Spectrus: Creating calibration curves for quantitative UV/Vis analysis

Description
Quantitative UV/Vis spectroscopy relies on calibration curves relating absorbance to concentration. Spectrus can process multiple standards and generate these curves using the Optical Workbook.

Solution

1. Prepare standards at known concentrations and acquire UV/Vis spectra for each standard under identical conditions.
   • Note: at least 3 standards will be required to generate a calibration curve.
2. In Spectrus, ensure that Optical Workbook is enabled under Options.
3. Collect spectra into a series by selecting all while holding the Shift key then selecting the Series icon.
4. Create a calibration dataset:
   • Go to Tools > Calibration > Data
   • Set X and Y axes and wavelength.
   • Go to Tools > Calibration > Auto to apply.
   • A calibration curve will appear with an equation relating sample number to peak intensity.
   • Using the known concentrations of standards, this will relate concentration to absorbance (according to Beer–Lambert law). Inspect fit quality and R².
   • Note: More settings (such as setting a zero intercept) can be found at Tools > Calibration > Options.
5. For unknown samples:
   • Record their spectra and measure absorbance at the same wavelength.
   • Use the calibration curve to calculate concentration from measured absorbance.