Selective comprehensive multi-dimensional separation for resolution enhancement in high performance liquid chromatography. Part I: Principles and instrumentation

An approach to enhancing the resolution of select portions of conventional one-dimensional high performance liquid chromatography (HPLC) separations was developed, which we refer to as selective comprehensive two-dimensional HPLC (sLC × LC). In this first of a series of two papers we describe the principles of this approach, which breaks the long-standing link in on-line multi-dimensional chromatography between the timescales of sampling the first dimension (1D) separation and the separation of fractions of 1D effluent in the second dimension. This allows rapid, high-efficiency separations to be used in the first dimension, while still adequately sampling 1D peaks. Transfer, transient storage, and subsequent second dimension (2D) separations of multiple fractions of a particular 1D peak produces a two-dimensional chromatogram that reveals the coordinates of the peak in both dimensions of the chromatographic space. Using existing valve technology we find that the approach is repeatable (%RSD of peak area <1.5%), even at very short first dimension sampling times – as low as 1 s. We have also systematically studied the critical influence of the volume and composition of fractions transferred from the first to the second dimension of the sLC × LC system with reversed-phase columns in both dimensions, and the second dimension operated isocratically. We find that dilution of the transferred fraction, so that it contains 10–20% less organic solvent than the 2D eluent, generally mitigates the devastating effects of large transfer volumes on 2D performance in this type of system. Several example applications of the sLC × LC approach are described in the second part of this two-part series. We anticipate that future advances in the valve technology used here will significantly widen the scope of possible applications of the sLC × LC approach.