Design Guidelines and Kinetic Performance Limits for Spatial Comprehensive Three-Dimensional Chromatography for the Analysis of Intact Proteins.

The design aspects of microfluidic chips for spatial three-dimensional chromatography featuring an interconnected channel network and targeting protein analysis are discussed, and the corresponding kinetic performance limits have been established using a Pareto-optimality approach. The pros and cons to integrate different separation mechanisms (IEF, CE, SEC, RPLC, HILIC, HIC, and IEX) are discussed considering development stages in the spatial domain (^<i>x</i>LC) in the first and second dimension and time domain (^<i>t</i>LC) for the third dimension. Based on Pareto-optimization, we discuss the considerations of the channel length, particle diameter, and the effect of number of second- and third-dimension channels on the resulting peak capacity of a spatial ^<i>x</i>IEF × ^<i>x</i>SEC × ^<i>t</i>RPLC device. Novel equations are proposed to determine the peak capacity in ^<i>x</i>SEC and to account for sample modulation affected by the number of second- and third-dimension channels. The corresponding Pareto fronts have been constructed demonstrating the resolving power, in terms of peak capacity and analysis time, considering current state-of-the-art prototyping methodologies. A microfluidic spatial prototype chip with an integrated channel layout (64 ²D and 4096 ³D channels) has been created, which has the potential to yield a peak capacity of 32,600 within only 44 min of the total analysis time, by implementing ^<i>x</i>IEF × ^<i>x</i>SEC × ^<i>t</i>RPLC separation stages.