Hi,
I am trying to separate supposedly small organic acids by HPLC. To do this I bought a C18 column with polar endcapping, since that approach has worked in the past. However, this column does not seem to be polar enough and I get close to no retention of my analytes.
My question is - does anyone have an idea about how to make the column act more polar? I'm thinking that since it already has polar sites maybe their impact could be enhanced by addition of a moderator to the eluent.
Looking forward to input on this matter!
best regards,
Maria Fanto
What solvent system are you using now?
If just using water/acetonitrile or methanol, try adding formic acid, trifluoroacetic acid, or acetic acid to the solvent system. lower pH tend to keep small organic acids as the free acid, which are less polar than their conjugate base. I've read up to 5% modifier.
Another possible approach is to make the mobile phase more polar by adding a salt, if your detector will permit doing so. ELSD and mass spectrometer detectors don't work very well with non-volatile salts. Possible salts include sodium chloride and phosphate salts, which can also act as buffers. This is essentially the "salting out" effect used for liquid extraction applied to solid phase extraction. Trying the acidic modifier mentioned above may make your mobile phase sufficiently polar to work.
A third possibility is to use aqueous normal phase using a diol, alumina, or silica column. The weak solvent is something like acetonitrile, methanol, ethanol, acetone, or another organic solvent miscible with water. The strong solvent is water. You may still want a modifier such as those mentioned for C18. The technique is also called HILIC.
Depending on what you are doing, you may need ion exchange columns.
Here are some links to aqueous normal phase (for flash chromatography):
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN78_HILIC_Purification_Strategies_for_Flash_Chromatography.pdf
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN84_RediSep_Rf_Gold_Silica_and_Highly_Polar_Solvents.pdf
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Strategies_for_the_Flash_Purification_of_Highly_Polar_Compounds.pdf
What solvent system are you using now?
If just using water/acetonitrile or methanol, try adding formic acid, trifluoroacetic acid, or acetic acid to the solvent system. lower pH tend to keep small organic acids as the free acid, which are less polar than their conjugate base. I've read up to 5% modifier.
Another possible approach is to make the mobile phase more polar by adding a salt, if your detector will permit doing so. ELSD and mass spectrometer detectors don't work very well with non-volatile salts. Possible salts include sodium chloride and phosphate salts, which can also act as buffers. This is essentially the "salting out" effect used for liquid extraction applied to solid phase extraction. Trying the acidic modifier mentioned above may make your mobile phase sufficiently polar to work.
A third possibility is to use aqueous normal phase using a diol, alumina, or silica column. The weak solvent is something like acetonitrile, methanol, ethanol, acetone, or another organic solvent miscible with water. The strong solvent is water. You may still want a modifier such as those mentioned for C18. The technique is also called HILIC.
Depending on what you are doing, you may need ion exchange columns.
Here are some links to aqueous normal phase (for flash chromatography):
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN78_HILIC_Purification_Strategies_for_Flash_Chromatography.pdf
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN84_RediSep_Rf_Gold_Silica_and_Highly_Polar_Solvents.pdf
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Strategies_for_the_Flash_Purification_of_Highly_Polar_Compounds.pdf
More aqueous phase in mobile phase will block the active polar site of HPLC column stationary phase and less will be the analyte retain.
Add 20mM tetraethylammonium chloride to your mobile phases, and hold in 100% aqueous phase for a few minutes before you start your gradient.
Hi Jack,
thank you very much for your extensive answer. The mobile phase I use today is acidic - 20mM fosfate buffer pH 2.5. I will try using a higher concentration!
About adding inorganic salts to the mobile phase - I was under the impression that salts should be avoided in the system since it can take a long time to flush them out. Is this incorrect?
best regards
Maria
The covalently bonded phases, whose polarity can be easily adjusted, constitute the base of the reversed phase polarity partition chromatography. Part of the Si-OH groups remain intact and can establish polar interaction. Endcapped C18 colums eliminate polar interactions as the residual silanol groups on the silica surface are capped with trimethylsilyl groups. For increasing polarity of the stationary phase it could be better to use non endcapped C18 colums.
The use of a mobile phase rich in water weakens the separation in a stationary phase of type RP-18. For this reason, as Jack said, phases presenting a residual polarity, such as Phenyl, Amine, Diol,... are preferred.
Why you do not try to use weak polar column instead of using end caped RP-C18? However, nonendcapped RP-C18 may have alternative appropraite choice.
Hi Seyed,
it is end capped, but it is a polar endcapping. The column is made to separate polar organic compounds.
Could you give more details regarding this "polar endcapping" to separate polar organic compounds?
Hi Luis,
knowing exactly how the stationary phase is made of is sometimes one of the tricky bits. I have not been able ot retrieve any information about what the endcapping consists of, only that it is a polar substance.
Hi Maria,
If the phase is polar, why you have selected the endcapped type. By this, as the polar end has been capped, therefore, the polarity of the phase has been reduced too, so, make the phase not to have an apprpraite polarity for your compound of interest. What I am thinking is that, by endcapping the phase, the functional group of OH has been capped. Am I right. Probably, I am not properly oriented to your problem!
@Sayed & Luis
Some types of C18 columns are end-capped with, instead of TMS, a more polar group that helps the column resist phase collapse. Most of these are proprietary end-cappings. The advantage of these columns is the issues of compounds interacting with both the C18 and the bare silanol groups is reduced, as with a methyl end-cap, but the column runs better in highly aqueous solvent systems.
Here is an app note showing how such an "Aq" column improves resolution for flash chromatography: http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN76_RediSep_Rf_Gold_C18A_for_Highly_Aqueous_Mobile_Phases.pdf
@Maria-
Since you are using a phosphate buffer, you are already using an inorganic salt. Your procedure for equilibration and washing that you are using now should suffice. After using your column, it is a good idea to wash out your buffer. I find doing so improves column life.
Thanks again, Jack.
I will keep trying. But if I increase the ion strength in the eluent - is there not a risk for phase collaps? Is such a collaps reversible?
Maria, increasing the ionic strength will not lead to phase collapse. Only running 100% aqueous buffer will cause this. you do have to worry however with high salt buffers salting out in the column.
Jack, this is an interesting discussion.
I have read RediSep Rf Gold C18Aq Application Note. How do the hydrophilic groups reduce tendencies towards phase collapse allowing better retention of highly polar samples?
What is the advantage of using RP-18 stationary phase with hydrophilic groups? This is a little bit contradictory, in my opinion.
Maria, when using high ionic strength mobile phases it is very possible to precipitate salts in the system particularly when raising the % organic in the phase.
Bruce!
If I add a permanent cation to the column - don't I risk to make it more hydrophobic by possibly irreversibly attract the nitrongen to polar sites? I have no info on what kind of polarity that the endcapping consists of.
@Luis-
The problem is that under highly polar and aqueous conditions, the C18 chains interact with each other rather than with the solvent. By placing a polar group in the end-capping, the interior environment is just as "uninviting" as the solvent. If the C18 starts to aggregate, there is a polar group down there as well as outside in the bulk solvent. I suspect, but don't know because I don't know exactly what is used in the end-capping, that the polar solvent molecules are better able to infiltrate the C18 chains. This would alter the environment between the C18 to reduce phase collapse as well.
@Maria- Phase collapse is reversible. The column simply needs any salts washed out (to prevent precipitation as mentioned earlier), then washed with your organic solvent. The solvent will interact with the C18 chains and the column is good again. The column you are using is resistant to phase collapse, but there is the possibility it will occur. The symptom of phase collapse is that peaks that were resolved suddenly elute quickly; washing the column as described above restores the elution.
In this case (adding the tetraethylammonium chloride) the column end capping may be irrelevant. I am suggesting forming a more hydrophobic ion pair with the organic acids to allow them to interact more strongly with the C18 phase. You want a stronger column interaction to be able to retain compounds that elute near the void, so add a hydrophobic ion pairing agent. The cation is not permanent the teraethylammonium chloride is very soluble in methanol and water and can be washed from the column. You should be able to buy this compound pure enough to do UV detection on the HPLC. Do not even consider trying this additive for LC/MS! If tetraethyl doesn't work you can always try longer chain combinations on the quaternary amine. The tetraethylammonium ion pair worked for me many years ago on a C18 column with UV detection.
@ Jack and Bruce
Thank you for your input. I learned a lot.
Best regards
Maria
Jack, from your answer I understood that the main advantage of using polar end-capping is to avoid the phase collapse. Your explanation is very clear. Thanks.
@ Jack Sílver
You said: ..."Another possible approach is to make the mobile phase more polar by adding a salt, if your detector will permit doing so.... (on a C18) ... sodium chloride ... salting out. "
When I think salting out I'm thinking high concentration salt. In this case - is it still up to 5% modifier that is recommendable?
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