Greener microvolume UV–Vis and synchrotron radiation circular dichroism workflows for pathlength correction, and pipetting validation and quantified uncertainty for reliable analytical outcomes

• Sustainable microvolume workflows require explicit integration of validation and uncertainty management. • Closed-well microvolume formats decouple optical geometry effects from meniscus-driven artefacts. • Low-volume dispensing error is a critical but under-reported contributor to analytical uncertainty. • Error-budget approaches provide a quantitative bridge between greenness and analytical reliability. • Material-efficient workflows can deliver major sustainability gains without sacrificing data integrity. Microvolume UV–Vis and synchrotron radiation circular dichroism (SRCD) offer clear sustainability benefits by reducing sample and reagent consumption, yet their adoption is often limited by concerns over optical pathlength variability and low-volume dispensing inaccuracies. Here, we benchmark a μDrop™ Duo microplate workflow implemented on both a Multiskan SkyHigh spectrophotometer and the B23 HTCD/SRCD platform against conventional cuvette-based measurements, using a combination of per-well pathlength calibration, baseline subtraction, and rigorous pipetting validation. Across 32 wells, relative pathlengths showed measurable well-to-well variation, confirming that a single global correction factor is inadequate. Once corrected, microvolume spectra demonstrated quantitative agreement with cuvette references, supported by regression analysis, narrow Bland–Altman limits of agreement, and low baseline-region noise. Gravimetric evaluation of a multichannel pipette revealed substantial volume-dependent error at 20 µL, underscoring the importance of matching pipette class to microvolume operation and verifying performance under actual environmental conditions. Together, these results show that microvolume UV–Vis and SRCD workflows can achieve cuvette-level analytical reliability when appropriate calibration and QC procedures are applied. The study provides a three-tier set of best-practice recommendations—essential, recommended, and aspirational—designed to help laboratories implement greener workflows without compromising metrological integrity. This work demonstrates that sustainability and analytical performance can be mutually reinforcing when validation is embedded at each stage of microvolume analysis. Validated Greener Microvolume UV-Vis/SRCD Workflow