[MorganFreeman]

Hi Everyone,

I have an issue with my column chromatography for a diene. The 70 mg of compound comes off the column (15 cm diameter, 6 inch of silica) over the course of 300-500 mL. I have tried using a skinny column (10 cm diameter, 6 inch of silica), and still the compound comes out over about 150 mL.

The separation is fairly close, so I need to keep my compound at an Rf of 0.2. I have tried dry loading on both silica or celite, and tried switching the solvent system from Hex:EtoAc to DCM:EtoAc or PetE:EtoAc.

Any suggestions would be much appreciated. I am thinking about running a very short wide column with a low Rf. I think the problem is band spreading and this might circumvent the issue.

Thanks in advance.

[Doc Oc]

1) If you can elaborate on the structures, that would help.  What does your diene look like, and what would the major impurity be?
2) What are the percentages of each solvent you're using?
3) Specifics of the silica would help.  Particle and mesh size?

Without any more knowledge than what you've written, if you really are using 10 or 15 cm diameter columns, those are WAY too big for 70 mg of compound.  Are you sure you aren't using 10/15 mm diameter columns?  Those would be more practical for a small quantity like that (though I'm not sure I've ever worked with anything as small as 10 mm diameter, aside from using a Pasteur pipet for a microscale column).

In general, if your compound is on the nonpolar end of the spectrum and has a close-running impurity, running a taller column with a highly nonpolar solvent system (hexane/EtOAc 95:5 or 90:10) would be a good start.

[Dan]

Yeah 10 cm diameter is far too big, I'd take something in the 1-2 cm range

I've never worked with dienes, so I don't know if they tend to be inherently streaky or if there is a standard trick, but I have found that switching to toluene as the weak solvent instead of an alkane can quite dramatically alter the separation - especially when aromatic compounds or alkenes have to be separated. Of course the change in separation is not always for the better, but usually worth a shot.

I would try some TLCs with toluene/EtOAC, toluene/acetone and toluene/Et₂O. You can also use toluene as the strong solvent if the substrate is running too fast (e.g. hexane/toluene).

I would also strongly suggest step gradient elution as opposed to isocratic. That is probably the most general way to reduce trailing.

[MorganFreeman]

The diene has benzyloxy groups on each end, otherwise is a straight chain. The major impurity is an vinyl boronate ester, which strangely enough is about the same Rf, just a bit lower (snowman spots)

Solvent systems: 20:1 PetE:EtoAc, 15:1 Hex:EtoAc

Yes you are correct, I meant to say 10-15 mm, not cm.

Particle size 32-63 um.

If only the product came out over 5 fractions max I would have a nice separation.

[Dan]

The diene has benzyloxy groups on each end, otherwise is a straight chain.

So it has enol ether functionality? Again, never worked with enol ethers but I've heard they can be acid sensitive and badly behaved on silica. Perhaps neutral or basic alumina would be a better option?

[MorganFreeman]

I am sorry for the confusion, there are saturated carbons between the benzyl oxy and the diene. The molecule is symmetric

[OC pro]

Switch ethyl actate to diethyl ether or tert butyl methyl ether. Try 10:1 mixture hexane:ether. Ethyl acetate is too polar. I got always better separation for nonpolar compounds when using ether instead of ethyl acetate. 100:1 mixture was the strangest I run. And I got separation because my product came eluted with 70:1.
I would suggest a column from 1.5 to 3cm. For better separation you could also collect very small fractions like 10ml.

 OT: I am a big fan of cc. Always got a small *peep* when having a good separation. Kind of miss it. Now I deal with compounds in the µ to nM range using SPE cartridges (for babies).

[MorganFreeman]

So then possibly a more nonpolar polar component would reduce the band broadening/tailing? I will have to try that out.

[OC pro]

I suppose the tailing comes from the oversized column.

[Dan]

So does it look streaky on TLC or is it only really on the column that you see it? If you only get it on the column then maybe it's an overloading issue. Is your product very crystalline? I've had massive trailing issues with very crystalline compounds before, and in those cases overcome it by picking a weak solvent in which the product is reasonably soluble (e.g toluene or chlorofom as opposed to hexane or petrol). 

I would definitely try out some other solvents on TLC to increase separation, and certainly give toluene a shot as a component. I'd also switch to gradient elution, personally I find it far superior to isocratic elution.

[MorganFreeman]

Toluene is a keeper. Makes the spots no longer touch, and I am hoping that it will dissolve my product. Previously ethyl acetate and hexane elution mixtures would not even dissolve the crude mixture and I had to resort to dry loading.

On the TLC the spots don't seem to be streaking. Maybe you are correct that I may be overloading the column. Based off Still, Kahn, and Mitra I should be using 15-20 mm column. 

[Dan]

Previously ethyl acetate and hexane elution mixtures would not even dissolve the crude mixture and I had to resort to dry loading.

That sounds like it could be a crystallinity issue causing the trailing. When you dry load, do you see the loaded material gradually dissolve over the course of the column?

Dry loading should give you sharper bands, but you don't have to wet load in the eluent - you can use a minimum amount of e.g. DCM to wet load and then push it out with a volume of weak solvent before running the column. For a thread on increasing separation see: http://www.chemicalforums.com/index.php?topic=50530.0

[nox]

This thread basically sums up my frustration with the last 1/3 of my project -- some of the substrates are so highly crystalline that dry loading leads to a complete disaster, yet for some of them the separation is so difficult that if I wet load with DCM (even if just a small amount) onto a column packed in 100% hexanes, I kill any hope of getting clean fractions.

Eventually for the really difficult cases I resorted to running 2 columns. First one I wet load in DCM and get the bulk of the crap out, then dry loading and running a second column with a hexane/DCM system. The one good thing about this system is that because DCM is such a good solvent, stuff crystallizing on top of the column no longer becomes an issue.

In a few cases I found that hex/DCM actually gave a far superior separation compared to hex/EtOAc. One thing about a hex/DCM system though is that the trailing problem can become really annoying. Had a couple cases where Rf was about 0.5 in the system I was running it in, yet it took forever for everything to come off. After running the initial TLC plate a couple more times, I found that while the product moves fast initially, it starts tailing off, and it took about 6-7 runs before the product hit an Rf of 0.8.

[MorganFreeman]

Previously ethyl acetate and hexane elution mixtures would not even dissolve the crude mixture and I had to resort to dry loading.

That sounds like it could be a crystallinity issue causing the trailing. When you dry load, do you see the loaded material gradually dissolve over the course of the column?

Do you mean at the top of the column? In general I cannot see any bands as the column is running, what exactly do you mean? Perhaps I should wet load it with DCM, but I think I did that before and it was still trailing.... I may need to retry this.

On a side not nox, I noticed the same with hex/dcm columns. The seperation for my compounds was much better, yet as soon as I use it as a solvent system I notice that it takes huge amounts of solvent for my compound to even show up. Very strange...

I have recently tried changing from a 10-15 mm column to one which is about 25-30 mm and noticed that my peaks were much more seperated, yet my compound was coming off over 400 ml (for 100 mg crude). This column was roughly 5 inches, so I am going to use the same solvent system/column, yet half the silica and see if that works.

[Dan]

Do you mean at the top of the column? In general I cannot see any bands as the column is running, what exactly do you mean?

Yeah, I'm asking about the dry material you load. Does it stay opaque or does it become semi-translucent (like the rest of the column) when you start to elute, or does it gradually go from opaque to translucent as you run the column?

I hope that makes sense, not sure how best to explain what I mean, but if it's only gradually dissolving this might explain the trailing issue. It's usually easy to tell by eye if the compound is remaining crystalline on the column. If high crystallinity is the issue you can either switch to a "better" weak solvent like DCM instead of an alkane as nox described (I've also found toluene and chloroform to fulfill a similar role), or recrystallise instead.