Linearity issue in C18 in "pass-through mode"

[Edde]

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.

[PeterS]

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

[Edde]

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.