Chromatography Forum

The aim of selecting the reference wavelength is to correct the signal obtained with the measured wavelength. Thus the reference wavelength chosen should be a wavelength where no one from the analytes analysed / mobile phase constituents absorbs on it.

So if you choose 210nm or 215nm as reference wavelength where background absorption is significantly high (especially when using methanol as modifier in the mobile phase), you'll get less sensitivity and also baseline that is prone to changes as mobile phase ratio changes.

Also using 280nm as reference wavelength when your analytes have absorption maxima near 280nm, will cause similar changes regarding sensitivity or noise.

Hope this helps

After all of all, I finally called Agilent for some explaination for the difference between VWD and DAD/MWD detectors. But I was told there should not be such difference between these two types of detectors. The tech support gentleman even did not strongly agree with what is stated in their detector manual (reference wavelength section). Even I emphasized that I have tried mutiple VWD and MWD/DAD detectors using same column and mobile phase, he still prefers to believe in hardware problems or method problems.

If he is right, saga is still on. I need to find a system with both DAD/MWD and VWD.

thanks

duantech

for rick1112:

First of all, are you sure it was a MWD (multi-wavelength detector), and not a DAD (diode array detector)? I may be wrong, but I was under the impression that most MWDs were true double-beam, with a beam splitter between the monochromator and the flow cell in the optical path, so that the "reference" is always the same wavelength being used for detection. This is impossible with a DAD because the flow cell is located in front of the polychromator in the optical path.

we found high noise in the analysis, when the reference wavelength was absent this noise level decreased (we got a very smooth baseline).
Could any one suggest the reason for this obervation??

To expand a bit on zokitano's explanation: the use of a reference wavelength corrects for certain kinds of noise: specifically noise caused by variations in the intensity of the light beam because these variations will be correlated in the two signals. It can exacerbate other types of noise: specifically electronic noise, because these variations will be uncorrelated in the two signals. So, depending on the cause of noise, use of a reference wavelength can either help or hurt.

And how do you set the reference wavelength nm???

I usually start by reading the instruction manual.