Wandering baseline in buffered MeOH pH 10.7 UV 254

[Calendula]

Hi, forum,

I have prepared some Methanol and Water mobile phases with pH 10.7 phosphate buffer (3.9 g/L HNA2PO4*2H2O brought to pH with NaOH). I am running this on a new Kinetex Evo core-shell C-18 with guard column and VWR detection at 254nm... trying to figure out why I am seeing the baseline that I am.

My mobile phases are
A) 10% MeOH, 90% phosphate buffer
B) 90% MeOH, 10% phosphate buffer

The gradient is
time
0: 100%A
20: 100%B
30: 100%A
35: 100%A

I'm also seeing that the back pressure follows the gradient, as you would expect, but instead of being highest with the highest percent aqueous, it is maximized at about 50%A/50%B.

I'm hoping there is no precipitation when the phases mix, but I'm wondering if this is possibly happening--and my guard column is catching it and causing the increased back pressure.

I still can't explain why the dramatic change in baseline, though.



Any ideas? This also happened at 277nm.

Thanks in advance!

[HPLCaddict]

I'm also seeing that the back pressure follows the gradient, as you would expect, but instead of being highest with the highest percent aqueous, it is maximized at about 50%A/50%B.

That's exactly what you should expect. In contrast to Acetonitrile, Methanol/Water mixtures have the viscosity maximum at ~50/50, so there's the pressure maximum, too. Pure methanol shows roughly the same backpressure as pure water.
Methanol is much more polar than acetonitrile, so you seldom see problems with precipitating buffers. Still, I'd be reluctant to go to 90% Methanol with a phosphate buffer.

Concerning the baseline drift, it seems your A eluent has much higher UV absorbance than your B solvent. Both phosphate and methanol should be rather transparent at 254 nm. Maybe some sort of contamination? Is this reproducible? If you didn't try yet, I'd prepare fresh mobile phases (in fresh containers!) and see if it persists.
You're not using a reference wavelength, I assume?

[Calendula]

Cool, thank you, HPLCaddict! I did not know that about methanol and water solutions.

Okay, so I will re-make my mobile phases and use less of the buffer salt and repost. Appreciate your reply!

[Andy Alpert]

Are you using HPLC-grade phosphate salt?

[danko]

Nothing to do with pressure!
The pressure aftere the column is negligible

Nothing to du with impure phosphate salt.
It's just light scattering whent the salt concentration is high i.e. at the begining of the chrom.
Light scattering is interpreted as absorbance by the detector due to the fact that some of the energy/light is scattered in all directions rather than directed into the detection unit.

Best Regrds