Conditioning sorbant in RP-SPE

[DJ]

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

[tom jupille]

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).

[HW Mueller]

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.

[Don Shelly]

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

[DJ]

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?

[Peter Apps]

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

[HW Mueller]

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.

[Don Shelly]

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

[DJ]

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

[Don Shelly]

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.

[HW Mueller]

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

[DJ]

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.

[tom jupille]

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.

[HW Mueller]

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.

[DJ]

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?