Chromatography Forum

I've used Dodecyl sulfate in my eluent, with a C18 column, How should i wash my column from this ion pair (regenerate the column)???

I'd use alcohol-water mix. Dodecyl sulfate is not retained on RP-18, should wash out.

I think dodecylsulfate is retained on C18, see e.g:

http://chromatographyonline.findanalyti ... rticle.pdf

But I agree that gradients of water:MeOH or water:ACN should wash it out.

Best

It will be washed out, but not 100%. Ion pair reagent is always like a surface modifier. Column should be used only for this application. Method development with such a treated column can cause major frustration when later a brand new column is used for the developed method.

Silica based C18 phase has un-reacted silanol groups on the surface which are usually deprotonated (or carrying negative charges). Thus anionic surfactant like SDS is retained by the net effect of RP intereaction from C18 minus electrostatic repulsion between the sulfate group of SDS and deprotonated silanol group on the silica surface. At the result, anionic ion-pairing agents won't stick to the standard C18 phase. In fact, the k' of SDS on the Acclaim 120 C18 column using MeCN/NH4OAc buffer 50:50 (v/v) is around 3. My firsthand experience indicates that washing the column with 90% acetonitrile or acetone for 15 column volumes is sufficient to remove SDS completely.
However, the use of cationic surfactants or ion-pairing agents is another story. Due to strong electro-static attraction between positively charged ion-pairing agent and the negatively charged silica surface, it is extremely difficult if not impossible to remove the ion-pairing agent completely. Therefore, the column should be dedicated to particular applications which is the frustration that Gerhard mentioned.

For removing ion-pair reagent wash the column with 100ml of 200mm phosphate buffer pH 6.0& methanol 50:50(v/v).

But try to dedicate the column for that analysis only. Instead of washing.

At low pH there are no silanolates (SiO-), so positively charged detergets should go easily at low pH. If I remeber correctly, we came to a conclusion before that this "permanent sticking of detergents" is a myth based on inproper handling.
On the other hand, I have seen much simpler ions and even uncharged material "stick" to columns for extremely uncomfortable lengths of times after grossly overloading the column.
Also, it seems obvious that there are some phenomena operating when solids are involved which are very different from normal solution chemistry. I am very much impressed, as just one example, how 22Na+, a positron emmitter, sticks to the start of silica based columns at pH over ~5 while in small molecule solutions it is seen extremely seldomly that sodium can have a partner to which it sticks like that. (The 22Na+ is washed out very fast even with only a bolus of strong acid or other positive ions).

I tend to agree with XL if we go into detail, but Gerhard has a point.

However, I am quite sure that it is a general experience and opinion that everything works much smoother (better reproducibility), and time consuming washing procedures are avoided when columns are dedicated for certain methods (or method development). A considerable amount of troubleshooting questions are actually raised because of columns used in various methods.

I'm surprised. I had thought of using SDS Sodium Dodecyl Sulfate to regenerate my reverse phase columns and wash protein and other biological material from C18 columns. All the literature I read said detergents like SDS do not wash out and permanently alter the column chemistry, thus cannot be used.

I have used SDS to clean columns off stubborn protein deposits, actually, it was a mixture of SDS and thiothreitol. It has been described here.