Studies of the improvement of RP-HPLC in a 2D-HPLC technique for top-down and bottom-up proteomics applications

Sudheer Sami

Absract:

Although 2D-HPLC technique where 1^st dimension is chromatofocusing and 2^nd dimension is reversed phase HPLC [RP-HPLC], offers more advantages over existing methods for proteomics applications, it has lower total peak capacity. In this study, effect of various parameters such as organic solvent, stationary phase morphology, maximum inlet pressure, diameter of particles, temperature on the peak capacity of RP-HPLC is studied and using the theory of kinetic plots, plate height equations, concept of separation impedence and theory of gradient elution, peak capacity of RP-HPLC is theoretically predicted. Theoretical predictions of peak capacity are then compared with the peak capacity obtained from gradient elution experiments.