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8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
1/17
Photosynthetic Pigments
Common Photosynthetic PigmentsChlorophylls:
Chlorophyll a
Chlorophyll b
Accessory Pigments:
Carotenoids -carotene
xanthophylls
Phycobilins phycocyanin
phycoerythrin
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
2/17
monochromator detector readout
lamp
lens
Student discussion question 1: In
spectrophotometry, what is the difference
between an absorption/transmission/scatteringspectrum?
Spectrophotometry
Wavelength (nm)400 700
A b s o r b a n c e
violet red
Absorption Spect rum
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
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Absorption spectrum is the plot of a pigment’s
absorption of light versus wavelength of light. Each
pigment has its own characteristic absorption spectrum.
Pigment YPigment X
Spectrum of pigment mixture
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
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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?
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
5/17
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?
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
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Organization of Photosynthetic Pigments-- the antenna systems
Antenna pigment
Molecules(Protein Pigment Complex)
Reaction Center
chlorophyll
Photon
Organization of Photosynthetic Pigments
Chlorophyll
Antenna System
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
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Cyanobacteria In Culture
Green, Red and Brown Algae
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
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Light and Dark Grown Barley
Methanol Extraction
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
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The Experiment
Factors affecting pigmentcomposition 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
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
10/17
Light-grown barley;
Dark- grown barley
Grind leaves in ~ 3 ml
methanol; transfer to
microcentrifuge tube
Spin 5 min.
Thin Layer
Chromatography
(Exp. 1A)
Spectrophotometry
(Exp. 1B)
Exp 1: Environmental effect
on pigment composition
Exp 2: Evolutionary hi story
on pigment composition
Cyanobacteria; Red algae;
Brown algae; Green algae
Retrieve provided
methanol extracts
Pipet 50 uL methanol extract
into cuvet containing
corresponding phosphate
buffer extract
Thin Layer
Chromatography
(Exp. 2A)
Spectrophotometry
(Exp. 2B)
pipet 500 uL of
the provided
phosphate
buffer extracts
into 6 cuvetsTransfer supernatant
to cuvet containing
corresponding phosphate
buffer extract
(50 uL of light-grown;100 uL dark-grown)
For both
Exp 1 & Exp 2
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)
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
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0.5 cm Solvent level (mobile phase)
silica gel on plastic sheet
(stationary phase)
Initial Spot (Sample)
Thin Layer Chromatography (TLC)- Experiment Setup
Solvent front
0.5 cm
C
X
B
Rf = B / X
Rf = C / X
Solvent level (mobile phase)
silica gel on plastic sheet
(stationary phase)
Initial Spot (Sample)
Thin Layer Chromatography (TLC)- Experiment Setup
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
12/17
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
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
What determines Rf value?
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
13/17
Thin Layer
Chromatography
(Exp. 1A)
Exp 1: Environmental effect
on pigment composition
Exp 2: Evolutionary hi story
on pigment composition
Thin Layer
Chromatography
(Exp. 2A)
Light-grown barley;
Dark- grown barley
Grind leaves in ~ 3 ml
methanol; transfer to
microcentrifuge tube
Spin 5 min.
Spectrophotometry
(Exp. 1B)
Cyanobacteria; Red algae;
Brown algae; Green algae
Retrieve provided
methanol extracts
Pipet 50 uL methanol extract
into cuvet containing
corresponding phosphate
buffer extract
Spectrophotometry
(Exp. 2B)
pipet 500 uL of
the provided
phosphate
buffer extracts
into 6 cuvetsTransfer supernatant
to cuvet containing
corresponding phosphate
buffer extract
(50 uL of light-grown;100 uL dark-grown)
For both
Exp 1 & Exp 2
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!
Procedure highlights TLC1
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
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Close top of capillary tube before
spotting, touch drop to paper.Lift capillary tube, remove finger
to allow liquid to descend.
How to spot
Light-grown
barley
Exp 1: Environmental effect
on pigment composition
Exp 2: Evolutionary hi story
on pigment composition
Cyanobacteria Brown algae
Red algae Green algaeDark- grown
barley
Initial Spot
(Sample)
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
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Report Writ ing (50 points; 6-8 pages)*reports are writ ten 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 textDiscussion :
Discuss two experiments in separate paragraphs
Give suggestions for further research
Lab
Safety
PPE required:
• Safety
glasses
or
goggles for
the
entire
lab• Gloves when handling any chemicals (remove for using the
spectrophotometer) – change gloves if you come in contact with
methanol.
• Lab coat for the entire time you are in the lab room
SOPs
referred
to
in
this
lab
:
•Methanol
•TLC solvent (petroleum ether + ethyl acetate)
•TLC plates (bonded silica)
Potential hazards: Methanol is a poison! May be fatal or cause blindness if swallowed. Can be absorbed through skin. Flammable. Irritant.
TLC solvent is highly flammable. Harmful if inhaled. Irritant.
Silica dust from TLC plates can cause irritation to skin, eyes and respiratory tract.
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
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Lab
SafetyWaste disposal:
Used methanol/phosphate
buffer
waste
must
be
decanted
into the hazardous waste jar
provided in the hood. Only
liquid in the jar! No plant
material or plastics.
The plant material and
emptied plastics
(microcentrifuge tubes,
cuvettes, pipet tips) can be
disposed of in the regular trash.
Gloves also
go
in
the
trash.
TLC solvent must be left in the
capped
tanks.
The
tanks
will
be
returned to the prep room for
disposal.
Capillary tubes (glass) and TLC plates
(silica)
must
be
placed
in
the
red
sharps
container in the hood. No sharps waste
in the regular trash!
Mortar and pestle must be washed
and returned to your work station.
Required
PPE
Lab coat
Gloves
Standard lab attire
Safety goggles
8/19/2019 For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14
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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.