Here is a "supersolvent" that Rod Levine has suggested
"We've done a fair bit of CNBr cleavage over the years, followed by C18
purification of the cleaved peptides. While we didn't do an exhaustive,
systematic study, we did adopt a prefered solution for redisolving the
peptides. As a general rule (always exceptions), it gives excellent
recovery of most peptides, even fairly hydrophobic ones. Around the lab
it
is known as "supersolvent":
6 M Guanidine HCl
500 mM Potassium Phosphate
pH 2.5
To make it up, start with the correct volume of phosphoric acid and use
KOH
to bring the pH up to the desired 2.5.
I don't pretend that there's a rational basis for the gemisch; it evolved
and seems to work for us.
We're only beginning to get into the LC/MS side, but so far we're pretty
happy with using this as the loading "buffer" for the peptides. We still
prefer the Vydac C18 columns, the 218TP series."
Rod Levine
NIH
Bldg 3, Room 106 MSC 0320
Bethesda, MD 20892-0320
Now, even if you get the peptide in solution, the next trick is to see if
it will elute from the column. I like to start out and run from Mobile
Phase A 0.1 % TFA, to Mobile Phase B 0.1% TFA, 90% Acetonitrile in 30
minutes and see if the peptide comes off the column--don't overload the
column and be sure to have a frit in-line to filter the injection sample
or the frit of your column will get clogged fairly quickly.
You can always start at a higher per cent acetonitrile for your gradient
to see if that works.
An even quicker way to see if your peptide will come off of a reverse
phase column is to run isocratically, say, try 40% acetonitrile or 50%
acetonitrile.
I've used some mixed solvent systems in the past--with some few percent of
isopropanol added to the TFA/acetonitrile mobile phases, but watch the
back pressure of the system.
If you are starting from scratch with this chromatography--i.e., you need
to buy a column, you should look at the information available on HILIC
chromatography columns. There has been quite a bit of discussion about
HILIC in the past few days on the ABRF "bulletin board". It is an ideal
system for purification of hydrophobic peptides--just go into the ABRF
home page and search for HILIC.
Hope this is of some help,
Deb McMillen
Institute of Molecular Biology
University of Oregon
Eugene OR
On Mon, 31
Aug 1998, laurent boyer wrote:
> Hi, everybody !
> I would like to run a HPLC with The following peptide : Poly(Ala)-Val
> Given it's very hydrophobic, it is not easily soluble in common
> solvents.
> Does anyone know which one I could use ?
> Thanks, bye,
> Laurent.
>