How to lower HPLC-UV LOQ?

[Edde]

I have an HPLC question and wonder if anyone could kindly help me out.

I currently use Agilent 1200 to detect derivatized nitrite by HPLC-DAD. The column used is ZORBAX Eclipse XDB-C18 4.6x150mm, 5u Analytical with 100 uL sample injection, analyte RT 3.6 min.

The LOQ is 1 ug/mL. But I want to lower LOQ as much as possible (preferably 220 fold lower to around 4.5 ng/mL). I can think of increasing injection volume and using analytical column of smaller particle size.

Which column do you recommend for use in HPLC and with high injection volume e.g. 200 uL? Any other ways to lower LOQ?

Please kindly comment. Thank you very much.

[unmgvar]

what you want is difficult
there is nothing for free
you can go to a 2.1mm ID column, that is adding 4.5 fold. but extra column volume effect can kill the resolution if it is important and increase easily the tailing and you will be closer to 4 or 3 folds only
you can increase the injection volume, but very fast also get peak shape problems and overload on the column
going to 3u might improve peak shape and get you also, maybe a fused core column? but the pressure will go up and you will need to prefilter everything 0.2 micron
cut the flow by half and you will get a bigger response.
do you have a standard UV? it can improve the result up to 7 fold because of noise reduction.
can you use another derivatisation agent? this can also improve the results. but you will need to see the stability of those samples again
what about using a fluorescence detector instead of PDA?

[Edde]

Many thanks.
In that case, I cannot obtain the required LOQ.
As you mentioned, I shall think about other derivatizing agent or other method.

[Mattias]

How much sample do you have?

If you have a column switch, you can inject as much as you have on a precolumn (several mL). Then you can switch and elute the analyte on a a analytical column, without any effect of the large injection volume.

[Hollow]

How much sample do you have?

If you have a column switch, you can inject as much as you have on a precolumn (several mL). Then you can switch and elute the analyte on a a analytical column, without any effect of the large injection volume.

=inline SPE

without a column switch, maybe you can do the same by manual SPE procedures, pre or post derivatization.
For a 200 fold increase, that would mean to concetrate about >20 ml, elute with and inject 100 µL.

Other techniques/instruments:
- fluorescence detection (?)
- voltammetric detection (?)

- ion chromatography
- voltammetric determinaton (->Metrohm has an application with a LOD in that range ->maybe contact Mr. M. Laeubli here)

[Edde]

Thanks all for your comment.
I don't have a large volume of sample. Actually, I am thinking of connecting an ion exchange column to the Agilent 1200 with UV detection.
Using 1mM phosphate buffer, pH 9, ?isocratic so that I can simultaneously detect nitrite and nitrate. Serum will be ultrafiltered and measured. Urine will be cleaned up by C18 SPE, then diluted for injection.
However, I am wondering if chloride and other anions will interfere.