Starting MeCN% in RP-HPLC

[DJ]

For RP HPLC of peptides and proteins on alkyl columns, I typically equilibrate the column at low MeCN (5% MeCN 0.1% TFA). After loading, I do a 5 min ramp to (for instance) 30% MeCN, 0.1% TFA, and run a 60 min gradient to 60% MeCN, 0.1% TFA.

Is there any benefit to loading the sample at low MeCN%, when the peptide/protein elutes around 45% MeCN, or, could I simply cut that part of the program out, and equilibrate, load at 30% MeCN with no ill effects?

[tom jupille]

Depends a bit on what volume you're injecting, but in general it should not be a problem.

[DJ]

Sample volume is large-- 1-10 mL for a 4.6 mm column, and several injections are often needed. If there is isocratic band migration/spreading during loading, would the band *compress* once the gradient is started?

Is column capacity/analyte binding favored more so at lower MeCN?

[Jiri Urban]

I always start with concentration below elution of first compound and end with the concentration just above the last one.

[tom jupille]

If your sample diluent is very weak (100% aqueous, for example), then you will not get any isocratic migration during loading, and you will see "on-column concentration"). In that situation, your proposal (which matches Jiri's suggestion) should work just fine.

If your diluent matches the strength of your initial mobile phase, then you will probably see some isocratic migration during loading. The gradient will sharpen the bands somewhat, but you will probably still see peak shape issues.