In an effort to help you and your students better understand spectrometer absorbance readings, we’ve collected a few commonly asked questions:
Why don’t absorbance readings have units?
Absorbance readings are unitless because they are calculated from a ratio of the intensity of light transmitted through the sample (I) to the intensity of light transmitted through a blank (Io). This ratio results in a unitless value.
Absorbance = log (Io/I)
Why are absorbance readings most accurate between 0.1 and 1?
Remember that absorbance is the logarithm of the transmission of light through a sample. Transmission (T) is the ratio of the intensity of light transmitted through the sample (I) to the intensity of light transmitted through a blank (Io). Therefore, absorbance = log (Io/I).
At an absorbance of 2 you are at 1%T, which means that 99% of available light is being blocked (absorbed) by the sample. At an ABS of 3 you are at 0.1% T, which means that 99.9% of the available light is being blocked (absorbed) by the sample. Such small amounts of light are very difficult to detect and are outside the meaningful range of most spectrometers.
Vernier array spectrometers and colorimeters have a useful absorbance range between 0.1 and 1.0. Any absorbance reading above 1 can be inaccurate. There are spectrometers that will report meaningful values at absorbance ranges above 1.0, but these are research instruments that are also quite expensive. In most classroom settings, the best option is to simply dilute your samples to ensure they are in this range.
How important is it to use a quartz cuvette for absorbance readings in the UV?
It depends on how accurate you want your absorbance readings to be. UV plastic cuvettes are less expensive and have practical applications when working with students, but they lose transparency quickly in the UV. Most are only rated to 280 nm. If you want the most accurate data possible below 280 nm, a quartz cuvette is the best option. Another unfortunate side effect of using UV-plastic cuvettes is that students commonly confuse them with visible-only plastic cuvettes. This cuts out all UV light, so data will be very poor. If you are going to use UV-plastic cuvettes, make sure you are using them for the proper applications.