AP Lab 4 Part 1: Leaf Chromatography

```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
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?
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