Chromatography Forum

The little progress is entirely a result of your guidance and instructions. Thank you very much.

It is interesting that the pH has a very significant impact on selectivity. I decreased the MP pH from 8 to 6 and no interference for nitrite. Now I am trying 5.5 to see if the nitrate interference can go away. The pKa of nitrite is 3.4 and I hope 5.5 can give better selectivity for nitrate without affecting nitrite.

The nitrite and nitrate peaks span 1.05 and 1.6 mins respectively. The theoretical plate no. for nitrite and nitrate are 3234 and 2305 respectively for 50 ppm spiked urine.

Flattery will get you everywhere.

That plate count is a bit on the low side, but not alarmingly so; I agree that pursuing selectivity is the way to go.

MP pH 5.5 really resolved the nitrate interference from nitrate peak. However, the RTs are prolonged. I am trying higher buffer concs.: 70 and 80 mM to see if the RTs can be shortened.

I have a question: I am just experimenting on 1 urine sample. Different urine matrices should have other interferences. How can I optimize the LC on all possible matrices?

Also, I will be validating the following sample types:
1. serum (after centrifuging thro' a 3kD filter)
2. urine (3kD filtration followed by C18 filtration)
3. vegetable (not yet tested, probably 0.45um filter followed by C18 filtration)

For urine, is it necessary for 3kD prior to C18 filtration? Or is the serum sample preparation sufficient?
I am worrying about contamination of the anion exchange column because I don't know any cleaning procedures for such column except passing higher organic after run (but I already used a near maximum tolerated organic content in analytical MP). The Waters guard column has a large adapter. I don't think it can be fitted inside of column compartment of Agilent 1200.

Sorry for asking so many questions. Thank you very much.