Chromatography Forum

I clean up urine for nitrite analysis by C18 (Bond elut 100mg, 1mL) in "pass-through mode" as follows:-
1. activate column by 2x1mL MeOH, then 2x1mL H2O.
2. pass 1mL urine and discard the flow-through.
3. pass 0.3mL urine and collect the flow-through. When sample stops dripping, apply suction at </=5mmHg.
4. Then dilute 1 in 10 for nitrite analysis.

However, I can't obtain a linear curve with fortified standards. The responses are not precise. Is there anything wrong with my C18 cleanup procedure? Is there any benefit if I dilute the urine sample before loading to C18 since this may lower the viscosity and hopefully enhance reproducibility?

I appreciate your comments.

Probably you should wash the column with water. The 0.3 ml is absorbed by the column and does not elute even with vacuum.

Peter

Thanks for your comment.
I tried passing 1mL 1 in 10 diluted urine and discarding the flow-through. Then applied 0.6mL 1 in 10 diluted urine and collected the flow-through. The curve was linear and reproducible now.