Troubleshooting Tips: Absorbance of Spice Extracts
Whether you’re measuring the absorbance of spice extracts or other solutions isolated in a cuvette, here are some basic tips to help ensure accurate measurements:
Good Measurement Practices
- Transmission may vary from cuvette to cuvette. It will also vary with the orientation of the cuvette in the sample holder if the cuvette does not have the same pathlength in all directions. For best accuracy, always use the same cuvette. If this isn’t feasible, make sure cuvettes are oriented the same way every time they are placed in the cuvette holder.
- Warm up the light source for the recommended amount of time (up to 30 minutes in some cases). Light source output will continue to change very slightly until the source is in thermal equilibrium, affecting measurements.
- Record frequent dark and reference measurements in software to reestablish an accurate baseline.
- Never turn off the light source or disconnect fibers to take a dark measurement. Instead, block the light source at its origin, or at the filter slot in the cuvette holder. Make sure the object used will block 100% of the light; metal works well.
- Set the spectrometer integration time so that the reference spectrum peaks at 80% to 90% of the full scale of counts. This takes advantage of the full dynamic range of the spectrometer, improving signal to noise (S:N).
- Set spectral averages as high as tolerable to improve signal-to-noise. The S:N improves with the square root of the number of averages taken. For example, setting averages to 9 improves the S:N by a factor of 3.
- Increasing the boxcar value can also smooth out noise in the spectrum. This is a moving average with wavelength, so a boxcar value of 2 will average an additional 2 pixels on each side (5 in total) and assign that average to the center pixel. If the boxcar value is set too high, it will begin to blur the spectral shape, so use this feature carefully. To smooth data without affecting resolution, set the boxcar value equal to the pixel resolution of your spectrometer.
Visit the Absorbance section in Measurement Techniques for additional tips.
Stray Light Considerations
Stray light refers to any light that reaches the detector via scattering. This can include light that did not travel through the sample, or photons of the wrong wavelength hitting a pixel. Stray light has many sources, including ambient light that leaks into the instrument, light that bypasses the sample (like light that gets wave-guided through a cuvette wall), higher-order diffraction from the grating, and scattering from optical surfaces inside the spectrometer