[opsomath]

Hi all,

(cross-posted from Reddit)

I'm an organic chemist who is starting to put a toe into the world of bioconjugate chemistry...meaning that all the reactions I'm used to doing in organic solvents, I now have to do in buffers. I made a derivative of a particular molecule (a dye molecule, as it happens) and it's extremely water-soluble and not really soluble in organics at all. The crude reaction mixture has a lot of water, and considerable salt (mostly NaCl) as well as some small molecule, low MW byproducts which are also very water-loving. Does anyone with experience in these things have some good ideas for initial cleanup steps, like gel filtration or anything? This is a very practical question so the more specifics you can give (like the part number of a gel filtration plug filter) the better the answer.

Thanks!

[Honclbrif]

When you've got really polar material, reverse phase is usually the way to go. The best option is prep HPLC. If that's unavailable, you can get C18 chromatographic media in bulk or pre-packed into cartridges, but it tends to be dearly expensive. Solvent is usually something aqueous, with methanol or acetonitrile as the organic phase. If you need to adjust pH, it can be done with acetic acid, or which ever amine is favorite.

Since you're trying to get a dye, your life will be a little easier. Just look for bands that are the right color.

[Dan]

If you can move it on a silica TLC plate with water/IPA/EtOAc 1:2:7, then you can column it on silica. That is one of the most polar systems I know for silica. If your compound is basic or acidic you can adjust the pH with NEt₃ or AcOH, though you may want to consider ion exchange in these cases, depending on the contaminants.

[opsomath]

I dug up some ion exchange (quaternary ammonium) solid-phase cartridges in our lab. I managed to elute the material onto one of them with DI water, then washed with several portions of water. Since the stuff is bright blue, it was obvious that it was remaining on the cartridge, but hopefully my nonionic polar byproducts are gone. (The stuff itself has two sulfonic acid groups, so it is anionic at all normal pH values so is easy to stick onto a positive SAX column) Eluting with 0.1M phosphate buffer got the material off the column. Now it's in buffer, but that might be good enough for my purposes; I think I'll try linking it to a protein next and see what happens.

[enahs]

My first thought.

Remove water by rotavap or pumping it off.

If your material it is not soluble in methanol, I would try loading it onto a thimble into a soxhlet extractor, and let it "extract" with MeOH for a few days. Hoping everything you do not want comes out with the methanol, and you are left with a purified product still in the thimble.
Almost like a reverse extraction.

NaCl is soluble in MeOH at ~ 14g NaCl/ 1kg MeOH. So, it will take a while, but it can get it that out too.

[zolarpwr]

Could you try a lipophilic cation, like tetrabutylammonium? Maybe then your dye can be extracted into organic solvent, leaving your salts behind.