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 1s. 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.

Selective comprehensive multidimensional separation for resolution enhancement in high performance liquid chromatography. Part II: applications.

In this second paper of a two-part series, we demonstrate the utility of an approach to enhancing the resolution of select portions of conventional 1D-LC separations, which we refer to as selective comprehensive two-dimensional HPLC (sLC × LC), in three quite different example applications. In the first paper of the series we described the principles of this approach, which breaks the long-standing link in online 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. In the first example, the power of the sLC × LC approach to significantly reduce the analysis time and method development effort is demonstrated by selectively enhancing the resolution of critical pairs of peaks that are unresolved by a one-dimensional separation (1D-LC) alone. Transfer and subsequent ²D separations of multiple fractions of a particular ¹D peak produces a two-dimensional chromatogram that reveals the coordinates of the peaks in the 2D separation space. The added time dimension of sLC × LC chromatograms also facilitates the application of sophisticated chemometric curve resolution algorithms to further resolve peaks that are otherwise chromatographically unresolved. This is demonstrated in this work by the targeted analysis of phenytoin in urban wastewater effluent using UV diode array detection. Quantitation by both standard addition and external calibration methods yielded results that were not statistically different from 2D-LC/MS/MS analysis of the same samples. Next, we demonstrate the utility of sLC × LC for reducing ion suppression due to matrix effects in electrospray ionization mass spectrometry through the analysis of cocaine in urban wastewater effluent. Finally, we explore the flexibility of the approach in its application to two select regions of a single ¹D separation of triclosan and cocaine. The diversity of these applications demonstrates the power and versatility of the sLC × LC approach, which will benefit tremendously from further optimization and advances in valve technology that specifically address the needs of this new technique.