[profmsg]

I'm trying to analyze purity of my organic triflate salt. Im  using gradient  system of 95:5 H2O: MeCN  to 5:95 at 25 min. Im getting very very broad peak. I have no idea if the organic cation and triflate anion elulte out separately or together?? I would be greatful if you could help me with this.

[discodermolide]

I'm trying to analyze purity of my organic triflate salt. Im  using gradient  system of 95:5 H2O: MeCN  to 5:95 at 25 min. Im getting very very broad peak. I have no idea if the organic cation and triflate anion elulte out separately or together?? I would be greatful if you could help me with this.

You will not separate the cation and the anion!
The triflate salt may be reacting with the water?

[profmsg]

You will not separate the cation and the anion!
The triflate salt may be reacting with the water?

so the salt should elute out as a single peak? I tried the same condition for the same organic compound without charge and got a very sharp peak. But once it is converted to salt, i got very broad peak. I tried to change mobile phase gradient, added 0.1% TFA, non of them seems to be working. Do you have any further advice?

[Arkcon]

Yes, by converting it to a salt, you've caused it to have multiple modes of separation, and that's why your peak is broad.  Also, is it also tailing badly?  That's another clue that you're getting multiple separation modes.  For analytical methods, you may get improvement with ion-pair reagents.  TFA is not good enough for this application, you may need triethylamine, or longer alkyl amines.  However, if this is for later workup or isolation, that would be a bad idea, ans they substances are sometimes difficult to purify away.  What potential impurities are you trying to separate?  Can you switch to some more advanced column chemistry?

[profmsg]

Yes, by converting it to a salt, you've caused it to have multiple modes of separation, and that's why your peak is broad.  Also, is it also tailing badly?  That's another clue that you're getting multiple separation modes.  For analytical methods, you may get improvement with ion-pair reagents.  TFA is not good enough for this application, you may need triethylamine, or longer alkyl amines.  However, if this is for later workup or isolation, that would be a bad idea, ans they substances are sometimes difficult to purify away.  What potential impurities are you trying to separate?  Can you switch to some more advanced column chemistry?

Thank you Arkcon. Exactly, it is tailing badly. This is only for analytical purpose. We do not intend to isolate. Small amount of methyl triflate  or starting material might still be present. So what % of  triethyl amine in mobile phase is good for this analysis?

[Arkcon]

The knee jerk response is 0.1 M TEAA.  You may get a better baseline if you also make the acetonitrile solvent 0.1 M TEAA, but you'll have to see.  You make also get away with as little as half, or a quarter that concentration of TEAA, especially if its in both eluent A and B.  Very low concentrations may be necessary, if you have to switch to LC-MS for any reason.  In fact, some people forbid the use of TEAA in a mass spec, its hard to get out.  Likewise, once you've used it on a column, it can't be easily washed out, and the column will always have a different separation profile.

I'm guessing you're using a typical silica-based C-18 bonded phase column to do reverse phase HPLC.  The charged molecule is interacting with exposed silanol groups, while the alkyl chain is interacting with the bonded C18 phase.  The two separation modes fight for the same molecule, so the peak widens, and tails.  There are newer column media types, that have embedded polar groups in the C-18 chain and embedded non-polar groups in the media, that may minimize the effect, without needing ion-pair reagent.

[profmsg]

The knee jerk response is 0.1 M TEAA.  You may get a better baseline if you also make the acetonitrile solvent 0.1 M TEAA, but you'll have to see.  You make also get away with as little as half, or a quarter that concentration of TEAA, especially if its in both eluent A and B.  Very low concentrations may be necessary, if you have to switch to LC-MS for any reason.  In fact, some people forbid the use of TEAA in a mass spec, its hard to get out.  Likewise, once you've used it on a column, it can't be easily washed out, and the column will always have a different separation profile.


I'm guessing you're using a typical silica-based C-18 bonded phase column to do reverse phase HPLC.  The charged molecule is interacting with exposed silanol groups, while the alkyl chain is interacting with the bonded C18 phase.  The two separation modes fight for the same molecule, so the peak widens, and tails.  There are newer column media types, that have embedded polar groups in the C-18 chain and embedded non-polar groups in the media, that may minimize the effect, without needing ion-pair reagent.

I tried using TEA .05% on both mobile phase. The noise level was so high that i could not run any sample. Why the fluctuation is so high when i was just running mobile phase. Is it suppose to stabilze after? it did not happen to me. I tried at 240, 260 and 460 nm absorptions

[Arkcon]

Yes, it will require a long initial equilibration, you effectively plugging the exposed silanols with the ammonium salt, leaving exposed triethyl groups.  As the conventional explanation goes.  Actually, its been determined that the actual process is much more complicated then that -- the TEAA coats everything, both column and analyte, in a sort of micelle, and those are separated by the solvent.  Anyway I digress ...

The fluctuations should disappear after a few column volumes at 50:50.  The baseline should lower after a couple column volumes at initial conditions.  Unfortunately, you will need a column volume or more between injections at initial conditions.  Modern HPLC methods are written with a vary rapid turnaround for consecutive injections, and that can really screw up reproducibility with ion pair reagents.  You may see a significant "baseline hump" as your gradient runs, as the solvent starts to elute ion pair reagent off the column.  So that's another thing you may have have to work around.

The TEAA has a pretty high UV cut-off, unfortunately, so you may be getting interference at short wavelengths.  Though I don't think 240 nm is all that low.  I routinely used 260 nm for this sort of analysis.  And 460 nm should work well.  That's assuming these are good wavelengths for your analyte -- 460 nm is near visible, is your analyte colored?

[profmsg]

Yes, it will require a long initial equilibration, you effectively plugging the exposed silanols with the ammonium salt, leaving exposed triethyl groups.  As the conventional explanation goes.  Actually, its been determined that the actual process is much more complicated then that -- the TEAA coats everything, both column and analyte, in a sort of micelle, and those are separated by the solvent.  Anyway I digress ...

The fluctuations should disappear after a few column volumes at 50:50.  The baseline should lower after a couple column volumes at initial conditions.  Unfortunately, you will need a column volume or more between injections at initial conditions.  Modern HPLC methods are written with a vary rapid turnaround for consecutive injections, and that can really screw up reproducibility with ion pair reagents.  You may see a significant "baseline hump" as your gradient runs, as the solvent starts to elute ion pair reagent off the column.  So that's another thing you may have have to work around.

The TEAA has a pretty high UV cut-off, unfortunately, so you may be getting interference at short wavelengths.  Though I don't think 240 nm is all that low.  I routinely used 260 nm for this sort of analysis.  And 460 nm should work well.  That's assuming these are good wavelengths for your analyte -- 460 nm is near visible, is your analyte colored?

Most of  my compounds are colored (either yellow or red) . DAD detector shows  the absorption bands at 240, 310 and 450 nm. Yes there is a baseline hump whenever i run the sample and no baseline hump in other sample without charge. You seems to have a very good experience in this type of analysis. Thank  you so much. I'll let it to equilibrate at 50:50 and let you know how things go. Thank you once again.