Chromatography Forum

I ran mix of four standard using PDA detectors it works fine( four Clearly resolved peaks) but when i ran the same standards using RI detectors its giving me huge peak in about 1.5 min and not other peaks. I am using the same method conditions. I have never worked with RI detector before.

Please advise what i am doing wrong..i washed my flow cell with pure methonol.

If your compounds have strong UV chromophores, RI will be much less sensitive. The huge peak at 1.5 minutes may simply be the baseline upset at t0. It's hard to say any more without information:
- what compounds?
- what column dimensions?
- what flow rate?

My standards are mixture of ethyl,methy,propyl,butyl benzoates at 5 ppm conc.I am injecting 10 ul. col is 100x4.6,5u, hypersil gold
Mobile phase is 60:40 MeOH:water at 1 ml/min. RI of all standard is about 1.5

Your chances of seeing 5 ppm with a 10 ul injection are fairly remote on most RI detectors.

Look at the manual for for your RI, it should have some sensitivity data and the sample volumes.
Calculate the differential RI of your samples and mobile phase, work out the amount you injected, peak width etc., and that will tell you if the peaks would be detectable under optimal conditions.

If you don't want to do that, prepare 50 and 500 ppm stds and run those, then dilute accordingly to determine your LOD and LLOQ. I would not be too surprised if the LOD is over 100 ppm on a 10 ul injection.

You may be able to improve sensitivity by ensuring your sample is dissolved in the mobile phase and increasing injection size to 50 - 100 ul, setting the detector time constants to be optimal for your peak width, and providing good temperature control ( eg premixing the solvents, degassing, and allowing to thermally equilibrate), ensuring no thermal drafts around the instrument etc., but in reality, RI may be the wrong detector for your application.

Bruce Hamilton

Given your column size and flow, the big peak at 1.5 minutes is amost certainly t0 noise. I agree with Bruce: you are *not* going to be able to detect 50 ng with an RI detector.

Next question: why not stay with the PDA ?

Thanks. I am trying to find RI for my standards they are ethyl,methyl,propyl,butyl parabens
Could not find in chemfinder

I am going to inject higher concentration on wednesday.I can not switch to PDA I need RI data only.

Personally, I'm not a big fan of resorting to sub-standard science to try to fit to existing instrumentation, especially when such instrumentation is so commonly used. Tell your boss to open his/her wallet. Do you make manual injections because boss won't let you get autosampler as well?

I really appreciate your concern but
Money is not issue here cause we do all have all kinds of detectors in our lab purpose is software testing with RI detector So wont be able to switch to any detectors