Hello,
I have a question to all of you with greater experience in rp-chromatograhy. I have a compound which elutes in 99% of ACN on Accucore Vanquish C18+ column. I tested also Ascentis C18 and is no good either. I cannot use NARP and strongly change the HPLC system in any other way. I have different C4, C8 and C18 columns and can slightly mix up with mobile phases.
Can you have some suggestions on what can I do to even slightly move this peak into the left?
Many thanks in advance! :)
Jack's answer is right on the money. You need to change the mobile phase polarity. Remember that you are partitioning, so you have to make sure that the compound you are interested in actually partitions between the stationary phase (in your case C18) and the mobile phase. Since C18 is highly non-polar, and your compound rather obviously likes a non-polar environment (highly lipophilic), you need to make it more attractive to your compound to be in the mobile phase.
Your C4 and C8 phases will probably help some; they don't tend to hold onto lipophilic compounds quite as strongly as C18. However, you gave us no other information about what else is in the run. I have to assume from your comments about not being able to make major changes that you have other compounds of interest but we don't know that. Remember, we can't help you with what you don't tell us, so make sure that you tell us the whole story.
Natalia Miliszkiewicz Instead of trying different columns, in your case I suggest you to go with ion-exchange chromtagraphy on selected column. As long as all of the molecules being analyzed are ionizable, retention on an ion exchange column should be possible.
And as a matter of fact, if you want early elution of drug, you need your mobile phase to be more similar to your drug (nonpolar) and your stationary phase to be more dissimilar (polar). In that case, C4 is more polar to C18. For moderate interaction/hydrophobicity, you can go with C8 for optimum retention times (not very early and not too late) and optimum capacity factor (4- 10)
Have you tried tetrahydrofuran instead of acetonitrile? It is a stronger solvent, and is miscible in water.
Jack's answer is right on the money. You need to change the mobile phase polarity. Remember that you are partitioning, so you have to make sure that the compound you are interested in actually partitions between the stationary phase (in your case C18) and the mobile phase. Since C18 is highly non-polar, and your compound rather obviously likes a non-polar environment (highly lipophilic), you need to make it more attractive to your compound to be in the mobile phase.
Your C4 and C8 phases will probably help some; they don't tend to hold onto lipophilic compounds quite as strongly as C18. However, you gave us no other information about what else is in the run. I have to assume from your comments about not being able to make major changes that you have other compounds of interest but we don't know that. Remember, we can't help you with what you don't tell us, so make sure that you tell us the whole story.
Just an off topic question, is it safe/recommended to use THF on HPLC system and/or LCMS platform? Is it one of the routine approaches to adopt in cases as reported in this thread?
Thank you very much for those hints. I will try with C4 column and maybe will change ACN to tetrahydrofuran.
I am just one of many people who run routine analyses on this system, so any big moderations are not welcome. For now, I'm focusing only on this compound.
Best!
Navid J. Ayon THF is used for HPLC. It will sometimes have preservatives added to prevent the formation of peroxides, and this may interfere with UV detection at some wavelengths. I tend to buy the THF without preservatives as I need it, and not keep any around after I am done. So, for an analytical UHPLC, I'd buy only a liter or two.
Good question, which adds to the conversation. The link below suggests that 90% THF in water is similar in strength to 100 % acetonitrile.
https://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/Acetonitrile-Methanol%20Substitution%20in%20C-18%20Reverse%20Phase%20App%20Note.pdf
You can also use IPA also instead of ACN (since it is a stronger non-polar solvent and is miscible with water). As I recall the wavelength extinction coefficient is 205 nm, which is much lower than THF.
I suggest you use the gradient elution, for which the mobile phase composition is systematically changed from a weaker solvent to a stronger
Hi @Natalia Miliszkiewicz, fortunately, in the HPLC system you've many parameters to work on. In your case, I suggest to either use a different solvent or even better; a solvent system (mixture of IPA or THF & ACN) isocratic or gradient (relying on the number and the nature of your eluents). Moreover, you can use the other columns you've got i.e. the C8 or C4.
All the changes, I've mentioned before, are depending on how far you want to change the parameters in your current system. If you want a reasonable change, then perform a reasonable change. Preferably, once at a time.
Hope this helps.
I think instead of 99% ACN you can try THF as a solvent or combination of it
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