This is a 3 part answer for proteins and peptide separations:
- Start with an existing method for a protein/peptide and use an equivalent column to what you are currently using. Run your existing gradient method or a generic gradient (5 to 65 % organic in 30 minutes) and use existing flow rate and temperature. The gradient can be optimized based on resolution of critical component and elution time. Adjust slope, initial and final % B based elution profile. Temperature and flow rate can be increased to improve peak shape and recovery if necessary. Column specific tips are listed in part 2 and 3.
- For protein separations on Aeris WIDEPORE: Proteins will generally elute earlier on the Aeris WIDEPORE column compared to existing fully porous 300 Å columns and thus the gradient and initial % B will typically be adjusted 5-10 % lower than existing methods. Temperature can get hot if necessary. To improve resolution, increase recovery, and improve peak shape temperatures up to 90ºC can be used. Flow rate can be increased up to 1.5x of existing flow rates (1.0-2.0 mL/min for 4.6 mm ID; 0.3-0.5 mL/min for 2.1 mm ID). Loading should be done in mobile phase A if possible otherwise use low organic in the diluent.
- For peptide separations on Aeris PEPTIDE: Peptide and peptide maps will elute only slightly earlier than fully porous media so gradient adjustment may not be necessary. Flow rate should be adjusted up (1.2-2.2 mL/min for 4.6 mm ID; 0.3-0.7 mL/min for 2.1 mm ID) to improve efficiency, especially if a 1.7 µm Aeris PEPTIDE column is being used. Peptide and peptide maps can be adjusted similar to any other columns; steeper gradient to shorten run time, shallower gradients to increase resolution. Longer columns and moving from the 3.6µ to the 1.7µ Aeris PEPTIDE column will improve resolution.
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