How to make an Alliance inert?

[Mattias]

Hi,

I have a problem with adsorption of 5-aminosalicylic acid (5-ASA) to my LC-system (Waters Alliance). The method uses a 10 µl injections and I need to inject up to 10 standards before the areas become stable (always increasing trend).

I have tested to condition the system with a 100 µl injection, but then the system seems to become oversaturated with 5-ASA (and I get a decreasing trend for several injections).

I have also injected samples without the column, and I seem exactly the same trends (i.e. the adsorption of 5-ASA must take place somewhere between the injection needle and the column inlet).

I have passivated the system with nitric acid, but it didn't help. Are there any ways to cover the stainless steel with something, to make it less prone to adsorption? Or do you have any other ideas?

[nonagon]

Try passivation by chelation - common procedure for metal pssivation sometimes used in bioanalyses.
Use EDTA Sodium salt (Merck 324503 or whatever) about 0.05M.
Run it slowly through your system without column, inject it a few times to take care of the needle and injector, wash with water, run your application and than tell me if it works because I have the same issue with a certain polypeptide and I am too scared of trying it myself.

Laughing, of course, I would never recommend doing something I have never tried myself.
I join Mattias in asking for help with passivation of HPLC wetted metal components.

[Mattias]

Try passivation by chelation - common procedure for metal pssivation sometimes used in bioanalyses.
Use EDTA Sodium salt (Merck 324503 or whatever) about 0.05M.
Run it slowly through your system without column, inject it a few times to take care of the needle and injector, wash with water, run your application and than tell me if it works because I have the same issue with a certain polypeptide and I am too scared of trying it myself.

Laughing, of course, I would never recommend doing something I have never tried myself.
I join Mattias in asking for help with passivation of HPLC wetted metal components.

Thank you for your reply!

I have actually tested this procedure as well, and it works really well to get a nice baseline. Unfortunately, it does not seem to affect the adsorption.

I got so frustrated that I pumped and injected a solution of Sigmacote (silicone in hexane) mixed with isopropanol though the system (5 ml of Sigmacote in 100 ml IPA). I have no idea if this can hurt the instrument, but I needed to do something.

It actually seems to work! After washing I injected five standards with an RSD of 0.07%. No increasing trend at all. The peak area of the standards increased also by 3%, compared to before the "treatment". I take this as a sign that the adsorption has decreased/disappeared.

Now I have to determine if the intrument can take this kind of treatment, and how long the effect will last.

[zokitano]

Hi Mattias,

If Sigmacote won't give you stability and intertness of the HPLC system for a longer period, i suggest to consider adding an EDTA (50-250 micromolar end concentration) in the final injection solution, together with your analyte(s) (in the standards and in the samples). I had similar problems with derivatives of salicylic acid and I succeeded to obtain sharper and more consistent peaks using an EDTA as injection solution additive.

Before, I tried to passivate the system using several procedures (haven't tried the Sigmacote), but quickly after passivization I observed the same peak shape and reproducibility problems.

Hope this hepls.
Best regards