ad do not forget to recalibrate On Thu, Jul 12, 2012 at 10:53 PM, Zhijie Li <zhijie...@utoronto.ca> wrote:
> Hi, > > Yes, it can be done by yourself. I repacked our 26/60 column a few years > ago with homemade apparatus. The column has been working for us since then. > Basically the loading apparatus was a tubing connecting the top of the > column and bottom of a reservoir (a flask with an opening near the bottom) > that contained 30%-50% suspended beads. The length of the tubing was about > 2m, and I left the reservoir on the highest shelf I could find in our lab, > so that the gravity of the buffer in the column and tubing will drive the > flow in a constant even manner. It took overnight to get the column nearly > filled. > The gel filtration columns need to be tightly packed with no > discontinuity. As I remember, the GE gel filtration packing manual said: > push the top filter 2mm further down, after the column has been fully > compressed by flowing buffer at 1cm/min flow rate. > The beads need to be gently suspended in the beginning, but constant > stirring is not necessary. The flow of the buffer will carry beads into to > column (because my reservoir has an opening at the bottom). The difficult > part is at the end, when the column is filled to the top. The beads will be > compressed quite a lot when you start pumping buffer, then you need to fill > in more beads. As I remember, I removed the upper filter on the inlet > connector so that I could load beads through the upper inlet tubing with a > 10mL or 30mL syringe, which generated enough pressure while loading the > beads - so that I didn't have to take the top off again and again. > > There is a manual from GE on how to pack superdex columns. You should be > able to find it online. The Superdex beads used in different sized columns > from GE have different diameters. The bigger the column is, the larger the > beads are. And the larger the beads are, the easier the packing will be. > 16/60 should still be fairly easy to pack. I checked the theoretical plate > number of our 26/60 after repacking, it was >11000, not as high as the > 13000 stated in the 26/60 new column manual, but it was good enough for us. > > Zhijie > > ------------------------------**-------------------- > From: "Peter Hsu" <hsuu...@u.washington.edu> > Sent: Thursday, July 12, 2012 9:51 PM > To: <CCP4BB@JISCMAIL.AC.UK> > Subject: [ccp4bb] offtopic: packing gel filtration columns > > > Hi all, >> >> Sorry for the slew of offtopic posts, but does anyone here have any >> experience repacking the large 120mL Superdex75/200 columns? Any >> advice/tips on doing it? I've got an older column that's gotten clogged >> while washing w/NaOH (can't go over 0.1mL/min w/o getting overpressure >> alarm), and not sure the bossman would be thrilled w/buying another column. >> >> Thanks for any ideas. >> >> Peter >> > -- Pius S Padayatti,PhD, Phone: 216-658-4528
