Experiment 1

Spectroscopy & Preparation of Solutions

Introduction

A spectrophotometer is an instrument for measuring the absorbance of a solution. Absorbance is a

useful quantity. Absorbance is therefore a measure of the portion of the light leaving the lamp that
actually makes it to the detector. The ability to measure absorbance at different wavelengths is
very useful, because the extinction coefficient of a compound varies with wavelength. In addition,
the absorbance spectrum of a compound can vary dramatically depending on the chemical
composition of the compound, and depending on the environment (such as the solvent) around the
compound.

Materials
Micropipette
Pipette tips
Parafilm
Water
1.0 M CuSO4 solution
UV cuvettes
Vis cuvettes
Quartz cuvettes
A CuSO4 solution of unknown concentration

Procedures
Use of Micropipette
Perform absorbance scans on the different types of cuvettes containing only water.

Simple Dilutions-1
1. Set the spectrophotometer to wavelength 700 nm and blank against air.
2. Prepare 1 mL of the following dilutions of the CuSO4 solution using water: 1:2, 1:5, 1:10, 1:50,
and 1:100.
3. Determine the A700 for each dilution.

Simple Dilutions-2
4. Assuming the CuSO4 solution is a 5x stock, prepare the following solutions: 0.5x, 1x, 2x.
5. Determine the A700 for each dilution.

1
Serial Dilutions
6. Prepare the following solutions of the CuSO4 solution using serial dilutions: 1:5, 1:25, 1:125,
and 1:625
7. Determine the A700 for each dilution.

Analysis of Results
1. Record the weight you measured for the three trials.
2. Average the three weights.
3. Calculate standard deviation for your average.
4. What is the standard deviation as a percent of the average value?
5. Which cuvettes do you need to use to measure absorbance accurately in this wavelength range?

Simple Dilutions-1
1. Give the volume of water and CuSO4 solution used for each sample dilution.
2. Plot the results on an A700 vs. Concentration of CuSO4 solution graph.

Simple Dilutions-2
1. Give the volume of water and CuSO4 solution used for each simple dilution.
2. Plot the results on an A700 vs. Concentration of CuSO4 solution graph.

Serial Dilutions
1. Describe how each serial dilution was prepared.
2. Plot the results on an A700 vs. Concentration of CuSO4 solution graph.
3. Calculate the extinction coefficient for aqueous CuSO4 solution.

Note: Many solutions used in biochemistry are prepared by diluting a more concentrated stock
solution. In preparing to make a dilution (series of dilutions), you need to consider both the desired
final concentration and required volume of the diluted material using the equation:

C1V1 = C2V2

C1 is the concentration of the initial solution

V1 is the volume of the initial solution available to be used for dilution (this may be a small
fraction of the initial solution)
C2 is the desired final concentration
V2 is the desired final volume

The Beer-Lambert law:

A = cl

A is the absorbance of the sample at a particular wavelength,

 is the extinction coefficient for the compound at that wavelength in (M•cm)-1,
c is the molar concentration of the absorbing species
l is the path length of the solution in cm.

Write the experimental report accordingly.

2
 Experiment 2

Photosynthesis: Paper Chromatography of photosynthetic pigments

Introduction

Pigments are substances that absorb light in the visible spectrum (400 -700 nm) and transfer the
light energy to electrons. There are two types of pigments: primary and accessory. Primary
pigments, i.e., chlorophylls a & b, are an integral part of Photosystems I & II and are used during
the light-dependent reactions to absorb photons of light. Accessory pigments (e.g. carotenes and
xanthophylls), on the other hand, act as magnifiers that capture and transfer light energy to
chlorophyll a.

Different pigments absorb different wavelengths of light as shown in Figure 1; chlorophylls

absorb light at wavelengths in the ranges of 425-450nm (blue) and 625-675 nm (orange-red)
whereas carotenoids have a much broader absorption peak, ranging from 400-525 nm (blue-
green). Also note that the color of a pigment originates from the wavelengths of light that are
reflected, i.e. those that are not absorbed. Chlorophylls a and b do not absorb light between 550
and 600 nm, the yellow-green portion of the spectrum, thus, organisms in which these pigments
predominate appear green in color.

Figure 1. Absorption spectrum of primary and accessory pigments

Because most photosynthetic organisms generally have more than one pigment, and many
pigments reflect similar wavelengths, it is not possible to judge which pigments a plant of
interest contains simply by noting its color. However, we can examine which pigments the plant
possesses by chemically extracting the pigments and then using paper chromatography to
separate them. Paper chromatography is based on the principle that different compounds adhere

3
to (adsorb) and move across the chromatography strip based on their molecular size, polarity and
solubility in a particular solvent. Applying a plant’s pigment extract to chromatography paper
causes the pigments to adsorb to the cellulose fibers on the strip. Subsequent immersion of the
strip in a solvent will cause the solvent to move up the strip as it becomes adsorbed by the fibers
in the paper. As the solvent moves up the strip, the pigments dissolve and move along with it.
The relative distance traveled by the solvent from the pigment origin compared to the distance
traveled by the different pigments in the extract (also measured from the same point) is used to
calculate the Rf (Retention factor) value of each pigment.

Retention factor, is calculated using the equation below, is dependent upon the compound’s
chemical properties; a small Rf value indicates a strong affinity of the compound for the
chromatography paper, i.e., the compound adsorbs strongly to the paper and will move very
slowly, while a large Rf value is indicative of a fast moving compound that demonstrates a
decreased affinity for the fibers in the strip. In addition, larger molecules tend to move slower
and adsorb more strongly to the chromatography paper than smaller ones. Under identical
conditions Rf values for each pigment should remain constant.

Rf = Distance moved by pigment

Distance moved by solvent

Objectives
 To separate photosynthetic pigments in spinach leaves
 To calculate Rf values of photosynthetic pigments

Chemicals and apparatus

 Sample (Spinach and carrot)
 Chromatography paper
 Cheese cloth
 Measuring cylinder
 Scissors
 stapler
 Blender
 Acetone
 Distilled water
 Beaker
 Glass test tube
 Gloves
 Pencil, ruler
 Rubber stopper
 Hook
 Micropipette

4
Safety NOTES:
a. The chromatography solvent is EXTREMELY dangerous and volatile.
b. All students MUST wear protective equipment including lab coats, safety goggles and gloves.
c. If any solvent is spilled, inform your lab staffs.
d. Dispense the chromatography solvent in the fume hood using a glass pipette.
e. At the end of the experiment, dispose of the used solvent in the bottle labeled
"chromatography solvent waste," also located in the fume hood.

Procedure A: Pigment extraction (performed by lab staffs)

1. Homogenize 2 handfuls of spinach leaves (Spinacia oleracea) in 100 mL of 80% acetone:

20% water in a blender.
2. Filter the homogenate through 4 layers of cheesecloth into a beaker.
Suggestion: The smell of the extraction solution is very strong so this step should be performed
under the fume hood.
3. Transfer the extract into a large capped glass test tube.
4. Repeat steps 1-3 using 2 handfuls (3oz) of carrot sticks (Daucus carota subsp. sativus).

Procedure B: Separation of Pigments by Paper Chromatography:

1. Wearing gloves, obtain 2 strips of chromatography paper from the front of the room. Make
sure not to touch the strips with your bare hands or else you will contaminate the paper.
2. Measure the chromatography strip to make sure that it fits properly in the tube when placed on
the hook. Keep in mind that you will have 2mL of solvent (9:1 petroleum ether and acetone) in
the tube which should not touch the pigment origin. You will need to make a hole at the top of
your strip for the hook to be placed. You can use a hole puncher.
3. Add 2mL of chromatography solvent to each of the two large glass test tubes at your station
and close each tube with the rubber stopper.
4. Using a pencil and a ruler, mark a line approximately 2cm from the tip of the paper as well as
a dot in the middle of that line, as shown in the figure below.
5. With the capillary tube at your station, apply the spinach extract onto your first
chromatography strip directly on top of the penciled dot and then blow on the strip to dry.
a. Repeat application of the extract at least 15 times to ensure that the extract is sufficiently
concentrated for separation to occur properly.
6. You should repeat step 5 but with the carrot extract using your second chromatography strip
and a different capillary tube.

5
 Figure 2. Labeling the chromatography and measuring Rf

7. Remove the rubber stoppers from the test tubes containing the chromatography solvent. Hook
each chromatography strip on the pin in each stopper so that the tip (but not the dot of plant
extract) of the paper is submerged in the solvent.
8. Place both test tubes in a test-tube rack and leave them undisturbed as you observe the
movement of the solvent up the paper.
9. Remove the strips before the solvent reaches the top of the paper (about 3-5 minutes).
Immediately, mark the position of the solvent with a pencil and set the strips aside to dry.
10. Dispose of the used chromatography solvent in the "chromatography solvent waste” bottle
which is found under the fume hood.
11. Once the strips are dry, calculate the Rf (see formula in the tables below) for each pigment
observed on each strip. Note: All distances are measured from the point of pigment origin.
Record your results in Tables 2.1 and 2.2 for the spinach and carrot extracts, respectively.
12. Staple your chromatography strips or draw it in the spaces provided.

Results:
Table 2.1: Spinach Extract

Distance solvent traveled in mm ____________

Pigment (observed colour) Distance Pigment Rf = distance pigment

Moved (mm) traveled  distance solvent
traveled
Carotene (yellow-orange)
Xanthophylls (yellow)
Chlorophyll a (blue-green)
Chlorophyll b (yellow-
green)

6
Chromatography Strip:

7
Table 2.2: Carrot Extract
Distance solvent traveled in mm ____________

Pigment (observed colour) Distance Pigment Rf = distance pigment

Moved (mm) traveled  distance solvent
traveled
Carotene (yellow-orange)
Xanthophylls (yellow)
Chlorophyll a (blue-green)
Chlorophyll b (yellow-
green)
Chromatography Strip:

Questions:

1. What does a small Rf tell you about the characteristics of the moving molecules?
2. Is it possible to have an Rf greater than 1? Why or why not?
3. The accepted theoretical Rf values for each pigment in a green plant extract are as follows:
carotenes = 0.98; chlorophyll a = 0.59; chlorophyll b = 0.42; xanthophylls = 0.67. Use these
values to calculate the percent error of your experiment separating the spinach extract. Show
your work in the table below.

8
 Percent error = experimental value – theoretical value x 100%
theoretical value

Pigment Percent Error Calculations

Carotenes
Chlorophyll a
Chlorophyll b
Xanthophylls

Write the experimental report accordingly.

9
 Experiment 3

Absorption spectrum

Introduction

A spectroscope is an instrument that separates white light into its component colors, ranging
from violet (400 nm) to red (740 nm). Lab staff will demonstrate how to use the spectroscope at
your station to observe this phenomenon. Wavelengths of light that are no longer visible have
been absorbed. Examine which colors are absorbed by the spinach extract by placing it between
the light and the spectroscope.

Questions:
1. Which color(s) are diminished or absent and therefore absorbed in the spinach extract?
2. Which color(s) are reflected in the spinach extract?
3. Complete the absorption spectrum below for both the spinach extract. For each color, estimate
the relative absorbance of that color by placing an X above the color name at the appropriate
position along the y-axis.
Absorbance

Violet Blue Green Yellow Red

Color of Light

Fluorescence (Lab staff Demo)

As light strikes a plant, its chlorophyll molecules become excited causing electrons to move into
higher energy orbitals. This process initiates the light-dependent portion of photosynthesis where
the energy of the excited electrons is passed through a series of pigment molecules until it
reaches chlorophyll a. However, if these energized electrons are unable to be passed onto the
next pigment molecule in either Photosystem, they fall back into their original lower energy
orbital and release the remaining energy as fluorescence. This excess energy is dissipated in the
form of light with a larger, less energetic wavelength.

10
Procedure:
1. Place the tube of spinach extract under a UV light.
2. View the tube from the side.
3. Note your observations in the space provided.

Observations:

Questions:
1. What color light does the extract fluoresce?
2. What wavelength does this color correspond to?

Write the experimental report accordingly.

11
 Experiment 4

Electron transport in chloroplasts

Introduction

The purpose of this experiment is two-fold: (1) to indirectly measure photosynthesis by

observing the rate of electron transfer, and (2) to demonstrate that the dark reaction is a separate
and independent event from the light reaction. In order for the light-dependent and light-
independent reactions to be disparate events, the electron transport chain should not require
carbon dioxide to fix carbon. You will illustrate this concept empirically by providing
chloroplasts with an alternate electron-acceptor, DCPIP, to transfer electrons. DCPIP (2, 6-
dichlorophenol-indolephenol) is a dye, which is dark blue in color when oxidized and colorless
when reduced, i.e., after accepting electrons. The rate of DCPIP decoloration depends on its
concentration and the rate of electron flow. Thus, by using DCPIP as an artificial electron-
acceptor, you will be able to monitor electron transport, in the absence of CO2 fixation.

Note: DCPIP is very dangerous. Use extreme care and wear gloves.

Procedure:

1. Label the test tubes with tape with the test tube number and your group number.
2. Add the buffer and water to each of the 4 test tubes with the amount designated on Table 4.1.
3. Then add 0.5mL of the chloroplast mixture provided to test tubes 1, 2, and 4.
4. Add 0.5mL of DCPIP as your last step into test tubes 2, 3, and 4.
5. Cap all 4 of the test tubes.
6. Wrap tube 4 completely with aluminum foil.
7. Mix the contents of each tube well.
8. Record the current colors of each test tube in Table 3.3 under “Before Observed Results”.
9. Place the 4 test tubes in front of the light. Have someone keep an eye on them as the reaction
will occur within minutes (approximately 3-5 minutes).
10. Record expectations for each tube in Table 3.4 while you wait.
11. After 5 minutes, record the “After Observed Results” in Table 4.1.

12
 Table 4.1:

Test tube Chloroplasts 0.1M PO4 H2 0 0.2mM

Preparation Tube # Buffer (pH 6.5) DCPIP
1 0.5mL 3mL 2mL 0
2 0.5mL 3mL 1mL 0.5mL
3 0 3mL 1.5mL 0.5mL
4 0.5mL 3mL 1mL 0.5mL
Foil wrapped

Table 4.2:
Tube Expected Results Before After Explanation
# Observed Observed
Results (color) Results (color)
1

4
Foil

Questions:
a. Which of the 4 tubes served as a negative control for chloroplast? Why?
b. Which of the 4 test tubes served as a negative control for DCPIP? Why
c. Which of the 4 test tubes served as a positive control? Why?
d. What was the purpose of covering tube 4 with foil?
e. Why was there some color change in the wrapped tube? (Hint: Does electron transport occur
without light?)
f. Are there electrons being accepted in tube #1 even though there’s no DCPIP present? Explain
your answer.

Write the experimental report accordingly.

13
*Preparing the chloroplast suspension

Materials needed

 Homogenization buffer
 polypropylene centrifuge tube
 15 ml Potter-Elvehjem homogenizer
 50 ml graduated cylinder
 Foil ice bucket
 100 ml beaker plastic funnel
 13mm test tube misc pipets

Procedure:

In the first part of the exercise, spinach leaf cells will be fractionated to yield a chloroplast
fraction stabilized in an isotonic solution. The homogenate and chloroplast suspension should
be kept cold (in an ice bucket) at all times. Homogenization buffer should be pre-chilled.
Homogenize the spinach leaves (this is done in batch for the whole class)

1. Fresh spinach leaves are washed with roH2O and then stored in the refrigerator overnight.
2. In a blender, 100 g of de-veined leaves will be combined with 300 ml of homogenization
buffer. (Homogenization buffer is isotonic to the cell cytosol, preventing rupturing of
the chloroplasts during preparation.)
3. The leaves will be homogenized with the blender set at its highest speed for 10 second
periods, ~four times, interrupted with brief pauses.
4. The homogenate is then passed through a cloth filter to remove larger particulate materials.
Each group will receive 40 ml of the filtered homogenate.
Centrifuge the filtered homogenate and resuspend the chloroplasts
5. Transfer the filtered homogenate to a polypropylene centrifuge tube.
6. Counterbalance your tube against that of another group and put the tubes opposite each other
in the centrifuge SS-34 rotor.
7. Centrifuge the filtrate at 2,000 x g (4,250 rpm) for 10 minutes. In this step
the chloroplasts in the homogenate sediment to the bottom of the tube.
8. Carefully pour off the supernatant from the centrifuge tube.
9. Add 2.0 ml of homogenization Buffer to the pellet, and resuspend the chloroplast pellet by
gently sucking it into and out of a Pasteur pipet.
10. Transfer the chloroplast suspension to a Potter-Elvehjem homogenizer.
11. Rinse the centrifuge tube with an additional 2 ml of Homogenization Buffer,
and then combine this with the rest in the homogenizer
12. Fully resuspend the chloroplasts with 3 - 4 passes of the pestle.
13. Using a Pasteur pipet, transfer the chloroplast suspension into a 13 mm test tube wrapped in
foil and place it in your ice bucket.

14
 Experiment 5

Uptake of carbon dioxide during photosynthesis

Introduction

Elodea, an aquatic plant, and a pH indicator, phenol red, will be used to demonstrate that plants
use carbon dioxide (CO2). Phenol red is red in alkaline (basic) solutions (pH > 7) and turns
yellow in acidic solutions (pH < 7). To prepare for this experiment, you will blow air into 3 tubes
containing phenol red, releasing carbon dioxide into the liquid in each tube. The carbon dioxide
will bind with the water in the phenol red indicator to form carbonic acid (H2CO3).

H2O + CO2 H2CO3 H+ + HCO3-

Water carbon dioxide Carbonic acid Proton Bicarbonate

Questions:
1. Before blowing into the test tubes, what color would you expect the solution to be? Explain.
You will then add Elodea to two of the tubes to begin carbon dioxide fixation process (i.e.
photosynthesis). As the amount of carbon dioxide decreases, the pH is going to change, causing
the phenol red indicator to change color.
2. In what direction (higher or lower) would you expect the pH to change as Elodea fixes the
CO2?
3. After blowing into the test tubes, what color should the phenol red indicator become? Explain.

Procedure:
1. Fill three small screw cap test tubes half way with the phenol red indicator at your station.
2. Label the tubes with tape with the test tube number and your group number.
3. Gently blow into each of the 3 tubes with a straw until the indicator turns yellow in color. You
should make sure that all 3 tubes are the same shade of yellow.
4. Add a piece of Elodea (approximately 7cm in length) to tubes 1 and 2 only.
5. Immediately screw the cap on each of the 3 tubes to prevent the escape of carbon dioxide.
6. Completely cover tube 2 with aluminum foil.
7. Record your expectations for each tube in Table 5.1.
8. Place all 3 tubes in front of the light source.
9. Observe the tubes every 15 minutes for a total of 45 minutes. Record your observations in
Table 5.1.

15
 Table 5.1
Tube # Contents Expected Results Observed
Results/Explanation
1. Elodea
Phenol Red
2. Elodea
Phenol Red
Foil
3. Phenol Red
4.

Questions:
1. What was the purpose of covering tube 2 with foil?
2. What was the purpose of tube 3?
3. Did tube 1 change color after the 45 minutes? Why?
4. Which one of the test tubes is considered the positive control? Why?

Write the experimental report accordingly.

16