Chromatography Forum

Hi
I'm looking for advice on how to remove sodium phosphate from a crude peptide prior to purification.

I have performed a disulphide bridge formation of Salmon Calcitonin in a pH 4 phosphate buffer at a concentration of 0.58mM.

Following full cyclization the solution was freeze dried and dissolved in 6ml Water for purification.

However, I'm worried that the remnant sodium phosphate, of which the concentration is now 5 times higher compared to the initial buffer solution, will
influence the recovery of the peptide.

I'm thinking of purchasing 1-time use SPE C18 cartdriges in order to remove the sodium phosphate, does that sound viable?

In advance thanks
Erik

So just to elaborate a bit. What I'm worried about is that my semi-prep column will be saturated with salt and hence
the peptide won't be retained on the column.
I'm running a linear gradient of 30%-70% 0.1% TFA/ACN - 0.1% TFA/H2O, and the sodium phosphate might not pose any problems, just a thought.


\Erik

A question or two come to mind, the first being what kind of separation is your semi-prep column? My thought would be size exclusion chromatography as long as your peptide is large enough to be excluded by a gel -- and it has been a long while since I've considered such, so I don't remember size cut off for gells. But a bit more detail about what you are doing in the next separation might help with the question of the possible effect of phosphate.

Sorry for not being more comprehensive, the purification is commenced by means of reverse phase on a C18, 5 um, 300A 10x250 column.
So what I plan to do is to inject the redissolved crude cyclized peptide (900 uL, ~3mM) and obviously separate it from impurities. But I'm uncertain whether
the phosphate will saturate the column and my peptide will go right through. Or is this phenomenon only seen when injecting more harsh solutions like 8M urea or 6M Guanidine hydrochloride, which have been mentioned in the literature?

\Erik

one thing you can do is desalt over a SEC column
we helped develop such a method only recently for a team that like you does peptides and proteins
at the beginning they wanted the method to do direct sample injection to LC-MS/MS
up to know they were doing it in an off-line mode.
they wanted to be capable of using a 01.% to 1.0% formic acid solution as mobile phase into the MS
the sample of course being salted on column (salts coming off at the end)
so we look around for a column that could stand well at pH 2.0 and the use of high ACN concentrations.
we took a Sepax Zenix column 2.1mmX150mm
after a while they took the same concept for their prep procedures as well.
the column has no problem unlike Tosoh, Thermo or phenomenex to run with a 0.1% TFA solution mixed up in water and ACN at various concentrations for a long period of time.
the desalting is done on column, that has no problems with high salts concentration coming from the samples.
for complex mixtures they do a second run over a RP column. for them it is great since the sample is already in a solution that is very close to the standard mobile phase composition with TFA.

Thank you Unmgvar, I'll have a look at the Sepax Zenix SEC columns.

\Erik