Expanding analytical horizons: 3D HPLC calibration surfaces for micromole scale self-optimizing flow reactors†

In this paper, we use the concept of the HPLC 3D calibration surface for evaluating reaction performance in micromole scale self-optimizing flow systems. This approach enables comparing the analyte and internal standard relative levels for a wide range of internal standard concentrations. This approach considers fluctuations in response factors and potential nonlinearities in the Beer–Lambert law, which arise from performing HPLC analyses at different concentration ranges. The concept is validated by constructing a calibration surface for a formal [3 + 3] cycloaddition. The robustness of the 3D calibration surface is assessed in an autonomous flow self-optimization in a non-steady-state regime, where a standard 2D calibration curve produces inconsistent results due to variable concentration HPLC injections.