For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14

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


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


3/17

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


4/17

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?


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?


6/17

Organization of Photosynthetic Pigments-- the antenna systems

Antenna pigment

Molecules(Protein Pigment Complex)

Reaction Center

chlorophyll

Photon

Organization of Photosynthetic Pigments

Chlorophyll

Antenna System


7/17

Cyanobacteria In Culture

Green, Red and Brown Algae


8/17

Light and Dark Grown Barley

Methanol Extraction


9/17

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


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


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


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)


11/17

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


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?


13/17

Thin Layer

Chromatography

(Exp. 1A)





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


buffer extract

Spectrophotometry

(Exp. 2B)

pipet 500 uL of

the provided

phosphate

buffer extracts

into 6 cuvetsTransfer supernatant

to cuvet containing


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


14/17

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





Cyanobacteria Brown algae

Red algae Green algaeDark- grown

barley

Initial Spot

(Sample)


15/17

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.


16/17

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


17/17

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.

Documents

For STUDENTS Intro Plus Pigments Plus TLC TA Presentation - Updated 2-14-14