Chromatography Forum

Why must RP SPE material be conditioned with methanol, followed by water before use? If not conditioned, is binding or elution/recovery affected?

Yes, because there will be a good chance that the cartridge bed is not thoroughly "wet" (i.e., you won't have good contact between the bed and your sample).

A long time ago I tried to improve HPLC of vasopressin via a SPE cleanup, using a C-18 cartridge. The vasopressin got hung up on the cartridge, so I gave up that cleaning step. A while later I bought vasopressin RIA kits that used exactly the same cartridge for the same purpose. It worked quite well, the only difference to what I did a few years earlier was a methanol washing step prior to sample application. I don´t know exactly why the peptide got hung up without the methanol washing step.

Was is the exact same C18 made by the same manufacturer?

By "hung up" do you mean recovery was poor?

What if I'm not interested in recovery, only removing an impurity from an aqueous solution?

I always thought that the methanol conditioning was to get the C18 phase "uncollapsed" - because the cartridges ship dry the phase is in the same state as a reverse phase column that has been run in 100% aqueous, the C18 chains are lying flat on the support and so do not have the capacity that they should.

Peter

With "hung up" I meant that it stayed all on the cartridge and that I couldn´t get it off with a bunch of solutions (don´t remember what I used, one of them was about identical to what they used in the RIA kit).
I used the same cartridge as the kit manufacturer, although I don´t expect the batch having been the same.

The reason that I was asking is because you should not see variation in sorbent performance when using the same manufacturer. (The company that I work for manufactures SPE sorbents) If you do see variation, the manufacturer has a big quality issue.

You will see variation from manufacturer to manufacturer, because no two companies make it the same way or use the same raw materials.

I agree with Peter's explanation of what the methanol is for.

Don

Interesting. Since polystyrene/DVB is a different animal than C18 altogether, I wonder if conditioning would have similar effect.

It helps, but it isn't as important as it is with silica based sorbents. The most important thing that it does is it ensures a clean product.

Don, I got different results, because I didn´t wash with MeOH. When I did this the washing step was not yet "invented".

Supposedly there are active sites onto which peptides irreversibly bind. It would be pretty discouraging, though, if this contributed to a measurable loss in recovery.

It would be pretty discouraging, though, if this contributed to a measurable loss in recovery.

Actually, it can, which is why you want to look at recovery when you are developing/validating a method.

Well, recovery was 0% before washing with MeOH. My guess was that the peptide caked out on the stat phase. One sometimes sees this in TLC also.

It would be pretty discouraging, though, if this contributed to a measurable loss in recovery.

Actually, it can, which is why you want to look at recovery when you are developing/validating a method.

What is the origin of these so-called "active sites"? Are these hydrolyzed silanols?