Chromatography Forum

Nice problem to be discussed here:D

Just some quick thoughts that came to my mind:
- Just to be sure that there are no undissolved excipients left, I'd add a filtration step via syringe filter. 0.45µm filters usually are fine, 0.20µm might be better to be on the safe side
- If anyhow possible, use your mobile phase as sample solvent (in the case of gradients, the initial mobile phase). You might prevent a lot of problems
- You wrote that your analytes of interest are similar to Boceprevir. Boceprevir has no ionazible groups. If the same is true for your analytes, you don't need any buffer or acid in your mobile phase. Good thing - your mobile phase is simpler. Bad thing - you loose the most powerful tool to tune separation selectivity, i.e. mobile phase pH. If, on the other hand, you impurities contain ionizible groups, you will see huge changes in selectivity when changing pH.
- For gradient separations, particle size of your stationary phase has much less impact on separation efficiency than with isocratic separations. If pressure is an issue, you might consider changing to a 5µm phase. With gradients, you usually don't loose that much efficiency.
- The columns you tested so far (ACE C18, XBridge C18, and YMC Pro C18) are all quite similar. They belong to the same group of very hydrophobic, well-endcapped columns. Therefore, chromatography usually will look quite similar on all of them. In your case, if it doesn't look good on ACE, chance is high it will not look much better on XBridge or YMC Pro. For REAL changes in column selectivity, you might want to look at different column chemistries, like polar-embedded C18s. And don't forget there other things than C18...
- It might me worth to try some other organic modifiers than just ACN and MeOH. THF and 2-propanole, for example, can give quite astonishing shifts in selectivity even when used in very small amounts.

If your samples are centrifuged at room temperature don’t use a cooled autosampler to avoid precipitation in autosampler vial.

Now I have seen your most recent chromatogram. Here are my first thoughts:
You have problems with re-equilibration. Problems with the pump are also possible. Or chemistry interactions are absolutely not sufficient for your substance.

In these test we are using three independent channels (for ternary mixture) in a Waters Alliance equipment.

I have tried this once long time ago and the results were absolutely useless. I cannot remember if GPV was broken or it was a general problem. Nevertheless mixing the solvents especially with a low-pressure pump can cause partially precipitation of the buffer salt. Please check your system.

XBridge, YMC Pro C18 columns did not bring better resolution when used with original and extended gradient.
From your experience and from the examples I have posted, do you agree on using original column ACE 4.6x150 mm, 3 um, but changing mobile phase (e.g. remove phosphate salt and include TFA), and other parameters?

Your column should be fine. If you have a 5 cm column you can shorten the runtime for your experimental runs by increasing the flow. As HPLCaddict mentioned buffers may be necessary for impurities or excipients. I would suggest 0.1 % TFA and 0.1% TFA in ACN for your mobile phase A and B and make four experiments (like needed for Drylab model) with different gradient time (e.g. 15 and 25 minutes for 15cm colum) and temperature (20°C and 60°C) to see how selectivity is influenced. You can also do four additional runs using methanol instead of ACN.

Nice problem to be discussed here:D

Just some quick thoughts that came to my mind:
- Just to be sure that there are no undissolved excipients left, I'd add a filtration step via syringe filter. 0.45µm filters usually are fine, 0.20µm might be better to be on the safe side
- If anyhow possible, use your mobile phase as sample solvent (in the case of gradients, the initial mobile phase). You might prevent a lot of problems
- You wrote that your analytes of interest are similar to Boceprevir. Boceprevir has no ionazible groups. If the same is true for your analytes, you don't need any buffer or acid in your mobile phase. Good thing - your mobile phase is simpler. Bad thing - you loose the most powerful tool to tune separation selectivity, i.e. mobile phase pH. If, on the other hand, you impurities contain ionizible groups, you will see huge changes in selectivity when changing pH.
- For gradient separations, particle size of your stationary phase has much less impact on separation efficiency than with isocratic separations. If pressure is an issue, you might consider changing to a 5µm phase. With gradients, you usually don't loose that much efficiency.
- The columns you tested so far (ACE C18, XBridge C18, and YMC Pro C18) are all quite similar. They belong to the same group of very hydrophobic, well-endcapped columns. Therefore, chromatography usually will look quite similar on all of them. In your case, if it doesn't look good on ACE, chance is high it will not look much better on XBridge or YMC Pro. For REAL changes in column selectivity, you might want to look at different column chemistries, like polar-embedded C18s. And don't forget there other things than C18...
- It might me worth to try some other organic modifiers than just ACN and MeOH. THF and 2-propanole, for example, can give quite astonishing shifts in selectivity even when used in very small amounts.

Thank you for your interest and feedback.

- In fact only with two "integrated" steps (centrifugation and filtration) we were able to clarify the "solution" (at the end it is a "suspension" of undissolved excipients). But not completely. A careful observation can still detect some "opalescence". Filters (0.45 um, 25 mm diameter) tend to block easily with excipients.
- When we tested mobile phase as solvent that provided a similar profile to the one obtained with methanol. Of course always using the original gradient.
- In fact the two different methods include phosphate buffer (pH = 7) (mehtod 1, the original shown here) and the other (alternative) with 20 mM of ammonium acetate (pH = 6.7). I'm not sure if we really need to fix pH around 7 to ensure separation/efficiency and avoid any possible degradation. This has to be further investigated.
- Ok. Probably we will test 5 um of column particle size.
- I understand your point in regarding to column chemistry. But don't you agree that we can keep ACE column and work through mobile phase/solvent/gradient?
- We have some methods (one is compendial) applying THF in mobile phase. With 2-propanol I have no experience. In any case don't you think, any of them can be "worst" and can accelerate the column degradation?

Thank you again for all your support.

If your samples are centrifuged at room temperature don’t use a cooled autosampler to avoid precipitation in autosampler vial.

Now I have seen your most recent chromatogram. Here are my first thoughts:
You have problems with re-equilibration. Problems with the pump are also possible. Or chemistry interactions are absolutely not sufficient for your substance.

In these test we are using three independent channels (for ternary mixture) in a Waters Alliance equipment.

I have tried this once long time ago and the results were absolutely useless. I cannot remember if GPV was broken or it was a general problem. Nevertheless mixing the solvents especially with a low-pressure pump can cause partially precipitation of the buffer salt. Please check your system.

XBridge, YMC Pro C18 columns did not bring better resolution when used with original and extended gradient.
From your experience and from the examples I have posted, do you agree on using original column ACE 4.6x150 mm, 3 um, but changing mobile phase (e.g. remove phosphate salt and include TFA), and other parameters?

Your column should be fine. If you have a 5 cm column you can shorten the runtime for your experimental runs by increasing the flow. As HPLCaddict mentioned buffers may be necessary for impurities or excipients. I would suggest 0.1 % TFA and 0.1% TFA in ACN for your mobile phase A and B and make four experiments (like needed for Drylab model) with different gradient time (e.g. 15 and 25 minutes for 15cm colum) and temperature (20°C and 60°C) to see how selectivity is influenced. You can also do four additional runs using methanol instead of ACN.

- Yes. We will define room temperature (20ºC) for autosampler.
- The use of three independent channels was only a faster way to screen several gradients using original components MeOH, ACN, and phosphate buffer and avoid the tedious need of preparing several and consecutive different mobile phases with different % components. But you're right. The focus would be to simplify the mobile phase composition and test different %s.
- Thank you again for your reccomendation for specific tests. We have to "fight against time", so I would follow your suggestions using the ACE column with 3 or 5 um.
- As soon as I have "new" data I will comment on that here for your evaluation.

In the meantime if you think any other test would be relevant please let me know.
Thank you very very much for your feedback. Very nice to have here this discussion.

This is the overlay for ACE (30ºC) vs. YMC (45ºC).
Ammonium acetate 20 mM gives a pH around 6.7
Pre-main peak region seems better but post-main peak zone seems to loose resolution/separation for the closest impurity.