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.