Selecting the best HPLC wavelength(s) to monitor during an analysis method for use in quantitation and/or purity determination requires both knowledge and careful attention. Here is the basic procedure to use:
Step 1. Create the Method. To determine which UV/VIS
detector wavelength(s) should be chosen for the analysis of your sample, you
will first need to create a general HPLC Method which retains and resolves the compound(s)
of interest on the column (goal is a K prime of >2.0, less than 10.0). Be
sure and utilize a scanning diode array detector in full scan mode (often
referred to as a photo-diode array detector, PDA or DAD) to scan all relevant
wavelengths of your samples (e.g. 210 to 450nm). Note: Your choice of mobile phase and detector settings will effect the S/N values.
Step 2. Determine the lambda max of the sample's spectra using the Data analysis software. Once you have completed the analysis, review the
spectral data to determine which prominent peak wavelengths have the
maximum signal to noise (S/N) ratio. These “peaks” can be used as the individual
wavelengths for integration and purity determination. By sure and double check
that any detector options which use a “reference wavelength" are turned ‘OFF’ when
running these methods (more info on “reference wavelengths” can be found on
this blog in another post). With the wavelength selected, chose an appropriate bandwidth for use (narrow).
Step 3. Edit the HPLC method to use the discreet wavelengths found in step 2. Whenever you run a real sample, continue to use the
full scanning mode of the detector so you will know about any other components
which absorb at wavelengths far away from and/or near the peak wavelengths. These
compounds can add or subtract signal from the main peak making it appear to be
more or less concentrated (or more or less pure) than it actually is. If you
only monitored the sample with a single wavelength detector, then you would
miss this vital information and make errors in your purity or concentration
determinations.
Conclusion. (1) Using a multi-wavelength, scanning HPLC detector such as a DAD is one of
the most important tools you can use to create accurate and reliable chromatography
methods. Always use a scanning DAD for method development to prevent errors. (2) Learning how to correctly use and set up the detector's settings, parameters, special features and
options may prevent false
or misleading results. Only after you have developed a reliable and repeatable method with good sample retention and peak shape can you begin to report accurate integration and concentration values (and/or make UV/VIS "purity" determinations).