Chromatography Forum

Is there any advice how to clean column after analysis- eluent is CH3CN:phosphate buffer 20 mM (pH 2.5).
Column is Varian Intersil 5- ODS-2 250 mm, 4,6 mm.
Is it ok to wash it with water and then with 100 % CH3CN?
For how long does it have to wash with water?Ovenight?

Any aditional advice on thios subject; column and eluent with buffer?


Thanks a lot!

be sure to read the column leaflet first, and abide by the directions therein.

50:50 Organic:Water is sufficient for most column washes, but if your mobile phase contained a high percentage of a concentrated buffer phase, you should up the water content to facilitate the washing. Always keep some organic in your column wash though, as it actually facilitates the water washing the pores.

an hour or less will be sufficient to remove the bulk of the buffered phase, then you can up the percentage organic to clean the column of organics. some people like to reverse the column here. again, keep some water in the strong column wash - it is recommended to keep the silanol groups hydrated

How about just using H2O, or a very high % H2O as a first wash after there is phosphate on the column to prevent the precipitation of phosphate (which could occur if one washes with high % organic). Many have suggested to use a pure aprotic organic like ACN to store the column without risking solvolysis. You can usually "see" the cleaning if monitored by a UV detector (not recommended if you have immense crud, that may stick to the detector cell, on the column).
adoyle, it shure would be instructive if you could provide some evidence or refs on some of your suggestions (always have some organics in the water, even though one follows this up with organic...., and always keep some water in the organic...).

Wash with a acetonitrile/water mix in roughly the same proportion as your acetonitrile/buffer mobile phase. About 10-15 minutes at 1 mL/min for a 4.6x250mm column. For short term storage that is good enough. For long term storage, about 5 minutes with the acetonitrile/water mix followed by 10 minutes with 100% acetonitrile. The initial solvent/water blend will remove the buffer salts with no risk of precipitation.

On always keeping some water in the column wash/storage solvent:
I have no references, just have been advised by my superiors more than once. Most reverse phase columns come stored in water/organic mixtures, so I think following the practices of the manufacturers is obviously following good practices.

On always keeping some organic in the column wash:
I very recently completed a three day intensive training course in HPLC.
There, the lecturer advised us that a totally aqueous phase can have problems in penetrating the relatively lypophilic pores. He actually went as far as to say the thermodynamics of the system sees the water eventually ejected from the hydrophobic pores of the stationery phase particles. The presence of organic facilitates the pore penetration, and subsequent pore washing.

Hope this is suitably instructive

On water in organics: Manufacturers have to sell columns.

On organics in water: There is quite a bit of info in this forum on dewetting. Modern RP columns usually don´t do this. Even if they do you probably got pores cleaned before dewetting happens, or you get it while following up with pure organic.
Anyway. I am lazy, so if I think it will do the job (one gets some experience in time) I just do the extremes : From buffered mobile phase to pure H2O to pure organic. Now if you have some tenacious ions (or whatever) hung up on your column (for instance, after injecting reaction media in ionic liquids) you will need more than pure water or org./water to clean out in acceptable times (lets say 1h). In a case of an ionic liquid medium an extensive wash with a low pH high concentration phosphate buffer did a good job.