Spectrophotometer Calibration with Caffeine - UV Vis Spectroscopy
Verification of Instrument Accuracy and Reproducibility
In this application note, caffeine is used to assess the wavelength accuracy and reproducibility, and the pathlength accuracy of Mettler-Toledo's UV/VIS Excellence spectrophotometers. For more information, please proceed to download the application note below.
Why should spectrophotometer calibration be performed?
The repeatability of the instrument as well as the accuracy of the pathlength and wavelength is vital in achieving reliable results. Caffeine is one of the most commonly used primary standards in UV Vis spectroscopy and is effective in spectrophotometer calibration because of the distinct peaks displayed in its spectrum. Specifically, this application verifies:
- Wavelength accuracy in the UV region at 275nm
- Mirco volume accuracy and repeatability using sample volumes of 1.0µL and 3.0µL
- Path length accuracy and repeatability at three different path lengths
How is spectrophotometer calibration performed?
Three different concentrations of standard caffeine solution ranging from 2-1000 mg/L are prepared, measured and used to create a calibration curve of absorbance versus concentration. When comparible results are obtained at different sample volumes and pathlengths, and the instrument is proven to satisfy the Lambert-Beer law within the concentration range then reliable, repeatable results can be achieved in subsequent measurements.
