On-line comprehensive two dimensional separations of charged compounds using reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography. Part II: Application to the separation of peptides

• On-line RPLC × RPLC was compared to RPLC × HILIC for the separation of peptides.
• A peak capacity of 3300 in 200 min was reached in RPLC × RPLC while 2600 in RPLC × HILIC.
• The sampling time was optimized with respect to overloading and injection effects.
• RPLC × RPLC leads to less overloading effects than 1D-RPLC due to steeper gradients.

In this second paper of a two-part series, on-line RPLC × HILIC is compared to on-line RPLC × RPLC through the separation of peptides. Our choices regarding the conditions are discussed. Injection effects and overloading effects are evaluated in both configurations. It is shown that whereas large volumes can be injected in the second dimension in RPLC × RPLC under HT-UHPLC conditions (>20% of the column dead volume), even small injection volumes (8% of the column the dead volume) have a detrimental effect on peak shapes in RPLC × HILIC. Advantages and limits of the two 2D-systems are compared through the 2D-separation of a tryptic digest of three proteins. A ten-fold gain in analysis time along with a significant gain in peak capacity are obtained with both systems compared to the most efficient one-dimensional separation of peptides recently published.