chromatography - CIP

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JackBean
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chromatography - CIP

Post by JackBean » Thu Feb 25, 2010 7:58 am

Hi everybody,

there isn't any appropriate forum for this, so I will ask here.

I'm running some chromatographies with crude extracts from maize on Octyl Sep and High Q and because the extract is really dirty (just yeasterday, the Octyl Sep was totally black :lol:, now it looks a little better...), I need to clean the columns often and efficiently. So, I was looking to manuals, how to do that, but I have still two question (more like why, insted of how)

1)in Octyl Sep's manual they write:
alternatively (to only 70% EtOH), wash the column with 1-2 bed volumes of 0.5% non-ionic detergent (e.g. in 1M acetic acid), followed by 5 bed volumes of 70% ethanol to remove detergent, and by 3-4 volumes of water.

The question is - why should I use the detergent in acetic acid? I understand, that detergent will compete with the contaminants, so they will be washed out, so for that I need as hydrophobic enviroment as possible, but if I add HAc, that is contradictory, isn't that?
Just another question came up. Because for the ethanol I need to use really low flow rate, I'm using 50% EtOH. Do you think, that's enough for removing of the detergent?

2) in High Q's manual there is:
Use high salt buffer for regeneration,
...
For lipids or hydrophobically bound contaminants, wash with ... or 10-30% acetic acid.

Remove additional contaminants with 0.4M NaCl in 1% acetic acid/1% phosphoric acid.


They evokes for me, to use high salt buffer, than 30% acid and than NaCl in acid. So, you have like high salt -> no salt -> little salt and no acid -> high acid -> little acid. Wouldn't be more logical to use first the NaCl in acid and than concentrated acid?

Thanks everybody :)
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