Chromatography Forum

help!

I accidentally mixed up my samples and injected plasma (untreated) into my column (Zorbax SB C-18). How can I fix/recover it?

There is no guarantee that you can. In general, backflushing the column with something that will solubilize protein is your best hope (perhaps something like isopropanol with a few percent DMSO?). The most cost-effective approach might be to simply get a new column, and then try to revive this one in you spare time.

Here is a "harsh" wash procedure designed for RP silica columns. Make sure you check the solvent, temperature, and acid compatibility of your column and HPLC system. I don't have that information for Zorbax columns and would be concerned that this procedure may negatively impact your column. This is a procedure we developed that works really well to increase the life of columns being used for crashed plasma samples. We have seen this significantly reduce back pressure on columns that were near the end of their useful life.

Start by inverting the column. I wouldn't recommend having any detectors inline during this procedure.

1. 20-50mL of DI water at 50 C. (to remove salts)
2. 20-50mL of 0.1% phosphoric acid (to remove water soluble basics and weak acids)
3. 20-50mL of DI water at 50 C to flush the acid
4. 20-50mL of 50/50 methanol water
5. 20-50mL of 70/30 IPA water
6. 20-30mL of 100 IPA
7. 20-30mL of Hexane (check the warnings on pump seals and injector seals) (this should remove any fatty substances
8. Repeat step 6
9. Repeat step 5
10. Repeat step 4

Mike has solvents/solutions in there which would precipitate proteins onto the column and/or frits (depending on where the protein is hanging). Apparently he had used samples which were essentially protein-free. I would recommend chaotropic solutions like 6+ molar urea.
There are many discussions here on more "stubborn" builtup of proteins.

hello all

I've done the exact same thing, albeit most of it precipitated.

I have been using a SCX column from Phenomenex (150 x 10mm; 10um particle size), to analyse C-11-choline in cancer patients' plasma. the reason for the large column is because I am without a rotovap, therefore I have to crash the protein with perchloric acid (2M) and inject everything onto the column. Initially I was advised to go with 75uL perchloric for every 750uL plasma, but as I found out, this wasn't enough.

I think the column is recoverable, but just wondering if there's a good cleaning/regen protocol for getting proteins off SCX column?
I was thinking just run a couple of tfa/propanol gradients, but all suggestions welcome

thanks

P.S. I've found a protocol that should do it...

- 20 column volumes (cv) 500 mM phosphate buffer (I've used sodium phosphate)

- 20 cv 10% acetic acid
- 10 cv water
- 20 cv phosphate buffer
- 10 cv water
- 20 cv 5M urea
- 20 cv water

I'm using urea to remove the protein - replace this with MeOH if not worried about proteins