Chromatography Forum

Hello there,

We have faced an issue regarding unstable recoveries. We transferred aliquot to several HPLC vials from the SAME stock solution. However, the results obtained have an about 1% variation, while the system was stable with %RSD of 0.1% by injection of the same HPLC vial 5 times. So we assumed that there may be a cause hidden behind the procedure between solution transferring and injecting.

We have tried glass vials from agilent and waters and pp vials from thermo, and all results were unstable. I wondered if anyone had ever faced a similar issue and how to resolve it? We plan to continuously rinse a vial to see if the peak area would change after each time rinse.

Thank you in advance.

Parameters:
HPLC: Waters
Wavelength: 302nm
Mobile phase/diluent: 0.15M KH2PO4 buffer: ACN: acetic acid= 750:250:7.5

Are the 5 replicates RSD area 1% or just average between vials is 1%? What is the area RSD on 5 reps of same vial?

Did you filter the solution into the vials? If so could it be the filters causing the problem.

A lot depends on what the target analyte is that is being affected and the solvent being used. Could there be adsorption of the analyte on the surface of the vial or the instrument being used to transfer the solution to the vial?

even stupid things like if the solution from which the vials were filled was a stock-bottle out of the fridge, where a little bit has evaporated and condensed round the lid, and whoever did it wasn't super-careful about mixing really thoroughly, then you have a potentially non-homogenous sample, and each vial gets a slightly different amount of nice clean condensed solvent. If the solvent is very prone to evaporation, you can even get a difference because of evaporation during the course of pipetting, if it takes a minute or two to get the lid back on. You only need to lose 2 uL out of a 200uL insert to get a 1% error.