October 24, 2021, 02:57:16 AM
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Topic: Menthol derivative tend to co-elute on column chromatography = Possible explanat  (Read 8664 times)

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Offline manishbiyani

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I have synthesized various diastereoisomers of menthol derivative. One thing in common with these compounds were their difficulty in separating on column chromatography, they tend to co-elute (streaking on column). Interestingly, these diastreoisomers could be easily differentiated on 1H-NMR AND 13C-NMR.

I was trying to speculate the possible reason for this kind of behaviour, i would appreciate if some one could assist me on those lines...

Has anyone come across similar problem?

menthol being intrinsically saturated compound doesn't offer pi-pi interaction or doesn't provide environment which could aid in providing different electronic environment.

P.S = Similar diastereoisomers of ethyl derivatives were easily separated.

Your suggestion will be appreciated.

Offline OC pro

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Have you already tried different eluent systems? Which solvents did you used in your separation?

In case you have solids you could also try crystallization.
 

Offline manishbiyani

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Thanks,

I have already tried various TLC system and recrystallization method with no success.

What I am looking over here is possible explanation for streaking and co-eluting of menthyl (or camphor sulfonyl derivative) on column?

Thanks in anticipation.




Offline lavoisier

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If the two diastereomers coelute because they streak on elution, i.e. not because they have very close Rf's, then your problem is not differentiating them, but achieving a higher resolution on LC, and there's no contradiction with the theory.

On the other hand, if they also have close Rf's, one possible explanation could be that the menthol bit is too big compared to the rest of the molecule, a bit like in the case of cryptochiral molecules.
This could account for the fact that the analogous ethyl derivatives were better differentiated.

Offline manishbiyani

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Thanks lavoisier,

I had never come across the term cryptochirality. Based on the defination and description of cryptochirality, my compounds doesn't fall in that category for following reason:

1) Cryptochirality is observed in enantiomer. 2) specific rotation is zero. In my case, compounds are diastereoisomers and has good specific rotation (especially when both isomers are racemic based on HNMR). The chirality introduced by the chiral auxiliary is near to the chiral centre (2 bonds away).

Rf's are close hence streaking is observed is not true: I agree.
 For example: Rf = 0.7 (menthol) and product Rf = 0.5 tends to co-elute to some extent. Various diastereoisomers, which  had Rf= 0.05 difference between two products were separated with great difficulty. In certain diastereoisomers no Rf difference was observed in various TLC system (similar compounds in literature and patents seemed to have separted with these system). Infact, i tried to induce pi-pi interaction by introducing aromatic ring, which made compound crystalline (easily recrystallizes), good separation of peaks on NMR but no difference in RF or fails to separate after twice recrystallization.

Based on my understanding, I want to suggest that it is the combined effect of mainly menthol (intrinsic nature menthol to co-elute) and basic nature of my compound, which is causing this effect. ALthough, i want to explain this in more scientifcally rigorous manner. what could be possible factors like..electronic nature, conformation, steric hindrance, other effect etc

Offline lavoisier

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I wasn't suggesting your compounds were cryptochiral, I was making an analogy with them.
Cryptochirality is sometimes observed when the molecule is very large, so that a small, single stereocentre, perhaps even buried inside shielding carbon chains, isn't able to impart strong, detectable chiral behaviour to the whole molecule.

Also, your reply confuses me a bit, because now you seem to be saying your compounds coelute with menthol. I thought your problem was coelution of 2 (or more) diasteromers. And you said the ethyl derivatives were well separated, but then you seemed to imply your compounds were not chiral before you introduced the menthol part. This is impossible.

So just to make myself clearer, I thought this was what you did: you took a mixture of diastereomeric compounds, let's call them RR and RS; you reacted them with a (S)-menthol-based chiral auxiliary, thus generating RRS and RSS; you observed that RRS and RSS were not well separated by LC.
My hypothesis was that IF the molecular weight of the menthol part is large compared to the MW of the rest of the molecule, the difference between the two diastereomers could be reduced rather than increased.

Menthol is indeed commonly used to separate stereoisomers, but the most frequent case is when you have two enantiomers R and S, and you convert them into diastereomers by reacting them with a fixed enantiomer of menthol. Then you have a pair of diastereomers, which, according to the theory, should be easily separated.

Offline manishbiyani

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Thanks for pointing out more details on cryptochirality,

Just giving you brief idea of what i did:

RR RS SS SR ETHYL DERIVATIVE  (RR AND SS) TRANS AND (RS AND SR) CIS GETS EASILY SEPARATED

INORDER TO SEPARATE TRANS ENANTIOMER (RR AND SS) AND CIS ENANTIOMER (RS AND SR)

I am introducing S-menthol step prior ion which generates these 4 diastereoisomer.


RR-S RS-S SS-S SR-S are generated
IN theory all 4 diastereoisomer should get separated, especially TRANS (RR-S AND SS-R) AND CIS (RS-S AND SR-S) should get easily separated as it was the case of ethyl deraivative
unfortunately, with great difficulty i separated cis and trans isomer in menthol  derivative.

Intrinsic nature of menthol:

say I am separating menthol and menthol derivative (apart from menthol itself there is no chiral molecule) then

menthol= say Rf = 0.7 and menthol derivative Rf = 0.5. Even such a huge Rf difference I had to separate them carefully.

I have obtained X-ray crystal of one such related derivative, it does show decent difference in atom-atom contact diffference. Also HNMR AND CNMR shows reasonable difference but again there was no difference in Rf and no separation.

Offline lavoisier

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OK, I understand a bit better now.

The suggestion of changing the eluent system, which some people gave you straightaway, might solve the problem from a practical point of view, but I seem to gather you're more interested in explaining why this happens from a theoretical point of view.

I'm still convinced my hypothesis could be correct.
I'm not surprised NMR detects the difference between the various stereoisomers: NMR, in a way, gives you a 'picture' of the nuclei in your molecule, i.e. it goes very deep into the structure, and the local environment of the nuclei counts more than the whole molecule, in most cases.
But when you have your molecules in solution and you let them interact with silica and a solvent, it's the overall, average properties that count.
Imagine you have two diastereomers RR and RS. Their ethyl derivatives RR-Et and RS-Et are well separated as you said, because the Et group is small enough, and the intrinsic difference in properties between RR and RS prevails. Now you make derivatives with a large moiety such as menthol, that we can call M, so you get RR-M and RS-M.
My idea is that M is so big that it overpowers the RR/RS part, so that the overall properties of either RR-M and RS-M will be more similar to M alone, making them more similar rather than more different.
Now, I know it doesn't work like this in reality, but just as an example imagine that the overall properties are proportional to molecular weight, and say that the RR moiety weighs 50. Ethyl weighs 28, whereas menthol weighs 155. So, the ethyl derivatives will weigh 78, the menthol derivatives will weigh 205.
In the ethyl case, the % of weight of your derivative that is contributed by RR is 50/78 = 64%, whereas in the menthol case it will be only 50/205 = 24%.
When you use menthol on two enantiomers, the fact that you generate asymmetry where there was symmetry is probably more powerful than these considerations.
But I might be totally wrong. This is just my hypothesis.

Offline lavoisier

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menthol= say Rf = 0.7 and menthol derivative Rf = 0.5. Even such a huge Rf difference I had to separate them carefully.

I just wanted to add something about this statement on Rf's.
I think most analysts will agree that Rf 0.5 and 0.7 isn't such a huge difference.
As you know, the linear separation of your peak maxima in terms of column volumes is roughly equal to the difference between the reciprocal of the Rf's, in this case (1/0.5 - 1/0.7) = 0.57. This is not much by any means, it's about half CV. I would feel more comfortable with at least 1.5-2 CV, if not more.
Of course you can't reduce the Rf's indefinitely, because the resolution of your separation is going to decrease when the Rf's get too small (the peak broaden and overlap more than they would do with a shorter elution time).
So there is an optimal value of Rf's that gives you the best separation, and in practice most of the time you want to have your main product at an Rf of 0.2-0.3.

Offline orgopete

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I agree that Rf of 0.5 and 0.7 are too high for an effective chromatography and lower values would be much better. In my experience, a separation of this magnitude would be easy. I think in the original paper on flash chromatography, they had smaller differences and had good separations.
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Offline manishbiyani

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Yes, i am looking for theoretical explanation for the co-eluting nature of menthol.

Although, i was aware of the ideal Rf appropriate for the product(0.2-0.3), i was not aware of the reason, so thanks for the details.


If, i combine your hypothesis and propose, vander Waal interaction and H-bonding effect of compound with silica in a particular solvent would be overall effect of the compounds, which decides the movement on column,and not depends on particular chiral region.


Although, i do believe that menthol do have some specific properties, which makes it co-elute with other compounds.

Offline orgopete

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I think there are two different properties that need to be taken into consideration, the theoretical nature and the practical nature of chromatography. The question implies that there may be a theoretical reason that menthol may co-elute with other compounds.

That is difficult to rationalize. In the theoretical model for chromatography, the analyte is in equilibrium with the solvent and stationary phase. In order for that to be true, the concentrations of analytes must be sufficiently low to not interfere with the separation. At the extreme, one would not expect a mixture of menthol and another compound to be separable on a chromatography column. Menthol would be the mobile phase and co-elution must occur (though fractions of pure menthol may also elute).

On the practical side of chromatography is the amount of stationary phase used per amount of analyte. While the best separation can be achieved with a large number, doing so requires larger amount of solvent and longer times to elute the column. Also, how a compound is loaded onto a column becomes important. It is increasingly difficult to load a polar compound (or one likely to crystallize) onto a column in a very non-polar solvent. Using a more polar solvent also causes greater mobility or co-elution and poorer separation.

So, if I understand the question correctly, it would not seem necessary to invoke van der Waal forces or hydrogen bonding to explain menthol co-eluting with your impurity. I can understand a lack of separation in many circumstances, but not ones in which an interaction of the mixture is greater than that of the mobile and stationary phases.
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Offline manishbiyani

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Thanks pete,

I used to normally practice with slightly longer column for this kind of separation, as you mentioned. Normally, i use to load the compound by preadsorbing on silica or minimum solvent (sand layer - around 2-3 cm). As i  mentioned earlier, i have comfortably separated diastereoisomers, i don't see why another diasteroisomer of similar nature should be different (apart from menthol).

Regarding, crystallization could be one of the factors. My compounds were moderately polar..pet ether/EA 8:2 was good enough to pull the compound slowly.

I still feel lavoisier suggestion holds good. I am not sure about additional proposal form my side would be appreciated on same lines though.

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