Guidelines for bioanalytical 2D chromatography method development and implementation.

2D chromatography is a rapidly evolving, very powerful tool for bioanalysis. Advances in the theory of 2D separations, instrument technology and data analysis strategies continue to complement each other and advance the state of the art. Separations of complex mixtures of biomolecules yielding several hundred peaks in practical analysis times (tens of minutes to several hours) are relatively common. However, this level of performance largely remains the domain of expert researchers and several practical limitations stand in the way of more widespread use of 2D separations among practitioners. While off-the-shelf instruments are increasing in number, the most effective 2D instruments are often home-built, and analysis of the extremely rich datasets resulting from these separations continues to be a serious bottleneck in the overall workflow. This review summarizes some of the most serious challenges in method development and describes best practices to help guide users in designing effective 2D separations for bioanalysis.