Chromatography Forum

Hello analyser,

I have a problem and I would very glad when I get some advise from you.

I am using a HPLC from Shimadzu and I have an oven which is able to cool down and heat up from 15 degree to 80 degree. After the oven there is a UV/VIS detector with a cell temperature of 40 degree. After the UV/VIS detector there is Refractive Index Detector with also a cell temperature 40 degree. My mobile phase consists of Sodium acetate 0,5 mol/l, Acetic acid 0,5 mol/l and HPLC water (Sigma Aldrich). The problem is that I am not able to reach a stable baseline with my RID. That means that the TOTAL EN value, which represents the light intensity of the photodiode, is not stable and goes up and down. Moreover I am not able to get measurements without noise. To solve this nasty problem I thought about some solutions:

1. The temperature gap between the cold fluid and the warm cell leads to this problem.

--> After I did some experiments with a fluid, which was 30/40/50 degree warm, there was no stabilization.

2. The temperature in the room is not constant and turbulences could cause this instability.

--> We have an air conditioner in the lab (24 degree), the HPLC is not near window, only other students are crossing the HPLC quite often. Could this leads to the problem, because the RID is quite temperature sensitive

3. The solvent which I am using shows up particles after a certain time in the stock solution. Before the solvent goes into the tube there is a 10 ul filter out of plastic and I have also another filter before the sampling.

--> After some test with 100 % Methanol there was also no improvement


I would be very glad for every help.

Thanks a lot

Merten

Temperature is a big issue with RID detector stability. Are you actually cycling the HPLC oven temperature during a run, don't understand that. I would troubleshoot with pure solvent only, no buffers. Also, RID is generally not as sensitive as UV-vis, do you have the RID sensitivity up too high? Have you flushed the RID reference side with the same solvent?

It might be helpful to know what the signal looks like. For the RI
what is the peak to peak noise with only mobile phase going thru?
Should be ~ 0.1 uRIU or so. And what is the noise of the UV
at a low wavelength... say 210 nm?

@mardexis : it is not in general a peak noise. There is also a noise when there is no sample going thru the detector. The detection for the UV/VIS is fine.

@CSG: the temperature in the oven is constant at 15 degree during the whole run. I have no idea which detector is more sensitve, but the RID is more sensible against temperature influence. Yes I have flushed the reference cell with solvent.[/img]