Chromatography Forum

Hi, in the software for the hplc-uv for the agilent 1100 there is an option to check a reference wavelength. How does this work in practice. IS there a reference electrode?
It seems to default to 360 nm on our instruments. Why? is this 360nm and should the bandwidth specified be the same as for the analyte wavelength
Thanks
Liv

There was a fair bit of discussion about that last October:
http://www.sepsci.com/chromforum/viewtopic.php?t=7081

The use of a reference wavelength is analogous to the use of a reference beam in a double-beam spectrophotometer. Like a reference beam, it can compensate for fluctuations in lamp intensity; in addition, it can compensate to a certain extent for refractive index variations. That's the good news. The bad news is that it can add noise to the measurement and you can sometimes see negative peaks if you have an analyte that absorbs more strongly at the reference wavelength than it does at your measurement wavelength.

360 nm is not sacred, but it is well out of the way of most chromophores. I didn't know you could specify the bandwidth of the detection and reference wavelengths independently, but probably best to leave them the same unless you know a good reason to do otherwise.

Thank you
Once again you have been a great help to me
Liv

I wanted to add a few comments to Tom's suggestions.

In my experience with the 1100 diode array, including the reference usually will result in a small improvement in the baseline noise. The amount of improvement seems to depend more on the system (age, pump health,etc.) than the actual settings. I have never seen an increase in noise, but as Tom mentioned, you can make the peak disappear if you choose the wrong settings.

The reference bandwidth is usually set to a large value (50 - 100 nm) so that some other types of noise are cancelled out. The actual value doesn't have much of an effect in this range, as long as nothing absorbs in this region.

The sample bandwidth should be set to the width of the absorbance band at half-height. (Think of it in the same way that you mesure chromatographic peak width, but in nm units.) This value gives you the best signal-to-noise ratio. Smaller values give you more selectivity, but not necessarily more absorbance. Larger values produce less selectivity, but also less absorbance. It is always a balancing act, and the value that you choose depends on the needs of your analysis.