By Ananda on Tuesday, April 30, 2002 - 01:06 pm:
Hello All,
I am forwarding here someone else's problem from AFBR forum. I thought most of the smart Chromatogrphers are here so I also can learn something out of this.So I will forward your answers to her. I also have used HFBA but only with ACN.
Thanks!
Ananda
"We are getting low yields when purifying amphipathic coiled coil peptides.
We are interested in trying out a new protocol using 0.05% HFBA and 90%
N-propanol as the mobile phase instead of using the standard acetonitrile
and TFA. Does anyone have any experience with these solvents? "
By Anonymous on Tuesday, April 30, 2002 - 04:30 pm:
The cause for the low yield may be the column, not the solvent.
By John B on Wednesday, May 1, 2002 - 04:49 am:
n-propanol is very viscous and at this % may exceed system pressure limits.
By Jess on Thursday, May 2, 2002 - 08:46 am:
Has anyone had any experience with 1:1 IPA/ACN as the buffer?
By Armando on Wednesday, July 3, 2002 - 06:17 am:
Hello Ananda,
I have used 1 and 2-P in protein purification and its advantage (in my short experience) is its better recovery of mass and biological activity. Resolution is not as good as with AcN, also 1 and
2-P are viscous so it is necessary to run at higher temperature to decrease the high pressure in the chromatographic system, specially when one uses analytical columns with id<=4.6mm.
I don't have experience with HFBA but I have read that it is more hydrophobic than TFA, acetic acid and phosphoric acid and maybe this is influencing your recovery because HFBA increases the strength of the interaction with the stationary phase. Try a weaker ion-pairing agent, for example phosphoric acid. It is recommended for very hydrophobic proteins. I'm used to clean RP columns with gradient from 25mM phosphoric acid/water to 25mM phosphor.acid/1 or 2-P pure or mixed with AcN. The mixture with AcN is useful since it decreases the pressure.