Troubleshooting ascorbic acid - Chromatography Forum

Troubleshooting ascorbic acid

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

6 posts Page 1 of 1
Hello everyone,

I am quantifying ascorbic acid in pharmaceuticals with a 100% aqueous mobile phase, pH 2.6 adjusted with acetic acid.

During the first 5 injections of standard solution, peak area increases 4-fold, then stabilizes, which has no obvious explanation...

Can anyone help? Could this be a matter of column equilibration or dilution solvent?
Ascorbic acid UV-spectrum (and peak area too) rely heavily on mobile phase pH so it may be column equilibration.
Depending on injection volume and mobile phase buffer capacity it may be solution pH too.
Best regards,
Dmitriy A. Perlow
I will try longer column equilibration time, I will decrease injection volume and let's see what happens...

Thank you!
Also , consider changing rotor seal of your injector.
The described phenomenon is due to unspecific binding of the analyte (ascorbic acid in this case) to the stationary phase.
When the binding sites (typically underived stationary phase support - maybe silica) are saturated with ascorbic acid, only specific binding takes place and the top areas become consistent.
If you are looking for a solution, you might 1. Dedicate one or two columns to this analysis so they do not need to be saturated every time you start an analytical series.
2. Alternative, you can load a large amount of standard 1-2 times so that the column gets saturated faster and thus ready for analytical runs.

Best Regards
Learn Innovate and Share

Dancho Dikov
You could try 10mM phosphate buffer adjusted to 2.8pH with a polar embedded column to enhance retention
6 posts Page 1 of 1

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