detergent removal from C18 column

[JohnDoe553]

hello all,

we have run an assay for a detergent on our hplc system (rp-hplc) and now all other methods are screwed up, it seems that it binds so strongly that we washed the system for days with pure acn, meoh, ipa, organic aqueous mixtures with salts but it still causes us problems. it seems that its not just in the column but also in the injector, lines etc..

how can u get this stuff out??? pls help

thank you

[Hollow]

I would say: "forget about that column, use a new one" (keep the actual one for this detergent method")

For the system: if possible (ask manufacturer), try strong acids like HNO3 6mol/L or phosphoric acid 30%.
Or the opposite, conc. bases like sodium hydroxide 5mol/L or ammonium hydroxide about 2 mol/L
then wash until neutral with pure water.

Attention: DO NOT collect such cleaning effluent in the "normal" waste container! Use a separate flask or bottle instead!

[unmgvar]

what was the sample pH?

[Gerhard Kratz]

Don't forget to wash the system with water after using acid or base washing step. My recommendation is to use hot water.
And keep the column as a souvenir, use a new one.

[HiteshInamdar]

" we have run an assay for a detergent on our hplc system (rp-hplc) and now all other methods are screwed up, it seems that it binds so strongly that we washed the system for days with pure acn, meoh, ipa, organic aqueous mixtures with salts but it still causes us problems. it seems that its not just in the column but also in the injector, lines etc.."

Hi,

If ur using mobile phase as a detergent (ion-pairing reagent) then accumulation of ion-pair will happen on the column bed. So, If you make a column dedicated to a specific sample or a product will make your analysis simple and effective. Second and the formost thing is Column Washing. When your are working on some Dirty sample or Ion-pair chromatography, column washing should be proper in order to get reprodusability. Its better to wash Column primarily with water (100%) for longer time. Then go for Water:MeCN or MeoH (50:50) and the finally while closing up of method use higher organic. This will make washing up of detergent from the column bed.

Even after doing so, the problem persist, then Wash the column with 3% OPA overnight and then with column washing cycle. This will Definitely cure your column.

If the number of injections are high then try the column regeneration.

[HPLCaddict]

If I understood correctly, the OP had detergent SAMPLES, not detergent in the eluent. So this is not about getting ion-pairing reagents off the column (and I really don't want to start a new thread about this, there have been some in the past; and I do not trust those general washing procedures like the one presented above...) but getting the analyte off the system.

Depending on the design of the autosampler (it is an autosampler, or ist it a manual injection valve?) just pumping whatever solvent through the system will reach only parts of the autosampler. You may have to switch the autosampler to "purge" or "flush" or manually switch the injection valve in order to wash the sample loop and/or injection needle. Consult the manual if there are any information provided. A series of high volume injections with the cleaning solutions might also be a good idea to clean the autosampler, but be careful with the serious stuff (nitric acid etc.) - verify if your autosampler can handle it. If the contamination is really sticky, it might be necessary to change the rotor seal.

Apart from that, first try to get some information about that detergent - is it acidic, basic or neutral? Solubility in different solvents? This might help you to choose an appropriate cleaning solvent. If you don't have any information, I'd suggest a "mashup" cleaning procedure of the ones already presented: hot water, acidic, water, basic, water.
Oh, yes, as already suggested: Dedicate that column to this procedure and use a new one for everything else.