Low-pH taling of bases in RPC

[kaavie0801]

Hello everyone
This figure shows the overlaid chromatograms for 0.05–1 μg of phenylethylamine in RPC. Column: Zorbax SB-C8 (150 × 4.6 mm i.d., 5 μm particles); mobile phase:10 mM H3PO4–acetonitrile 95:5 (wwpH 2.2) at 1.5 mL min−1.

my opinion is the amine had an overload tailing. however, a referee commented that "the tailing was caused by the interactions between protonated analytes and surface silanols, rather than by column overload, because the concentrations of the analyte was very small." Would you please give your comment?
Thanks
Kaavie

[Gerhard Kratz]

I agree with your referee. Can you run at a pH around the pK of your compound?
At more alkaline pH tailing must be reduced.
Good luck.

[kaavie0801]

I'm confused about the various explanations in literature for the peak tailing of ionized bases in RPC.
What on earth are the reasons for the peak tailing, kinetic or thermodynamic?

[Gerhard Kratz]

Please don't be confused, that's our daily bread and butter.
Maybe your question will be answered in more detail if you google for this article: LC Troubleshooting, Why Do Peaks Tail?
John W. Dolan, BASi Northwest Laboratory, McMinnville, Oregon, USA.
Or you will be even more confused.
To continue with your work don't ask why tailing, ask how can I get rid of Tailing. Good luck

[artsjeroen]

The use of end capped bonded phases or a different type of column (CN or Zr - column) might give better results. Mobile phases with high-ionic strength or amine modifiers as additive in the mobile phase might help to decrease the peak tailing on silica based columns.

[kaavie0801]

Thank you for your replies, Gerhard and artsjeroen.
I have no trouble eliminating the peak tailing problem.
However, according to a recent review on this topic (D. V. McCalley, "The challenges of the analysis of basic compounds by high performance liquid chromatography: Some possible approaches for improved separations," Journal of Chromatography A, vol. 1217, p. 858–880, 2010. ), the peak tailing problem I encountered can be attributed to column overloading. So I just have no idea how to answer to the referee properly because I'm not quite sure who is right.
Can someone explain it to me in detail？

[Gerhard Kratz]

Looking on your chromatogram it is not overloading!

[artsjeroen]

http://www.crawfordscientific.com/Chrom ... rload.html

This shows a similar situation as you describe. I didn't have time to read it completely so I hope it might be useful.

[kaavie0801]

http://www.crawfordscientific.com/Chrom ... rload.html

This shows a similar situation as you describe. I didn't have time to read it completely so I hope it might be useful.

Thanks for providing the information. According to this paper, ionized amines easily overload reversed-phase columns because of solute–solute electrostatic repulsion，leading to nonlinear tailing. This explanation generally agrees with that of McCalley.
I would welcome further discussion.

[HPLCaddict]

Column: Zorbax SB-C8 (150 × 4.6 mm i.d., 5 μm particles); mobile phase:10 mM H3PO4–acetonitrile 95:5 (wwpH 2.2) at 1.5 mL min−1.

however, a referee commented that the tailing was caused by the interactions between protonated analytes and surface silanols, rather than by column overload, because the concentrations of the analyte was very small. Would you please give your comment?

At a pH of ~2.2 all "normal" surface silanols should be protonated. AFAIK the silanols pka is in the range of 4-5. Protonated silanols do not lead to strong ionic interactions with protonated bases.
That leaves the more acidic silanols, e.g. silanols activated by metal impurities, for these interactions. Zorbax-SB AFAIK is a moderately modern type B column which should not have much of these metal impurities. I'd therefore conclude that classic silanol interactions are NOT the main cause of the tailing.

[kaavie0801]

I'd therefore conclude that classic silanol interactions are NOT the main cause of the tailing.

Many thanks for your valuable discussion. I fully agree with that. Then what's your view of the cause and nature of the tailing?

[Klaus I.]

I’m thinking that you are right. The picture looks like a classical example for overload. Some parts of the analyte have no chance to interact with the stationary phase, since the stationary phase is occupied by other molecules of your analyte. Therefore many of the molecules stay in the mobile phase and are eluting earlier. Simple ‘chemical tailing’ would give more delay compared to the symmetric peak with low concentration (I would assume that this was meant by your referee).

Since you are analyzing a charged molecule under reversed-phase conditions it is normal, that the capacity of your RP-column for such interactions is very limited. Most of the RP-column surface is constructed for non-polar interactions.

[kaavie0801]

I’m thinking that you are right.

Thanks for the good discussion, Klaus. Now it seems that more of you are against the referee's point and I'm encouraged

[Klaus I.]

... and I'm encouraged

I apologize, that is not what I wanted

To receive the agreement from others for the own considerations is at the end often not very helpful for the scientific process.

Have you planned more experiments on this topic? I’m just curious how the chromatograms will look when you replace the H3PO4 with TFA or replace the column with a Zorbax SB-C18.

You provided us a really nice example about the possible polar/ionic interactions with a C8 column although the column is claimed with steric protection. Thank you.

[M Farooq]

Hello everyone
This figure shows the overlaid chromatograms for 0.05–1 μg of phenylethylamine in RPC. Column: Zorbax SB-C8 (150 × 4.6 mm i.d., 5 μm particles); mobile phase:10 mM H3PO4–acetonitrile 95:5 (wwpH 2.2) at 1.5 mL min−1.

my opinion is the amine had an overload tailing. however, a referee commented that "the tailing was caused by the interactions between protonated analytes and surface silanols, rather than by column overload, because the concentrations of the analyte was very small." Would you please give your comment?
Thanks
Kaavie

I am afraid that I do not agree with the reviewer. This is a mass overload effect (Langmuirian isotherm). Since the peak shape at low concentration is perfectly symmetrical it implies that there are no (or few) secondary interactions.

As the name of your stationary phase implies, it has large steric groups (probably isopropyl) which protect the silica suface. Even if there are silanols, they must be shielded. One can guess that silanols are partially protonated at low pH (pKa ~ 3) so the question of cation exchange does not even arise to a significant extent. To confirm the silanol activity, I usually inject benzyltrimethyl ammonium chloride and see the peak shape for detecting silanol activity. If the peak shape is symmetrical and the elution time is not too high, it means there is little cation exchange going on. You can convince the reviewer by a similar experiment.