Lab Practical #2 - Bio110

.19 mg/L of dissolved oxygen

1 mV =

more

450nm wavelength photon has ______ energy than a 650nm wavelength photon

inversely

A photon is _________ related to its wavelength

light absorbance as a function of wavelength

A spectrophotometer measures

to help make glucose

ATP is used in Calvin-Benson cycle

absorbance/time increases

As more chloroplasts are added

decreases

As time increases, absorbance with .1, .2 and .4 chloroplasts

remains constant at one

As time increases, absorbance with no chloroplasts

light energy well

Blue end of absorbance spectrum absorbs

400

Blue wavelength range

ethanol

Carotene affinity

a member phylum Chlorophyta, an aquatic photosynthetic unicellular organism

Chlorella is

Petroleum ether

Chlorophyll A affinity

methanol

Chlorophyll B affinity

light of particular wavelengths

Chlorophyll absorbs

Chlorophyll a and b have absorption peaks in the blue and red regions. This means that chlorophylls transmit green light.

Chlorophyll is green; how does this relate to its absorption spectrum?

Absorbance

Dependent Variable: Photosynthesis- Chlorophyll Extraction and Absorption Spectrum

Rate of water oxidation

Dependent Variable: Photosynthesis-Oxidation of Water

rate at which Chlorella produces oxygen

Dependent Variable: Photosynthesis-Production of Oxygen

Not much

Does chlorophyll absorb in the green region of the spectrum?

Light alone accounts for the majority of the color changes of DPIP because light alone causes greatest loss of blue color

Does light alone appear to be needed to drive the DPIP reduction?

light energy poorly - reflects

Green end of absorbance spectrum absorbs

500

Green wavelength range

Chlorophyll only absorbs light in this region, a plant should carry out photosynthesis

How much photosynthesis would a plant carry out if it were illuminated by only blue light?

Not much because chlorophyll cannot absorb much light energy in the green region

How much photosynthesis would a plant carry out if it were illuminated by only green light?

Chlorophyll only absorbs light in this region, a plant should carry out photosynthesis

How much photosynthesis would a plant carry out if it were illuminated by only red light?

Electrons are used to help make sugar come from water that Photosystem II oxidizes; if pII can't deliver electrons, sugar production will stop and plant will die

How will DCMU negatively affect the production of glucose in a plant?

Lower the rate of oxygen production when compared to the rate produced by the same PFR and no DCMU; will fall because it is dependent on electron-deficient Photosystem II.

How would DCMU affect the production of oxygen in plants?

Lose blue color; absorbance at 10 minutes should be less than at 0 minutes

If DPIP spontaneously loses color, what should have happened in the "no light, no heat" control?

Lose blue color, absorbance at 10 minutes should be less than at 0 minutes

If heat is necessary for the DPIP to change color, what should have happened in the "no light, heat" control?

Greater loss of blue color in "light and heat" than the "light" alone

If light and heat are required for DPIP to lose its color, how should the change in absorbance be different between the "Light and heat" control and the "light, no heat" condition?

Chlorella were consuming oxygen

If the "no light control" rate was negative, what was happening to oxygen in the dark Chlorella tube?

More chloroplasts, faster the dye reduction; direct relationship between rate of DPIP reduction and amount of chloroplasts indicates the dependence of water oxidation/dye reduction on active chloroplasts

In the experiment where the amount of chloroplasts was varied, what do your results show about chloroplast function?

C. Change in absorbance over time

In the oxidation of water lab that utilized the blue dye DPIP, which of the following represents a measured (dependent) variable? A. Light intensity. B. pH. C. Change in absorbance over time. D. Wavelength of light. E. Temperature

B. The PFR was varied by adjusting the distance between the light source and the tube containing Chlorella

In the oxygen production lab, how was PFR varied? A. The PFR was varied by adjusting the offset control on the amplifier. B. The PFR was varied by adjusting the distance between the light source and the tube containing Chlorella. C. The PFR was varied by adjusting the color of the light source. D. The PFR was varied by moving the light source. E. The PFR was varied by adjusting the temperature of the light source.

A. A tank of water was placed in the light path to absorb heat.

In the oxygen production lab, what compensation was made for the heat generated by the lamps? A. A tank of water was placed in the light path to absorb heat. B. An amount was subtracted from each oxygen production rate to account for temperature effects. C. A heat-resistant strain of Chlorella was used. D. All of the above represent means that were used to compensate for the heat. E. Only A and B above were used to compensate for the heat.

Wavelength

Independent Variable: Photosynthesis- Chlorophyll Extraction and Absorption Spectrum

the amount of chloroplasts added to the reaction mixture

Independent Variable: Photosynthesis-Oxidation of Water

light quantity (measured as PFR)

Independent Variable: Photosynthesis-Production of Oxygen

light energy well

Red end of absorbance spectrum absorbs

600

Red wavelength rage

light source, monochromator, test tube, light detector, amplifier, readout

Six parts of a spectrophometer

400 to 700 nanometers

Spectrophotometer operates in the wavelength range of

C. The carotenes extracted in our lab were contaminated with chlorophyll; the carotenes used in the textbook were not contaminated with chlorophyll.

The absorption spectrum of carotenes as determined in lab sometimes showed an absorption peak in the red region of the spectrum. However, the absorption spectrum of carotenes shown in the textbook did not show such a peak. What caused this difference? A. Hot ethanol was used to extract pigments in our lab; the extraction procedure outlined in the textbook used cold ethanol. B. The spinach used in lab was briefly boiled in water; the procedure outlined in the textbook did not use boiling water. C. The carotenes extracted in our lab were contaminated with chlorophyll; the carotenes used in the textbook were not contaminated with chlorophyll. D. The spectrophotometer used in our lab was able to emit light in the red region of the spectrum; the one used in the textbook could not. E. In our lab we used the proper amount of carotenes; in the textbook the amount of carotenes was inadequate to measure absorption in the red region.

oxidize water

The chlorophyll-based photosynthetic machinery of chlorplasts is able to

the removal of electrons from water and the shuttling of the electrons and protons

The photo energy absorbed by chlorophyll permits

B. The oxidation of water

The reduction of DPIP by chloroplasts was coupled to which reaction below? A. The oxidation of carbon dioxide. B. The oxidation of oxygen. C. The oxidation of water. D. The oxidation of NADPox. E. The oxidation of sodium bisulfite.

electrons and protons

The shuttling of _____ drive the generation of ATP and reduce CO2

1. Oxygen, protons, and electrons are produced 2. Helps make ATP 3. Helps reduce CO2

Three important things that happen as a result of oxidation

place a sample of Chlorella culture at various predetermined distances from a light source

To vary PFR

Carotenoids - more soluble in 95% ethanol Chlorophylls- more soluble in petroleum ether

What are the relative solubilities of carotenoids and chlorophylls a and b in petroleum ether and 95% alcohol?

Chlorophyll a higher solubility in petroleum ether Chlorophyll b higher solubility in methanol

What are the relative solubilities of chlorophylls a and b in petroleum either and 92% methanol?

Oxidize sugars that they have to make

What do plants use oxygen for?

Higher the PFR, greater the oxygen production (linear)

What is the relationship between PFR and oxygen production rate in Chlorella?

Exploited the different solubilities of the pigments in petroleum ether, ethanol and methanol.

What major property of chlorophyll a, chlorophyll b and carotenoids did we take advantage of in order to separate them from one another?

D. To eliminate pH as a variable in the experiment

What was the purpose of using a buffer for the isolation and function assessment of chloroplasts? A. The buffer maintained a constant temperature in the chloroplast preparation; this was necessary because the lights used in the experiment produced heat. B. To maintain a constant amount of light (about 500 foot-candles) in the chloroplast preparation. C. To maintain a constant amount of carbon dioxide in the chloroplast preparation. D. To eliminate pH as a variable in the experiment. E. To maintain a constant amount of oxygen in the chloroplast preparation.

Maximum absorbance in the blue & red regions, blue or red wavelengths are most effective in reduction

What wavelength do you think would have been the most effective for driving the reduction of DPIP?

Green wavelengths; least well absorbed

What wavelength would have been least effective for driving reduction of DPIP?

Chlorophyll is only useful if it absorbs or captures light energy which is used to excite electrons that are used to produce ATP and reduce NADPox. Chlorophyll that doesn't absorb light would be useless in photosynthesis.

What would be the consequences to photosynthesis be if the chlorophyll molecule were transparent to light?

Transparent and colorless chlorophyll would not absorb light; it would transmit all the light that hit it. The absorbance at visible wavelengths would be 0/

What would the absorption spectrum of a colorless, transparent substance look like?

loses its blue color

When DPIP is reduced it...

remove electrons from water

When Photosystem II absorbs light energy it is able to

Water

Where are the electrons that reduce DPIP coming from?

Given to CO2 to reduce it; reduction of CO2 to sugar is essential to photosynthesis

Where do electrons end up when photosynthesis is occurring?

E. Only A and B above. (Sodium bisulfite, illuminated chloroplasts)

Which of the following are capable of reducing DPIPox? A. Sodium bisulfite. B. Illuminated chloroplasts. C. Potassium Chloride (KCl) D. All of the above. E. Only A and B above.

C. Sodium bisulfite and chloroplasts may cause DPIP that is initially blue in solution to become colorless

Which of the following do chloroplasts and sodium bisulfite hold in common? A. Sodium bisulfite may break down into DPIP and chloroplasts may produce DPIP. In either case the DPIP produced may oxidize water. B. Sodium bisulfite may break down into DPIP and chloroplasts may produce DPIP. In either case the DPIP produced may reduce water. C. Sodium bisulfite and chloroplasts may cause DPIP that is initially blue in solution to become colorless. D. Sodium bisulfite and chloroplasts may cause DPIP that is initially colorless in solution to become blue. E. Sodium bisulfite and chloroplasts both may oxidize water by adding electrons to the water.

D. Comparing the absorbance changes in separate tubes in which increasing amounts of chloroplasts were added.

Which of the following exercises that you performed in lab best shows that chloroplasts play a crucial role in the color change of DPIP? A. Determining the difference in the absorption spectra of chlorophylls a and b that were removed from chloroplasts by boiling ethanol. B. Comparing the change in absorbance of an illuminated tube completely covered with aluminum foil with the change in absorbance in an identical illuminated tube that was not covered with foil. C. Checking the absorption spectra of oxidized versus reduced DPIP. D. Comparing the absorbance changes in separate tubes in which increasing amounts of chloroplasts were added. E. Determining the difference in the absorption spectra of chlorophyll a and carotenoids that were removed from chloroplasts by boiling ethanol.

A. Measure its absorbance over a range of different wavelengths of light

Which of the following is done to determine the absorption spectrum of a substance? A. Measure its absorbance over a range of different wavelengths of light. B. Plot the rate of absorbance change versus time. C. Measure its absorbance at one wavelength over time as the substance is reduced. D. Measure its absorbance at one wavelength over time as the substance is oxidized. E. Subtract the absorbance of the substance when it is reduced from the absorbance of the substance when it is oxidized.

E. Only B and C are true

Which of the following is/are true of the absorption spectrum of chlorophyll b as studied in lab? A. Chlorophyll b had an absorption peak in the green region of the visible spectrum. B. Chlorophyll b had an absorption peak the red region of the visible spectrum. C. Chlorophyll b had an absorption peak the blue region of the visible spectrum. D. All of the above are true of the chlorophyll b absorption spectrum. E. Only B and C are true of the absorption spectrum of chlorophyll b.

A. Carotene is more soluble in ethanol than in petroleum ether

Which of the following is/are true of the solubilities of the pigments studied in lab? A. Carotene is more soluble in ethanol than in petroleum ether. B. Chlorophyll a is more soluble in methanol than in petroleum ether. C. Chlorophyll b is more soluble in petroleum ether than in methanol. D. All of the above are true concerning the pigments studied in lab. E. Only B and C are true of the pigment solubilities.

A. 600

Which of the following wavelength settings is best for having a Spec 20 detect the absorbance of light by a substance that is blue in solution? A. 600. B. 425. C. 450. D. 475. E. 400.

To control the pH; necessary to maintain a constant external environment for the chloroplasts; helps them survive; held pH steady

Why was a buffer used in the Oxidation of Water lab?

Bisulfite reduced the DPIP, blank had everything in it that the experimental tube has except blue color

Why was bisulfite added to the "blank" tubes in your experiments with chloroplast function?

absorbance is greatest at 400s wavelength, drops a lot in the 500s, raises again in late 500s, levels off in 600s

ethanol graph of absorbance

low in the 400s, increases in 500s and levels off, drops in 600s then levels off again

methanol graph of absorbance

absorbance is second highest in the 400s, drops significantly in late 400s and levels off in 500s, increases again in 600s

petroleum ether graph of absorbance

Absorption spectrum

the pattern of absorption by a substance