Dissolving hydrophobic compounds for RP column

[lemon]

I've just joind this forum and this is my first post, so first of all - HELLO to everyone!

I'm not very experienced in chromatographic techniques therefore I would appreciate any help.

My first problem is:
I have a dichloromethane extract of some natural compounds which, as I expect, contains non-polar or weakly polar compounds. I would like to separate this sample by RP (first, preliminary fractionate it using solid-phase extraction, and then by HPLC). For this I should disolve my sample in polar solvent, like MetOH. I'm afraid however, than some non-polar compounds present in the sample can aggregate when increasing polarity. Do you have any tips to avoid the problem?

One more question, probably very basic:
If I wanted to separate the same sample in normal phase, should I expect that non-polar compounds will be weakly bound to the stationary phase or rather they will pass through the column?

Thank you.

[Alex Buske]

In terms of RP-LC Methanol is the LESS POLAR solvent. Injections of samples dissolved in MeOH will give suboptinal separation - even if its possible to dissolve everthing in MeOH.

I would fractionate first more generally: liquid-liuid separation DCM: Heptane and DCM:water to separate the most unpolar and most polar substances. alternatively do a normal phase open column separation. For this transfer your extract on silica gel (rot evaporator) and place on top of a silica gel column. Then extract with hexane, ethyl acetat, methanol and water or mixtures of that. Monitor by TLC.

[lemon]

Hi Alex,

Thank you for your reply.

[quote]In terms of RP-LC Methanol is the LESS POLAR solvent. Injections of samples dissolved in MeOH will give suboptinal separation - even if its possible to dissolve everthing in MeOH.[/quote]

What I was meaning is that methanol is more polar than dichloromethane in which is my sample. What I'm afraid of is that if I dry my dichloromethane sample and reconstitute it in methanol, I can loose some important hydrophobic compounds because they will not resuspend in the more polar solvent.

About normal phase separation I'm just wondering if non-polar or very low-polarity compounds have any affinity to the polar stationary phase, which would enable their separation, or they just pass through the column.

[mardexis]

If you can beg borrow or steal access to a GC/MS then you should do that. This is perfect for GC/MS. If you are stuck with LC then Normal phase seems better suited than RP.

[Uwe Neue]

The simplest thing is to try things out. Note that in most cases, a low sample concentration in te injection solvent is not a problem. If you do not see undissolved parts of your sample in the methanol, you can inject it.

Second, there are some special techniques that have been developed for the case where the MeOH, which is a strong solvent in RP, will create peak distortion. The technique is called "at-column dilution". I can describe it to you and/or send you some literature, if you need it.

SPE is a crude separation technique (I assume that you use silica SPE). In your first fraction (MeCl2), you will get anything that is less polar than MeCl2, typically aliphatic or polyaromatic hydrocarbons without or with few weakly polar functions. To elute the more polar material, you can increase the polarity of your elution solvent. I would go with the following steps (but these are wild guesses): MeCL2. then MeCl2 with 20 % MeOH, then MeOH, then MeOH/water 50/50. After you have done this for the first time, you will see which steps are not useful, and you can simplify things.

[lemon]

@mardexis
Thank you very much for your advice. I'm afraid, however, that GC/MS is not a solution for me. What I want to achievie is to purify a compound in bigger amounts for further assays, not for analytical purposes only.

@Uwe Neue
Thank you very much. I will search internet for the technique you've mentioned about but if know any good links, I would appreciate if you provided me with.

You are right that the best way is trying things out, but it also should be planned resonably, so I really appreciate any advice in this matter, especially that I'm not a chemist.

I have different types of SPE mini-columns to try (free samples from one of the suppliers): RP, normal phase, ion exchange, as silica as well as polymer-based. I assume, once I find appropriate conditions to roughly separate compounds on SPE, I will also get some useful information for further HPLC purification on similar kinds of beads.

[Alex Buske]

[quote="lemon"]Hi Alex,

What I was meaning is that methanol is more polar than dichloromethane in which is my sample. What I'm afraid of is that if I dry my dichloromethane sample and reconstitute it in methanol, I can loose some important hydrophobic compounds because they will not resuspend in the more polar solvent.
[/quote]

If they don't dissolve, they don't come out of a RP column. Then NP is the better solution. Yould have to dissolve your sample in an unpolar solvent like heptane however.

Separating mgs of sample with HPLC is possible but laborous. Believe me. Prep. LC equipment is expensive, not to mention freeze driers etc to get your sample out of the aqueous solvent.

Extract a portion of your extract with hex- or heptane and then try Uwes SPE separation (on silica gel).

Alex

[lemon]

Thank you Alex.

[Uwe Neue]

Two references:
U. D. Neue, C. B. Mazza, J. Y. Cavanaugh, Z. Lu, T. E. Wheat, “At-Column Dilution for Improved Loading in Preparative Chromatographyâ€

[lemon]

Thank you Uwe. I've sent you an email message.