Tailing question

[Mike H.]

What steps can be taken to minimize peak tailing besides pH changes?

I'm currently working on an HPLC method to resolve 6 compounds. I have adequate K', good separation, but the main peak is tailing somewhat ~1.8. I have tried pH changes basic and acidic. (7.6 PPD - 0.1% H3PO4) with no effect on tailing. Does anyone have any suggestions?

[Andy Alpert]

1) What is your starting mobile phase and what's the solvent that your sample is in when injected?

2) Does your compound have any ionizable groups? Be specific. If it does, then what electrolytes are in your sample solvent and in the mobile phase?

[Gerhard Kratz]

In addition to Andy I ask, what column (C18??) are you using? Remember: C18 is never the same!

[tkubowicz]

Hello

You didn't give any details about your compounds (neutral/bases).
Triethylamine is common additive to mobile phase to reduce peak tailing.

Please provide more information about your compounds, solvents and method.

Regards

Tomasz Kubowicz

[Mike H.]

I made a mistake in thinking I was limited in pH due to stability of sample. After increasing pH to 8.0 the tailing was 1.1. FWIW I was using a Phenomenex EVO column, 20mmol PPD pH 8.0, standard ACN gradient, 60° and the compound is basic.

Although separation is adequate (critical resolution pair is 1.8 ) the separation was better under acidic condition, except for the bad tailing of course. I have one question, possibly retarded. Can an additive such as TEA be added to a acidic buffered mobile to control tailing. I know this sounds counter intuitive, but an Agilent tailing guide seemed to suggest this was an option. Thx.

[Andy Alpert]

You used only 20 mM salt because you're used to thinking that salt doesn't promote elution in reversed-phase. Accordingly, the amount you have won't affect retention much. That's true of retention, more or less, but it's not true of peak shape. A charged analyte migrates through a chromatography column with a counterion(s). Here, that would be an anion. If more than one type of anion is present (e.g., one salt in the sample solvent and a different one in the mobile phase), then different molecules of the analyte will have different counterions. The resulting ion pairs will differ in polarity and will migrate through either a reversed-phase or a HILIC column at different rates. In a worst-case scenario, you'll get multiple peaks for a single compound. Less severe cases manifest as skewed or tailing peaks. For an example of an extreme case of this, see Fig. 14 in the following paper: http://pubs.acs.org/doi/pdf/10.1021/ac070997p
The solution is to have the same anions in both the sample solvent and in the mobile phase and to use enough of them. Try repeating your acidic conditions run with 2x or even 3x the concentration of salt.

I speculate that the Agilent tailing guide says to add TEA because with an adequately buffered solution you won't change the pH much but you will increase the salt concentration.

[Mike H.]

This is simple enough to do. Thanks for the suggestion.

[Mike H.]

Using an NH4Fm concentration of 200mmol tailing was down from 1.8-1.3 at pH 4.8. Does this imply that pH is no longer important with respect to tailing if you can using increasing buffer concentration?

Is there a rule of thumb ceiling for buffers such as PPD,PPM? I was always told to limit the concentration of these buffer as much as possible. Would 50mmol of potassium phosphate dibasic be too much for a separation?

[Andy Alpert]

It's probably too broad a statement to say that pH is never important regarding peak shape, but certainly increasing the salt concentration helps things regarding analytes with a high charge-to-mass ratio.

Phosphate salts: Did your instructors/supervisors say why you should keep their concentration as low as possible? I suspect it's a matter of convenience. Use of salts requires occasional flushing of the system and could produce accumulating salt deposits if there's a minor leak someplace (e.g., behind a seal that's less than pristine). Also, with the widespread use of mass spectrometry these days (and corona discharge or ELSD detectors), even volatile salts must be kept below certain limits. That's a factor affecting the detector, though, not the chromatography. Usually 50 mM salt suffices to confer all the benefits you're going to get in some mode of chromatography other than ion-exchange or HIC. Extreme cases (e.g., an aminoglycoside antibiotic) may require concentrations as high as 125 mM. Let me know if you want references.