Investigations on Photosynthesis Ib Biology Lab Report

Plants are capable of capturing light energy because they have photosynthetic pigments. In this investigation we will utilize the paper chromatography technique to separate and identify these pigments.

Experiment I:

Separation of photosynthetic pigments by paper chromatography

Background

Plants are capable of capturing light energy because they have photosynthetic pigments. In this investigation we will utilize the paper chromatography technique to separate and identify these pigments.

Research question

Is chromatography an accurate method of separating and observing the various colors of plant pigments?

Hypothesis

Using paper chromatography, the pigments that give a leaf its color can be separated and observed to determine the Rf value of each pigment and their function during photosynthesis.

Material required

• Pestle and mortar
• Filter funnel
• Muslin
• Beaker (100c.c.)
• Jar
• Stopper
• Scissors
• Acetone (pure)
• Chromatography solvent (petroleum ether/acetone, 9:1)
• Fresh spinach leaves
• Chromatography paper

Procedure

• Cut a strip of chromatography paper sufficiently long to reach almost to the bottom of a jar and narrow enough that the edges will not touch the sides of the tube.

• Rule a pencil line across the strip of paper 2 cm from one end. Fold the other end through 90 and attach it to the stopper with a pin.

• With a pestle and mortar, grind up fresh healthy spinach leaves in pure acetone, producing as concentrated a pigment solution as possible. Filter this through muslin into a small beaker.

• Remove the paper from the jar and, using a very fine capillary pipette, place a drop of the pigment solution at the center of the pencil line. Repeat this procedure several times, building up a small area of concentrated pigment.

• While preparing your pigment stain, pour some chromatography solvent into the jar to a depth of not more than 1 cm. Seal the jar with a stopper for about 10 minutes so that the atmosphere inside becomes saturated with vapor.

• Now suspend the strip of paper in the jar. The bottom edge of the paper should dip in the solvent, but make sure that the pigment is not immersed.

• The solvent front will rise rapidly and the pigments will separate in about 10 minutes. When the solvent is close to the top of the paper, remove the strip, rule a pencil line to mark the solvent front, and dry the paper.

• Identify the pigments by their colors and by their RF values, where

RF= (distance traveled by solute)/ (distance moved by solvent)

Data collection

Qualitative data

After leaving the bottom edge of the chromatography paper in the solvent for 10 minutes, 4 stains of different colors are observable at different locations on the paper. From bottom to top the colors of the stains are: green, blue green, brown yellow and yellow.

Shot 1*:strip of chromatography paper showing different colored stains*The smell of the solvent in the Erlenmeyer spreads very quickly after removing the lid for only a few seconds.

Quantitative data

Distance moved by solvent front: D=8.45cm

Table 1: Distances traveled by each stain

Color of stain

Distance traveled (cm)

yellow

7.93

yellow-brown

6.25

blue-green

5.12

green

3.65

Data processing and presentation

We know that chromatography is used to separate the different solutes in a mixture. In this case the different colored stains are the pigments within the plants that capture sunlight. Hence each stain contains different pigments

RF= (distance traveled by solute)/ (distance moved by solvent)

Sample calculation of the RF of green stain

*Photo retrieved from http://regentsprep.org/Regents/biology/units/homeostasis/processes.cfmRF1= 3.65/8.45 = 0.43

Table 2: Colors and RF values for the pigments in a typical leaf, separated in an acetone/petroleum ether mixture

Color of pigment

RF

yellow

0.94

yellow-brown

0.74

blue-green

0.60

green

0.43

Conclusion and evaluation

We know that chromatography is used to separate the different solutes in a mixture. In this case the different colored solutes are the pigments within the plants that capture sunlight. Hence each stain contains a different pigment.

Since a solute has the same Rf (if present alone or in a mixture) each time we submit it to separation by chromatography, then by comparing the different RFs obtained to those given, we can identify the pigments present in each stain.

By comparing our results to this table, we can identify the pigment present in each stain.

Table 3: Colors and Rf values for the pigments in a typical leaf, separated in an acetone/petroleum ether mixture

Pigment

Color of pigment

RF

carotene

yellow

0.95

xanthophyll

yellow-brown

0.71

chlorophyll a

blue-green

0.65

chlorophyll b

green

0.45

Since the yellow-brown stain has an RF of 0.94 which is extremely close to that of carotene (0.95), we can state that this stain contains carotene.

Since the yellow-brown stain has an RF of 0.74 which is extremely close to that of carotene (0.71), we can state that this stain contains xanthophyll.

The blue-green stain has an RF of 0.60 which is extremely close to that of chlorophyll a (0.65); hence we can conclude that this stain contains chlorophyll a.

The pure green stain has an RF of 0.43 which is extremely close to that of chlorophyll b (0.45); hence we can conclude that this stain contains chlorophyll b.

Petroleum ether is highly volatile; which explains why its smell spread so rapidly. It has a boiling point between 25 and 70ºC.

Paper chromatography showed to be a precise method of separating and view the various colors of plant pigments. The pigments dissolved in the solvent and traveled upward. The Rf value of each pigment was determined by dividing its migration by the migration of the solvent. It was determined that 4 pigments were present in the original stain --- carotene, xanthophyll, chlorophyll a, and chlorophyll b. Carotene was the most soluble since it migrated the most, while chlorophyll b was the least soluble since it moved upwards the least.

Experiment II:

What do plants do with sunlight?

Background

Plants are photosynthetic autotrophs. They capture sunlight energy and store it in glucose molecules. The glucose molecules are then almost immediately bound together into chainlike molecules of starch. We will test for the presence of starch by using iodine solution which turns blue-black in the presence of this polysaccharide.

Research question

Will cells in a leaf produce glucose in the absence of light?

Hypothesis

In the absence of sunlight, cells within a leaf cannot produce carbohydrates (glucose and starch).

Material required

• Geranium plant
• Aluminum foil
• Dark construction paper
• Scissors
• 1000 ml beaker
• 250 ml beaker
• 3 Petri dishes
• 95% ethyl alcohol
• Medicine dropper
• Hot plate
• Forceps
• Iodine solution
• Paper clips

Procedure

Process A: Preparing the plant

• Completely cover one of the leaves on the plant with aluminum foil; you may need to secure the foil with a paper clip. On a second leaf, paper clip a piece of construction paper that has been cut in a particular shape of your choice. This procedure should take place 5 days before commencing with the rest of the investigation.
• Identify each leaf you are using with a masking tape label that gives your group number.
• Place the plant in bright light.

Process B: Testing the leaves

• On the day of the investigation, remove 3 leaves from the plant: the two experimental leaves covered beforehand, and one leaf that was not covered., remove the masking-tape labels, but in order to identify the leaves, cut their petioles different lengths according to the following;
• No petiole-uncovered leaf
• Half petiole-leaf with paper shape
• Full petiole-leaf covered with aluminum foil
• A water bath should be set in the lab in which alcohol is heating (on a hot plate). Drop all three leaves into the boiling water for 1 minute.
• Remove the leaves from the boiling water with a forceps and drop them in the hot alcohol in the inner beaker. Keep them in the hot alcohol until they have lost most of their color.
• Remove the leaves from alcohol with forceps and dip them into hot water again for a few seconds.
• Place the leaves into three different Petri dishes, and by means of a medicine dropper cover each of the leaves with iodine solution. After one minute, pour off the excess iodine solution and record your observations.

Data collection

Qualitative data

Table 1: colors of experimental leaves and uncovered leaf after process A

Leaf

color

Completely covered

Brown but mainly dark yellow

Semi covered (with part of it exposed with the shape of a heart)

Uncovered part: normal green

Covered part: dark yellow

uncovered

Normal usual green color of leafs

Table 2: colors of experimental leaves and uncovered leaf after submitting them through

process B and adding starch

Leaf

color

Completely covered

brown

Semi covered (with part of it exposed with the shape of a heart)

Uncovered part: dark blue

Covered part: brown

uncovered

dark blue

Shot 1*:semi covered leaf before and after submitting it to process B and adding iodine

*Photo retrieved from

http://www.accessexcellence.org/LC/TL/filson/lab.html

Conclusion and evaluation

Iodine is used as an indicator that turns blue in the presence of starch.

We know that green plants produce glucose by photosynthesis. To do this plants need carbon dioxide, water, sunlight and photosynthetic pigments. The equation illustrating this reaction is: light

6CO2+6H2O C6H12O6+6O2

In green plant cells, most of the glucose which is produced is metabolized into starch (the plant cells metabolize glucose into this polysaccharide in order to store it as an energy compound).

In the absence of light no glucose can be produced and hence no starch can be metabolized from this carbohydrate.

The totally covered leaf backs up this statement since it did not react with the iodine which indicates that there is no starch present. This means no photosynthesis took place. The uncovered leaf reacted completely with the iodine giving off a dark blue color which indicates the presence of starch. This must indicate that photosynthesis also took place. In the semi covered leaf, the covered part did not react with the iodine; however the uncovered part did, giving also a dark blue color. Hence the leaf's dark blue color indicate that photosynthesis must have taken place in order to produce the glucose that was metabolized into starch (the plant cells metabolize glucose into this polysaccharide in order to store it as an energy compound).

The leafs were placed in boiling water to destroy the cell membranes, the high temperature causes the proteins in the membrane to denature. Then by dipping the leafs in alcohol we removed its pigments from the cells since they are more soluble in this solvent.

These results confirm our hypothesis, since we can conclude from our observations that photosynthesis does not take place during absence of light.

Resources

http://sps.k12.ar.us/massengale/chromatography_of_simulated_plan.htm

http://regentsprep.org/Regents/biology/units/homeostasis/processes.cfm

http://www.accessexcellence.org/LC/TL/filson/lab.html