Supplementary Appendix 2 to A Practical Approach to Modelling of Reversed-Phase Liquid Chromatographic Separations: Advantages, Principles, and Possible Pitfalls

This information is supplementary to the article “A Practical Approach to Modelling of Reversed-Phase Liquid Chromatographic Separations: Advantages, Principles, and Possible Pitfalls” that was published in the March 2018 LCGC Europe issue.

Flow diagram for modelling based on the manual approach

Optional determination of V_m V_d, ECBB and confirm linearity of gradient profile

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Select appropriate samples (that is, forced degradation, mother liquors etc.)

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Select suitable retention model (for example, log(k )= q₁ + q₂ F + q₃ F².)

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Select appropriate input experiments (t_G, T etc.)

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Write methods and sequence (allowing sufficient time to equilibrate the new conditions)

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Collect input data for fitting and evaluation of models (typically performed overnight)

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Perform peak assignment / tracking (using MS, UV DAD, or peak areas), determine peak area, width, and asymmetry

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Produce Excel tables for each input experiment, copy, and paste into retention software input section

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Enter chromatographic conditions used

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Combine each individual input run into one large combined table

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Transfer input data to modelling software and fit models

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Evaluate resolution as a function of the operating variable examined (that is, gradient shape and T)

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Establish operational parameters fit for purpose (that is, identify robust conditions)

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Collection of data for confirmation of optimal conditions

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Optional in silico evaluation of other column formats, particle size, and flow