Measurement of Light Absorption, Reflection and Transmission Measurements with a Spectrophotometer
In absorption, reflection and transmission spectroscopy you direct light from a broadband light source onto a sample. Then you take a spectrum I(λ) of the light that is transmitted through or reflected from the sample. In order to get meaningful results that are independent of the light source, you also take a spectrum of the light source LS(λ) as a reference. The spectral transmittance T(λ)or reflectance is then simply the fraction of transmitted or reflected light:
This formula gives values in the range between 0 and 1. The transmittance is usually written as a percentage. The spectral absorbance λ is defined as:
A(λ) = log LS(λ)/ I(λ)
Please note that the absorbance is sometimes defined as the base 10 logarithm and sometimes as the natural (base e) logarithm. In Waves, you can choose in the Options window from the File menu which logarithm to use. In order to measure absorption, reflection or transmission you need to take a reference spectrum of the light source first. If your measurement setup is not in complete darkness, it is also generally recommended to take a background spectrum before the measurement.
To take a background spectrum, turn off the light source, set an appropriate exposure time and click the “Take Background Spectrum” button on the Exposure Toolbar. In order to minimize noise, you may also want to use temporal averaging. When you’ve taken a background spectrum, you can select the “Use Background Spectrum” button. If you take a spectrum afterwards, the background spectrum is automatically subtracted from the measured spectrum, eliminating unwanted background signals.
To take a reference spectrum for transmission or absorption spectroscopy, turn on the light source again, remove the sample, set an appropriate exposure time and temporal averaging and click the “Take Reference Spectrum” button. After selecting the “Use Reference Spectrum” button, you can take spectra of your sample for measuring absorbance or transmission.
To take a reference spectrum for reflection spectroscopy, you need a white reference sample to calibrate the setup for 100 % reflection. However, all real-world reference samples have a reflection of less than 100 % and an uneven spectral distribution. Therefore, for accurate measurements you also need to supply a reference spectrum for your reference sample. To take a reference spectrum, place the white reference sample where you’ll later place the sample to be measured. Then set an appropriate exposure time and temporal averaging and click the “Take Reference Spectrum” button. In the following dialog window, you can also specify a data file that includes a calibrated spectrum of your reference sample. After selecting the “Use Reference Spectrum” button, you can measure reflectivity spectra. The y axis of the spectrum now shows the relative intensity of the measured spectrum compared to the reference spectrum. In the “Options” dialog from the File menu you can choose different units for displaying the spectra:
- Ratio shows the ratio of the measured spectrum to the reference spectrum as in
the formula for T(λ) above.
- Percentage shows the same value T(λ) , but written as a percentage (i.e. multiplied by 100).2 This unit is usually used for transmittance.
- Absorbance shows the spectral absorbance A(λ) according to the formula above.
You can choose between natural and base 10 logarithm. These options only determine how to display the spectra in the diagram. The do not modify the actual spectrum data. If your light source does not cover the full range of the spectrometer, the calculated spectrum may show large amounts of noise outside of the wavelength range of your light source. This is normal and reflects the fact that the measurement has a large uncertainty at these wavelengths.
These measurements are quite sensitive to changes of the sample illumination. If in doubt, you may want to take another reference spectrum to check if it is still valid.