[zarhym]

Greetings,

I am having a problem with this product. This chemical is synthezed from buchwald coupling as shown. After reaction, the mixture is purified through a silica column (200-300 mesh silica, PE/EA) and then trituration in hot EtOAc/DCM/PE mixture to afford 17g white powder. On TLC, it is a fluorescence dot under UV light(Rf=0.3, DCM/EA=1:1), which looks very pure. However, on RP-HPLC the purity is about only 90%, while the impurities has higher retention time (less polar). We tried different chromogenic agents such as I₂ and phosphomolybdic acid. Still no impurity is observed on TLC.

Thus, we did a second silica column (300-400mesh silica, DCM/EA) and then trituration in hot EtOAc/DCM mixture. However, the purity on HPLC does not change a lot, even the first 3g elute was cut on column.

The only explaination I have is this weird isomerization. But I cannot convince myself and my client about this isomerization.

Do you think this is possible?

Thanks a lot.

[zarhym]

BTW, I have checked our starting materials, both of them are pure enough on H-NMR.

[rolnor]

How does it look on NMR? Should you not se the isomeres?

[mjc123]

As the isomerisation only involves proton shifts, I would expect it to be fast, and would not expect to see more than one peak in the HPLC. But I may be wrong.

[zarhym]

The attachment is the HNMR after the first column and trituration. The corresponding HPLC indicates 85.58% purity under 254nm and 89.34% purity under 220nm.

I'll post the HNMR after second column/trituration next week.


Thanks.

[pgk]

All the proposed isomers have similar polarity and structure and consequently, the ought to have the same or at least, similar retention time (Rt) in HPLC.
Thus, the HPLC peak at Rt = 7.360 min is the desired product and at Rt = 8.471 min, is obviously the chloro-(deamino guanine) starting material, which happens to have the same Rf in TLC.
You can repeat the TLC by adding a few drops of triethylamine in the mobile phase (EtOAc/DCM/PE/Et3N drops). This is a common practice in column/plate chromatography, for the separation of amines and which may work in your case. 
The third peak at Rt = 9.030 min is probably the dimer of the chloro-(deamino guanine) starting material, which derives from the simultaneous arylation of the pyrrole ring and which is the less polar and whose the surface area of the HPLC peak, seems to fit with the difference of integration between pyrrole N-proton and the pyrrole aromatic proton in the 1H-NMR spectrum.
The yield of the desired product might be increased by working with longer reaction time and/or at higher temperature by choosing another solvent, e.g. diglyme (bp = 162oC) that can be removed by washings with water.
Good Luck.

[phth]

Greetings,

I am having a problem with this product. This chemical is synthezed from buchwald coupling as shown. After reaction, the mixture is purified through a silica column (200-300 mesh silica, PE/EA) and then trituration in hot EtOAc/DCM/PE mixture to afford 17g white powder. On TLC, it is a fluorescence dot under UV light(Rf=0.3, DCM/EA=1:1), which looks very pure. However, on RP-HPLC the purity is about only 90%, while the impurities has higher retention time (less polar). We tried different chromogenic agents such as I₂ and phosphomolybdic acid. Still no impurity is observed on TLC.

Thus, we did a second silica column (300-400mesh silica, DCM/EA) and then trituration in hot EtOAc/DCM mixture. However, the purity on HPLC does not change a lot, even the first 3g elute was cut on column.

The only explaination I have is this weird isomerization. But I cannot convince myself and my client about this isomerization.

Do you think this is possible?

Thanks a lot.

If the structures are in equilibrium then thier ratios will not change. Can you run more than an analytical sample through a HPLC? If the peaks are in equilbrium with one another then the same NMR, and HPLC pattern will be observed for all the retention times.

[zarhym]

We have comfirmed that this is caused by impurities in the sample. These impurities can be observed on HNMR. They are just very robust during purification process.
We will try solve this problem by using prep-HPLC.

Thanks for you help.

[hypervalent_iodine]

We have comfirmed that this is caused by impurities in the sample. These impurities can be observed on HNMR. They are just very robust during purification process.
We will try solve this problem by using prep-HPLC.

Thanks for you help.

Pgk gave some great suggestions you could try first. For example, have you tried the suggested method of adding TEA to your mobile phase? I usually pretreat the plate by running solvent containing 5% TEA, and then run my samples with 1-2% TEA. It’s a good option to try and can help a fair bit with separating these sorts of compounds. Another option is basic alumina if you have it.

Prep HPLC is great, but you seem to be working with a lot of compound and purification by this method could prove to be quite the bottleneck if you need to carry multigram amounts forward.

[zarhym]

Yes we tried.
Here are the TLC plates.

[wildfyr]

Have you given recrystallization a shot? Sometimes it can exclude impurities in the crystal formation, leading to purifications not possible by column.

[zarhym]

Have you given recrystallization a shot? Sometimes it can exclude impurities in the crystal formation, leading to purifications not possible by column.

Recrystalization is pretty much the same principle with trituration. The chemicals are completely dissolved in recrystalization, while in trituration they are partially dissilved.

[kriggy]

Have you given recrystallization a shot? Sometimes it can exclude impurities in the crystal formation, leading to purifications not possible by column.

Recrystalization is pretty much the same principle with trituration. The chemicals are completely dissolved in recrystalization, while in trituration they are partially dissilved.

But the impurity might be locked in the solid structure of your product therefore not being possible to triturate

[pgk]

After a careful look at the proposed isomers:
1). The top and the middle one are conjugate forms of the your aromatic compound, which means the same compound, same Rf and same Rt.
2). The bottom one is an oxidized form of your compound (one hydrogen less), which cannot be formed under the non-oxidant conditions of the preparation reaction.
3). Your compound is a purine derivative that forms strong hydrogen bonds with other purines and amines, like your starting materials are (hint: compare with DNA structure).
4). The use of aprotic solvent only, favors the intermolecular hydrogen bonding between the product and the starting materials, leading to an unsuccessful separation.
Taking into account all the above:
1). You use methanol or ethanol that destroy intermolecular H-bonds, as a component of your solvents mixture for both TLC and column chromatography.
2). You add triethylamine in your (alcohol containing) solvent mixture for both TLC and column chromatography.
3). You pre-treat not only your TLC plate but also, the packed chromatographic column with a triethylamine containing solvent mixture, as Hypervalent-Iodine suggests.
4). After the (column) chromatographic separation, you recrystallize the product with an alcohol containing solvent mixture (methanol alone might work), as Wildfyr suggests and Kriggy explains.
5). I wish the best.