Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1228, Issue -, Pages 31-40Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2011.06.035
Keywords
Two-dimensional; Multi-dimensional; HPLC; Focusing; Resolving power
Funding
- Camille and Henry Dreyfus Foundation
- Howard Hughes Medical institute
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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 x 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 (D-1) separation and the separation of fractions of D-1 effluent in the second dimension. This allows rapid, high-efficiency separations to be used in the first dimension, while still adequately sampling D-1 peaks. Transfer, transient storage, and subsequent second dimension (D-2) separations of multiple fractions of a particular D-1 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 x 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 D-2 eluent, generally mitigates the devastating effects. of large transfer volumes on D-2 performance in this type of system. Several example applications of the sLC x 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 x LC approach. (C) 2011 Elsevier B.V. All rights reserved.
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