The HiPrep 16/60 Sephacryl S-100 column that we use is clogged with some dark coloured particles at the very top. I tried cleaning up the column with 0.5M NaOH followed by distilled water. This removed few coloured compounds but not all. Is there any other way to clean the column or can I reverse the flow of the column to elute those particles out?
Dear Soundaraya
the positive result of unclogging will depends a lot from the nature of the coloured particles that block your coloumn.
Are these protein precipitates, salts, metal precipitates (e.g sulfurs?) Which samples, or buffers were loaded in the coloumn before that this precipitates were observed?
Were this precipitates presents in your sample or buffers (that you do not filtered 0,22uM) or this were formed once that the samples was injected in the coloumn?
If you think that this precipitates are proteins you can try to wash the coloumn with UREA 6M Ph=8
IF you think that are sulfurs of some metal, yon can try to pass HCl 1M (some metal form soluble complexed with cloride and is it possible that the sulfurs will be slowly slowly re-solubilized
Of course you can also certainly try to wash the coloumn in the opposite direction.
I'm not sure that one of this solution will work, but before true away the coloumn you can certanilly lost some times to test it.
OF course if the problem will be solved, run a standard sample to check if the coloumn resolution and the peak shape are still the same.
good luck
Manuele
Manuele Martinelli The precipitates are from some plant extract samples that were not filtered properly. It looks brownish in colour.
Dear Soundarya
it is difficult to told you with this sample which is the best strategy. Probably if i have to follow my gut feeling, i will try to wash it with 6m urea in opposite direction at low flow for at least 3 cv and afterwards wash it extensivelly with water and check if there are some improvment. But it is a feeling.. non a scientific truth.
good luck
Manuele
Urea wash (as suggested)- water-buffer wash; next in line is 1mg.ml pepsin, 0.1M Nacl, 0.1M Acetic acid - upside down - Leave overnight, next day wash extensively with water and buffer - Good to go. Filter - 0.45 - repeat 0.45- last 0.2.
I tried reversing the column but the pressure alarm was triggered due to increase in pressure. Just to make sure if it is the column or system I tried removing the column. There was a block in the flow restrictor. We manually purged all the tubings and the flow restrictor. Now is it advisable to run the column in reverse?
i think you can proceed. however with a coloumn with a so low pressure limit 0.15 MPa you can avoid the use if the flow restrictor. it is needed when you have coloumns as supedex 10/30 the work at pressure higher >1MPa
Manuele
Your best bet IMHO will be to unpack the column, discarding the coloured material. Then thoroughly wash the remaining gel, Manuele Martinelli suggestion of urea is reasonable. Do not use a magnetic stirrer or any other method that breaks the gel beads, an end-over-end mixer is probably best. Add new gel to replace what you have discarded, decant any fines and then repack the column, using a reservoir. The procedure is described in https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-gel-filtration.pdf.
I think the reverse flow is a good idea.
I have several proposals:
1. Our standart column wash: 6M Gu-HCl -MiliQ-NaOH-MiliQ-2%SDS-MiliQ.
In any combination, there needs to be a thorough MiliQ wash betwen GuHCl
and 2%SDS!
2. 50mM acetic acid with 1% triton X-100.
3. 30% isopropanol.
4. And I also suggest 0.1 M HCl. This can help with some reduced, precipitated
substances. I would place this solution in superloop. And then wash the loop
and column thoroughly with MiliQ.
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