Dear Tim

Please make your problem still more clear, on instrumental conditions and configurations.

Tim,

Additional details are necessary to comment on the problem. What type of chromatography are you performing. You mentioned both conductivity and absorbance. 1 N NaOH is considered very high pH. Is your stationary phase stable under that condition?

Please elaborate as much as you can.

Thanks.

SC

Hey guys,

Thanks for answering

So, I see 1 N NaOH has a high absorbance at 280 nm during anion exchange chromatography runs using both membrane supports and resin beads (Sepharose FF Q). All the membranes utilize the Q functional group. I use the NaOH to sanitize the column/membranes and pass 4-6 CV/MV at a flow rate of 1 CV/MV/min. The flow rate  varies by what column/membrane I'm using, but its between 0.6 and 1.5 mL/min.  I use 50 mM Tris buffer pH 8 as my equilibration buffer and 1 M NaCl as my elution buffer.

I also see it when I'm cleaning the Akta Avant system using 1 N NaOH and when I'm flowing NaOH through the system bypassing the column. I don't know the path length of the uv-vis detector. I don't see absorbance in any of my other solutions and all parts (connectors, pumps, tubing) are made from PEEK and are sourced from GE.  I know abs. at 280 nm is often from aromatic rings. My only guess is that the filter is letting in small bacteria, which are then being denatured by the NaOH, but I would imagine that would be a common problem that I could find others with this issue. Also, the pH is high, around 12ish. Let me know if there is any more details I should provide. 

Thanks,

Tim Brantley

While not being 100% sure I would assume there is 2 possibilities a) you solution as such has the absorbance (check with a spectrophotometer) or b) 1N NaOH is pretty harsh to anything that might be left in your system from any old samples in your flow path (I assume there is a reason why you statred washing the system with NaOH). if B is the case one would expect the absorbance going down after a while. . On top of things it is not uncommon that there devellop biofilms in chromatographic systems that are run 100% aquaous.

concerning your issue ,for the chemical stability in chromatography, cleaning is performed at low flow rates using sodium hydroxide (500 mM sodium hydroxide for Superdex and Superose columns and 200 mM sodium hydroxide for Sephacryl columns). Note that the column should never be stored in sodium hydroxide. Equilibrate the column immediately after the cleaning with water followed by running buffer. If cleaning using sodium hydroxide is not sufficient, additional cleaning using for example 500 mM acetic acid can be useful.  Check the instruction for your specific column on details of the cleaning procedure. Also the following solutions can be used for cleaning : up to 30% acetonitrile, up to 1M NaOH, up to 70% ethanol, up to 70% acetic acid, up to 30% isopropanol or up to 0.1M HCl.

I hope this  these words help you in your study.

Thanks

NaOH has a significant absorption at 280.