Chromatography Forum

Hi,

I had been trying to build a calibration curve but was unsucessful, so I checked the RSD of my system and it was as follows:

275 - 235 - 220 - 215 - 285 (nm)
2.5 - 1.1 - 4.0 - 6.4 - 3.4 (%RSD)

This was calculated from the peak area of a single peak (no other peaks in chromatogram) across 25 samples, where there was 5 injections of 5 samples all taken from the same stock solution, total loop fill of 1ul. wavelenghts were selected from spectrum as 275 maxma, 235 minma, 220-285 flat reigons.

Long story short

I have now got the following:

275 - 235 - 220 - 215 - 285
0.6 - 0.6 - 0.5 - 0.6 - 0.6

Again across 25 samples in the same protocol.

Should I be happy with these RSD's or should I continue to try and improve them? If I should improve them what areas should look at improve the RSD?

I have a Agilent 1100 system but with a Gilson sampler and rheyodyne loop. I have already replace the the flow cell, lamps, rheyodyne and seals of injection port needle, needle.

I have lots more infomation if needed

First, check the specifications for your autosampler. As long as you are comfortably within spec, you should be good to go. (For context, the spec on mine is 0.8 %RSD; the last qualification came in at 0.2 %RSD.)

Given that your first set showed different %RSD at different wavelengths makes me think that you might have had either contamination or peak integration problems. The second set, being consistent, suggests that you have eliminated that problem.

Gilson specify:

0.5% RSD for a 200ul total loop fill across 10 injections by HPLC

<0.9% for a 20ul total loop across 30 injections (using fluoresence as detection).

I suppose 0.5% for 1ul by HPLC is OK?

Normal sampling loop is 20-50 uL. You should inject a higher volume to have a lower RSD (how about 20uL?). In our tests, we usually inject 5uL or more. (3uL is very rare)
If injection volume is 1uL, RSD may exceed 2%.
Alfred.

I don't do a partial loop fill, I have a 1ul loop so do total loop fill.

I could change up to a 5ul loop, again total fill an dilute my sample more.

Thanks for the advice

Hi Ytterbium

How good does the rsd need to be for your analyses to provide useful data ? As long as it meets that target you do not need to make it any better.

Peter

<1% is pretty much universally acceptable for any assay using an 1100 and any decent autosampler. Sure, you can do better, but nobody I know would worry about rerunning a set w/ <1% RSD.

The variance across wavelengths is actually indicative of a detector problem - wavelength accuracy/consistency - were not up to par. Evidently the new lamp & flowcell fixed that.

I hope you changed one component at a time so that you don't spend more $ than is needed next time you have these issues...

Thanks for the advice everyone, I'll try to do a calibration curve and check the result for that.