Molecule soluble in chloroform

[chem_tr]

Hello,

I have a molecule soluble in chloroform. I've tried to learn its pureness with thin lead chromatography on silica gel. After several tries, I've discovered that my molecule is best eluted with 9:1 CHCl₃:MeOH (for now). I am not satisfied with the elution, the molecule does not advance very well as progressed with a visible spot; it is a tailing spot. But I want it to be a single spot. I want to share my findings with you, and to expect your answers.

I've found dielectric constants of the solvents I used, and here reads 4,8 for chloroform, and 33,6 for methanol. If we prepare a solution containing %90 chloroform by volume, what would the overall dielectric constant of the mixture? I did a simple calculation (4,8*9+33,6*1)/10=7,68. Is that a logical approach? Secondly, I tried to approximate all my different solvent mixtures to this value by appropriate mixing. But none of them worked, for one exception, methanol:toluene:chloroform. This is as nice as the initial one. I think that it is not about the final value; chloroform should be much to dissolve the compound, and the additional solvent is needed to drive the spot higher as pure chloroform does not drive it upwards.

Sorry for long post, but I think you understood the event.

 

[Bystander]

Hello,

and here reads 4,8 for chloroform, and 33,6 for methanol. If we prepare a solution containing %90 chloroform by volume, what would the overall dielectric constant of the mixture? I did a simple calculation (4,8*9+33,6*1)/10=7,68. Is that a logical approach? .

First order? Looks good. Worth niggling about non-ideal details, mixing volumes, molecular complexes, and the rest? Uh-uh.

Secondly, I tried to approximate all my different solvent mixtures to this value by appropriate mixing. But none of them worked,

What mixtures didn't work? That can be more useful information than knowing the mixtures that do work.

 

[chem_tr]

Hello, thank you for your interest.

It is interesting that only chloroform:methanol mixtures work well, even chloroform:dmf doesn't work. So I decided to stick to chloroform:methanol and will play with the mixture ratio.

There is another weird thing, that my tail-like spot has a continious other tail from the beginning to the end (solvent line), but this tail gives fluorescence at 313 nm, whereas my spot has a significant green color with no fluorescence. Can this be used for an efficient removal?

Thanks again,
chem_tr

 

[movies]

What is the molecule?

You might try dichloromethane:methanol, that sometimes streaks less. Also ethyl acetate:methanol. There are other additives that help with streaking, but it depends on the molecule you are trying to separate.

 

[chem_tr]

Thank you for your help,

My molecule is an ester composed of pyridinecarboxylic acid and an octakis(hydroxyethyl)-substituted compound. The starting alcohol does not advance with chloroform:methanol, but the ester does. You are right, I haven't tried dichloromethane, as the molecule dissolves in it, too. It is less harmful than chloroform, in addition.

Thanks for this reminder, maybe it will be better in CH₂Cl₂. Although there are eight ester groups, ethyl acetate failed to drive the spot, but I have only tried one mixture according to my "method".

I have another question: Since my molecule's endgroups are basic due to pyridine nitrogens, what adsorbent should I use? Silica gel is acidic, and aluminium oxide is basic. I am using silica gel, and it is driven to R_f 0,33 with 9:1 CHCl₃:MeOH, but stops then. It goes with the solvent line on aluminium oxide in a nearly similar manner, I mean, no significant adsorption occurred.

Regards and thanks to everyone who contributed to this topic.
chem_tr

 

[movies]

First off I would try adding 1% triethylamine. That will minimize streaking when you are using basic compounds like pyridines. Be aware that this will significantly affect the eluting power of the solvent. You might try backing up to maybe 2:1 ethyl acetate:hexanes + 1% TEA. You can use 1% ammonium hydroxide and achieve essentially the same effect. When you use this kind of solvent system, it's often a good idea to "pre-treat" your plate with the eluent by running it up before spoting your reaction mixture on it. That should minimize the acidity of the silica.

The three most common solid phases for chromatography are silica, alumina, and Florisil. Silica you are obviously aware of, alumina as well. They do make acidic, neutral, and basic alumina. One of these might better suit your needs. You should be able to get them from any chromatography supplier. Florisil is less polar than alumina (which is, in turn, less polar than silica) so that might be an option as well.

 

[chem_tr]

Thank you, Movies. I'll try these and turn back to you if it helps.

But I've not fully understood one thing; do you recommend me that I use basic alumina instead of acidic silica? Because you suggested that I treat the solid phase with basic compounds like triethylamine to pull back its acidity.

Thank you anyway, you've given me a great idea.

 

[movies]

Silica is acidic as is. You can't buy various acidities of it as far as I know. You can buy different acidities of alumina, however. I would first try treating silica gel plates with TEA and if that doesn't work, maybe try basic alumina plates. Basic alumina plates are essentially "pre-treated" so you could use normal solvent systems and get pretty much the same effect as if you added TEA to silica gel plates, but the polarity of alumina is different from the polarity of silica, so sometimes one will work better than another.

I hope that makes sense. It is a bit confusing.

 

[Chemicalsuperfreak]

What's the Rf in 9:1 chloroform:methanol?

Have you tried 100% EtOAc, or AcOH in EtOAc?

 

[chem_tr]

I've found the Rf as 0,33 in 9:1 Chloroform:Methanol. Only ethyl acetate has not eluted anything but some impurities (this may be used to purify on column, and then drive the spot by gradient elution, etc.). I haven't tried acetic acid in ethyl acetate; I'll give it a try.

By the way, I tried 9 CH₂Cl₂:1 EtOH in the presence of 0,16% triethylamine, as movies recommended. I haven't adapted the system fully, but it seemed to work. Lots of spots occurred, but I think this is due to non-pre-exposure of silica to the eluent.

Thank you all,
chem_tr

 

[chem_tr]

Today, finally. I tried the system, i.e., 9:1 Dichloromethane:ethanol in the presence of 5% triethylamine with pre-treatment of the thin lead. It worked, with tolerable amounts of streaking (from the beginning to the end, unfortunately). The system seems to be suitable for column chromatography, but I appreciate your discussions anyway.

 

[movies]

You might try a little less TEA and maybe methanol instead of ethanol to prevent streaking. Ethanol often brings a lot of water with it, which can dissolve the silica. Methanol can dissolve silica too, but it's usually okay if you use less than 10% MeOH.

 

[chem_tr]

...maybe that's why ethanol didn't worked well as methanol... You've given me a great feedback. Thanks! I'll try these tomorrow.

 

[chem_tr]

Thank you Movies, your help provided me an efficient way to separate my molecule from impurities. By the way, please say something about my last findings (I welcome any comments of other members, too).

I have found that my molecule is best eluted in 19:1 CH₂Cl₂:MeOH in the presence of 1% acetic acid on alumina column (basic alumina, I think). I applied the same elution on the blank thin lead before spooting my mixture, but forgot to change the eluent after applying the blank thin lead; so it was eluted faster than the first one as evidenced by R_f values. The first was about 0,5; but the last one was near 0,7. It is simply because of change in elution composition, of course.

1) What should I do to keep my eluent composition the same during chromatographic separation?

2) Is there another efficient way to separate this molecule, instead of column chromatography? I am told that column chromatography causes large amounts of analyte to be lost during the process, with yields about 30% only. I'm seriously thinking of an alternative in which you prepare the adsorbent on glass plates and immobilize it by putting the adsorbent-applied glass to an oven. After immobilization, you place your sample as a line, and apply the eluent. We say "preparative column chromatography" to this technique, I am sure you are aware of it. What do you think about this technique?

3) May I consider gradient elution with more polar eluent, since impurities advance faster than my molecule, after I discard these invisible things to the naked eye (UV-active at 254 nm and fluorescent at 365 nm), will it be a wise choice to increase the polarity of my medium with something to drive my green-colored molecule faster?

Thank you for your interest, I'll welcome any suggestions.

 

[movies]

I'm not sure that I completely follow the problem you had with different eluents. Did you expose the plate to the eluent, then let it dry, then spot your mixture on the plate and elute again with the same eluent? If that's what you did, then there shouldn't be any problems.

The eluent composition shouldn't change significantly during elution. Are you using flash chromatography or some sort of gravity driven chromatography? Flash chromatography is very efficient. I have used it and recovered purified material in nearly quantitative yield. Typically the lower recovery is due to a low reaction yield, not a problem with chromatography.

The method you describe in part 2 sounds like preparative TLC to me. It's essentially like a large TLC plate. These are very useful when working on small scale or with materials that do not separate well. The drawback is that you can only purify a rather small amount of material at a time (the amount varies depending on the thickness of the silica/alumina on your plate).

Solvent gradients are a great way to conquer a difficult separation. When I run a solvent gradient for flash chromatography I usually begin with an eluent that gives an Rf of about 0.1 or less. I will typically run about 3 column volumes of solvent through with this eluent. Then I will use a solvent that gives an Rf of about 0.15 and run through about 2 column volumes, then a solvent system that gives an Rf of about 0.2 (2 column volumes), and finally a solvent that give an Rf of about 0.25 or 0.3 and run through another 2 or 3 column volumes of solvent. That will ensure that your material is off of the column. A typical gradient I use would be 4% ethyl acetate/hexanes --> 6% ethyl acetate/hexanes --> 8% ethyl acetate/hexanes --> 10% ethyl acetate/hexanes. However, it all depends on how your compound behaves. I have had good luck with the gradient 2:1 hexane:ethyl acetate --> 1:1 ethyl acetate:hexanes --> 100% ethyl acetate when purifying very polar compounds.

Basically, try a lot of different conditions. If you can get a separation on TLC where your product spot doesn't overlap with the impurity (even if the impurity is very close) you should be able to purify it with a solvent gradient. To ensure a that you can remove the impurity without using a gradient you need significantly better separation; an Rf of difference of about 0.2 is usually enough to ensure that you don't need a gradient.

 

[obirai]

Can anyone help me here? A friend needs to know the standards to use for for Carpaine, dehydrocarpaine, pseudocarpaine, benzyl glucosinolate and tannin in HPLC analysis. She will like to know the suppliers of such standards as well as their brand names.

Thanks
Joe