Quantification with different wavelength

[strutter527]

Dear all.

We would like to know which is the proper way to perform the quantification using a different wavelength from which the sequence was run (in a equipment LC-PDA detector), saying that the wavelength is within the range.

Thank you in advance.

My best regards.

[Daniel Mentlik]

Dear Andres,
the process of quantifying on a different wavelength signal than that was used for acquisition is not overly straightforward, for future analyses I will advice to try ensuring that the quantification wavelength is collected aside of the PDA spectra - depending on the detector, such signals may have better resolution and/or frequency so they are better suited for quantification than a signal extracted from the spectra. Now, let me describe the necessary process:

• Open the PDA Chromatogram window with the chromatogram in question opened.
• Use the Chromatogram - Add Signal function to manually add a desired signal to the chromatogram file. You can specify the bandwidth for the signal, so it may actually be a signal collected over several Wavelengths. Specify a signal name in the topmost field.
  • You can't replace any signal present in the chromatogram at the moment of its measurement, but you may replace similarly added signals created after the chromatogram was measured.
• Switch to the Chromatogram window and save the chromatogram.
• Create a new calibration, or switch to the already existing calibration. Open the chromatogram in question as a standard.
  • If the calibration does not exist yet, you will need to add the information on peaks, responses, etc. You can do so using typical processes described in the user guide.
  • If the calibration already exists, it has fewer signals than those in the target chromatogram; as signals are mapped first in chromatogram to first in calibration, second to second, ..., your added signal does not have a counterpart in the calibration created, you will have to copy the information from the signal that has the information to the signal added.
    • Switch to the manually added signal - should be the last one through toolbar.
    • Manually check used checkbox for peaks you want to quantify. At the same time, you may want to uncheck the checkbox on the same peaks on different signal(s) - depending on your choice.
    • Copy the response values from the signal you already have and which contains the responses in the calibration to the freshly added signal.
  • Save the calibration file and link it to the chromatogram.
• Adjust the added signal integration - as a newly added signal, it will have default (and probably inappropriate) integration information.

The results will be visible in the Result Table of a Chromatogram window when switched to the newly added signal, or in the All Signals Result Table.
This process can't be automated, so it will be necessary to perform it on each required chromatogram manually. However, if a series of chromatograms is using the same calibration and the same wavelength, modifying the calibration will only be needed once. Thinking ahead of the measurement on which signals will be needed later for quantification makes the situation much more straightforward.