Chromatography Forum

Hi all!
A newbie but important question for.
For a purity method on meds, if the detector used to measure peaks is satured for the main peak at a define wavelength, can we use another wavelength where the response is lower without changing concentration to evaluate the whole profile?
In a general way, for a impurtity HPLC method, does the concentration be adjusted to avoid detector saturation at any wavelength, or can we adjust the concentration at a define wavelenght without taking care about the others wavelengths?
As said above, a newbie question...

Best regards

If you are "off-scale" good practice is to dilute and re-inject.

In most cases, the "defined wavelength" will represent an absorbance maximum for your compound of interest. In effect, you are measuring on top of a "hill" in the spectrum, and small errors in wavelength (or shift in the spectrum) will have relatively little effect on the signal. If you try to measure on a "slope", then small errors can generate big errors in your results.

If your compound has several peaks in it's absorbance spectrum, then you could shift to a different wavelength (on one of the other "hills"), but you would have to run a calibration curve at that wavelenth, which strikes me as more work than diluting and re-injecting a single sample.

Also, unless your specific absorption is extremely high (at the wavelength causing saturation) you may have an interaction of the analyte such that the linear relationship between absorption and concentration doesn´t hold (at any wavelength). Go with Tom and dilute.

you say that your aim is to do a purity method.
if i understand correctly then in effect you are looking at the smaller impurity peaks in the chromatogram, not the main peak.

if this is the case then depending on your specifications and the chromophores of your impurities it is possible that you cannot move to another maxima region or dilute since you will get less signal for the small peaks, which have a lower concentration. anyway it is common that the main peak be overloaded in such methods in order to be able to see the impurities

doing a combined assay and impurity test because of these is many times hard since you either overload the main peak or bring the impurities below QL. in any case in combined tests such as these you still need most of the times 2 calibration curves especially if the specs for your impurities are way low. one for the assay of the main compound one for the impurities.

unless you can really combine in order to run the same sample for both tests then create a shorter method for the assay at a lower concentration, it will be easier overall

one of the things thou that helps to combine, are newer UV detectors were the UV linearithy range is above 2-2.5 AU