dry gel filtration column

[Pepa Florez Pérez]

Hello,

I have discovered today that one of our prepacked gel filtration column is completely dried out.
We have tried to make it work by re-hydration and the pressuere has decreased, but it is stil very high.

Do you know if it is possible to make it work in the same way as before?

The column seem wet now, but we cannot run it to the same flow as before.

Are the beads damaged somehow irreversibly when they become completely dried?

Thank you very nuch

Pepa

[Wolfgang Schechinger]

Hi Pepa,

probably you just have some air trapped inside and possibly buffer salts
have precipitated in the tubings.

The simpliest thing to try is to pump large volumes of buffer (10 to 25
or even more column volumes of PBS with azide or just DD water/azide) at
a low flow rate through the column (inlet at the bottom of the vertically
mounted column!). From time to time, increase the flow rate to check if
the performance of the column improves.

If that does not work, try to suck the buffer through with gentle vacuum.
Lower pressure will make the trapped bubbles bigger and might get them
out from the pores easier.

If the column may be opened without destroying it, just pour the gel into
a beaker with buffer and remove the air by applyig vacuum in e.g. an
exsiccator. When all the air has gone out, repack the column.

If you have a spare empty refillable column, you might consider breaking
the selaed column, too.

At least for sephadex based materials, this works well. Don't know about
other stationary phases like sephacryl, however.

If, additionally, the column packing has suffered (visible "cracks"), you
could try to make it even again by frequently reversing the flow
direction and applying lots of pressure (flow) changes.

Or you may simply pretend that the column has been eaten by molds and
bugs and ask your PI for a new column for your precious samples...

HTH

Wo

[Dr Engelbert Buxbaum]

In article <MMnIq.30896$2e7....@newsfe18.iad>,
d...@no.email.thankstospam.net says...
>
> In article <mailman.34.132441...@net.bio.net>,
> =?iso-8859-1?B?UGVwYSBGbG9yZXogUOlyZXo=?= <snipe...@hotmail.com>

> wrote:
>
> > have discovered today that one of our prepacked gel filtration

> > column is ompletely dried out. e have tried to make it work by
> > re-hydration and the pressuere has decreased but it is stil very
> > high.
> >

> > o you know if it is possible to make it work in the same way as
> > before?
>

> If it is sugar polymer-based (agarose, dextran) then no, a massive dry
> out is irrepairable. The reason is, even though modern matrices are
> highly cross-linked, upon drying granules collapse and that is pretty
> much it.
>
> Dried silicagel or resin columns (used mostly in HPLC) would be no
> problem - wash extensively with degased buffer with the outlet up for
> the air to escape, then wash with methanol that will very efficiently
> dissolve air still trapped.

If the particles dried out, yes, the gel is lost. However, that takes
quite a while (weeks or months). Usually, only the fluid in the space
between gel particles is replaced by air, and then the column can be
rescued. However, it needs to be unpacked, the gel resuspended in
buffer, the "fines" removed, remaining gel de-aerated with vacuum, and
re-packed. This needs to be done according to manufacturer's instruction
for the gel (packing funnel, correct flow rate/pressure etc.). You may
need an adjustable end-piece, as you are likely to loose some part of
the gel volume.

Do not try to just pump buffer into the dried-out gel bed! It is
unavoidable that air bubbles and cracks remain, this will seriously
lower the resolution of the column.