The Visible Spectra of Plant Pigments

C2PRISM Teacher Development Workshop

Dino Magou

Overview

• Classic chromatography experiment• Description and review of Vernier Colorimeter• Using Colorimeter to measure pigment

absorption at different wavelengths• Description of alternative method using a

Spectrometer

Chromatography

• Use of a solvent (mobile phase) to separate a mixture of pigments (stationary phase)

• Solvent is attracted to paper– This is how it moves up

the paper

• Pigments separate based on how soluble they are in the solvent

Procedure

What you should expect to see

Xanthophyll (yellow)

Carotene (yellow to yellow green)

Chlorophyll a (bright green to blue green)

Chlorophyll b (yellow green to olive green)

Solvent moves upthe paper

Calculating Rf

Mark where solvent stops moving

Measure the distance each pigment travelled from the starting point

Starting point

Calculating Rf

Starting point

Mark where solvent stops moving

Colorimeter

• Vernier Colorimeter provides 4 wavelengths of light– 430 nm– 470 nm– 565 nm– 635 nm

How the colorimeter works

Colorimeter

• Beer’s Law (Chemistry)• Photosynthesis

(Biology)• Cell Membrane vs.

Alcohol (Biology)• Cell Membrane vs

environmental stresses (Biology)

• Population dynamics with yeast (Biology)

The visible spectra of plant pigments

• Make a rough plot of absorbance peaks for different plant pigments– Spinach– Carrots

What you need

• Fresh spinach• Fresh carrots• Mortar and pestle• Grater• 70% Isopropanol• Acetone or petroleum ether• Vernier Colorimeter kit

Procedure (Prep)

This can be done by the students or by the instructor before class depending on your time constraints

1. Measure out 0.5 g of fresh spinach. Tear the spinach into tiny pieces and grind them with a mortar and pestle. Add 20 mL of 70% isopropanol (IPA) and transfer the mixture to a small beaker. Allow the mixture to sit.

2. Measure out 0.5 g of carrot slices (or shavings) and place them in a container. Add 20 mL of either acetone or petroleum ether to the flask and stopper it.

Procedure (Colorimeter)

1. Calibrate the colorimeter using the solvent used in your sample

2. Place prepared spinach sample in cuvette

3. Record absorbance at 430 nm, 470 nm, 565 nm, and 635 nm

4. Repeat step with carrot sample

5. Have students plot absorbance as a function of wavelength of light

SpectroVis Plus

• Vernier SpectroVis Plus will plot the complete spectra automatically

Possible topics include:• Kinetics of a Bleach

Reaction• Explore Fluorescence

Spectroscopy• Study Neurotrasmitters• Study Proteins

Sample Spectra

Questions?