Download Chromatography lab - Castle High School

Plant Pigments
Overview
In this laboratory you will separate plant pigments using chromatography. You will also
measure the rate of photosynthesis in isolated chloroplasts. The measurement technique involves
the reduction of the dye, DPIP. The transfer of electrons during the light-dependent reactions of
photosynthesis reduces DPIP and changes its color from blue to colorless.
Objectives
At the completion of this laboratory you should be able to:
* Understand the principles of chromatography.
* Calculate Rf values.
* Design an experiment in which chromatography is used as a separation technique.
* Describe how light intensity, light wavelength, and temperature can affect photosynthesis.
* Design an experiment to measure how light intensity, light wavelength, and temperature can
affect photosynthetic rates.
Plant Pigments
Paper chromatography is a useful technique for separating and identifying pigments and other
molecules from cell extracts that contain a complex mixture of molecules. The solvent moves up
the paper by capillary action, which occurs as a result of the attraction of solvent molecules to the
paper and the attraction of solvent molecules to each other. As the solvent moves up the paper, it
carries along any substances dissolved in it, in this case plant pigments. The pigments are carried
along at different rates because they are not equally soluble in the solvent and because they are
attracted to different degrees, to the cellulose in the paper through the formation of hydrogen
bonds. Examination of the molecular structure of the pigments may reveal chemical functional
groups that help determine the pigment's solubility in the solvent.
Four pigments are typically found in leaf extracts from plants. They are: Chlorophyll a
(bluish green), Chlorophyll b, (yellow to olive green), carotene (orange-yellow), and xanthophyll
(lemon yellow). The molecular structures of each pigment are shown on the next page. Note
that the difference between chlorophylls is a slight difference in the functional group on the ring.
Carotene and xanthophyll are also similar chemically, differing only slightly in their functional
groups.
Chromatography of Plant Pigments
Procedure
Students will work in groups of two in this section. Each group should do the following:
1. Each student should obtain a 6-10 cm strip of chromatography paper. Using a pencil (not
pen), draw a baseline approximately 1.5 cm from the bottom of the paper. Try to touch the
paper as little as possible because skin oils can interfere with the chromatogram development.
2. Using a fine-tip paint brush or capillary pipette, streak the plant extract (prepared by the
instructor) along the baseline.
3. Repeat the application of the pigment several times until a dark streak is present. Allow the
paper to dry between applications and try to keep the streak as narrow as possible.
4. Each group will be given a chromatography development jar. Keep the jar sealed until ready
to use. (Caution - avoid inhaling the solvent fumes from the jar). The solvent used for this
experiment is a mixture of petroleum ether, a highly lipophilic solvent and acetone.
5. Hang the chromatograph paper from the hook in the jar's cork. The chromatograph paper
should hang so that the bottom touches the solvent in the bottom of the jar, but the pigment
strip must be above the solvent. Hold the strip next to the jar and determine if the length is
satisfactory before opening the jar. Trim the top of the paper if necessary to shorten the
paper's length. Each jar will have two hooks to hold the group's papers.
6. When the solvent is about 1 cm from the top margin of the paper (several minutes), remove
the paper and immediately mark the location of the solvent front in pencil before it
evaporates. Your instructor will help you identify the spots. Label your pigment spots (1-4)
from top to bottom. Reseal the jar after removing the paper.
Analysis of Results
Calculate the Rf (Rate of flow) value for each of the four pigments using the formula:
Rf = distance pigment migrated
distance solvent front migrated
Note - because Rf is a relative value, there are no units.
Report your data by completing Table 1. Use the colors, Rf values and molecular structure to
match each spot with a pigment name.
Table 1.
Spot Number
Color
Rf Value
Pigment Name
1
2
3
4
Answer the following questions on a separate sheet of paper:
1. What factors are involved in the separation of the pigments? Does molecular weight appear to
have a role in pigment separation?
2. What functional group differences separate the almost identical pigments of Chlorophyll a
and Chlorophyll b?
3. Is it possible to have an Rf value greater than 1.00? Explain.
4. Would you expect the Rf value of a pigment to be the same if a different solvent were used?
Explain.
Photosynthesis
Objectives
At the completion of this laboratory you should be able to:
* Describe how light intensity, light wavelength, and temperature can affect photosynthesis.
* Design an experiment to measure how light intensity, light wavelength, and temperature can
affect photosynthetic rates.
Light is a part of a continuum of radiation or energy waves. Shorter wavelengths have greater
amounts of energy (e.g. high-energy ultraviolet rays can harm living cells). Wavelengths of light
within the visible part of the radiation spectrum are used in photosynthesis. Visible light has
enough energy to be useful but not enough energy to break chemical bonds.
The photosystems found in the chloroplasts of leaf cells contain pigments that absorb light.
These chloroplasts have two different kinds of pigment systems, Photosystem I (PS I) and
Photosystem II (PS II). PS I contains a specialized type of chlorophyll a molecule called P700
(its absorption spectrum peaks at 700 nm), and PS II contains a specialized chlorophyll a
molecule called P680 (its absorption spectrum peaks at 680 nm).
When light is absorbed by leaf pigments, electrons within each photosystem are boosted to a
higher energy level and the energy is captured in the chemical bonds of ATP and NADPH.
These high-energy products are then used to incorporate CO2 into organic molecules.
In this experiment, a dyereduction technique will be used.
The dye-reduction experiment tests
the hypothesis that light and
chloroplasts are required for the light
reactions to occur. In place of the
electron-accepting compound
NADP, a dye DPIP (2,6dichlorophenol-indophenol), will be
substituted. If the dye accepts
electrons from the chloroplasts, it
will become colorless.
In this experiment, chloroplasts
are extracted from spinach leaves
and incubated with DPIP in the
presence of light. As DPIP is
reduced and becomes colorless, the resultant increase in light transmittance can be measured over
time using a colorimeter.
The experiment requires:
1) a colorimeter blank containing everything but the DPIP
2) an experimental test with chloroplasts incubated in the light (positive control)
3) an experimental test using a variable chosen by the group
4) an experimental test using no chloroplasts incubated in the light (negative control)
The experimental design matrix is presented in Table 2.
Table 2.
Cuvette 1
Cuvette 2
Cuvette 3
Cuvette 4
Blank
Light
Phosphate Buffer
1 ml
1 ml
Distilled Water
2.5 ml
1.5 ml
DPIP
_____
1 ml
1 ml
Normal
Chloroplasts
5 drops
5 drops
_____
variable
No Chloroplasts
1 ml
1.5 ml + 5 drops
Procedure
Students should work in groups of 3 individuals. Read the directions carefully before starting
the experiment in order to avoid mistakes. The steps of the experiment are as follows:
1. Set up the colorimeter with a laptop computer. Set the absorption to 635nm.
2. Your instructor will prepare a chloroplast suspension from spinach leaves.
3. Set up an incubation area using the gel light boxes as the light source.
4. Your instructor will provide the class with the chloroplast suspensions. The suspensions
should stay on ice at all times. The chloroplasts do tend to settle over time so make sure to
pipet from the bottom or gently stir before adding to cuvettes.
5. At the top rim of the cuvettes, place labels numbered 1 - 4 respectively. Remember to handle
the cuvettes only near the top (try to touch only the corners) and use lens tissue to clean the
outside walls of each cuvette.
6. Add the phosphate buffer, water and DPIP to each cuvette as indicated in Table 2. Gently
mix the regular chloroplast suspension and transfer five drops to cuvette 1.
7. Calibrate the colorimeter with cuvette 1. This may need to be repeated intermittently
throughout the experiment as well.
8. Transfer five drops of the regular chloroplast suspension to cuvette 2. Mix immediately and
insert the cuvette into the sample holder. Read the % transmittance and record. Remove
cuvette 2 from colorimeter and place in the light. Take and record additional readings at 5, 10,
and 15 minutes. You are taking point readings NOT collecting data over time! Mix the cuvette
prior to taking each reading. Remember to use cuvette 1 occasionally to check and adjust the
colorimeter to 100% transmittance.
9. Transfer chloroplast suspension to cuvette 3 following the procedure written by the team for
the chosen variable. Mix immediately and insert the cuvette into the sample holder. Read the
% transmittance and record. Take and record additional readings at 5, 10, and 15 minutes. Mix
the cuvette prior to taking each reading.
10. Mix the contents of cuvette 4. Insert it into the sample holder and read the % Transmittance.
Place Cuvette 4 in the light. Take and record readings at 5, 10, and 15 minutes. Mix the
cuvette prior to taking each reading.
Comment - With adequate planning and team cooperation, it is possible to stagger the readings
so that treatments 2, 3, and 4 can be run simultaneously, saving lab time.
Table 3.
Time in Minutes
Treatment
0
5
10
15
Light - 2
Variable - 3
No Chloroplasts - 4
Analysis of Results
Graph - Plot the data from the three cuvettes (2-4) with the independent variable (Time) as the X
axis and the dependent variable (% Transmittance) as the Y axis. Be sure to label the axis and
give their units. Construct a key to identify each treatment (colored line, symbol type etc.).
Answer the following questions on a separate sheet of paper (or the back of the lab sheet) using
complete sentences:
1. What is the effect of the chosen variable on the reduction of DPIP? Explain.
2. What is the effect of another chosen variable (from another group) on the reduction of DPIP?
Explain.
3. What would you expect to happen if only green light had been used to incubate the
chloroplasts in the light treatment? Explain.
4. What is the purpose of the phosphate buffer in the experiment?
5. What molecule found in chloroplasts does DPIP "replace" in this experiment?