Chemical Forums
Chemistry Forums for Students => Organic Chemistry Forum => Organic Chemistry Forum for Graduate Students and Professionals => Topic started by: kriggy on February 27, 2014, 05:18:43 AM
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Hi guys Im having bit trouble with my lab work.
We are trying to make the mono-substitued cycle(the one on right side) but we are getting the mixture of mono and bis-substitued
(https://www.chemicalforums.com/proxy.php?request=http%3A%2F%2Fi.imgur.com%2FCLCp38I.png&hash=3812c77537e835265f54b5ccd8781d13efa8ddf5)
We did try column chromatography with CHCl3:MeOH 20:1 but there is always the bis-substitued going first but then after that there is mixture with mono-substitued and if I increase the polarity of MF the mixture of mono-substitued and unsubstitued is elued. I triend about 10 different mobile phases but the compounds are just not separating. Preparative HPLC is kind of out of the question because we need this compound in >g amounts and our column has capacity of +- 100 mg so it would require 10 or more cycles. I tried just puting the mixture into water so the bis-substitued might precipitate but it didnt work.
Does anyone has eperience with separating compounds like those ? (ie. diferenting in 1 functional group).
I was thinking we could synthesise it by alkylating the amino-ether which gets coupled to pyridine-dicarbaldehyde and then react it like this
(https://www.chemicalforums.com/proxy.php?request=http%3A%2F%2Fi.imgur.com%2Ff1Ltg8K.png&hash=b369c4451db5a20d1fc4e66075b5b1e8f9fd6be6)
But this still doesnt solve the problem with getting mixture of mono and bis alkylated compound.
Any ideas or thoughts about this?
Thanks you!
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I wonder if this sort of compound would form a complex with some transition metal, Cu Co or the like. If it does you may be able to separate them by crystallisation.
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Yeah we want to make the complex with manganese but for that we need it to be pure mono-substitued for kinetics measurment. I think both of those will make the complex but its worth trying I guess. Do you have any paper where procedure like this was used?
All I have about this alkylations is alkylation of cyclene but the compound they want just precipitates out of the water :-\
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Sorry, I don't have any such literature.
If you need the Mn complex anyway make it and try a crystallisation. You can always re-crystallise to get the purity up.
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I triend about 10 different mobile phases but the compounds are just not separating.
What did you try?
I have never had success with difficult separations with chloroform/MeOH or DCM/MeOH.
I find that at this end of the polarity spectrum, WIPE (water/IPA/EtOAc) performs a hell of a lot better.
Try a TLC in 1:2:7 water/IPA/EtOAc and see what you get. You can adjust this ratio to the appropriate polarity for column chromatography (reduce the water/IPA content if it's running too fast - e.g. 1:5:45).
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Thanks I will give it a try.
I tried:
pure CHCl3, CHCl3+MeOH 20:1 and 40:1
CHCl3:THF, CHCl3:isopropyl alcohol
CHCl3+MeOH 20:1/40:1 + drop of Et3N
CHCl3:isopropyl alcohol + drop od Et3N
pure THF
pure ethyl acetate
ethyl acetate + MeOH
pure acetone, pure CH2Cl2
acetone + CH2Cl2
The CHCl3:MeOH 20:1 is kind of OKish. It elues the disubstitued cycle but it takes a lot of solvent. And I always get the tail while trying the TLC with those mobile phases.
I tried like all solvents we have in a lab but to be honest, I feel I dont have enough practice doing chromatography to be able to choose a good mobile phase.
I will try the one you suggested.
Thanks for help
edit: one more thing which makes me confused is that in the 1st fraction I got 2 spots on TLC but in the 2nd and 3rd I got only 1. I was expecting the bis-substitued to be elued 1st in such a low polar mobile phase so I cant figure out what might be the other spot.
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With amines I use a mobile phase with triethylamine (1-5%) present, so a drop might not be sufficient. Another option if you are using MeOH in your mobile phase is to swap it with MeOH:conc. ammonium hydroxide (9:1).
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I forgot that one.. I used MeOH:NH3 50:1 and 5:1 but then everything got washed ouf of the column.
I will try more triethylamine tommorow.
Thanks for ideas
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Why is 10 cycles of pHPLC out of the question? That would take a full day chronologically, and maybe only an hour or two of total effort, right?
I do this frequently for difficult to separate compounds.
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Im not realy sure. But the thing is we dont have method for this compounds, they dont separate on HPLC either (using acetonitrile water gradient).
Im just a student so it doesnt go my way anytime I want
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I used to do a lot of carbohydrate chemistry, which would invariably lead to mixtures, and developed the following procedures....
1. With silica gel TLC plates, find a solvent (e.g. starting at EtOAc/Hexane, 1:5 v/v) that just moves products above baseline. Adjust mobile phase, if necessary, so that products have Rf values in range ~0.1-0.6 after 4-5 developments of the same plate. Make sure plate is thoroughly air-dried between sequential developments. The idea is that multiple developments represent APPROXIMATELY the behavior to be expected on a column.
2. Perform dry-column chromatography of the rxn. mixture on silica gel, using as eluent the same mobile phase from above. Note, the rxn. mixture, as a solution, is mixed with silica gel which is rotovapped to dryness; dry, free-flowing powder is then applied as thin band to top of column.
This general procedure has worked well for me with complex mixtures; have also used solvent mixtures of the type recommended by Dan (i.e. EtOAc/EtOH/H2O, 10:3:2 or 45:5:3).
Please let us know what works for you (or not).
Good Luck
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Im not sure if I understand your procedure. I mix the reaction mixture with silica and evaporate the solvent thats clear. The I use this silica gel as a stationary phase and elute it with mobile phase right? How the compounds separate? If I put this silica into the column, the products are somehow distribued /lets say evenly/ evenly in the volume of silicagel..
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Note, the rxn. mixture, as a solution, is mixed with silica gel which is rotovapped to dryness; dry, free-flowing powder is then applied as thin band to top of column.
While you have been rotovapping down to a dry powder, you have also been packing a chromatography column with DRY silica gel. Then add your dried powder from the rotovap flask to the top of the silica gel column; cover that layer with a thin layer of sand or silica gel to disperse liquid flow and get even wetting of your rxn. mixture band. Start mobile phase flow and collect fractions.
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I see It makes sence now. You dont wash the silica with mobile phase before you add the sample? (in general)
Do you have an explanation why it worked for you this way but not the traditional way?
The problem is, IMHO, just the lack of the right mobile phase. I tried the one Dan suggested on TLC and sample which had only the bis-substitued (from MS we know its almost pure) gave me 2 spots on TLC but the one where is mixture of products gave me only one. Could it be that the sample was so diluted that I cant see the other spot ?
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Hmmm. Try a toluene based mobile phase, such as toluene/acetone. I find toluene can give drastically different mobility compared to aliphatic solvents.
Are you getting any streaking? It's probably worth seeing if triethylamine doping turns the tables in your favour.
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Streaking you mean like the spot on TLC had some kind of "tail"? If so then yes. I addded few drops of Et3N to the mobile phase (probably 3-4 ml of mobile phase total) and it helped with streaking but not with separation. I got one spot for reaction mixture and one for bis-substitued with same Rf. Without Et3N i got also same Rf for mixture and bis but with the tail behind. So it seemed that by using Et3N the compounds which are less mobile are moved together with the more mobile ones. I have pictures im my phone of this so I can upload them when I get home.
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Second the toluene suggestion! I completely forgot about that trick.
Aromatic solvents like benzene and toluene can pi stack with compounds containing aromatic moieties, often helping with tricky separations.
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Im gonna try it next time.
I mean, I already had the mixture on column and doing the CHCl3:MeOH before I asked here. But I slowly increased the amount of MeOH in mobile phase so it worked OK-ish (IMO much better then 1st time). THe thing is quite large amount of CHCl3 required. I will have MS next week. But I guess I will have to go even more polar because there is still quite a lot left on column.
At least I got some nice crystals of bis-substitued ligand from before :D
BWT you guys posted so many ideas I dont know If I will have enough time to try them all ;D
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Hi guys I managed to almost separete the compoounds by CHCl3 + MeOH. I tried toluene MeOH 1:1 and acording to miscibilita table they are miscible but on TLC it seems that there are 2 mobile phases when the 2nd one moves with Rf 0,8 compared to the faster one. It seems that the compounds separate well but the 2 fronts of MF are bugging me. Any ideas how they are formed?
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So the solvent system looks miscible in a bottle when mixed, but on TLC you are getting two distinct solvent fronts?
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In your drawing is there supposed to be a methylene group in between the nitrogen atoms and the carbonyl group?
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So the solvent system looks miscible in a bottle when mixed, but on TLC you are getting two distinct solvent fronts?
Yes. I was told that this could be because one of the solvets in the mixture is evaporating much faster than the other.
In your drawing is there supposed to be a methylene group in between the nitrogen atoms and the carbonyl group?
yes but i can see the drawing being not clear enough
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Hrmm... what sort of container do you do your TLC's in? If you aren't already doing so, try doing it in a sealed vessel of some sort (I use a 30mL glass vial) with a bit of kimwipe up one side. This usually alleviates any sort of evaporation problems you will have.
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I did it in beaker with piece of glass over it so it might evaporate a bit but I didnt have any similar problems with different mobile phases.
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Hi guys I managed to almost separete the compoounds by CHCl3 + MeOH. I tried toluene MeOH 1:1 and acording to miscibilita table they are miscible but on TLC it seems that there are 2 mobile phases when the 2nd one moves with Rf 0,8 compared to the faster one. It seems that the compounds separate well but the 2 fronts of MF are bugging me. Any ideas how they are formed?
I've never come across that one before, but I've never attempted to run toluene/MeOH. Try swapping MeOH for EtOH, i-PrOH or acetone.
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Hi its me again :)
Ok because the compounds still doesnt separate well - im getting mostly pure bis-substitued, then some amount of mono (which doesnt yield anything after evaporation so its realy low amounts) and then mixture of mono and unsubstitued I decided to try the prepareative HPLC way. Or at least get some information. I told you that the compounds doesnt separate in analytical HPLC but actualy it wasnt HPLC chromatogram but it was from MS showing us how much of the compound goes into the ionization chamber over time because the sample was measured for minute or longer.
Anyway, the thing is that our preparative HPLC doesnt have gradient elution which is little disapointing. (and doesnt have autosampler so doing 10 cycles is eh.. time consuming I guess ;D)
What are the differences between prep. HPLC and column in terms of elution strenght? I mean for example when I use pure methanol on column all products get elued is it same on HPLC or when I use MF that doesnt elue the compounds at all in collumn, is it possible that it will work in HPLC? How does the high pressure change things compared to normal column?