Peak shape issues at the apex (top) of a peak are typically related to detector saturation. This symptom can be observed as flattening and/or noise at the peak apex.
Peak disturbances are commonly observed in preparative separations. Such a symptom is often considered permissible when the chromatogram is used only as a time marker for fraction collecting, rather than quantification purposes.
For quantitative purposes, this will be a problem as the peak area/height will fall above the linear calibration range of the detector.
Injecting a lower concentration within the calibration range will be necessary. If subsequent impurities present in low amounts cannot be quantified at their required levels with a lower injection concentration, a “high-load:low-load” injection scheme could be used.
With a “high-load:low-load” injection scheme, the same sample is injected at the high concentration for the impurity quantification and at the low concentration for assay level quantification.
The Beer-Lambert Law relates the absorption of light to the properties of material through which the light is traversing.
For UV/Vis detection in chromatography, it is often simplified to A=εcl, where A is the absorbance (inversely proportional to the transmittance that is measured), ε is the extinction co-efficient (for a particular molecule eluted in a given set of conditions), c is the concentration of the molecule(s), and l is the path length through which the light is traveling.
The extinction co-efficient and path lengths are therefore considered constant, leaving the absorbance directly proportional to the concentration.
If this is observed under method conditions previously established without the problem, likely contributing causes could be an incorrect dilution factor in the sample preparation, a different detector with a lower range being used, or an otherwise higher than expected sample concentration in the detection path.
As seen in the Beer-Lambert equation, changing the detection path length with a different sized flow cell could also change the absolute responses.