Chromatography Forum

Hi all,

this is the problem. I have a compound that I have synthesised that is soluble in chlorinated solvent such as DCM or chloroform and I would really like to purify it! It turns out that it has a really high affinity for silica so that as soon as I put it on a silica column (not hplc but flash column) it sticks to it and won't come off even with water or everything I tried.

I would like to purify it using hplc. The set up that is in my lab has a c18 column and acetonitrile water mixture as eluent. My question is can I inject it? and in what injection solvent? It's not soluble in water/acetonitrile, and I am afraid that if I inject it (after being dissolved in dcm) it will just precipitate in contact with the water/acetonitrile eluent and clog the system or anyway do something bad to it.

Any suggestion is much appreciated!

Man

What is it?

It's a novel organic molecule soluble in organic solvents to which I have linked another molecule that, on its own, is water soluble. So I have the final molecule that has a big apolar body and a water soluble tail.
I hope this helps,

Man

Dear all,

no one interested? thanks for your attention anyway, I thought this would be a challenging question but you seem to be more interested to answer questions from complete newbies.

if it's not soluble in ACN at all, then how do you want to chromatograph/elute it with ACN?
so you have to change your eluents anyway.
And then maybe you will find something that works for your molecule.
Maybe something like Isopropanol or THF or find some ternary one-phase system together with DCM?

You know how your molecule looks like and have it on your hand to make the experiments.

I don't think it is a matter of lack of interst, it is a matter of the problem presented. The compund soluable in chloroform or DCM, not soluable in AcN/water and sticks to sillica - and you want a separation.

You tried to strip the compund off the sillica with water. So is the compund soluable in water? And is there pH sensitivity to the soluability?

It suggests going to some other stationary phase than sillica and a mobile phase that is or includes a chlorinated solvent.

There are other stationary phases than sillica and C18, starch comes to mind. This may not work well in HPLC, but paired with the chlorinated solvent may help to purify the compund.

For difficult molecules, I have ocasionally resorted to THF as a solvent.

But without details, it is hard to give an answer rich in details - or as you have noticed with the newbies, much of an answer at all.

Back to basics.
I might try doing some normal phase chromatography on a cyano phase.
You might want to TLC to see what mobile phase it moves on before spending a lot of time on HPLC or flash
If the aqueous soluble portion is ionic suppress ionization if possible otherwise ion pair.

Cool guys!

now I really feel like a newbie!

I know how to use and HPLC and I am the one running it in our lab but I am not an expert at all. I am more of an organic/inorganic chemist.

For KDF:
I have notice that we do have some Phenomenex cyano HPLC columns and I will let you know later which ones to be precise. To be honest I understand they are more polar so opposite to c18 (or almost right?) but I have no idea where to start as for the choice of eluent mixture in normal phase.

I have tried TLC on silica only and believe me when I say that even the most polar mixture I know hasn't moved the spot at all, I even tried to dissolve the silica to which my compound had been adsorbed and extract it with dcm with no success.

The water soluble portion is not ionic but anyway how should I suppress ionisation? buffer?

Thanks to everyone that has joined the conversation!

Man

A cyano column is a bit of a chameleon because of it's "intermediate" polarity. Used with something like Methanol/water mobile phase, it will be a reversed-phase column (solvent more polar than column). Used with something like hexane or DCM, it will behave as a normal-phase column (solvent less polar than column). Since your molecule is soluble in DCM, one possibility is to use a cyano column with non-polar solvents. I would not recommend using the *same* physical column for both modes (getting complete wash-out is a pain!), but the same type would work.

A good starting point is to run a gradient starting with 1% isopropanol/hexane running up to 100% isopropanol. When (if!) you get elution, the composition will give you a reasonable guess as to solvent strength. If your compound is unretained, run another gradient from hexane to DCM.

Your compounds solubility in DCM suggests that it has a mode of substantially hiding its polar nature.
I do normal phase separations for a living, generally by supercritical fluid chromatography (SFC) which uses CO2 [about the polarity of hexane] and methanol as the mobile phase.

The fact that you stated the compound is permanently retained on silica leads me to believe the polar aspect is not totally hidden.
Again in SFC, this would be dealt with by addition of an acidic or basic additive [<0.5%] to a CO2:Methanol mobile phase to compete with active silanol groups of the silica. In liquid normal phase you would have to use hexane:ethanol with an acidic or basic additive [generally <0.5%] since methanol is not very miscible.
I suggested cyano since it is nearly the weakest normal phase, but I was not kidding about TLC. You can run tons of combinations of mobile phases on Silica or cyano plates just to see what moves the compound. Once it moves off the baseline, you can get a separation.

If this works and you are doing preparative cleanup [e.g. flash], choose an additive with a boiling point lower than the solvent to get rid of it easily by rotovap.

Good luck

I wanted to add one more thing. If you do decide to go back to TLC don't waste time. Try a mobile phase of 74%hexane:25%Isopropanol and 1% concentrated ammonia in one case [basic]. Replace the ammonia with 1% TFA in the other case [acidic additive]. You can also. of course, use DCM with an acid or base additive. If you still can't get any movement, you will have to use a bonded phase.

When you go to HPLC, any residual water will tear up your column. Ammonium hydroxide is not recommended, Use an amine such as n- or iso-propyl amine if your compound will not react with it. otherwise a secondary of ternary amine is needed.