Chromatography Forum

I know there has been a lot of discussion here about the choice of different columns which show better retention/selectivity for polar compounds in reversed phase mode, however I've come across a problem with my flash chromatography cartridge for analyzing a mixture of polar and nonpolar compounds and you know that for flash cartridges/sorbents there is not that much choices .
(I'm studying two compounds, one is polar, which elutes first the other one non polar which elutes late)

Unfortunately the polar fraction, which one of my compounds is in, tend to elute so fast with solvent front and other impurities, this has been with 100% water. Manufacturer suggest that there is silanol activites, but seems to not to be that effective.
Any idea how can I increase the retention? I'm not sure about the moiety of the compounds (trying to find out) but guess they might be acidic.

change in pH? any additive, prefer not as it is going to be further analyzed with MS and NMR ? using C8 cartridge instead of C18 to increase the polar interaction with resin, phenyl functionalized to resin to decrease hydrophobicity and increase polar retention possibly?

Flow rate is already at minimum ( 5ml/min) with a 14 ml /10 g cartridge.
Sorry for the the long post and thanks in advance for coming advices!

Hi Alireza,

It’s quite understandable if one keeps some details undisclosed in certain situations. But the side effect of that is: We don’t know how to help you.
If you share some details (type of compounds f. ex. functional groups, Mw, stationary phase type etc.) then some of the members of this eminent board might have valuable suggestions for you.
One thing is almost certain: Lower flow rate won’t increase the effective retention. Actually it will increase the retention generally, but it will affect the retention of the other impurities (those you’re not interested in) as well as the solvent front. So, what you need is retention, combined with selectivity. But as mentioned above some details are needed for further thoughts/suggestions.

Best Regards

P.S. BTW when considering the flow rate, the most important parameter is the diameter of the column. The mass and the volume are of less importance in this context.

Dear danko,

I was going to be dissapointed of getting any reply after that long wait! seems it's been my fault maybe, any way, the nature of my research is to try to find out whatthese compounds are, and I do not have much information to give to be diiscussed in first place!

The only think I can add is their hydrophilic nature, probably heterocylclic structure (by some rough results from Mass Spec) and they can generally be classified as metabolomics, as they have derived from microorganisms,

The stationary phase, as I pointed out later in the post, is typical C18, which runs under 100% water. The issue , as siad as well, is co-elution of impurities with product in solvent front, and I think of how can I retain one compound and let the others go, but as you suggested this may need further information about the structure which unfortunately is not available.

The only indication of polarity is when I run the sample on the synergi-fusion type C18 (Phenomenex) which has improved retention for polar with same mobile phase, and the impurities tend to elute with solvent front, and my compound after that, which may suggest is a bit less polar comparing to impurities.

Thanks

I see. Well it seams to me it is going to be the hard way you’ll have to solve this problem (i.e. try and error).
You can hope the polarity of your compounds is partially due to one or several acidic groups. If so, acidifying the mobile phase should give you some indications of that (i.e. increasing retention time). For a start you can try 10 – 20 mM Sodium Phosphate buffer at pH 2.2 – 2.5.
If that doesn’t help, there might be some amino groups on your compound/s. And that was the reason I asked the “stationary phase questionâ€

Hi Alireza,

1 Not all C18 are suitable for running with 100% water. That could result in "dewetting" or "phase collapse", which both describe a loss of retention. If you are not sure that your phase tolerates 100% water try to store it over night in 100% organic , equilibrate and run with maybe 5% or 3%organic.

2. If that doesn't work or apply and you need just samples for NMR and MS try to separate on your Fusion column. Analytical columns can be loaded with up to 1mg sample (maybe precleaned with flash). Put your sample in the NMR and recover the substance for MS (could give nice effects due to H to D exchange).

3. try HILIC. Thats basically a MeOHor ACN to water gradient on pure silica (without any derivatisation).

4. If you play with pH make sure you use a volatile buffer (TFA would be ok if your compound is acidic). Be aware that TFA concentrations can rise during evaporaion of the solvent and cause reactions.

5. Keep having fun.

Alex

Hi Alireza,
I am sure that you can significantly increase the retention of whatever polar analyte you are analyzing by simply using 2% acetonitrile along with 0.05% heptafluorobutyric acid + 0.1% TFA in loading mobile phase. That always helps, at least for my analytes (broad variaties). If that does not yield with satisfactory result you can always use some ion pairing agents like octansulfonic acid. however, you must take care that octansulfonic acid is not compatible with the MS
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