Photosynthesis Lab

I. Chromatography

Introduction:

Chromatography paper allows for the separation of pigments and other cell molecules within cell

extracts. Solvents are able to move up the chromatography paper via capillary action and as the

solvent moves up it carries with it any substance that have been dissolved in it. Since different

substances are not equally soluble, they move up at different rates. The objective of this

experiment is to determine the relative hydrophobia of the pigments within the chloroplast of

plants based on their Rf value.

Hypothesis:

If a line of chloroplast is marked on a piece of chromatography paper that is dipped in a

hydrophobic organic solvent, then the carotenoid pigments will have the largest Rf value and

chlorophyll b will have the slowest.

Material:

This lab requires spinach leaves, a quarter, a hydrophobic organic solvent, a glass cup, and

chromatography paper.

Procedure:

First use the quarter to extract a chlorophyll mixture out of the spinach leaves. Use the

chlorophyll mixture to draw a line across the chromatography paper about 1 inch above the

bottom. Pour the hydrophobic organic solvent into the glass cup about half an inch from the

bottom then place the chromatography paper in the glass cup. Wait approximately 20 minutes

then remove the chromatography paper. Record the distance of every pigment, using the color

description to identify each band by color, and the distance of the solvent front starting from the

original chloroplast line. Calculate Rf of each pigment using the formula: Pigment

Distance(mm)/Solvent Front) Distance(mm).

Data Analysis:

Pigment Distance (mm) Rf (Pigment Distance(mm)/Solvent Front)

Distance(mm)

Carotenoid 45 0.714

Chlorophyll a 29 0.460

Chlorophyll b 13 0.206

Solvent Front 63 1

Conclusion:

The constant, Rf, of each pigment is the quotient of the distance each pigment travels and the

distance of the solvent front, therefore the larger the distance the higher the Rf. The rate of the

pigments distance is determined by its solubility to the solvent. Since the solvent was

hydrophobic, the more hydrophobic a pigment is the faster it will travel up the chromatography

paper. Because of this we can conclude that carotenoid is the most hydrophobic of the three

pigments, with chlorophyll a in second and chlorophyll b last.

1. The solubility, the pigment’s attraction to the chromatography paper, and the particle

sizes are all involved in the separation of the pigments.

2. Yes because Rf is a constant value for every pigment, therefore as long as the solvent is

hydrophobic Rf will not change.

3. The reaction center contains Chlorophyll a, while the other pigments function to transfer

energy to chlorophyll a and capture different light waves.

II. Transmittance

Introduction:

Photosynthesis is a process that depends on light waves to excite the electrons within its

photosystem to begin the process. Without photosynthesis, plants would be unable to convert

light energy into usable energy. The purpose of this experiment is to determine how varying light

conditions affect a plant’s ability to perform photosynthesis effectively.

Hypothesis:

If chloroplast solutions are placed in beakers of distilled water, a phosphate buffer, and DPIP

then the chloroplast that is unboiled and kept in the regular light will have the largest change in

transmittance percentage.

Material:

This lab requires 10 mL of phosphate buffer, 34 mL of distilled H2O, 8 mL of DPIP, 6 mL of

unboiled Chloroplast solution, 1 mL of Boiled Chloroplast solution, 10 cuvettes, a sharpie, a

green light lamp and a spectrophotometer.

Procedure:

First label all 5 cuvettes with a different number 1-5. Then add 1 mL of phosphate buffer to all

cuvettes, 4 mL of distilled water to cuvette #1, 3 mL of distilled water to cuvettes #2-5, 1 mL of

DPIP to cuvettes # 2-5, 0.5mL of Unboiled chloroplast solution to cuvettes #1-3, and 0.5mL of

boiled chloroplast to cuvette #4. Then shake each cuvette well and measure its transmittance

percentage with a spectrophotometer. Place cuvette #2 inside a cabinet and leave the rest outside

in the light. Finally re-measure the transmittance every five minutes for 10 minutes and record

your data. Repeat the experiment but leave cuvettes #1, and #3-5 under the green light lamp.

Data Analysis:

Transmittance percentage over times in different conditions (White Light)

Condition 0min 5min 10min Total Change in Transmittance

percentage

Control 22 22 22 0

Unboiled/Dark 22 23 23 1

Unboiled/Light 22 48 62 40

Boiled Light 22 27 29 7

No Chloroplast 22 14 15 -7

Transmittance percentage over times in different conditions (Green Light)

Condition 0min 5min 10min Total

Control 20 20 20 0

Unboiled/Dark 20 24 24 4

Unboiled/Light 20 31 39 19

Boiled Light 20 22 22 2

No Chloroplast 20 33 43 23

Conclusion:

Since the thylakoids of the chloroplasts within the solution were mechanically disrupted, the

Electron Transport Chain no longer functioned properly, allowing the DPIP to accept the excited

electrons from the chlorophylls. DPIP is a blue electron acceptor that turns clear as it accepts

electrons, meaning that if photosynthesis is occurring productively then the transmittance

percentage of a test tube will increase. Based on this information and the data it can be concluded

that the best conditions for photosynthesis are unboiled and in white light. This is because light is

required to excite the electrons in the magnesium molecule of the chlorophylls that begins the

process of photosynthesis and because boiling the chloroplasts denatures the photosynthetic

enzymes, rendering the entire process void. White light is preferable to green light, because the

chlorophyll pigments a and b are unable to capture green light, therefore it is unable to use green

light for photosynthesis.

1. The DPIP becomes clear as it accepts electrons, therefore since faster rate of

photosynthesis means more electrons used its purpose is to provide the experiment with a

way to measure the relative rate of photosynthesis within each cuvette.

2. DPIP replaces the role of NADP.

3. The thylakoid was disrupted so that the electron transport chain no longer functioned

properly, allowing the DPIP to accept the excited electrons from the chlorophyll.

4. Spectrophotometer measures the change in percent of transmission, which should

increase as the DPIP becomes clear as it receives electrons.

5. Darkness will mean a slower rate of photosynthesis. Therefore less electrons will be

available for acceptance at any given moment, slowing down the rate at which DPIP

clears.

6. Boiling the chloroplast denatures the photosynthetic enzymes, rendering the process of

photosynthesis discontinued. Without photosynthesis the DPIP will not be able to

function as an electron acceptor and clear.

7. In the dark photons were unable to power the photosystems within the chloroplast, thus

preventing the chloroplast from undergoing photosynthesis. Without photosynthesis the

DPIP will not be able to function as an electron acceptor and clear. The chloroplast in the

light faced no such problem, so the DPIP was able to accept the electrons from

photosynthesis and clear.

8. Cuvette 1 is acting as a control to show that without DPIP the percent transmittance will

not change.

Cuvette 2: To prove that the rate of photosynthesis is slower without light and therefore

depends on light.

Cuvette 3: The ideal condition for photosynthesis is in direct light.

Cuvette 4: To show that boiling the chloroplast denatures the photosynthetic enzymes,

thus preventing photosynthesis from occurring.

Cuvette 5 is acting as a control to show that without chloroplast DPIP will be unable to

accept electrons and therefore the percent transmission will not change significantly.