Chromatography Forum

I'm running forced degradation on a sample using Empower 3. I need to calculate peak purity and am having difficulty getting the absorbance below 1 AU. I know that I can adjust my wavelength range, injection volume, or sample concentration.

My understanding is that based on Waters peak purity training the sample absorbance needs to be below 1 AU in order for the peak purity calculation to be accurate. I'm wondering specifically why this is the case. Does anyone know why the absorbance needs to be below 1 AU? Any help would be great!

Spectral peak purity check is an unreliable way of confirmation that the separation method is selective enough. This purity check cannot prove that there are no hidden peaks of impurities under the main peak. Low concentrations of impurities and similarity of their spectra to the spectrum of the parent substance may result in the absence of any detectable spectral differences among the various parts of the main peak (these differences are sought in the peak purity check).

I agree with the previous post, but it kind of sidesteps the actual question about "what's magic about 1.0 AU".

It has to do with the fact that absorbance detectors don't actually measure absorbance -- they measure transmittance (the % of light that gets through the cell). Absorbance is then calculated as the negative log of transmittance. That means that when we see a "high" signal, we're actually seeing a very low amount of light getting through; 1 AU = 10% transmission; 2AU = 1% transmission. With that little light getting through it's difficult to accurately see small differences.

https://support.waters.com/KB_Inf/Empower_Breeze/WKB85832_Empower_3_PDA_What_is_Purity_1_Angle_Purity_1_Threshold_How_is_the_peak_purity_determined
4. Poor purity of the standard
High purity standard samples can be determined to be of poor purity mainly for the following reasons.
・The concentration is too high
Even if the absorbance exceeds 1 AU only at partial wavelengths, the absorbance linearity will be lost and the spectrum will be distorted, and the purity is measured to be poor. Please reduce the concentration so that the absorbance at the maximum absorption does not exceed 1 AU, and perform the measurement again.
・High background absorption of the mobile phase

https://support.waters.com/KB_Inf/Empower_Breeze/WKB11215_How_do_you_set_up_the_Purity_Thresholds_in_Empower_3
2. Record the maximum spectral absorbance (MSA) of this standard.
Tip: To use AutoThreshold, Waters recommends that the MSA of the standard be less than 1 AU. Since photometric error increases above 1 AU, Peak Purity measurements become significantly less sensitive to the presence of co-elution when the MSA of a compound exceeds 1 AU.