Journal
ANALYTICA CHIMICA ACTA
Volume 654, Issue 1, Pages 77-84Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.aca.2009.06.042
Keywords
Comprehensive two-dimensional liquid chromatography; On-line two-dimensional liquid chromatography; Peak capacity; Analysis time; Orthogonality; Peptide analysis
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A novel strategy is described for designing optimal second dimension (D-2) gradient conditions for a comprehensive two-dimensional liquid chromatography system where the two dimensions are not fully orthogonal. Using the approach developed here, the initial and final organic modifier content values resulting in the highest coverage of separation space can be derived for each D-2 gradient run. Theory indicates that these values can be determined by adapting D-2 gradient operation to the degree of orthogonality. The new method is tested on a comprehensive two-dimensional liquid chromatography system that uses reversed phase (RP) columns showing different selectivities in the two dimensions. A comparison between analyses carried out using normal and optimized D-2 gradients showed that the latter allow a more efficient use of analysis time. This can result either in an improved peak capacity or in decreasing total analysis time, depending on the final goal of the experiment. In the latter scenario, the number of separated peaks is comparable to that obtained using gradients spanning a wide range of organic modifier but, now, in half the time. As test samples complex mixtures of peptides were analyzed. (C) 2009 Elsevier B.V. All rights reserved.
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