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) Solvent moves up the paper Chlorophyll a (bright green to blue green) Chlorophyll b (yellow green to olive green) Calculating Rf Mark where solvent stops moving Measure the distance each pigment travelled from the starting point Starting point Calculating Rf Mark where solvent stops moving D R = D p f Starting point s 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?