TLC Separation of Nucleosides

[mchen518]

Hello, I'm fairly new to organic synthesis, and I have a fairly basic question to ask about solvent/stationary phase choice--

I'm trying to purify a nucleoside (7-8 isomers of the nucleoside are present in the same mixture) using either silica or alumina stationary phase, but I cannot get my nucleoside to move far off of the baseline. Additionally, I get an Rf of up to 0.2-0.3 with no discrete bands--it is one continuous smear. I've tried various polar solvents for silica phase, including 5-20% MeOH in DCM. On alumina, I've tried every mixture of acetonitrile, methanol, and water, including 100% ACN and 100% MeOH. The literature seems to be fairly sparse on this subject. By the way, the nucleobase the sugar is attached to is rich in amines, so I'm putting 0.5-2% triethylamine into the mobile phase.

I have seen that people will use RP-TLC to purify these nucleosides, but do any of you have suggestions on how to potentially accomplish separation on NP silica or alumina? I really would like to avoid buying a reverse stationary phase as it is significantly more expensive.

Thanks!

[tom jupille]

The literature seems to be fairly sparse on this subject.

One possible explanation for that is that it's not really a good approach. There is a reason why you see them mostly done by reversed-phase .

That said, if you wanted to keep trying, the best shot would be to exploit a "HILIC" type of mechanism. That would involve the use of acetonitrile with about 10-40% of an aqueous buffer (the aqueous buffer is the "strong" solvent in this case. The catch is that I'm not at all sure it would work with TLC because of the need to have a stable equilibrium between bound water on the silica (which is the actual stationary phase) and your mobile phase (TLC is inherently a non-equilibrium process). You might get away with it by equilibrating your plates in a moist atmosphere ahead of time.

[mchen518]

Thanks for your response Tom, I appreciate it. I'll try to see if this HILIC approach will work.

In the meantime, I found one paper that separated similar compounds on a silica TLC plate using a somewhat strange mobile phase: 1-propanol: MeOH: NH4OH (33%): H2O (45:15:30:10).

I was always told that silica TLC plates were incompatible with water and that the max amount of MeOH on these plates should be ~15-20%. I gather that this solvent composition is not useful with flash chromatography, but maybe prep TLC could work.

Just in case any of you are wondering, I'm not using RP-HPLC because most of our instruments are tied up right now and the separations I'm getting are not good enough for prep scale.

[krickos]

althoug "extreme" it makes some sense, with a few hydrogen bindig groups you normally can use as you stated a few % of an aminine or saturatiung phase with ammonia (removing water) etc to balance the silanol groups, but considering the amount of hydorgen binding groups on nucleosides,well harder to get them moving..

[Jack Silver]

Thanks for your response Tom, I appreciate it. I'll try to see if this HILIC approach will work.

In the meantime, I found one paper that separated similar compounds on a silica TLC plate using a somewhat strange mobile phase: 1-propanol: MeOH: NH4OH (33%): H2O (45:15:30:10).

I was always told that silica TLC plates were incompatible with water and that the max amount of MeOH on these plates should be ~15-20%. I gather that this solvent composition is not useful with flash chromatography, but maybe prep TLC could work.

Just in case any of you are wondering, I'm not using RP-HPLC because most of our instruments are tied up right now and the separations I'm getting are not good enough for prep scale.

You might have issues with the binder used for the TLC plate in this solvent system. Keep in mind that silica does dissolve under basic conditions in water, but I don't know if 40% water (the 30 NH4OH and 10 water in the system) is polar enough to allow the silica to dissolve. Alumina won't have the pH problem, but the binder may not be compatible with water.

Regarding flash chromatography, I've had good results to 100% methanol, and have run silica HILIC.