BIO 111 - Lab 8: Spectrophotometry and Photosynthesis

Spectrophotometry and Photosynthesis Lab purpose

observing how light energy powers chemical reactions inside chloroplasts by using an artificial electron acceptor (DPIP) to make these reactions visible

What does the DPIP color indicate?

DPIP changes color from blue to clear - reveals the rate of photosynthesis

Photosynthesis

Plants use the sun's energy to convert water and carbon dioxide into sugars

DPIP replaces what?

replaces NADP+ as the electron acceptor

Why does DPIP turn clear?

DPIP starts out blue but turns clear as it gains electrons during photosynthesis

What happens as the photosynthesis lab progresses?

DPIP becomes clear, the sample absorbs less light, and the absorbance value decreases

What does a faster decrease in absorbance mean?

photosynthesis is happening more quickly

What is the spectrophotometer doing?

detecting how much light passes through the sample

What are the two stages of photosynthesis?

light reactions and carbon fixation reactions

Chlorophyll

Green pigment in plants that absorbs light energy used to carry out photosynthesis

thylakoid membranes

Site of light reactions in chloroplasts.

Light Reactions

Chlorophyll in the thylakoid membranes absorbs energy to produce ATP and NADPH (stores energy and electrons)

Light Reactions formula

Light + H2O --> NADPH + ATP + O2 (water is split)

Carbon fixation Reaction

the enzyme RuBisCo uses ATP, NADPH, and CO2 to produce sugar

Carbon fixation Reaction Formula

CO2 + ATP + NADPH = C6H12O6

Carbon fixation Reaction Steps

1. CO2 molecule combines with RuBp = 6 carbon molecule

2. Enzyme rubisco turns 6 carbon molecule into two 3-carbon molecule = 3-PGA

3. ATP and NADPH provide the energy and electrons to convert 3-PGA into G3P

4. G3P is used to make glucose

Formula for DPIP

DCPIP+ (oxidized - blue) + 2H+ + 2e- = DCPIPH (reduced - clear)

Prepare an outline showing how each cuvette system was set up for this experiment

B. 2mL dH2O2 (hydroegen peroxide) - control group

1. 2mL DPIP

2. 2mL DPIP and 70 μL thylakoids

3. 2mL DPIP and 70 μL thylakoids (no light)

4. 2mL DPIP and 70 μL thylakoids (boiled)

Light Reaction Steps

1. light energy excites and loses electrons in chlorophyll at the reaction center

2. water is split to replace electrons, releasing oxygen

3. energized electrons move through an electron transport chain producing ATP

4. Electrons are then recharged to produce NADPH

What does a decrease in DPIP absorbance indicate?

a decrease in DPIP absorbance indicates that DPIP is becoming clearer and is being reduced (gaining electrons) - light reactions of photosynthesis are occurring and electrons are being transferred

Why is one cuvette kept in the dark?

control group

How does light intensity affect the rate of photosynthesis?

higher light intensity increases the rate of photosynthesis

Which cuvette system showed the greatest decrease in absorbance and why?

DPIP + unboiled thylakoids in light due to functional chloroplasts and active energy

What is the purpose of the thylakoid extract?

the thylakoid extract contains the photosystems, chlorophyll, and the electron transport chain protein

Would the boiled thylakoid extract be able to carry out photosynthesis? Why or why not?

no, boiling denatures the proteins and destroys the structures

Chromatography

a method used to separate and isolate a mixture based on hydrophobic and hydrophilic propertied

What is the purpose of using chromatography in this experiment?

to separate and identify the multiple pigments in a leaf

What the pigments in photosynthesis?

chlorophyll is the primary pigment (chlorophyll a is main, and chlorophyll b), accessory pigments - carotenoids and xanthophyll

What is the purpose of the solvent, and why must its level be kept below the pigment line when setting up the chromatography?

the solvent (ethanol) is the mobile phase (moves up the paper and carries pigments with it), the level must stay below the pigment line so the pigments don't dissolve and wash away

How many different colors appear in the chromatogram, and what does each color represent?

faint yellow/orange - carotenoids, yellow - xanthophylls, green - chlorophyll

Why do plants contain more than one pigment?

Plants have multiple pigments to absorb a wider range of light wavelengths (improves photosynthesis efficiency)

Why were the chromatography setup and procedure performed inside a chemical fume hood?

ethanol is volatile (will evaporate quickly) producing vapors that can irritate eyes and nose