Broad HPLC thiamine peaks

[kiska]

I injected several wine samples (10 times diluted) into my HSS C18 (waters) column and now I have broad thiamine peak, labeled thiamine peak and also HET (2-(1-hydroxyethyl)thiamin) all the other vitamins (nicotinamide, nicotinic acid, pyridoxine etc) are ok. I washed needle system with acn and water (about 100 times) and also the column with acn+0.1% formic acid and with water+0.1%FA about 2 days at flow 0.2 ml/min but no change

[carlos.teixeira]

Hello Kiska,
1) Did you verify your mobile phase pH?
2) Did you use any amine like Triethylamine on your mobile phase composition?

[kiska]

Hello Kiska,
1) Did you verify your mobile phase pH?
2) Did you use any amine like Triethylamine on your mobile phase composition?

Hi Carlos,
1) yes I did, mobile phase pH is 2.8 +/- 0.05 (water + 0.1% formic acid)
2) no, we don´t use triethylamine

[danko]

The pH is low enough for thiamine to be protonated (positively charged) but not low enough to protonate (neutralize) the free silanol groups on the stationary phase. You're experiencing non specific interactions (electrostatic to be more precise).

Best Regards

[kiska]

The pH is low enough for thiamine to be protonated (positively charged) but not low enough to protonate (neutralize) the free silanol groups on the stationary phase. You're experiencing non specific interactions (electrostatic to be more precise).

Best Regards

Thank you for explanation!
Can I do anything about it?

[danko]

Try and lower the pH, by increasing the formic acid concentration - say to 0.2 %.
Another possibility is utilizing an ion pairing reagent, but it should be the last option.
Third possibility is adding a substatial amount of some salt (maybe drop the formic acid and use 0.1 M Sodium Phosphate buffer at pH 2.3.
Fourth possibility is another (more inert) column. There are some of them on the market. I know of at least one

Best Regards