Automated device for continuous stirring while sampling in liquid chromatography systems

High- and ultra-performance liquid chromatography are widely used analytical tools for detecting and identifying components in complex mixtures through sampling and chromatographic separation. Stirring is fundamental to ensure sample homogeneity and, in systems forming large assemblies, mechanical agitation can influence behavior via breakage of assemblies. The authors developed a UPLC stirring device that replaces a standard sample holder and enables battery-powered magnetic stirring inside the instrument, allowing continuous stirring during automated, time-resolved measurements without additional sample preparation or human intervention. The device’s utility is demonstrated on a system highly sensitive to stirring, exhibiting exponential growth enabled by a fiber elongation/breakage mechanism. The development aligns with open-source and do-it-yourself trends, leveraging 3D printing and readily available parts.

The work builds on prior studies establishing UPLC as a key analytical method and on systems chemistry examples where mechanical agitation affects self-assembly and self-replication kinetics via fiber elongation/breakage mechanisms. It also connects to the growing literature on open-source, low-cost scientific instrumentation and applications of 3D printing in analytical chemistry, biotechnology, and custom laboratory devices.

Device design: The stirring device matches the footprint and vial positions of a standard UPLC sample holder, enabling drop-in replacement. A motor rotates an internal plate with magnets to drive standard Teflon-coated magnetic stir bars inside vials. The control module is separated from the stirring module. Stirring speed is adjustable from 200 to 1200 rpm in 100 rpm steps, controlled by an onboard microcontroller with feedback regulation to maintain set speed. An OLED display shows battery status and rotation rate. Deviations greater than 10% from the set speed lasting over 60 s trigger an on-screen error indicator; upon restart, a summary of past errors is displayed grouped by deviation ranges. The device is battery powered. Fabrication: Components were 3D-printed (Creatr Duel Extruder, nozzle 0.35 mm; da Vinci 1.0 Pro, nozzle 0.40 mm) using 1.75 mm PLA at 205 °C nozzle and 40 °C bed, low print speed (main body print times ~13 h for sample holder, ~10 h for control unit). Magnets used are 25 × 8 × 1 mm. Control is via an Arduino Pro Mini 3.3 V 8 MHz microcontroller with a magnetic sensor for rotational feedback. Design files are provided as STL (Supplementary Dataset 2), and firmware as Arduino code (Supplementary Dataset 3). The device profile is ~4 mm higher than the Waters Acquity UPLC sample holder (catalog 700005209), requiring UPLC needle height adjustment. Chemical system and sample preparation: A 2.0 mM aqueous stock of a peptide-based dithiol building block (sequence Gly-Leu-Lys-Phe-Lys) was prepared by dissolving 1.2 mg in 607 µL borate buffer (50 mM in boron, pH 8.12). Samples comprised 250 µL stock plus 750 µL borate buffer in 12 × 32 mm UPLC vials; a 5 × 2 mm Teflon-coated stir bar was added to designated samples and vials sealed with Teflon-lined screw caps. UPLC analysis: Samples were placed within the device in a Waters Acquity UPLC-H Class with PDA detector, using a reversed-phase column (Aeris Peptide 1.7 µm XB-C18, 2.1 mm, Phenomenex). Column at 35 °C; sample chamber at 40 °C. UV monitored at 254 nm. Mobile phases were H2O and acetonitrile (UPLC grade) with 0.1% TFA. Gradient and integration details are in Supplementary Notes 1–2. Samples were periodically analyzed automatically, enabling 60–90 consecutive measurements per sample over about a week. Pre-oxidation experiments: To decouple oxidation from replication, samples were pre-oxidized with 0.50 equivalents sodium perborate (NaBO3) to oxidize 50% of the building block, then stirred at 200 or 1000 rpm and analyzed as above. Validation measurements: Kinetics of hexamer macrocycle formation (autocatalytic fiber-forming system) were monitored with stirred vs unstirred conditions. Device positions used were C2, C7, D2, and D7. A video analysis measured actual stir-bar rotation at positions C2, D2, E2, F2, F3, A4, F4, A5, F5, C7, D7 at set speeds 200 and 1000 rpm to assess accuracy and spatial uniformity.

• Stirred vs unstirred kinetics: After ~100 h, stirred samples reached 79 ± 5% of building-block mass in hexamer, while unstirred samples reached 13 ± 5% (n = 4 per condition), demonstrating consistent stirring and its strong effect on the system.
• Homogeneity during repeated sampling: Total UPLC peak area remained well conserved over time with relative standard deviations of 2.27% and 2.30% in two independent stirred experiments, indicating reliable sample homogeneity during 60–90 consecutive automated injections.
• Stirring rate dependence (pre-oxidized): Time to reach 50% hexamer was 8.17 ± 0.86 h at 1000 rpm and 17.2 ± 2.2 h at 200 rpm (four repeats each), confirming faster exponential growth at higher stirring rates consistent with fiber elongation/breakage mechanisms.
• Stir-speed accuracy: Video analysis showed actual stir-bar rotation speeds averaging 0.99 and 0.98 times the set speeds at 1000 and 200 rpm, respectively, for positions surrounding the central motor, indicating ~1% deviation from setpoints.
• Spatial performance: Positions near the center performed reproducibly; positions in columns 1 and 8 exhibited diminished reproducibility due to weaker magnetic field at the periphery.
• Operational endurance: Starting from fresh batteries, battery level typically decreased to ~35% after ~72 h of continuous stirring, at which point batteries were replaced.

The device reliably enables continuous magnetic stirring within a UPLC autosampler, allowing unattended, time-resolved measurements of systems whose kinetics and phase behavior depend on agitation. The strong enhancement of hexamer formation under stirring, the clear rate dependence on stirring speed after pre-oxidation, and the conservation of total chromatographic signal across many injections collectively demonstrate that the device maintains homogeneous suspension and delivers reproducible agitation over extended runs. Video-based validation confirms that actual stir-bar rotation closely tracks setpoints, supporting the accuracy of feedback-controlled speed regulation. These results directly address the need to perform kinetic studies of agitation-sensitive systems without manual intervention, providing a robust platform for investigating dynamic self-assembly and other complex chemistries within chromatographic systems.

A battery-powered, 3D-printed device that replaces the standard UPLC sample holder enables continuous stirring during automated chromatographic sampling. Validation with an agitation-sensitive self-replicating system showed reproducible, homogeneous stirring over extended periods, accurate control of stirring speed, and clear kinetic dependence on agitation rate. The device facilitates automated, time-resolved UPLC measurements for studies such as kinetics and dynamic combinatorial chemistry and can benefit analytical laboratories broadly. The design can, in principle, be adapted to other chromatography platforms and expanded via microcontroller programming to support more complex operational scenarios.

Performance is position dependent, with diminished stirring reproducibility at vial positions farther from the device center (e.g., columns 1 and 8) due to weaker magnetic fields. Experiments were conducted at an elevated sample-chamber temperature (40 °C), which made oxidation by air comparable in rate to replication, complicating observation of stirring-rate effects without pre-oxidation. The device profile is ~4 mm higher than the standard Waters Acquity UPLC sample holder, necessitating needle height adjustment. Battery operation requires replacement after approximately 72 h of continuous use (battery level ~35%). Validation focused on a specific chemical system and a particular UPLC platform, which may limit immediate generalizability without adaptation.