You're right in assuming there will be problems. We've been making these
peptides for a couple of years and we still haven't figured out a general guide
for each tail length. You're waging the war of trying to get the peptides off
the column without sacrificing too much resolution. The war is much more
difficult if the peptide itself has a certain propensity for secondary
structure. You'll also have to deal with aggregation issues. We started out
using dialkyl tails but abandoned them due to aggregation. The best advice is to
use the shortest tail you can get away with to induce the activity you need.
The shorter carbon chains are usually not much of a problem (C6-C10). The longer
chains (we've gone up to C14 or C16) can cause some problems. We've tried
different conditions such as using a C4 column rather than a C18 or using IPA as
a mobile phase rather than ACN. In the beginning we also tried higher flow
rates, but this was quickly discarded (partly because the grad student working
on the project had a nasty habit of not paying attention to solvent and waste
levels). We also found that switching the mobile phase to IPA gave better
resolution than running ACN at 10 mL/min rather than 5 mL/min.
Another warning is what you see with a particular mobile phase on a particular
column for analytical HPLC is not always what you'll see using the same
conditions for prep HPLC (I just lost a peptide to that last week).
This message may sound like a bunch of warnings with no real help, but that's
the best I can do. We find great reproducability with a specific peptide and a
specific tail length, but if you change either segment you've got a new ball
game. Good luck and let me know if you have any other questions.
Jeni
Janelle Lauer-Fields
Department of Chemistry and Biochemistry
Florida Atlantic University
777 Glades Road
Boca Raton, FL 33431
Phone: 561-297-2094
FAX: 561-297-2759