Category: Curriculum Map

Chromatography Plant Pigments

 

Chromatography of Plant Pigments

INTRODUCTION:

Chlorophyll often hides the other pigments present in leaves. In Autumn, chlorophyll breaks down, allowing xanthophyll and carotene, and newly made anthocyanin, to show their colors.
The mix of pigments in a leaf may be separated into bands of color by the technique of paper chromatography. Chromatography involves the separation of mixtures into individual components. Chromatography means “color writing.” With this technique the components of a mixture in a liquid medium are separated. The separation takes place by absorption and capillarity. The paper holds the substances by absorption; capillarity pulls the substances up the paper at different rates. Pigments are separated on the paper and show up as colored streaks. The pattern of separated components on the paper is called a chromatogram.

PRELAB PREPARATION:

Gather leaves from several different plants. CAUTION: Avoid poisonous plants. Autumn leaves from deciduous trees are especially interesting. Sort the leaves by kind (maple, etc.) and color. Review a diagram of a plant cell . Find the grana and the chloroplasts of the cell.

MATERIALS:

Safety goggles
Chromatography solvent (92 parts Petroleum ether to 8 parts acetone)
Chromatography paper (or filter paper) about 1 cm x 15 cm
Ethyl alcohol
Fresh spinach
Test tube
Test tube rack
Scissors and Ruler
Fresh leaves of plants
Glass stirring rod
Paper clip
Cork (to fit test tube)
Mortar and pestle
Sand (optional)
10-ml Graduated cylinder

PROCEDURE:

Leaves should be grouped by kind (maple, etc.) and color. Work with a spinach leaf and with one or more other types. CAUTION: Chromatography solvents are flammable and toxic. Have no open flames; maintain good ventilation; avoid inhaling fumes.

1. Cut a strip of filter paper or chromatography paper so that it just fits inside a 15-cm (or larger) test tube. Cut a point at one end. Draw a faint pencil line as shown in figure 1. Bend a paper clip and attach it to a cork stopper. Attach the paper strip so that it hangs inside the tube, as shown. The sides of the strip should not touch the glass.

2. Tear a spinach leaf into pieces about the size of a postage stamp. Put them into a mortar along with a pinch or two of sand to help with grinding. Add about 5 ml ethyl alcohol to the leaf pieces. Crush leaves with the pestle, using a circular motion, until the mixture is finely ground. The liquid in which the leaf pigments are now for paper chromatography dissolved is called the pigment extract.

3. Use a glass rod to touch a drop of the pigment extract to the center of the pencil line on the paper strip. Let it dry. Repeat as many as 20 times, to build up the pigment spot. NOTE: You must let the dot dry after each drop is added. The drying keeps the pigment dot from spreading out too much.

4. Pour 5 ml chromatography solvent into the test tube. Fit the paper and cork assembly inside. Adjust it so that the paper point just touches the solvent (but not the sides of the tube). The pigment dot must be above the level of the solvent. Watch the solvent rise up the paper, carrying and separating the pigments as it goes. At the instant the solvent reaches the top, remove the paper and let it dry. Observe the bands of pigment. The order, from the top, should be carotenes (orange), xanthophylls (yellow), chlorophyll a (yellow-green), chlorophyll b (blue-green), and anthocyanin (red). Identify and label the pigment bands on the dry strip. Write the species of leaf on the strip as well.
Record the species, external color, and chromatogram pigments in the DATA TABLE of your report sheet.

5. Each pigment has an Rf value, the speed at which it moves over the paper compared with the speed of the solvent.

Rf = Distance moved by the pigment / Distance moved by the solvent

Measure the distance in cm from the starting point (pencil line) to the center of each pigment band. Then measure the entire distance traveled by the solvent. Remember, the starting point for the solvent is also the pencil line and the ending point for the solvent is the top edge of the paper. Do the required divisions and record your Rf values in the DATA TABLE of your report sheet.

6. Wash the mortar and pestle thoroughly, using a little alcohol to remove any remaining pigment.

7. Repeat steps 1 through 6 for each species.

DATA TABLE:

Chromatography Data
Leaf Type (species) External color Chromatogram Pigments
Colors from the Top Pigment Names Rf Values

 

DNA Quiz 2

Name: 

Mendel’s Genetics

 

 

True/False
Indicate whether the sentence or statement is true or false.
1.
The law of segregation states that two or more pairs of alleles separate independently of one
another during gamete formation.
2.
Cells that contain a single set of chromosomes are said to be haploid (N).
3.
Crosses involving a study of one gene are called monohybrid crosses.
4.
A dominant allele masks the effect of a recessive allele.
5.
Mendel concluded that the patterns of inheritance are determined entirely by the environment.
6.
A Punnett square represents the phenotype of an organism.
7.
The physical appearance of an individual organism, as determined by the genes it has inherited from its parents, is called its genotype.
8.
Individuals must exhibit a trait in order for it to appear in their offspring.
9.
In codominance, two alleles are expressed at the same time.
 

Multiple Choice
Identify the letter of the choice that best completes the statement or answers the question.
10.
In fruit flies, the gene for long wings, L, is dominant to the gene for short wings, l.  A heterozygous long wing male and a short wing female produce many offspring. The possible genotype(s) among the long-winged offspring is (are)
a.
Ll only
c.
LL and Ll
b.
ll only
d.
Ll and ll.
11.
In drosophila, curled wing is recessive to straight wing. If a homozygous straight-winged fly is mated with a curled-wing fly, how many different phenotypes will be produced?
a.
1
c.
3
b.
2
d.
4
12.
If an organism has two identical alleles for a trait, it is
a.
homozygous.
c.
homozygous dominant.
b.
heterozygous.
d.
heterozygous recessive.
13.
Alleles for the same trait separate during
a.
fertilization.
c.
meiosis I.
b.
mitosis.
d.
meiosis II.
14.
The inheritance of genes that determine one trait (hair color) is not affected by the inheritance of genes that control another trait (tongue rolling).  Which of Mendel’s rules apply to the above statement?
a.
the rule of dominance
c.
the rule of independent assortment
b.
the rule of segregation
15.
In fruit flies, the gene for straight wings, C, is dominant to the gene for curly wings, c. Two flies, when bred, produced 98 straight-winged and 102 curly-winged offspring.  What was the genotype of the curly-winged offspring?
a.
CC
c.
cc
b.
Cc
d.
straight-winged
16.
All homozygous individuals have:
a.
the same genotype
c.
two alleles exactly alike
b.
the same phenotype
d.
a hybrid genotype.
17.
If a family has three daughters, the probability that the next child will be a girl is
a.
1/4.
c.
1/2.
b.
1/3.
d.
3/4.
18.
Mendel explained the reappearance of recessive traits in the F2 generation in his principle of
a.
independent assortment.
c.
dominance.
b.
segregation.
d.
blending inheritance.
19.
If any offspring from a test cross show a recessive phenotype, the parent with the unknown genotype is
a.
heterozygous dominant.
c.
heterozygous recessive.
b.
homozygous dominant.
d.
homozygous recessive.
20.
A homozygous black rabbit is mated with a heterozygous rabbit. If black is dominant over white, they should produce:
a.
all white rabbits
b.
all black rabbits
c.
half black and half white rabbits
d.
one pure dominant and heterozygous individual.
21.
Mendel’s finding that the inheritance of one trait had no effect on the inheritance of another became known as the
a.
law of dominance.
c.
law of segregation.
b.
law of universal inheritance.
d.
law of independent assortment.
22.
The phenotype of an organism
a.
represents its genetic composition.
b.
reflects all the traits that are actually expressed.
c.
occurs only in dominant pure organisms.
d.
cannot be seen.
23.
In humans the ability to taste PTC paper is dominant to non-tasting and hair color shows incomplete dominance (Dark hair x blond hair => brown hair).  A brown haired man who cannot taste PTC paper marries a woman with brown hair and who can taste PTC paper.  Their first child had brown hair and could not taste PTC paper. What are the chances that their next child will be a brown taster?
a.
1/4
c.
1/8
b.
1/2
d.
3/8
24.
Two long-furred cats were mated and produced 25 percent short-furred cats.  The parents were probably:
a.
pure recessive individuals
b.
pure dominant individuals
c.
heterozygous individuals
d.
one pure dominant and heterozygous individual.
25.
When certain types of black roosters are crossed with white hens, speckled chickens result. These chickens, which have a mixture of black and white feathers, show
a.
dominance.
c.
polygenes.
b.
codominance.
d.
recessive
26.
codominance : both traits are displayed::
a.
probability : crosses
c.
homozygous : alleles are same
b.
heterozygous : alleles are the same
d.
Punnett square : chromosomes combine
27.
A Punnett square is used to determine the
a.
probable outcome of a cross.
c.
result of segregation.
b.
actual outcome of a cross.
d.
result of meiosis I.
28.
If a family has four sons, the probability that the next child will be a boy is
a.
1/2.
c.
1/5.
b.
1/4.
d.
4/5.
29.
Suppose that on Mars green creatures are dominant over red creatures and that 3-eyes are recessive to 4-eyes.  Assume that inheritance of traits on Mars occurs the same way as on Earth.  A cross between 2 GgEe Martians would result in what fraction of the offspring being red-3-eyed Martians?
a.
1/16
c.
4/16
b.
2/16
d.
9/16
30.
The fact that a man and woman, both of whom have wavy hair, could have children with curly hair, wavy hair, or straight hair is best explained by the phenomenon called
a.
codominance.
b.
dominance.
c.
incomplete dominance.
d.
None of the above; this would be impossible.
31.
A Punnett square does not show the
a.
genetic makeup of the eggs.
c.
genetic makeup of the sperm.
b.
probable outcome of a cross.
d.
actual outcome of a cross.
32.
The “father” of genetics was
a.
T. A. Knight.
c.
Gregor Mendel.
b.
Dr. Judd.
d.
None of the above
33.
What is the probability that the offspring of a homozygous dominant individual and a  homozygous recessive individual will exhibit the dominant phenotype?
a.
0.25
c.
0.66
b.
0.5
d.
1.0
34.
Which of the following is the designation for Mendel’s original pure strains of plants?
a.
P
c.
F1
b.
P1
d.
F2
35.
F2 : F1 ::
a.
P : F1
c.
F1 : P
b.
F1 : F2
d.
dominant trait : recessive trait
36.
The passing of traits from parents to offspring is called
a.
genetics.
c.
development.
b.
heredity.
d.
maturation.
37.
homozygous : heterozygous ::
a.
heterozygous : Bb
c.
dominant : recessive
b.
probability : predicting chances
d.
homozygous : BB
38.
The phenotype of an organism
a.
represents its genetic composition.
b.
reflects all the traits that are actually expressed.
c.
occurs only in dominant pure organisms.
d.
cannot be seen.
39.
If an individual has two recessive alleles for the same trait, the individual is said to be
a.
homozygous for the trait.
c.
heterozygous for the trait.
b.
haploid for the trait.
d.
mutated.
40.
Tallness (T) is dominant to shortness (t) in pea plants. Which of the following represents a genotype of a pea plant that is heterozygous for tallness?
a.
T
c.
Tt
b.
TT
d.
tt
41.
How many different phenotypes can be produced by a pair of codominant alleles?
a.
1
c.
3
b.
2
d.
4
chp_9_web_tutorial_files/i0440000.jpg
42.
Refer to the illustration above. The genotype represented by the cell labeled “2” is
a.
GgIi.
c.
GI.
b.
GGIi.
d.
Gi.
In rabbits, black fur (B) is dominant to brown fur (b). Consider the following cross between two rabbits.
      chp_9_web_tutorial_files/i0460000.jpg
43.
Refer to the illustration above. Both of the parents in the cross are
a.
black.
c.
homozygous dominant.
b.
brown.
d.
homozygous recessive.
44.
Refer to the illustration above. The genotypic ratio of the F1 generation would be
a.
1:1.
c.
1:3.
b.
3:1.
d.
1:2:1.
45.
In pea plants, yellow seeds are dominant over green seeds. What would be the expected genotype ratio in a cross between a plant with green seeds and a plant that is heterozygous for seed color?
a.
1:3
c.
4:1
b.
1:2:1
d.
1:1

 
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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

 

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:

 

Chordates

Chordates
All Materials © Cmassengale  

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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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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