Rapid development of chromatographic processes relies on effective high-throughput screening (HTS) methods. This article describes the development of pseudo-linear gradient elution for resin selectivity screening using RoboColumns V R . It gives guidelines for the implementation of this HTS method on a Tecan Freedom EVO V R robotic platform, addressing fundamental aspects of scale down and liquid handling. The creation of a flexible script for buffer preparation and column operation plus efficient data processing provided the basis for this work. Based on the concept of discretization, linear gradient elution was transformed into multistep gradients. The impact of column size, flow rate, multistep gradient design, and fractionation scheme on separation efficiency was systematically investigated, using a ternary model protein mixture. We identified key parameters and defined optimal settings for effective column performance. For proof of concept, we examined the selectivity of several cation exchange resins using various buffer conditions. The final protocol enabled a clear differentiation of resin selectivity on miniature chromatography column (MCC) scale. Distinct differences in separation behavior of individual resins and the influence of buffer conditions could be demonstrated. Results obtained with the robotic platform were representative and consistent with data generated on a conventional chromatography system. A study on antibody monomer/high molecular weight separation comparing MCC and lab scale under higher loading conditions provided evidence of the applicability of the miniaturized approach to practically relevant feedstocks with challenging separation tasks as well as of the predictive quality for larger scale. A comparison of varying degrees of robotic method complexity with corresponding effort (analysis time and labware consumption) and output quality highlights tradeoffs to select a method appropriate for a given separation challenge or analytical constraints.

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SummaryThe applicability of confocal laser scanning microscopy is limited, e.g. by attenuation of the excitation and the fluorescence emission beam. As a prerequisite for further processing and analysis of the obtained microscopic images, a new method is presented for correcting this attenuation. The correction is based on beam modelling and on a differential form of the modified Beer–Lambert law. It turns out that the intensity decay can be modelled as a double convolution of the microscopic image with the intensities of the excitation semibeam and the emission beam. Under weak assumptions made for the intensities of the fluorescent radiation and the detected signal, formulas for the attenuation correction and the attenuation simulation are derived. The method traces back to that one published by Roerdink which is modified concerning a more realistic beam modelling, avoiding the so‐called weak attenuation expansion and considering fluorescence excitation throughout the light cone of the excitation beam. The applicability of the method is demonstrated for synthetic examples as well as microscopic images of chromatographic beads. It is shown that the new method can be successfully applied for reconstructing the true fluorophore distribution in specimens even if the microscopic images are affected by strong attenuation.Lay DescriptionThe applicability of confocal laser scanning microscopy is limited by attenuation of the excitation and the fluorescence emission beam. As a prerequisite for further processing and analysis of the obtained microscopic images, a new method is presented for correcting this attenuation. The correction is based on modeling the excitation as well as the emission beam and on a modified Beer‐Lambert law for beam attenuation. The applicability of the method is demonstrated for synthetic examples as well as microscopic images of chromatographic beads. It is shown that the new method can be successfully applied for reconstructing the true fluorophore distribution in specimens even if the microscopic images are affected by strong attenuation.