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Photosynthetic Pigments

Common Photosynthetic Pigments


Chlorophylls:
Chlorophyll a
Chlorophyll b
Accessory Pigments:
Carotenoids

-carotene
xanthophylls

Phycobilins

phycocyanin
phycoerythrin

Spectrophotometry

monochromator

detector

readout

lens
lamp

Student discussion question 1: In


spectrophotometry, what is the difference
between an absorption/transmission/scattering
spectrum?

Absorbance

Absorption Spectrum

400
violet

Wavelength (nm)

700
red

Absorption spectrum is the plot of a pigments


absorption of light versus wavelength of light. Each
pigment has its own characteristic absorption spectrum.

Pigment X

Pigment Y

Spectrum of pigment mixture

The action spectrum is a plot of biological activity


(e.g., photosynthesis) versus the wavelength of light.

Student discussion question 2: Why


do plant leaves look green?

Chlorophyll is plant pigment that absorbs light of the


blue and red ends of the spectrum.

Student discussion question 3:


What is the endosymbiotic theory?

Organization of Photosynthetic Pigments


-- the antenna systems

Reaction Center
chlorophyll
Photon

Antenna pigment
Molecules
(Protein Pigment Complex)

Organization of Photosynthetic Pigments

Chlorophyll
Antenna System

Cyanobacteria In Culture

Green, Red and Brown Algae

Light and Dark Grown Barley

Methanol Extraction

The Experiment

Factors affecting pigment


composition in a plant

Experiment 1: Environment
Presence or absence of light
Daily light change
Seasonal light change

Experiment 2: Evolutionary History


Closely related organism share the
same suite of photosynthetic
pigments

Exp 1: Environmental effect


on pigment composition
Light-grown barley;
Dark- grown barley

Exp 2: Evolutionary history


on pigment composition
Cyanobacteria; Red algae;
Brown algae; Green algae

Grind leaves in ~ 3 ml
methanol; transfer to
microcentrifuge tube
Spin 5 min.
Thin Layer
Chromatography
(Exp. 1A)
Transfer supernatant
to cuvet containing
corresponding phosphate
buffer extract
(50 uL of light-grown;
100 uL dark-grown)

Retrieve provided
methanol extracts
For both
Exp 1 & Exp 2
pipet 500 uL of
the provided
phosphate
buffer extracts
into 6 cuvets

Thin Layer
Chromatography
(Exp. 2A)

Pipet 50 uL methanol extract


into cuvet containing
corresponding phosphate
buffer extract

Spectrophotometry
(Exp. 1B)

Spectrophotometry
(Exp. 2B)

Thin Layer Chromatography (TLC)


- used to separate organic compounds
Stationary phase (often polar)
- paper
- glass
- aluminum
- plastic with silica gel
Mobile phase (often nonpolar)
-- organic solvent mixture
- petroleum ether and ethyl
acetate (1:1)

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Thin Layer Chromatography (TLC)


- Experiment Setup

silica gel on plastic sheet


(stationary phase)

Solvent level (mobile phase)

0.5 cm

Initial Spot (Sample)

Thin Layer Chromatography (TLC)


- Experiment Setup
Solvent front
silica gel on plastic sheet
(stationary phase)
X

Rf = B / X
Rf = C / X

B
C

0.5 cm

Solvent level (mobile phase)


Initial Spot (Sample)

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What determines Rf value?


Background
Stationary phase: polar
Mobile phase: nonpolar
Nonpolar molecules dissolves in nonpolar solvent first
Rf is determined by
Solubility of molecules to solvent
Affinity of molecules to stationary phase
Size of molecule

What determines Rf value?


Rf value is a unique combination of its
- organic compound
- mobile phase
- stationary phase
Rf value is reproducible only when all
three variables are the same

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Exp 1: Environmental effect


on pigment composition

Exp 2: Evolutionary history


on pigment composition
Cyanobacteria; Red algae;
Brown algae; Green algae

Light-grown barley;
Dark- grown barley

Grind leaves in ~ 3 ml
methanol; transfer to
microcentrifuge tube
Spin 5 min.
Thin Layer
Chromatography
(Exp. 1A)
Transfer supernatant
to cuvet containing
corresponding phosphate
buffer extract
(50 uL of light-grown;
100 uL dark-grown)

Retrieve provided
methanol extracts
For both
Exp 1 & Exp 2
pipet 500 uL of
the provided
phosphate
buffer extracts
into 6 cuvets

Spectrophotometry
(Exp. 1B)

Thin Layer
Chromatography
(Exp. 2A)

Pipet 50 uL methanol extract


into cuvet containing
corresponding phosphate
buffer extract

Spectrophotometry
(Exp. 2B)

Procedure highlights TLC1


Grind leaves of barley thoroughly!
2-4 leaves of green barley
4-8 leaves of yellow barley
each in ~ 3 ml of methanol
Spin down cell debris for 5 min
- light-grown: Extract very green, leaf fiber pale
- dark-grown: Extract deep yellow, leaf fiber pale
Spot extract on TLC paper
- Make sure the spot is dry before repeating application!
- Spot should be small and intense!

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How to spot

Close top of capillary tube before


spotting, touch drop to paper.

Exp 1: Environmental effect


on pigment composition

Lift capillary tube, remove finger


to allow liquid to descend.

Exp 2: Evolutionary history


on pigment composition

Initial Spot
(Sample)
Dark- grown
barley

Light-grown
barley

Cyanobacteria
Red algae

Brown algae
Green algae

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Report Writing (50 points; 6-8 pages)


*reports are written individually*
Introduction:
Describe background of both experiments
Give a null hypothesis for experiment 1
Give a hypothesis for experiment 2
Cite two scientific articles, one related to experiment 1 and
one related to experiment 2
Materials & Methods:
Highlight important materials and procedures
Results :
Describe two experiments in separate paragraphs;
all graphs should be addressed in text
Discussion :
Discuss two experiments in separate paragraphs
Give suggestions for further research

LabSafety

SOPsreferredtointhislab:
Methanol
TLCsolvent(petroleumether+ethylacetate)
TLCplates(bondedsilica)

Potentialhazards:Methanolisapoison!Maybefatalorcauseblindnessif
swallowed.Canbeabsorbedthroughskin.Flammable.Irritant.
TLCsolventishighlyflammable.Harmfulifinhaled.Irritant.
SilicadustfromTLCplatescancauseirritationtoskin,eyesandrespiratory
tract.

PPErequired:
Safetyglassesorgoggles fortheentirelab
Gloves whenhandlinganychemicals(removeforusingthe
spectrophotometer) changeglovesifyoucomeincontactwith
methanol.
Labcoat fortheentiretimeyouareinthelabroom

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Wastedisposal:

LabSafety

TLCsolventmustbeleftinthe
cappedtanks.Thetankswillbe
returnedtothepreproomfor
disposal.
Capillarytubes(glass)andTLCplates
(silica)mustbeplacedintheredsharps
containerinthehood.Nosharpswaste
intheregulartrash!

Usedmethanol/phosphate
bufferwastemustbedecanted
intothehazardouswastejar
providedinthehood.Only
liquidinthejar!Noplant
materialorplastics.
Theplantmaterialand
emptiedplastics
(microcentrifugetubes,
cuvettes,pipettips)canbe
disposedofintheregulartrash.
Gloves alsogointhetrash.
Mortarandpestlemustbewashed
andreturnedtoyourworkstation.

RequiredPPE

Safetygoggles

Labcoat

Gloves

Standardlabattire

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Rat dissection next week!


Topics to present for next week

Explain the directional anatomical terms listed in


the lab manual (ventral/anterior, dorsal/posterior,
cranial/superior, caudal/inferior). How do they
differ in rats and humans?
Describe the flow of digestion in the rat. Which
organs are involved?
What is the function of the caecum? Why is it
important to handle it carefully?
Every student must present once during the quarter.
Presentations are brief and help the rest of the class
understand the concepts in the lab.

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