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Topic: TLC: increasing spot separation  (Read 20729 times)

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Offline 408

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TLC: increasing spot separation
« on: May 30, 2011, 01:38:49 PM »
I have not had to run a collumn in a while now, and I find my compound and impurity eluting at almost exactly the same rf, giving peanut-shaped TLC spots.  Any advice on how to increase the separation?  Other than "use a very long thin collumn", ideally I would be able to purify multiple grams at once...

Thanks,

Offline azmanam

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Re: TLC: increasing spot separation
« Reply #1 on: May 30, 2011, 01:54:28 PM »
switch the polar solvent.  Try acetone:hexanes, ether:hexanes, etc.  That usually works for me.
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Offline Dan

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Re: TLC: increasing spot separation
« Reply #2 on: May 30, 2011, 02:36:11 PM »
I also find switching the non-polar solvent to toluene can give very nice results - acetone/toluene has come to my rescue on several occasions.

Also try MeOH/DCM.

For columns, I always run a step gradient - it keeps the bands narrow. I find this gives me better separation than using a single solvent ratio and is much more economical in terms of time and solvent volume. For tougher separations I use a shallower gradient and dry-load the crude (otherwise I wet load because it's quicker). I was taught this years ago by a postdoc in the lab at the time and have never looked back.

The "run it in 100% hexane on a two metre column all day wasting litres and litres of solvent" approach I've seen people use has never given me significantly improved separation, it just wastes time and money in my opinion. Even a second purification of the mixed fractions in a practical solvent system is quicker and less wasteful than a very long and non-polar column if you ask me.
« Last Edit: May 30, 2011, 02:57:07 PM by Dan »
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Offline asa029

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Re: TLC: increasing spot separation
« Reply #3 on: May 30, 2011, 10:57:53 PM »
When I try to find a good system for collumn, I make a few TLC-plates and try different systems. I usually start from a weakly polar system like hx:etOAc (about 3:7) and either increase or decrease polarity. This is much less of a hassle if you make really tiny TLC-plates. Also remember that if you overload the plate, the spots will become much larger and two spots that might in fact be properly seperated, might look like just one spot.

Offline bolo

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Re: TLC: increasing spot separation
« Reply #4 on: May 31, 2011, 06:36:06 AM »
You can employ a simple syringe (e.g. of 20 mL volume for max. 300-500 mg) as your column and do an elution under high pressure from another syringe. It is very similar to flash column chromatography but the only difference is that you are manually controlling the flowing rate as well as the composition of your eluent. Believe me or not, it is very useful and effective when you don't have a flash at your laboratory as it gives often much better separation.

Because the increase of the pressure causes the equilibrium mobile:silica is weaker, so you only have to start with much weaker (polarity) solvent, such like hexanes:EtOAc - 50:1 and then make a gradient elution. I use it every time I have to isolate my product by a column chromatography since my first use of it. It may look non-pro but if it runs very well, it eventually doesn't matter how it looks like :).

Offline fledarmus

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Re: TLC: increasing spot separation
« Reply #5 on: June 13, 2011, 06:00:45 PM »
It really depends on your products. There are all sorts of tricks for adjusting the solvents to spread out two spots, and sometimes you just have to test a lot of solvent combinations. You can also try adding small amounts of weak acids or bases to the solvent system, eg 0.1% TEA in a MeOH/DCM mixture, or 0.1% AcOH in an EtOAc/Heptane mixture. Sometimes using a more branched solvent will help, sometimes an aromatic solvent, sometimes acetonitrile is the magic bullet.

If you know what your compound and impurity are, it makes it easier to pick an appropriate solution  ;)

Offline bmorvan

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Re: TLC: increasing spot separation
« Reply #6 on: June 15, 2011, 06:24:26 AM »
The easiest is to make several runs with the same plate and the same eluate.

Offline azmanam

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Re: TLC: increasing spot separation
« Reply #7 on: June 23, 2011, 02:01:52 PM »
ps, read this paper
doi: 10.1021/jo00408a041
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Offline the.khemist.ds

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Re: TLC: increasing spot separation
« Reply #8 on: June 23, 2011, 07:55:27 PM »
It's a common misconception that longer columns lead to better separation. Infact, this is often not the case, there is an optimum length - everyone has a different figure but it is usually between 15 and 20cm.
As you make a column longer it means that the components that you are trying to separate spend longer on the column. As the material moves down your column the bands of the eluent(s) become wider (whether this is due to diffusion or just the fact that the eluents are constantly adsorbing and desorbing from the stationary phase I wouldn't like to say for sure). The longer the material spends on the column the greater the band-broadening effect - which tends to cancel out the benefits of running the column longer.

As others have said, DCM based solvent systems can often give good separation. I'd sound a note of caution on the use of ether based solvent systems, they can often cause 'column cracking' which will significantly impair the separation profile - having said I have had occasional success with ether containing solvent systems

Offline OC pro

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Re: TLC: increasing spot separation
« Reply #9 on: June 24, 2011, 02:49:12 PM »
It's a common misconception that longer columns lead to better separation. Infact, this is often not the case, there is an optimum length - everyone has a different figure but it is usually between 15 and 20cm.
I'd sound a note of caution on the use of ether based solvent systems, they can often cause 'column cracking' which will significantly impair the separation profile - having said I have had occasional success with ether containing solvent systems

When spots tend to co-elute, a longer column can help. Last week I had a column with 870g crude product. It was a 95/5-mixture of diastereoisomers along with some other crap. Column was 1m high, diameter was 0,3m and total package of silica gel was 20kg (one whole can  ;D). Separation was very good (I rotavaped 100 fractions having 0,5l each).
In case of very unpolar compounds I tend to use tert-butyl mether ether (or diethyl ether) instead of ethyl acetate. Gives often a better separation.
 

Offline azmanam

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Re: TLC: increasing spot separation
« Reply #10 on: June 24, 2011, 02:56:28 PM »
WOW.  Kg-scale reaction, and you opted to run a column.  This is in one of those hoods with a garage-door as a sash, isn't it?

I hope column isn't your preferred method of purification on kg scale...  How much solvent did you go through.

(also, I think the.khemist was referencing normal-people-scale reactions.  Running a 500 mg reaction through a 1m column is too long of a column.  for 1 kg-scale, 1m is probably about right.  Biggest column I've ever run was 20-30 gram scale on ~18-20 in silica in a column w/ ~8-10 cm inner diameter.  you can see a picture of it here)
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Offline OC pro

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Re: TLC: increasing spot separation
« Reply #11 on: June 24, 2011, 03:20:22 PM »
I just wanted to impress you guys  8) . But this is also not normal for me. Normally, crude products have a 100g size which one can separate on 2kg of silcia gel. And we are also not doing kilo scale. It was only a 600g batch  ;D.
At 700ml/min the solvent cans get empty very fast (even the 25l cans  :P). Were about 150l of ethyl acetate which ran through the column in about 4 hours. I had my 100 fractions in 2l-flasks on three normal laboratory rotavaps. It took 10 hours to remove the solvent.

The bad thing was that the compound was a sticky OIL!! Column was the only option. Otherwise, I would have done a recrystallization.

Offline 408

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Re: TLC: increasing spot separation
« Reply #12 on: July 16, 2011, 03:52:06 AM »
Finally pulled it off.  Took 4 solvents  ::) ???  Hooray for random mixing until something worked...
hexane:benzene:MeCN;EtOAc 4:1:1:2

Offline OC pro

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Re: TLC: increasing spot separation
« Reply #13 on: July 16, 2011, 04:42:40 AM »
Looks like trial and error.
Quite unusual mixture  ;D

Offline 408

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Re: TLC: increasing spot separation
« Reply #14 on: July 16, 2011, 10:53:09 AM »
Hex: EtOAc
desired spot still had crap in it
Add random solvents till spot had separated into multiple spots
Adjust proportions until desired separation
desired spot still had crap in it
add new solvents
adjust proportions for desired separation
BINGO

It was madness, but with a method!   (Other than the fact that the only reason I tried benzene was because I happened to be using it on another reaction, and figured why not :P)

Totally worth it.  I am a little aroused by the compound I finally isolated, unfortunately it is a little sensitive.

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