A novel graphical method for assessing the compromise between conditional peak capacity and separation speed for packed bed columns under gradient conditions has been developed and applied to the separation of peptides. This approach is analogous to and complements the conventional "Poppe plot" used to study plate count in isocratic separations. The use of the new plot can assist the design of appropriate column formats (e.g. particle size and column length) for both dimensions in gradient elution two-dimensional liquid chromatography (2DLC). Particularly for the second dimension of 2DLC, we find that smaller particles provide faster separations even though fast separations based on particles smaller than 2 μm are practically limited by the required miniscule column length. We also find that high temperatures strongly enhance the kinetics of peak capacity production whereas higher pressures help achieve larger absolute peak capacities albeit at the cost of longer analysis time.
Bibliographical noteFunding Information:
The authors acknowledge the financial support from the National Institute of Health (Grant #5R01GM054585-09) and Agilent Technologies for the donation of the Zorbax SB-C18 column.
- Gradient elution
- Kinetic performance
- Peak capacity
- Poppe plots