Chromatography Forum

Hello, I would like to do HPLC analyses with a UV/Vis detector. The sugar to be determined has its absorption maximum at 190 nm, but so does the solvent water. Now I wonder if I can still do my measurements at 190 nm because I read the following: "At small wavelengths around 200 nm, many substances absorb, but usually so does the eluent. The absorption maximum of the analyte should be longer wavelength than the eluent absorption."
Can I still perform my measurements at 190 nm? I also use water as a mobile phase. Doesn't this take water off as a blank anyway?
Thank you!

Hi Clinephd,

Getting an accurate signal of any compound at the wavelength 190 nm will be hard due to, as you noted, the absorption coming from your mobile phase (and anything else that may be in there, additives, buffers). I'd look for another wavelength to observe your sugars at, or figure out a different method by which to detect them if 190 nm is your only option. As for your comment about "taking the water off as a blank", I'm not sure exactly what this means? If you're assuming that when you inject a blank of water that the system will subtract that blank from your future analysis then that is not true.

Hope this helps,
TS

Hi TylerSmith123,
thank you very much for the quick reply.

Getting an accurate signal of any compound at the wavelength 190 nm will be hard due to, as you noted, the absorption coming from your mobile phase (and anything else that may be in there, additives, buffers). ...
As for your comment about "taking the water off as a blank", I'm not sure exactly what this means? If you're assuming that when you inject a blank of water that the system will subtract that blank from your future analysis then that is not true.

A few more comments on this: I use water as a mobile phase. I use the same water to dissolve my sugar samples. Apart from the single sugar and the water, there are no other additives (no buffer or similar). Therefore, I thought that water is not visible in my chromatograms and therefore, the overlay does not cause a problem. Or do I have a mistake in thinking here?

Hi, if your run is isocratic (does not use a gradient between 2 mobile phases and instead runs at initial conditions with 1 mobile phase) then yes the software will blank the system at the start of the injection and any increase in absorbance will be detected so providing your sample is concentrated appropriately you should see the peak. If you are running a gradient with any organic modifiers however you will have problems with an increasing baseline at that wavelength which could potentially obscure your analyte peak.

If you could share more of your method parameters that could be useful,

thanks and good luck
Chromavore

Can I still perform my measurements at 190 nm?

You can. The sensitivity is poor in this case, but if the signal-to-noise ratio and the peak area precision are sufficiently high for your particular task (your particular concentration levels), you can perform the analysis. You can try to use slightly higher wavelength (e.g. 195 nm) and different bandwidth. However, usually sugars are detected with a refractive index detector.

Thank you everyone for your input

What just amazes me is that my predecessor did his measurements at 209 nm. At this wavelength, the peaks were of course much smaller.

If you could share more of your method parameters that could be useful,

The method is quit simply. I only use water as mobile phase (no gradients, no more substances than one sugar and water). So I think it could work?

I rarely used low wavelengths for anything. But we had RID, ELSD, conductivity detectors too.

I rarely used low wavelengths for anything. But we had RID, ELSD, conductivity detectors too.

^those detection modes are all more appropriate for sugars than UV.

I rarely used low wavelengths for anything. But we had RID, ELSD, conductivity detectors too.

^those detection modes are all more appropriate for sugars than UV.

You meant amperometry, didn't you?

uv will not work for sugars in general. Like what are mentioned above, RID, ELSD, CAD, And (IC)PAD are suitable for sugar analysis.