. The retention times and peak widths under different gradient elution programs were predicted by three solvent strength models and compared with the experimental results. The relative errors of prediction for the retention times and peak widths were below 14 % and 12 %, respectively. The results showed that the solvent strength models could be utilized for predicting the retention times and peak widths under gradient elution in the microchip LC system and that the gradient elution program for pillar array columns worked efficiently. The prediction by the retention model promises to be a potential tool for essential compound identification in biological samples.
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