UNI Plant Physiology 2005 www.uni.edu/berg

Measuring Light Absorbance

UNI Plant PhysiologySpring 2005

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

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

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

UNI Plant Physiology 2005 www.uni.edu/berg

No dye (blank)

Absorbance of Dye Solution

Absorbance = 0

(set to 0)Dilute dye

Concentrateddye

Absorbance low

Absorbance high

IN THE SPEC

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

UNI Plant Physiology 2005 www.uni.edu/berg

Absorbance of Dye SolutionIN WHITE ROOM LIGHT, SEEN BY

YOUR EYES

LOTS OF LIGHT GETS THROUGH

THE RED DYE, JUST NOT BLUE LIGHT

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)