Chromatography Forum

Hello all,

I am using a z-HILIC column to separate an extract containing a bioactive compound. I collected fractions and when I assess each fraction for activity, I see some non-specific inhibition against my cells, which I assume is from the solvent/buffer system.

I am using 98% ACN and 98% H2O, both with 7mM Ammonium Acetate as my buffer. I dry down samples with N2 (compound is most stable with this method of drying) after fraction collection. The fraction volume collected is 100x greater than the volume I must resuspend in to detect biological activity.

I noticed between 10-20 minutes of my run (when H2O is a larger portion of the gradient), a large amount of what appears to be ammonium acetate salt remains at the bottom of my tube (and it smells strongly). I have gone as far as drying these fractions 3x to remove the buffer, but I am still seeing the inhibitory effect of acetate on my cells.

Why can't I remove the volatile buffer?

Are you heating?

I would suggest that you re-suspend each solid residue sample in about 5 mL of water, transfer to a round-bottom flask, freeze the aqueous solution in a thin layer around the bulb of the RB-flask and apply to a lyophilizer.

Take down to dryness; repeat dissolution in water and lyophilize again; repeat as necessary.

Do not use heat on your bio-active samples!

Regards,
JMB

The thing is, my compound is not stable in the lyophilizer, so thats why i resorted to N2 drying.

What about resuspending in ACN to cause precipitation due to low solubility... then collect supernatant and dry down and resuspend in H2O?

Any other suggestions?

Still wondering why ammonium acetate isn't readily being removed. Maybe forming another salt with my matrix solution?

Maybe you can provide more info/details on your (chromatographic) process, your molecule?
What do you know about the structure of your target molecule?

I'm not familiar with the z-hilic but when it elutes with high water content in your mobile phase, then it may be enough lipophilic to be retained on a C18-SPE cartridge? Then the buffer could just be washed off, then eluted in a small volume.

If your target molecule is non-ionic, then maybe full-deionizing with serial cationic+anionic exchange resins (e.g. as SPE cartridge) maybe an option. (Maybe the 7mM is too high ionic load, but just providing some thoughts)

If your target is ionic, then it may be separated from the buffer by direct ionexchange chromatography?

Depending on the molecule size, maybe size exclusion or molecular sieves may be another options (trapping the ammonium acetate, while your target just flow through)

Maybe you can provide more info/details on your (chromatographic) process, your molecule?
What do you know about the structure of your target molecule?

I'm not familiar with the z-hilic but when it elutes with high water content in your mobile phase, then it may be enough lipophilic to be retained on a C18-SPE cartridge? Then the buffer could just be washed off, then eluted in a small volume.

If your target molecule is non-ionic, then maybe full-deionizing with serial cationic+anionic exchange resins (e.g. as SPE cartridge) maybe an option. (Maybe the 7mM is too high ionic load, but just providing some thoughts)

If your target is ionic, then it may be separated from the buffer by direct ionexchange chromatography?

Depending on the molecule size, maybe size exclusion or molecular sieves may be another options (trapping the ammonium acetate, while your target just flow through)

I do not know too much about my metabolite, as it is an unknown/novel compound. I do think it is cationic, as it does not bind at all to a C18/HLB cartridge if the solution is acidified (does this assumption sound correct?)

If I acidify the solution and pass it through a mixed anion exchange column, do you think I could just collect the unbound flow-through. But at low pH the acetate wouldn't be ionized. Acetate is toxic to the cells, but not sure how ammonium would affect the cells and is something I would want to get rid of.
It may be simpler just to dry just and resuspend in acetonitrile to cause salt precipitation? Solubility will be <10mM, which is not problematic. I can just centrifuge the precipitate?

If i use a cation exchange and acidify the solution, will acetic acid bind to the reverse phase in mixed-mode exchange? Then what can i wash it with to ensure the acetic acid is removed but my cationic compound is retained, keeping in mind the wash solvent should be non-toxic to cells.