Low absorbing wavelength = lower response but better S/N

[djacqo]

Hi everyone,

I need to develop a method to quantify low levels of impurities in a certain compound. I know the impurities have the same response factor so I am going with a purity based on simple peak area %.

The column I found that is able to separate the components unfortunately seems to be responsible for high baseline noise compared with other columns with all other parameters unchanged. I found that if using 210nm (near max absorption), I cannot detect any of the impurities. However, if I use the smaller max (290nm), the response is significantly lower, less than 10% of the lower wavelength signal, but I am able to detect and quantify the impurities. The amount of material I load onto the column ends up saturating the detector at 210nm but is within the linear range at 290nm (peak shapes do not suffer) to get a nice strong signal at 290nm with good S/N.

Monitoring a weakly absorbing wavelength to reduce S/N while saturating the max wavelength seems a bit unconventional, and I worry that I'm missing out on something that could be giving me unreliable data despite that I checked the linearity of the wavelength I use and the peak shapes are good. Does anyone have any thoughts on this?

Thanks,
dja

[Gerhard Kratz]

A column, I guess brand new, cannot be responsible for baseline noise, except that it is not equilibrated with the MP. Baseline noise is definetively lower at Lambda 290nm than at 210nm. Did you run UV spectra of each of the 6 compounds dissolved in mobile Phase?

[HPLCaddict]

As Gerhard already wrote, the column should not be responsible for higher baseline noise.
Some thoughts:
- Is it possible you're using a mobile phase with significant absorption at 210nm? Any acetate or formate, possibly? This will decrease sensitivity at this wavelength (as the baseline is "higher"). Possibly that's the reason why you're not seeing your analytes at 210nm.
- If you're fine at 290nm, why bothering how the chromatogram looks like at 210? I guess with nearly every impurity method that uses detection at higher wavelength you'll be saturating the detector at low UV...
- Concerning response factors, I'd be careful. Are you sure your analytes have the same response factors over the entire wavelength range? Just because they are comparable at 210nm doesn't mean they are at 290, too.

[djacqo]

Good morning, and thanks for the thoughts.

I was interested if people ever take this approach of looking at higher wavelengths to increase S/N in general. It's weirding my colleagues out, so I'm looking around for other cases.

Baseline noise from the column: This column is not at all new and I don't know its previous care, so the best I could do is give it a full cleaning procedure. Is it not possible that irregular flow (pressure is rock solid steady though), strongly retaining species constantly eluting in trace quantities, or something else could have an effect? When I get some time I'll look into this again.

Regarding response factors: I have a DAD, and the impurities have identical UV/VIS spectra (signals of the impurities are high enough to tell because I'm actually injecting a lot of sample, just monitoring a weak wavelength). Unfortunately, I do not have pure samples of the impurities to actually calculate response factors, but they are suspected stereoisomers.

I appreciate the time for your comments.

dja

[Consumer Products Guy]

I was interested if people ever take this approach of looking at higher wavelengths to increase S/N in general. It's weirding my colleagues out, so I'm looking around for other cases.

We do just this, frequently. Low wavelengths such as 210 nm are more "universal" but lots of stuff has some absorbance there. So if a component of interest has absorbance say at 280 nm or 300 nm, even if it's a trace level assay, the background/matrix absorbance and the signal to noise can be greatly enhanced.

I think that moving to a wavelength that's better for your analyte overall is a great strategy. Think: what's best overall for your detection.

We "inherited" a certain test method from a company when its products were acquired. That test procedure used 210 nm detection because the scientists there figured the active level was so low, that they needed the highest sensitivity. Unfortunately, the sample matrix often interfered at the wavelength. Moving to a higher wavelength eliminated the matrix effect, so even though the absolute absorbance of that peak was smaller, signal to noise, reliability, precision, and accuracy was improved. That method has since been GLP validated.

[juddc]

I concur with CPG.

Put succinctly, it's not so much the amplitude of the signal that matters, it is signal to noise and lack of interference. If your assay works better at a higher wavelength - and this can be objectively measured - then using the higher wavelength is better. Assuming you're using a photodiode array detector, you're not actually changing the method, you're using a different set of data for your calculations.

CJ