After several runs with samples containing proteins and gold nanoparticles I noticed that backpressure progressively increased. Further, the baseline of the detectors (fluorimeter and spectrophotometer) are not stable in showing signal spikes. I think the problem is the presence of some particulate in the column although I always filtered the elution solvent and centrifuged all samples runned.
Dear Matteo
An increase in back-pressure is generally due to blockage of your column matrix, and in my experience it could be due to particulate, sample precipitation, and or fatty residue from biological samples. I found that the most efficient way to revive any column with back-pressure problems is to turn the column around ("wrong" way flow) and wash it overnight at about 20% of you normal flow with you normal wash buffer or solvent. The reason for this is that most of the blockage issues would most probably be in the first part of the column and in reversing the flow would prevent the "blockage" moving through the whole of the column matrix. You can also inject a small volume of DMSO on to you column to remove the aggregated proteins. You can also wash the column with the most non-polar solvent it can accommodate, followed by water and then possibly 1 M NaCl to compete with metals contaminating the column.
Hope it helps.
Regards
Marina
Thanks for answer. I will try with DMSO and let you know.
Best regards,
Matteo
Dear Marina, DMSO work in cleaning the column but back pressure remains too high. However i will try to use it as a good candidate. Thanks
Dear Matteo
Glad to hear DMSO helped.This is the a first step to get rid of the proteins and possibly most of the biological contaminants. Now the question is what else is blocking you column. Could it be metal aggregates or is it fibrous material from dust/filters that is sitting in the fret or beginning of the column? If it is metals you can "reverse" wash your column with whatever solution/solvent that work best when you are preparing the metals for the nano-particles and that is compatible with the column. If it is the latter, you can try a bit risky last resort option - take the column of your system, remove the tubing and suspend just about 5 mm of the inlet part of the column in analytical quality filtered water or methanol and sonicate for 1-2 minutes, then put it back on you system in reverse and wash slowly with water or methanol of mixture. You can repeat this 2 to 3 times, but be aware that too much sonication can lead to matrix degradation and failure.
Good luck!
Regards
Marina
Effectively, problems in backpressure came out when i started using gold nanoparticles combined to my protein. However, prior to unmount the column and wash the frits, i will try some other "strong" solution. If this does note work, i'll do as you suggest!
- Badges
- Science method