Researchers Compare Configurations for Two-Dimensional LC for the Analysis of Peptides
In new research, separation scientists from the University of Lyon, France, have compared different configurations of on-line two-dimensional liquid chromatography (2D LC) for the separation of charged compounds such as peptides. In a recent paper (1) in the Journal of Chromatography A, Sabine Heinisch and Amélie D’Attoma presented work that compares the use of reversed-phase LC in both dimensions to the use of reversed-phase LC in the first dimension followed by hydrophilic interaction liquid chromatography (HILIC) in the second dimension.
Heinish and D’Attoma found that both systems could dramatically reduce the analysis time while increasing the effective peak capacities compared to one-dimensional separations. They also found pluses and minuses to both setups.
They obtained a lower peak capacity in reversed-phase LC x HILIC configuration, but the peak coverage was better in that configuration compared to using reversed-phase LC in both dimensions.
The researchers observed a severe deterioration of peak shapes in reversed-phase gradients with formic acid as an additive when the gradient time was increased. The phenomenon occurred with core-shell particles as well as with totally porous particles, although it was less pronounced in the latter case. Heinisch and D’Attoma noted that overloading conditions are very detrimental to 2D data processing because the peak shape in the second dimension becomes dependent on the solute concentration, which varies according to the first dimension peak fraction. As a result, they recommend very short sampling times in the second dimension, as overloading conditions can be overcome when using reversed-phase LC in both dimensions by employing very fast gradient conditions in the second dimension.
In contrast, the authors noted, no peak shape distortion due to overloading was observed with HILIC gradients, thus making HILIC potentially attractive for a second dimension. However, they did observe severe injection effects when using HILIC in the second dimension when injection volumes exceeded 9% of the column dead volume. When using reversed-phase LC in both dimensions, no additional peak band broadening was observed in the second dimension, even with injection volumes as high as 20% of the column dead volume.
This paper was the second of a two-part series. In Part I of the series, published last year, the authors looked at the degree of orthogonality and practical peak capacity considerations in 2D LC separations (2).
(1) A. D’Attoma and S. Heinisch, J. Chromatogr. A 1306, 27–36 (2013).