I have a HiLoad 16/60 Superdex 75 pg size exclusion chromatography column that is becoming yellowish on top (inlet). Does anyone know of possible causes? Can this affect column performance? How can I get rid of it? The column has been used to separate 500 mM imidazole-containing protein fractions originating from a first step of metal-chelate affinity chromatography (nickel) of his-tagged proteins overproduced in E. coli. Thanks
This is insoluble or strongly bound material. It will slightly affect your column performance. Isopropanol or EDTA water solution could be helpful.
Get rid of that upper gel, may be contaminated by organic material and microbial growth.
Refill your column with new and clean gel. Wash your column (saline buffer will be ok). Re-equilibrate your gel with running buffer.
No color should be present in any part of the column.
Hector Nolasco
You can try to wash column by 0.5V sodium hydroxide if it doesn't help try to wash 10% acetonitrile.
Some pigments can bind very strongly. It can also be denatured proteins sticking to the resin. Do a good cleaning wit NaOH (up to 1 M is OK for short term exposure, but do not store the column in NaOH). 2-3 column volumes at a flowrate of around 20 cm/h. Equilibrate with buffer immediately - preferably with a buffer also containing some NaCl. If this does not remove the colour wash with 30% isopropanol.
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