Chromatography Forum

I use IC-Pak anion HC 4.6 x 150mm, isocratic with 70mM KH2PO4 10% ACN, pH 5.5, flow 1mL/min to analyze nitrite and nitrate in urine.
Urine was filtered, then passed through C18 SPE, the flow-through was diluted x2 or x10 for analysis.
However, I can't get proportional increase in urine spiked with increasing amount of standards. Sometimes, the response in blank urine is even higher than that of spiked same urine matrix.
I suspect the various ions in urine is affecting unpredictably the column ion exchange mechanism.
I regenerate the column with butanol with acetonitrile in water. There seems some improvement but the condition worsened after some injections.
I intend to install a guard column and regenerate the column more frequently but I don't know if this is the cause for non-linearity.

Please comment.

Or is UV 214nm appropriate for urine nitrate, nitrite?
I tried in serum and the method was validated in serum.

Are you following a known application?
I do not have experience with that column. But I assume that the eluent is quite high in concentration.
UV should work as well.

Thanks for your comment.
Yes, I follow an published article but I increased the conc. of KH2PO4 after experiments. In this conc., the interference peak can be separated from nitrate peak.
Is that conc. of salt detrimental to the HPLC system?

Its not the bare concentration. that should be OK.
But I doubt whether the eluent is not already too strong.
What is the composition of the standard eluent (from the test chromatogram) of that column?

The Waters IC-Pak anion column insert recommends the following eluent for efficiency testing:
2% borate gluconate (3.4% boric acid, 1.2% d-gluconic acid, 0.86% lithium hydroxide monohydrate), 2% n-butanol, 12% ACN in water.

I'm not sure if this eluent is used for conductivity detection rather than UV detection or for both.

Thanks.