1. UNI Plant Physiology 2005 www.uni.edu/berg Measuring Light Absorbance UNI Plant Physiology Spring 2005

2. UNI Plant Physiology 2005 www.uni.edu/berg Water Red light passes through Blue NOT absorbed Green light passes through Blue light passes through Green NOT absorbed Red NOT absorbed

3. UNI Plant Physiology 2005 www.uni.edu/berg Red Dye Solution Red light passes through Green light passes through Blue light doesn’t pass through Red NOT absorbed Green NOT absorbed Blue IS absorbed

4. UNI Plant Physiology 2005 www.uni.edu/berg Inside the Spectrophotometer Light source Light beam Wavelength selector Measurement beam Spec tube holder Spec tube with solution Light detector measures how much light gets through tube

5. UNI Plant Physiology 2005 www.uni.edu/berg No dye (blank) Absorbance of Dye Solution Absorbance = 0 (set to 0) Dilute dye Concentrated dye Absorbance low Absorbance high IN THE SPEC

6. UNI Plant Physiology 2005 www.uni.edu/berg Absorbance and Concentration Right color (wavelength) for the dye Blank has zero absorbance (we set it) Low concentrations have low absorbances High concentrations have high absorbances You can see this with your eye with the right color light

7. UNI Plant Physiology 2005 www.uni.edu/berg Absorbance of Dye Solution IN WHITE ROOM LIGHT, SEEN BY YOUR EYES LOTS OF LIGHT GETS THROUGH THE RED DYE, JUST NOT BLUE LIGHT

8. UNI Plant Physiology 2005 www.uni.edu/berg Why does the spec say the tube is dark, but our eyes say it is light? The spec is only looking at the blue light that passes through (not much) Your eye sees all the colors that pass through (lots of red and green, though not much blue)