I am developing a method to synthesize amines using organometallic catalysts and my reaction yield these two diastereomeric amines ($\ce{R2N}$ is, in this case, a morpholine group).


With both being formed in approximate equal amounts, making it almost impossible to identify their structures by NMR without separating them. All the NMR spectra look like a mess since some signals get duplicated and some not. I have a hint that they are actually these two amines because they show the same molecular ion in the MS spectrum and the methodology used for synthesis offers little space for doubt, since it always forms the predicted structure.

I would like to know, if there is a practical way of separating those two compounds without using something super fancy like chiral column chromatography decorated with gold and antibodies kind of stuff. I have tried column chromatography but couldn't find a solvent system that could separate them. I also tried recrystallization (though I don't know exactly why I tried it), it also didn't work.


2 Answers 2


When you mentioned column chromatography I had to assume you meant flash chromatography or biotage, which by implication is normal phase. When people mention HPLC they are normally defaulting to reverse phase. Now, if you can get reverse phase TLC plates (C18 are available) , you could make some advances. The issue though is then getting the silica and you also mention large quantities.

Large lab scale to me is say, 50g plus. For this, I would advocate crystallisation, but to guarantee some differentiation, I would make salts with organic acids. Nitrobenzenesulfonate salts are renowned for giving well defined crystals.

If you needed something to better differentiate again the two amines, I would try a camphorsulfonate salt (pick an enantiomer). These tend not to be too expensive. The salts then have different solubilities and the right solvent becomes a screening exercise. Otherwise, tartaric acid is very cheap and easy to salt break after crystallisation.

  • $\begingroup$ Thanks for the advice @Beerhunter , by large amounts I mean about 5g of the mixture... At least for the compounds I work with this is a HUGE amount haha. I was trying to avoid crystallization at all costs since it is the most boring procedure ever to be done at a laboratory, but if I'm not successful with chromatography and preparative HPLC then I will have no better option than making crystals. $\endgroup$ Oct 3, 2017 at 3:19
  • $\begingroup$ Raul, for me there was nothing more satisfying than having a nice swirl of crystals after a crude solution had been made, and ending up with clean solid on a filter. While more is better, a few grams still plenty to play with to develop the crystallisation. It's all good scale up fodder for your new technique. $\endgroup$
    – Beerhunter
    Oct 3, 2017 at 6:42

Separating the two compounds should not be the issue. They are diastereomers, meaning they show different chemical properties which should be different enough to allow separation. The more difficult question is how to locate the separated substances.

I assume very strongly that standard, average HPLC should do the separation trick. It is commonly used to separate very similar compounds. There is no need for a chiral stationary phase as you already have diastereomers. The issue remains how to identify the products since they likely won’t show any significant absorption at any accessible wavelength.

You did mention that you performed MS measurements. Most MS measurements I have seen were actually HPLC-MS or GC-MS. If you have access to a HPLC-MS, you can check whether the standard gradient gives two peaks or a double peak in the graph that shows the total mass caught by the mass analyser. (Careful: these peaks are not quantitative!) If you already have two peaks, you should be able to directly port the separation method to preparative or semi-preparative HPLC. Be sure to collect a fraction after every ten or so millilitres (assuming a column designed for up to $\pu{60mg}$ and a flow rate of about $\pu{7ml/min}$). Then, analyse all these fractions by HPLC-MS again to identify which contain the first eluting and which the second eluting peak.

If you observe a double peak in the HPLC-MS, you will first need to improve the gradient to allow for separation. For close peaks it is often a good idea to choose an isocratic method over a gradient method. The rest remains the same.

If you have access to other, more rapid methods of identification (i.e. you can tell the two compounds apart on TLC) you can of course use these rather than running tens of HPLC-MS experiments and thereby take strain off your colleagues’ nerves. (However, if you have an autosampling mechanism you might run the experiments overnight instead.)

  • $\begingroup$ Thanks Jan, I mentioned chiral column decorated with gold and antibodies as a joke, but I know I don't need any of these to separate such compounds. TLC was the hell for me, I tried every possible combination of solvents and proportions but nothing worked. HPLC is probably the best way out, but I don't have access now to a preparative HPLC, and I need to separate big amounts of the two amines. $\endgroup$ Oct 2, 2017 at 14:00
  • $\begingroup$ @RaulLuciano Did you try re-evolving the TLC, i.e. use only apolar solvent (e.g. pentane, hexane) and do the TLC several times. Often this does the trick for me if the compounds are very similar. $\endgroup$ Oct 2, 2017 at 14:13
  • $\begingroup$ @RaulLuciano In those tricky cases where I had to separate isomers, it often helped to do a normal-phase gradient column, e.g. start with pure pentane, and then slowly mix in EtOAc. Alternatively, you could try to do a reversed-phase TLC. That would be a nice test to see if you get separation in RP. $\endgroup$ Oct 2, 2017 at 14:18
  • $\begingroup$ @deusexmachina yes, I tried all types of gradients... starting with pure hexane, mixing EtOAc, then a gradient of dicholoromethane and finally incresing methanol up to 100%. I can separate the amines from everything else, but they are still eluting together... I tried to run preparative TLCs multiple times with hexane, haxane/EtOAc, pure EtOAc, CHCL2, none worked. I feel terrible because I had no problem separating the other amines I obtained using the reaction, this pair is killing me though. $\endgroup$ Oct 2, 2017 at 15:11
  • $\begingroup$ There is also a terrible feature to these two amines, they are not visible under UV light. I can only see them using a GC or spraying a small TLC plate with ninhydrin. However, I can't use ninhydrin on a preparative TLC to see where the amines band are since it would destroy my product. That's why TLC didn't work for me even though I have separated all the other amines using only TLC. $\endgroup$ Oct 2, 2017 at 15:16

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