- The absence of any spectral differences across the peak is not an indication of actual purity;
- Compounds similar to your sample may have similar absorbance profiles;
- The relative concentration of actual impurities may not be high enough to detect;
- The compounds / impurities may not absorb light at the wavelengths scanned;
- The HPLC method used, the software settings and the parameters that you chose in the ‘Peak Purity’ software menu have a huge effect on the results obtained. Inputting poor quality settings or using a poor quality method often leads to misleading purity results.
- The peak of interest must be retained on the column (K prime > 2) and resolved apart from any observed peaks. Don't use peak purity to analyze peak which elute at or near the column void volume (demonstrates lack of a method and specificity, fails validation).
When configuring the Peak Purity parameters for your sample, you must start with a very high quality HPLC method (A "validated method" is not necessarily a high quality method). The correct detector sample rate, signal wavelength and bandwidths need to have been selected and used (Reference Wavelength OFF). The peaks shown in your chromatogram should have excellent symmetry with good on-column retention (K-prime), baseline separation (> 2.0) and very low baseline noise levels. The two spectral reference points should be manually selected and placed at times before and after the peak of interest in clear baseline areas where no other peaks or spectra are seen. Select at least 7 spectra from the sample peak for comparison (more detail can be provided with more spectra, but be careful not to select spectra near the noise limits). If your method and chromatogram are not of the highest quality, then please do not use the automated peak purity analysis feature, instead spend time improving your method.