NH4OH with PLRP, proteins/peptides
Posted: Sat Feb 13, 2010 8:49 am
I'm interested in trying different mobile phase as a way to improve selectivity in a multi-step purification scheme.
Peptide standards look fine when run on PLRP with 0.1% TFA and a linear gradient 5-60% MeCN. However, peak shape dramatically deteriorated when 15 mM NH4OH was used in place of TFA. What would cause this?
Perhaps the equilibration time must be longer?
Loading sample on a column equilibrated with 5% MeCN 15 mM NH4OH
My sample volume is 0.4 mL in 10 mM NH4OAc + 250 mM NaClO4, 5% MeCN.
After loading the 0.4 mL sample on to this 1 mm ID PLRP column at 50 ul/min, how long will it take to re-equilibrate with ammonia before beginning the gradient?
Peptide standards look fine when run on PLRP with 0.1% TFA and a linear gradient 5-60% MeCN. However, peak shape dramatically deteriorated when 15 mM NH4OH was used in place of TFA. What would cause this?
Perhaps the equilibration time must be longer?
Loading sample on a column equilibrated with 5% MeCN 15 mM NH4OH
My sample volume is 0.4 mL in 10 mM NH4OAc + 250 mM NaClO4, 5% MeCN.
After loading the 0.4 mL sample on to this 1 mm ID PLRP column at 50 ul/min, how long will it take to re-equilibrate with ammonia before beginning the gradient?