Chromatography Forum

Hey all,

I am running some vitamin c sample in hplc and I noticed that with each injection, the peak area is decreasing. I could never get a similar peak area if I prepared two standard of the same concentration level. Although the calibration curve for the standards has a okay R squared value of around 0.9998. Each standards or sample are done in triplicates, the %rsd between injections is less than 1%. However, I am unsure if I could use the calibration curve to do any accurate quantification with varying peak areas. I run the vitamin c at 20C isocratically with 0.5% acetic acid and methanol.

Does anyone have any idea why this may be the case? Any ideas how to solve this problem?

Thank you

With vitamin C you'll need to take measures to prevent oxidation (add reducing agents, minimize exposure to light,..). This is likely the cause of the decreasing peak area.

Check literature and application notes, there's no need to reinvent the wheel