2nd Semester 28 - BIOLOGY JUNCTION

Chlorophyll Fluorescence

Chlorophyll Fluorescence

INTRODUCTION

When a pigment absorbs light, electrons of certain atoms in the pigment molecules are boosted to a higher energy level. The energy of an absorbed photon is converted to the potential energy of the electron that has been raised to an excited state. In most pigments, the excited electron drops back to its ground-state, or normal orbit, and releases the excess energy as heat. Some pigments, including chlorophyll, emit light as well as heat after absorbing photons.
In the chloroplast, these excited electrons jump from the chlorophyll molecule to a protein molecule in the thylakoid membrane, and are replaced by electrons from the splitting of water. The energy thus transferred, is used in carbohydrate production.
This release of light is called fluorescence. Chlorophyll will fluoresce in the red part of the spectrum, and also give off heat. In this lab, you will observe this fluorescence by separating the chlorophyll from the thylakoid membrane.

MATERIALS

Spinach leaves	Flashlight or small lab light
Mortar and pestle	Test tube
Acetone	Filter paper
25-mL graduated cylinder	Funnel
Ring stand or funnel rack	Safety goggles

PROCEDURE

1. Grind the spinach leaves using a mortar and pestle.

2. Add acetone to the ground leaves, using enough acetone and spinach leaves to get between 10 and 15 mL of extract.

3. Set up your filtering apparatus, and using proper filtering technique, filter the extract to a test tube. NOTE: Use a small amount of acetone to wet the filter paper, to hold it into place, instead of water.

4. Shine a flashlight, or other similar light source, through the test tube and extract.

5. Observe the fluorescence of the chlorophyll at a 90 degree angle to the flashlight.

Chromatography Lab

Chromatography Lab

Problem: How do you separate the different pigments in a plant?

Materials:

Cone-type (size 4) coffee filter paper (or Whatman #1 chromatography paper)
large glass jars
acetone
distilled water
capillary tubes
fresh spinach
mortar and pestle
clean sand

Introduction:

In this activity you will be experimenting with a technique called chromatography which will allow you to visually demonstrate that the pigment in leaves is a combination of several different colored pigments.

This technique is useful in that it can separate and identify the various components of mixtures, such as those contained in plant pigments. A pigment is a substance that absorbs light at specific wavelengths, chlorophyll is one of these pigments. Its green-yellow in color is due to the absorption of red, orange, blue, and violet wavelengths and the reflection of the green and yellow wavelengths. This occurs when white light (containing all of the light wavelengths, or the entire spectrum of colors) shines on the leaf surface, all of the wavelengths are absorbed except for the ones you see, which are green-yellow, those are the portions of the spectrum being reflected.

If the conditions are identical, the relative distance moved by a particular compound is the same from one mixture to another. This is why chromatography can be used to identify a compound. The actual identification requires a simple calculation as shown below:

Rf = distance moved by compound from original spot divided by the distance moved by solvent from original spot

It is important to remember that several factors can influence the reliability of the Rf value, these include humidity, temperature, solvent, pigment extract preparation, and the amounts of the material present. Values are comparable only when the extracts are prepared in the same way and the chromatograms are prepared identically and developed together in the same container.

Acetone is flammable (even the amount found in nail polish remover), keep it away from sparks or open flames. Wear eye protection, especially if using pure acetone.

Procedure

1. Each lab group (or individual if not working in groups) will need 4 strips of filter paper, approximately 6 inches long and 1 inch wide, 2 chromatography development containers (500 ml beakers or large fruit jars work well), 2 large rubber bands (able to stretch around the vessels from the mouth to the bottom of the vessel), 2 solvents, water and either pure acetone, or nail polish remover.

2. Do the following with both fresh spinach leaves; tear leaf material and place in a glass container, cover with acetone (this should be done the day before the actual lab activity). An alternative pigment extraction technique is to use a
mortar and pestle. Place plant material the vessel, add a little clean sand, some acetone and then grind until a dark green liquid appears. Both techniques yield very dark pigments with which to work. Be certain to keep the pigments apart throughout the entire activity.

3. Place one of each solvents (water and acetone, or nail polish remover) in the chromatography vessels and stretch a rubber band length-wise around each vessel. The rubber band will be the mechanism for hanging the chromatography strips.

4. Make a pencil mark on each of the 2 chromatography strips, in the center, directly above the point of the strip, about 1 inch from the tip of the paper. Using a capillary tube, or tooth pick, apply the plant pigment to each filter paper strip. This is done by touching the tooth pick or capillary tube which has been dipped in the pigment, to the pencil mark. Make an application, then wave the paper gently to dry it a little before the next application. Be patient, you will need 12 to 15 applications.

5. By now you should have 2 strips with spinach pigment. Suspend one of each in each of the chromatography development vessels. You can attach them with paper clips, or simply fold over a portion of the end and it should hang in place. The tip of each strip should just touch the solvent.

6. Wait 20 to 30 minutes for the chromatograms to develop. Remove the chromatograms. Mark with a pencil (NOT a pen) where the solvent stopped as it moved up the chromatogram. This is called the solvent front. Mark also where each pigment stopped moving up the chromatogram. Using the equation below, determine a reference number for each pigment on the chromatograms. Depending on which chromatogram you are viewing, you should see greens, yellow/yellow orange, and red. All measurements should be in mm. (Any material which did not move from the
pencil dot is insoluble).

Rf = distance moved by compound from original spot divided by the
distance moved by solvent from original spot

Note: each pigment has a special name,
green = chlorophyll a or b
yellow/yellow orange = carotene
red = anthocyanin
brown = xanthophyll

The reference numbers for the chlorophylls in this activity are:
0.28 = chlorophyll a, 0.18 = chlorophyll b (spinach). You need these
numbers so that you can determine one chlorophyll from the other.
Calculate reference fronts for all of your pigments.

See if your calculations come close to those above for chlorophyll a and b.

Note: You can use different solvents such as mixtures involving petroleum ether
to do this sort of paper chromatography.

To view notes and a graphic showing a separation of plant pigments involving
paper chromatography, click here. Can you calculate the Rf values for the
pigments separated in this graphic?

Conclusion Questions

1. What reference numbers (Rf) did you calculate for chlorophyll a and chlorophyll b?
2. With what you have discovered about pigments, what conclusions can you
make regarding the changing color of leaves in autumn?
3. What adaptive purpose do different colored pigments serve for a plant?
4. Why do some pigments move farther up the chromatogram than others?
5. What are some possible sources of error in this lab?

Paper chromatography is a technique used to separate a mixture into its component molecules. The molecules migrate, or move up the paper, at different rates because of differences in solubility, molecular mass, and hydrogen bonding with the paper.

For a simple, beautiful example of this technique, draw a large circle in the center of a piece of filter paper with a black water-soluble, felt-tip pen. Fold the paper into a cone and place the tip in a container of water. In just a few minutes you will have tie-dyed filter paper!

Separation of black ink pigments

The green, blue, red, and lavender colors that came from the black ink should help you to understand that what appears to be a single color may in fact be a material composed of many different pigments —and such is the case with chloroplasts.

chromatography setup

In paper chromatography the pigments are dissolved in a solvent that carries them up the paper. In the ink example, the solvent is water. To separate the pigments of the chloroplasts, you must use an organic solvent

pigment separation

Analysis of Results I

If you did a number of chromatographic separations, each for a different length of time, the pigments would migrate a different distance on each run. However, the migration of each pigment relative to the migration of the solvent would not change. This migration of pigment relative to migration of solvent is expressed as a constant, Rf (Reference front). It can be calculated by using the formula:

Chromatography of Plant Pigments 3

Chromatography of Plant Pigments

Introduction:

Can chromatography be used to separate mixtures of chemical substances? The purpose of this experiment is to answer this question. In paper chromatography, a liquid sample flows down a vertical strip of absorbent paper, on which the components of a mixture are deposited in specific directions and locations. Chromatography is a tool used to examine and separate mixtures of chemical substances. Chromatography is essential to the separation of pure substances from complex mixtures. Separation results in a chromatographically pure substance. Chromatography allows you to determine the properties of chemical substances.

The relationship between the chromatography paper, mixture, and the solvent is very important in all chromatographic separations. The solvent has to dissolve the mixture that should be separated. The paper must also absorb the components of the mixtures selectively and reversibly. The substances making up the mixture must be evenly dispersed in the water. Chromatography is a simple and inexpensive tool for separating and identifying chemical mixtures if all these things are done.

Hypothesis:

Paper can be used to separate mixed chemicals.

Materials:

The materials used in this lab are filter paper, test tube, rubber stopper, paper clip, metric ruler, black felt-tip pen, pencil, calculator, and water.

Methods:

First, bend a paper clip so that it’s straight with a hook at one end. Push the straight end of the paper clip into the bottom of a cork stopper. Then, hang a thin strip of filter paper on the hooked end of the paper clip and insert the paper strip into the test tube. The paper should not touch the sides and should almost touch the bottom of the test tube. Next, remove the paper strip from the test tube. Now draw a solid 5-mm-wide band about 25 mm from the bottom of the paper, using a black felt tip pen. After this, use a pencil to draw a line across the paper strip 10 cm above the black band. Then, put the filter paper back into the test tube with the bottom of the paper in the water and the black band above the water. Observe what happens as the liquid travels up the paper and record the changes you see. When the solvent has reached the pencil line, remove the paper from the test tube. Let the paper dry on the desk. Finally, with a metric ruler, measure the distances from the starting point to the top edge of each color. Record the data in a data table and calculate a ratio for each color by dividing the distance, the color traveled by the distance the solvent traveled.

Results:

The results of the chromatography experiment are shown in a chart and a graph.

Color of ink (list in order)	Distance traveled by each color (mm)	Distance solvent traveled (mm)	Ratio traveled = distance color moved divided by distance solvent moved
Yellow	70	108	0.65
Orange	85	108	0.79
Pink	95	108	0.88
Violet	102	108	0.94
Blue	108	108	1.00

Questions:

1. How many colors separated from the black ink? Five colors separated from the ink: yellow, orange, pink, violet, and blue.

2. What served as the solvent for the ink? Water served as the solvent for the ink.

3. As the solvent traveled up the paper, which color of ink appeared first? Dark blue appeared first.

4. List the colors in order from top to bottom that separated from the black ink? The colors separated in the order of: blue, violet, pink, orange, and yellow.

5. In millimeters, how far did the solvent travel? The solvent traveled 108 mm.

6. From your results, what can you conclude is true about black ink? Black ink is a mixture of several different colors.

7. Why did the inks separate? The inks separated because black ink is a mixture of different pigments that are soluble in water, have different molecular characteristics, and travel different distances.

8. Why did some inks move a greater distance? Some inks move a greater distance because molecules in ink have different characteristics, like how readily they are absorbed by paper. This means that the ink least readily absorbed by paper will travel farthest from the starting mark and the ink most readily absorbed by paper will be the closest to the starting mark. All of the different color inks that were separated were different in how readily they are absorbed by paper.

Error Analysis:

There are a few errors that could have changed the results. First, there could be inaccurate measurements of how far every color traveled or how far the water traveled up the filter paper. Another error could occur when calculating the ratio traveled, Rf value. Also, a longer test tube could have been used by different groups which would make the filter strip longer. This means that a group could have detected another color because they had more room on their filter paper. This also could have affected the ratios. Finally, the groups could have put different amounts of black ink on the filter paper.

Conclusion:

The hypothesis that paper can be used to separate mixed chemicals was correct. The different colored inks mixed together give the black its color. The five colors that separated from the black ink were blue, violet, pink, orange, and yellow. Blue appeared first and then was followed by violet, pink, orange, and yellow. The colors separated the way they did because they have different molecular characteristics, like how readily they were absorbed by the paper and their solubility in water. Blue was most readily absorbed by the paper and soluble by water, while yellow was the least.

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Chromatography of Simulated Plant Pigments

Chromatography of Simulated Plant Pigments

Introduction
This experiment is conducted to investigate the components Plant Pigments separating visibly. There are a couple of different types of components in plant pigments, and they became clearly visible during this lab. The most important and abundant chemical pigment found in plants is chlorophyll. This pigment exists in two forms; chlorophyll a and chlorophyll b. Chlorophyll absorbs two main colors from light quite well. These are blue, and red. The chlorophyll reflects green light very well, however, the two different types of chlorophyll have their maximum absorption at different wavelengths of light. Chlorophyll a, being the main photosynthetic pigment, has a primary purpose to convert light energy to chemical energy used by the plant itself. Chlorophyll b absorbs light in a region of the spectrum apart from the dominant chlorophyll, and transfers the energy it produces to chlorophyll a. Along with chlorophyll b in transferring their energy produced to the dominant chlorophyll, two other pigments that are found in plants are carotenes and xanthophylls, which are orange and yellow respectively. Since chlorophyll is such a dominant pigment in green plants, this domination hides the color of the carotenes and xanthophylls in the leaves. This causes most plant leaves to appear green most of the time. During the autumn, however, the chlorophyll starts to break down, causing the carotenes and xanthophylls to show their bright red, orange and yellow colors.
These brilliant colors can be separated another way. This different technique, known as paper chromatography, separates mixtures in a liquid into individual components. The technique is based on the fact that each substance in a mixture has a specific affinity for a solid surface and a specific solubility in different solvents. By this method, the solid surface is the cellulose fibers in the chromatography paper, and the solvent is the solution that was placed in the bottom of the developing chamber.
This separation takes place through a process of absorption and capillary action. Just a small drop of the mixture, in this case plant pigment to be separated, is placed at the bottom of the strip of chromatography paper. The chromatography paper is then placed in the developing chamber with a solvent, which wicks up the paper, pulling the solvent up the paper by capillary action, and the mixture of pigments is dissolved as the solvent passes over it. The different components of the mixture move upward at different rates. A compound with greater solubility will travel farther than one with less solubility. The pigments then show up as color streaks on the chromatography paper. These substances have formed a pattern called a chromatogram on the chromatography paper.
The Rf values for each pigment is calculated to establish the relative rate of migration for each pigment. This value represents the ratio of the distance a pigment traveled on the chromatogram relative to the distance the solvent front moved. Scientists use the Rf value of a sample to identify the molecule. Any molecule in a given solvent matrix system has a uniquely consistent Rf value. The formula for this value is as follows:

Rf = Distance each pigment traveled ¸ Distance solvent front traveled

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.

Materials
For this experiment the following items are used — one chromatography reaction chamber, one paper chromatography strip, one capillary pipette, a pencil and paper, calculator, ruler, 50 ml beaker, colored pencils, approximately 10 ml of solvent depending on the size of the reaction chamber, scissors, and simulated plant pigment.

Procedure
Use scissors to cut the bottom of the chromatography paper to a tapered end. Measure the strip and cut the length to equal slightly longer than the reaction chamber. Draw a faint pencil line at the bottom of the tapered end and use a capillary pipette to add some simulated plant pigment to this line. Add 5-10 ml of solvent to the reaction chamber. Extend the chromatography strip through the slit in the lids of the reaction chamber and carefully lower the strip into the chamber so the tapered end is in the solvent and the pencil line is above the solvent level. Make sure the strip does not touch the walls of the chamber and do not bump the chamber as the pigments begin to separate. After the pigments have completely separated and the solvent front has reached the top of the chamber, remove the strip and mark the solvent front with a pencil line before it evaporates. Measure and record the distance the solvent and each pigment traveled. Use a calculator to determine the Rf values for each pigment.

Data

Table 1
Band #	Pigment	Color	Migration distance (mm)	Rf value
1	Carotene	Orange	59mm	.94
2	Xanthophyll	Yellow	56mm	.89
3	Chlorophyll a	Light green	29mm	.46
4	Chlorophyll b	Dark green	14mm	.22
Solvent			63mm

Questions
1. Describe what happened to the original spot of simulated plant pigments? The solvent separated the original spot by wicking up the solvent while dissolving the various pigments in the spot.
2. List some other uses of chromatography? Chromatography can be used to separate various mixtures of subtances, liquids and gases.
3. Which of the 4 pigments migrated the furthest and why? carotene ( orange) because it was the most soluble in the solvent
4. Which type of chlorophyll was the most soluble? chlorophyll a
5. Explain why leaves change color in the fall? In Autumn, chlorophyll starts to break down which allows the other brilliant plant pigment colors to show. These pigments include the red, orange, and yellow colors.
6. What is the function of plant pigments in photosynthesis? Plant pigments trap light energy and convert it into chemical energy that can be used by the plant to make glucose or sugar.

Error Analysis
The chromatography paper touched the sides of the chamber during the waiting time which caused the migration to go slightly to the side instead of straight to the top. Also the strip was bent at the top so there could have been a slight error in measuring the migration of the solvent front.

Conclusion
Paper chromatography proved to be an accurate method of separating and observing the various colors of plant pigments. The pigments dissolved in the solvent and migrated upward. The colors were observed and their migration distances measured & recorded. 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 spot — carotene, xanthophyll, chlorophyll a, and chlorophyll b. Carotene was the most soluble, while chlorophyll b was the least soluble.

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Chordates

Characteristics of Chordates

All chordates have a notochord, dorsal nerve cord, pharyngeal pouches, & postanal tail at some time in their life
Notochord is a firm, flexible rod of tissue located on the dorsal side of the body that becomes part of the endoskeleton in vertebrates
Dorsal nerve cord is a hollow tube lying dorsal to the notochord that becomes the brain & spinal cord in vertebrates
Pharyngeal pouches are small outpockets of the anterior part of the digestive tract that become gills in aquatic chordates & jaws, inner ear, & tonsils in terrestrial chordates
Postanal tail consists of muscle tissue & lies behind the posterior opening of the digestive tract

Subphyla of Chordates

The Phylum Chordata includes all of the vertebrates, as well as two groups of marine animals that lack backbones and are called invertebrate chordates
The phylum is divided into three subphyla, determined by the development of the notochord
Subphylum Cephalochordata contains about 24 species of blade-shaped animals known as lancelates that retain the notochord, dorsal nerve chord, pharyngeal pouches, and postanal tail throughout their life
Subphylum Urochordata contains 2,000 species commonly called tunicates because their bodies are covered by a tough covering, or tunic
* Called sea squirts because they shoot out a stream of water when touched
*Sessile, barrel-shaped, filter feeding animals that live on the sea bottom
*Adults have a pouch-like pharynx with slits
*Adults do not have a notochord, dorsal nerve cord, or postanal tail
Subphylum Vertebrata is the largest subphylum in which the notochord is replaced with vertebrae
* Skeletons consist of bone &/or cartilage
* Brain is protected by a cranium
* Well developed 4 chambered heart with a closed circulatory system
* Includes fish, amphibians, reptiles, birds, & mammals

Fish, Amphibians, Reptiles, Birds, and Mammals
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Table 1