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.
There is really nothing special or magical about Superdex or Pharmacia
columns. Good gel-filtration column packing can be tricky but it's been
done well decades ago and can still be done today.
Things to keep in mind:
1. De-fine the matrix before packing.
2. Strictly vertical column, no local heating from one side.
3. Lowering surface tension helps packing ==> add 0.05-0.1% Triton X-100.
4. Prevent electrostatic repulsion of beads through residual charges ==>
use 100-200 mM NaCl in packing buffer.
5. Avoid particles sorting by size ==> have slurry that is about 65-70% matrix
6. Avoid discontinuities whenever possible ==> use packing reservoir if
possible and pour definite excess of the matrix.
7. Avoid bubbles like plague ==> degas the slurry and packing buffer before
pouring, pour in one nice careful motion, over a glass rod inserted inside
(letting the slurry slide on it down rather then dropping and making
bubbles). Do not allow sedimentation - connect to pump and start packing ASAP.
8. Pack at as high flow rate as possible ==> use absolute maximum that the
matrix is spec'ed for (andthe column, of course; for Superdex prep grade it
means using medium pressure columns). Pack until matrix level no longer
changes.
9. Pay extra attention inserting an upper adapter without bubbles - right
to the top of the matrix. Pack until matrix level no longer changes, always
moving the adapter down to the matrix.
10. Compress the column: *slowly* (in several stop and go steps of
compressing/running buffer through) compress by moving an upper adapter
down by a total ~ 2-3% of the column height.
- Dima
