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-carotene
xanthophylls
Phycobilins
phycocyanin
phycoerythrin
Spectrophotometry
monochromator
detector
readout
lens
lamp
Absorbance
Absorption Spectrum
400
violet
Wavelength (nm)
700
red
Pigment X
Pigment Y
Reaction Center
chlorophyll
Photon
Antenna pigment
Molecules
(Protein Pigment Complex)
Chlorophyll
Antenna System
Cyanobacteria In Culture
Methanol Extraction
The Experiment
Experiment 1: Environment
Presence or absence of light
Daily light change
Seasonal light change
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)
Spectrophotometry
(Exp. 1B)
Spectrophotometry
(Exp. 2B)
10
0.5 cm
Rf = B / X
Rf = C / X
B
C
0.5 cm
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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)
Spectrophotometry
(Exp. 2B)
13
How to spot
Initial Spot
(Sample)
Dark- grown
barley
Light-grown
barley
Cyanobacteria
Red algae
Brown algae
Green algae
14
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
15
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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