Thank you both for your answers! *Upasana* - the chromatogram was randomly selected to ilustrate the point - we do a careful equilibration of the column and degas the mobile phase(s) before starting. I'm not really sure what you mean by oligomeric state of the protein - we are working with complex proteic mixtures that are not characterized, hence the need for fractionation. *Matthias* - your advice is spot on! I will give it a try and keep you posted.
On Mon, Jun 3, 2019 at 10:24 AM Barone, Matthias <bar...@fmp-berlin.de> wrote: > Hi sorin > The problem in your elution profile is that you cannot define a global > threshold as the baseline is heavily bent (relative to the peak heights) > and starting to fractionate at 6' is not the problem, telling the script to > stop once you reach baseline at 13' is. > I would try what you proposed: Given that your peak elution time points > are reproducible, I'd actually work with the "watch" function and turn it > on while watching OD for the given time windows. fractionate the first > block when OD exceeded a certain threshold and then turn the watch function > off after, say 13'. Turn it back on at 20' and fractionate into another > well. Use the time as base and keep the Äkta pumping at constant speed. > Curious if someone knows a better strategy! > Best, matthias > > ------------------------------ > *From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Sorin > Draga <sor.dr...@gmail.com> > *Sent:* Monday, June 3, 2019 8:29:40 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] AKTA FPLC fractionation peak by peak > > Dear all, > > We have an old akta system, running Unicorn 4.0, recently donated to our > lab. While attempting to fractionate a protein mixture, I was unable to > convince Unicorn to fractionate within given limits (say, for ex peak A > between RT a and b and peak B between retention times c and d). All that I > could manage to do is fractionate at a fixed time interval. > For clarity, I have attached a picture below (orange square would be what > we actually want to fractionate) > Any help would be appreciated, as we are pressed for time ( I am certain > that we are missing something simple) > > Thank you! > [image: image.png] > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
