Piperidine derivatives - extra peak in pure compounds

[Radek1990]

Welcome

I need help with solving a certain problem. Maybe someone had the same problem as mine and have a tip.
I have to prepare a calibration curves and make pharmacokinetic analize piperidine derivatives, which were synthesied. Purity all this compounds at least is 98%. They are free basis/acids or their hydrochlorid salts. During the preparation of the calibration curves compounds as a free form basis/acids, I noticed the appearance of an additional peak in the dead volume. Peaks area of extra peak increase with injected volume.
All hydrochlorid salts eluted as a one peak in adequate elution time.
Concentration probes: less than 0,2 mg/ml.
Solvent used: for salts: water or water/ACN 1:1; for free: MeOH (they don’t disolve in water or water/MeOH mix and water/ACN mix)
Injection volume: 1-10 µl.
Column: Kinetex 2.6µm PS C18 100A, 100 ×2,1 mm
Solvent A: water with 0,2% TFA (initial TFA concentration was 0,1%, after reading one topic increased)
Solvent B: ACN with 0,2% TFA (initial TFA concentration was 0,1%, after reading one topic increased)
Flow rate: 0,5 ml/min
Also i tried on other column: Accucore RP-MS 100×2,1 mm
Solvent A: 5% water/ACN with 0,2% TFA
Solvent B: ACN with 0,2% TFA
Flow rate: 0,5 ml/min
Probabilities:
- add buffer
- solvent is so strong
- altering the colmun but I don’t know which one will be better.

Please help.

[DR]

If it's in the dead volume, you really cannot count it as an additional peak.
If you cannot get the "extra" peak to elute later by lowering or holding at your beginning ACN concentration, it's not worth worrying about.

[Radek1990]

Unfortunately, I need to worry about because results from hplc analysis will be published in the future. If my MS worked properly, probably I would be calm albout results. Reviewer maybe accept this part of work.

I came up with an idea yesterday. Maybe i should add some buffers as a eluent. Probably all this synthesised compounds are "simmilar" to aminoacids. I know, that pKa compound is very important in HPLC analysis and scientist should worry about pH solution. But i have not realise that inappropriate pH cause compound elution at the beggining, without any affinity to stationary phase, like uracil.

[DR]

Knowing the pKa of each of your analytes is very important as having your MP's pH too close can result in peak splitting or smearing (different from having no retention in the stationary phase unless your peaks are poorly retained in the first place).

If you're using a mass spec for detection, you should not use any buffers that are not volatile.

The more you don't tell us, the more we cannot help you.

[LuccasLN]

Hi Radek,

I hope you are doing fine. let me first start by saying that DR is right when he says that it might not be worthy to check this out. i have failed to understand why this is an issue since you can quantify your active compound. you could probably try to point the other peak as "other" or "reaction byproduct" and keep your synthesis study. I can assure you i have lost an enormous amount of time trying to understand structures and origins of impurity signals from my synthesis and it has never had a rewarding ending. Anyway, if this is really important to you here are some strats you may find interesting as well:

- you are correct: changing mobile phase selectivity (buffers and solvents) may help you, just be aware of buffer-detector compatibility, like DR has said
- change stationary phases as well, if possible to a totally porous particle. i know SPP phases are told to be fast and efficient... but you are using quantities that are probbably way too high for an SPP, i would say that TPPs are more suitable for the kind of application you are looking for. also i do not like the idea of analyzing something very new in a PS column as first choice, you could be excluding molecules due to electrostatic repulsion for example. finally, since you are not really sure of what you have or may have for a synthesis product it is always good to have a column toolbox that involve moderate to high retention columns...not fast ones. you may want to have 100% water compatible C18 too.
- you mention a dilution with metanol and a mobile phase with ACN. and you have never mentioned detection. i mean, is it UV? what WL? can you confirm the diluent is different in comparison to the mobile phase? i think it would be nice to inject just diluent or any other reagent you may use for sample prep... maybe it is just a ghost peak
- Once you confirm it is not on any blank/reagent control. you could see if you can collect that in the outlet of your HPLC system, check if you have it and try to figure out what it is (once more, this is very difficult and payback is most of the times not worthy, but it is my duty to tell you this is something that people do.)
- You could run a final "unit operation" step on your synthesis procedure where you eliminate this impurity. example: a gravity chromatography column packed with a silica based stationary phase. load the sample, eliminate the early eluter, elute your API, analyze it!

i really hope this helps you.

best regards,