Station 1 – Extracting enzymes from potato

tissue and making water baths

Materials:
-​ 4 x 10 mL graduated cylinders
-​ 100 mL graduated cylinder
-​ 400 mL beaker
-​ 2 x 600 mL beakers
-​ 3 medium test tubes that fit the stopper from the gas pressure probe
-​ test tube rack
-​ ice
-​ 10 mL of 3% hydrogen peroxide
-​ 1 potato
-​ knife
-​ cutting board
-​ blender and soft spatula
-​ cheesecloth or coffee filter
-​ thermometer or temperature probe
-​ pH paper or pH probe
-​ hot plate
-​ Vernier lab quest and pressure probe with test tube attachment
-​ timer / stopwatch / chronometer
-​ Balance

Procedure (if potato filtrate is available, please skip steps

1 – 4)
1.​ Mass the potato and record. __________ g
2.​ Wash and cut the potato into 4 pieces using the knife and cutting board.
3.​ Add the potato quarters to the blender with 200 mL of distilled water. Blend until smooth.
4.​ Pour the liquid through cheesecloth or coffee filter and collect the filtrate in the 400 mL beaker.
5.​ Measure 2.0 mL of filtrate into each of 3 test tubes. Place one in a 600 mL beaker half full of ice
water temp: ____ °C, place one in a test tube rack at room temperature: _____ °C, and place one
in a 60°C – 70°C hot water bath temperature actual temp: __________ °C (made with the hot
plate and a 600 mL beaker half full of tap water).
6.​ Measure 3 x 2.0 mL of 3% hydrogen peroxide into 10 mL graduated cylinder and bring them to
the same temperature as the potato filtrate.
 7.​ Measure with a thermometer and record the temperatures of all three set-ups.
8.​ Get the pressure probe and lab quest ready to record pressure differences every second for 30
seconds.
9.​ For each of the temperature set-ups: invert the hydrogen peroxide into the test tube containing the
potato filtrate and IMMEDIATELY add the pressure probe stopper and press start. Record the
initial pressure and final pressure after 30 s.
10.​ Monitor pH by measuring with pH paper (or probe) all three set-ups. Record.
11.​ (If time had allowed: for the best experiment, we would choose at least two more temperatures
and do at least 5 trials of each temperature).

To hand in:
1) What are the manipulated, responding, and controlled variables for
the above experiment?
MV: heat of solution (Potato Filtrate & Hydrogen peroxide)
RV: pressure change
CV’s: Amount of potato filtrate in each cylinder, amount of hydrogen peroxide in each cylinder, type of
hydrogen peroxide, time for reaction

2 A data table or data tables that show data (with reasonable

uncertainties) that is requested to be recorded. Insert the table(s) here (on
the word doc on D2L).

Temperature Initial Pressure (+- 0.005) Final Pressure (+- 0.005)

Cold 89.46 89.51

Room Temperature 89.57 89.51

Hot 89.74 89.16

2 Design an idealized (if time is not an issue to collect sufficient data to
do stats) procedure to investigate the effect of pH on the enzyme
catalase. You may copy and modify relevant items from this procedure
you performed (make sure you reference it!).
Problem: How do different pH levels affect the enzyme catalase?
Materials:
-​ 30 mL of Potato Filtrate (Assuming we already have this since we did have it last time)
-​ 2 x 10 mL Graduated Cylinder
-​ 5 medium test tubes that fit the stopper from the gas pressure probe
-​ test tube rack
-​ ice
-​ 30 mL of 3% hydrogen peroxide
-​ 3 x pH Buffer (4, 6, 7, 8, 10)
-​ pH probe
-​ Vernier lab quest and pressure probe with test tube attachment
-​ Stopwatch
Procedure
1.​ Measure 2.0 mL of Potato Filtrate using a 10mL Graduated Cylinder into each medium test tube.
2.​ Measure 2.0 mL of pH buffer using a 10mL Graduated Cylinder into each medium test tube (2
mL of pH 4 Buffer in one, 2 mL of pH 6 buffer in another…)’
3.​ Ensure pH is accurate using pH probe
4.​ Measure 2.0 mL of 3% hydrogen peroxide into each test tube
5.​ Use the Vernier LabQuest and the Pressure Probe to measure the pressure difference every half
second for 30 seconds immediately after putting in the Hydrogen Peroxide
6.​ Repeat steps 1-5 two more times

4 Safety & Environment: Comment on some precautions that you took

for experimenter safety and how you minimized materials and disposed
of them safely
Some precautions we took during this lab was ensuring that the hydrogen peroxide was carefully
handled and didn’t touch anybody. We minimalized materials by only using the amount needed,
and we made sure to dispose materials properly in waste containers and thoroughly cleaned all
materials

Station 2 – diluting, determining the isotonic

point of a potato, calculating % change
Material
1 potato
knife & potato corer

plastic ruler

balance

1.0​ M sucrose solution

6 small test tubes

test tube rack

plastic wrap or parafilm

paper towel for blotting

tweezers for removing potato

Procedure
1) Take a 1.00 M sucrose solution and dilute it to obtain 5.0 mL of the following sucrose concentrations:
1.00 M, 0.80 M, 0.60 M, 0.40 M, 0.20 M, 0 M by using a 10 mL graduated cylinder and distilled water
(for example – 0.80 M is 4.0 mL of 1.00 M sucrose and 1.0 mL of distilled water).

2) Use a potato corer or cut an approximately 1.0 cm wide strip of potato from the middle. Use a ruler to
slice 6 approximately 1.0 x 1.0 cm cubes of potato. Blot each potato cube lightly, mass it and record its
initial mass before placing it in a marked test tube full of a designated sucrose concentration.

3) Cover the 6 test tubes with plastic wrap or Parafilm and place them in a safe location overnight.

4) The next day – remove each potato chunk one at a time. Blot them lightly and measure and record
their final mass.

To hand in:
a)​Data and Analysis combined table with initial masses, final
masses, and % change in mass

Sucrose Initial Mass (g) Final Mass (g) % Change in Mass

Concentration (mol)

1.0 0.38 0.21 -45%

0.8 0.48 0.26 -46%

0.6 0.36 0.35 -3%

0.4 0.45 0.31 -31%

0.2 0.40 0.50 25%

0.0 0.35 0.47 34%

b)​Get % change data from four other groups so you can calculate
means & standard deviations. Make a table of the 5 trials, the
mean, and the standard deviation.
Table 1: Quantitative Observations Recorded For a Cube of Potato Left Overnight
in 1.00 M of Sucrose Solution

Initial Mass (g) Final Mass (g) % Change in Mass

Trial 1 0.12 0.09 -25%

Trial 2 0.10 0.06 -40%

Trial 3 0.7 0.03 -96%

Trial 4 0.38 0.21 -45%

Trial 5 0.37 0.35 -5%

Average 0.33 0.15 -42%

Table 2: Quantitative Observations Recorded For a Cube of Potato Left Overnight
in 0.80 M of Sucrose Solution

Initial Mass (g) Final Mass (g) % Change in Mass

Trial 1 0.10 0.09 -10%

Trial 2 0.11 0.09 -18%

Trial 3 0.08 0.05 -38%

Trial 4 0.48 0.26 -46%

Trial 5 0.43 0.36 -16%

Average 0.24 0.17 -26%

Table 3: Quantitative Observations Recorded For a Cube of Potato Left Overnight

in 0.60 M of Sucrose Solution

Initial Mass (g) Final Mass (g) % Change in Mass

Trial 1 0.11 0.10 -9%

Trial 2 0.14 0.11 -21%

Trial 3 0.09 0.08 -11%

Trial 4 0.36 0.35 -3%

Trial 5 0.33 0.31 -6%

Average 0.21 0.19 -10%

Table 4: Quantitative Observations Recorded For a Cube of Potato Left Overnight

in 0.40 M of Sucrose Solution

Initial Mass (g) Final Mass (g) % Change in Mass

Trial 1 0.10 0.09 -10%

Trial 2 0.12 0.11 -8%

Trial 3 0.09 0.08 -11%

Trial 4 0.45 0.31 -31%

Trial 5 0.41 0.43 5%

Average 0.23 0.20 -11%

Table 5: Quantitative Observations Recorded For a Cube of Potato Left Overnight

in 0.20 M of Sucrose Solution

Initial Mass (g) Final Mass (g) % Change in Mass

Trial 1 0.15 0.15 0%

Trial 2 0.14 0.15 7%

 Trial 3 0.07 0.07 0%

Trial 4 0.40 0.50 25%

Trial 5 0.37 0.45 22%

Average 0.23 0.26 11%

Table 6: Quantitative Observations Recorded For a Cube of Potato Left Overnight

in 0.0 M of Sucrose Solution

Initial Mass (g) Final Mass (g) % Change in Mass

Trial 1 0.12 0.11 -8%

Trial 2 0.10 0.13 30%

Trial 3 0.08 0.06 -25%

Trial 4 0.35 0.47 34%

Trial 5 0.33 0.31 -6%

Average 0.21 0.22 5%

c)​Graph of sucrose concentration vs. average % change in mass of a

potato with isotonic point determined graphically and shown – add
in standard deviation error bars.
Isotonic point is going to be the ‘x’ intercept, which was calculated by creating a quartic
equation (degree 4, with a r2 = 0.9647) in order to most accurately represent the data, and then
calculating the intercept. The isotonic point is when there is 0.3269275 moles of sucrose. Error
bars was made using standard deviation calculated on the google spreadsheet below.

Data

Station 3 - Comparing cellular respiration of

germinating and non-germinating seeds in a
respirometer

Materials
Carbon dioxide or oxygen probe, 250 mL plastic bottle for probe and Vernier lab quest

25 Germinating and 25 non-germinating seeds of the same species

Procedure
1)​ Program the lab quest so that it will take carbon dioxide concentration (or oxygen) measurements
every 30 seconds for 5 minutes.

2)​ Choose 25 undamaged dry seeds of _________________________ species and add them to the
dry plastic bottle.

3)​ Press start and make a data table recording the CO2 concentrations every 30 s for 5 minutes (no
need to record this table).

4)​ Use the range in answers for number 3 to determine the uncertainty in the carbon dioxide (or
oxygen) probe for the data table.

5)​ Choose 25 undamaged germinating seeds to add to an empty plastic bottle and record the carbon
dioxide (or oxygen) concentration every 30 s for 5 minutes. Make a simplified data table that
records initial and final concentrations.

To hand in
a)​Data table with uncertainties, Calculate rate of cellular respiration
per seed (show calculations and units).

Type of seed Initial (%) Final (%)

+-0.005% +-0.005%

Non Germinating 0.28 0.29

Germinating 0.29 0.27

Rate of Cellular Respiration

Non Germinating: 0.00008 per seed per minute

Germinating seeds: 0.00016 per seed per minute

b)​Discuss 2 sources of error or limitations and 2 improvements or

extensions (list one strength too!)
Sources of error:
-​ Number of trials were limited
 -​ There is a lot of variation within seeds and how much they have grown.

​ Improvements:

-​ Increase trials to 5
-​ Newer or more secure lab tech.​

One strength

-​ The seeds were kept properly and the lab tech worked efficiently and gave us a
quality graph.

What were our units for the germinating seed?

% was our units. Change in pressure percentage.

Station 4 – Measuring the Rates of

Photosynthesis
4a) Leaf Disk Assay with spinach, with and without bicarbonate

(lots of youtube.com videos to help as it is an AP Biology Lab)

Procedure modified from http://www.elbiology.com/labtools/Leafdisk.html

Mini Background Information:

Leaf disks float, normally. When the air spaces are infiltrated with solution the overall density of the leaf disk
increases and the disk sinks. The infiltration solution includes a small amount of sodium bicarbonate. Bicarbonate
ion serves as the carbon source for photosynthesis. As photosynthesis proceeds oxygen is released into the interior
of the leaf which changes the buoyancy--causing the disks to rise. Since cellular respiration is taking place at the
same time, consuming oxygen, the rate that the disks rise is an indirect measurement of the net rate of
photosynthesis.
Materials:
●​ 0.5 % sodium bicarbonate with a drop of liquid soap added

●​ Liquid Soap

●​ Plastic syringe (10 cc or larger)

●​ Leaf material (Spinach)

●​ Hole punch​ ​ ​

●​ 100 mL graduated cylinder

●​ Timer

●​ Light source

●​ pH probes or pH paper

Procedure:
●​ Use approximately 50 mL of 0.5% bicarbonate solution with soap

o​ The soap wets the hydrophobic surface of the leaf allowing the solution to be drawn into the leaf.
It’s difficult to quantify this since liquid soaps vary in concentration. Avoid suds. If your solution
generates suds then dilute it with more bicarbonate solution.

●​ Cut 10 or more uniform leaf disks for each trial using a hole punch.

o​ Single hole punches work well for this but stout plastic straws will work as well.

o​ Choice of the leaf material is perhaps the most critical aspect of this procedure. The leaf surface
should be smooth and not too thick; avoid major veins.

●​ Infiltrate the leaf disks with sodium bicarbonate solution.

o​ Remove the piston or plunger and place the leaf disks into the syringe barrel. Replace the plunger
being careful not to crush the leaf disks. Push on the plunger until only a small volume of air
remains in the barrel (< 10%).

o​ Pull a small volume of sodium bicarbonate solution into the syringe. Invert, shake and tap the
syringe to suspend the leaf disks in the solution.

o​ Holding a finger over the syringe opening, draw back on the plunger to create a vacuum. Hold this
vacuum for about 10 seconds. While holding the vacuum, swirl the leaf disks to suspend them in
the solution. Let off the vacuum. The bicarbonate solution will infiltrate the air spaces in the leaf
causing the disks to sink. You will probably have to repeat this procedure 2-3 times in order to get
the disks to sink.

o​ Pour the disks and solution into a clear graduated cylinder. Add bicarbonate solution to a depth of
about 3 centimeters. Use the same depth for each trial.
 ●​ For a control infiltrate leaf disks with a solution of only water with a drop of soap--no bicarbonate.

●​ Place under the light source and start the timer. At the end of each minute, record the number of floating
disks (there can be an uncertainty in counts!!). Then swirl the disks to dislodge any that are stuck against
the sides of the graduated cylinder. Continue until all of the disks are floating.

To Hand in:
a)​We had a control experiment at this station. Explain what a
“control” means and how it is different from controlled variables
​ A control is something that you know every time will produce either a negative or
positive result. Controlled variables are used to ensure that the experiment follows the same
environment so that there is only one manipulated variable, while controls are used to validate
the results given by the experiment. Negative controls help prevent false positives.

Station 5 – Separating pigments in Spinach

Using Paper Chromatography

Materials (experiment to be conducted under

fumehood)
Spinach leaves

Filter paper

Test tube

Cork with paper clip hook

Petroleum Ether Solvent (and waste bottle)

Procedure
1.​ Pick a few fresh leaves of spinach.
 2.​ Place a filter paper strip with a tapering notch (triangle) towards one end of the strip.

3.​ Horizontally trace a line with a pencil that is ~2 cm from the notch’s tip (at the base of the triangle).

4.​ Crush the spinach leaves onto the midsection of the line.

5.​ In the fume hood, pour the ether acetone solvent into the test tube so that it will just cover the tip of
the filter paper.

6.​ Suspend the strip in the test tube from the hook on the cork.

7.​ The loading spot should remain about 1 cm above the level of the solvent.

8.​ Allow the solvent front to move up the filter paper, bringing the pigment with it.

9.​ Once the solvent gets close to the hook it is suspended from, carefully take the strip off.

10.​ Use a pencil to mark a line at the limit of travel for each pigment and the solvent front.

11.​ Measure the distances traveled by each pigment and the solvent front.

To Hand in:
a)​ Chromatograph and measurements recorded in a data table (use uncertainties)

a.​

Substance Distance (+,-0.05mm)

solution 5.0

spinach 4.5
b.​

b)​ Rf calculations which can be compared with theoretical using % error.

a.​ pigment travel

Rf = 4.5/5= 0.9, (+,- 0.10)

Solvent front distance

c)​ Safety and Environment Section

a.​ We used a fume hood in order to reduce the risk of inhaling gasses

b.​ We safely disposed of materials in a waste container

Theoretical Rf’s
You will find different Rf values on different websites. They vary depending on the method and the solvent used. I
found these ones from: https://www.depts.ttu.edu/ciser/science.../plants/Plant_Pigment_Chromatography.pdf .

β-carotene - 0.99

chlorophyll a - 0.30

chlorophyll b - 0.13

violaxanthin - 0.40

lutein - 0.68

(violaxanthin, and lutein are carotenoids)

Marking/Rubric
Lab Part Mark Person Responsible
Potato Enzyme Variables SK1
Variables
Table SK2
Procedure Reasonable pH range SK1
Use of pH buffers SK1
Measured / Controlled SK1
Temperature etc…
Had replicates SK1
Overall /6

Potato Osmosis Table Precision / units for SK3

mean
Standard deviation SK4
units
Sample Calculation SK4
Potato Osmosis Graph Isotonic Point on SK3
Graph
Correct Error Bars (and SK4
identified as stddev)
Graph Title SK3
Overall /6
Germinating Seeds Correct Rate SK3
Calculation/Table
Reasonable SK2
Uncertainties
Units & Precision SK2
Germinating Seeds Strength SK3
Evaluation
2 Weaknesses / SK3
Limitations
2 Improvements / SK3
Extensions
Overall /6

Photosynthesis in Control vs. Controlled SK1

Spinach Leaf Disks Variable
Separation of Spinach Data Table SK2
Leaf Pigments
Rf calcs SK3
Uncertainties SK3
Safety SK1
Environment SK1
Overall /6

SK1 /8 SK2 /4 SK3 /9 SK4 /3

Copy of Full Assignment

LAB STATIONS SK marks
Lab Part Mark Person Responsible
Potato Enzyme Variables SK1
Variables
Table SK2
Procedure Reasonable pH range SK1
Use of pH buffers SK1
Measured / Controlled SK1
Temperature etc…
Had replicates SK1
Overall /6
 Potato Osmosis Table Precision / units for mean SK3
Standard deviation units SK4
Sample Calculation SK4
Potato Osmosis Isotonic Point on Graph SK3
Graph
Correct Error Bars (and SK4
identified as stddev)
Graph Title SK3
Overall /6

Germinating Seeds Correct Rate SK3

Calculation/Table
Reasonable Uncertainties SK2
Units & Precision SK2
Germinating Seeds Strength SK3
Evaluation
2 Weaknesses / Limitations SK3
2 Improvements / Extensions SK3
Overall /6

Photosynthesis in Control vs. Controlled Variable SK1

Spinach Leaf Disks
Separation of Data Table SK2
Spinach Leaf
Pigments
Rf calcs SK3
Uncertainties SK3
Safety SK1
Environment SK1
Overall /6

SK1 /8 SK2 /4 SK3 /9 SK4 /3

Biochemistry, Osmosis, Cellular Respiration & Photosynthesis Lab Stations

(copy into Google docs and assign one group member to be responsible for each lab, all
answers MUST be inserted EXACTLY where the questions are). Labs must be saved as either a
word doc or PDF for submission to D2L.

Station 1 – Extracting enzymes from potato tissue and making water baths

Materials:
4 x 10 mL graduated cylinders 3 medium test tubes that fit the stopper
100 mL graduated cylinder from the gas pressure probe
400 mL beaker test tube rack
2 x 600 mL beakers ice
10 mL of 3% hydrogen peroxide
1 potato pH paper or pH probe
knife hot plate
cutting board Vernier lab quest and pressure probe with
blender and soft spatula test tube attachment
cheesecloth or coffee filter timer / stop watch / chronometer
thermometer or temperature probe balance

Procedure (if potato filtrate is available, please skip steps 1 – 4)

1.​ Mass the potato and record. __________ g
2.​ Wash and cut the potato into 4 pieces using the knife and cutting board.
3.​ Add the potato quarters to the blender with 200 mL of distilled water. Blend until
smooth.
4.​ Pour the liquid through cheesecloth or coffee filter and collect the filtrate in the 400
mL beaker.
5.​ Measure 2.0 mL of filtrate into each of 3 test tubes. Place one in a 600 mL beaker
half full of ice water temp: ____ °C, place one in a test tube rack at room temperature:

_____ °C, and place one in a 60°C – 70°C hot water bath temperature actual temp:

__________ °C (made with the hot plate and a 600 mL beaker half full of tap water).
6.​ Measure 3 x 2.0 mL of 3% hydrogen peroxide into 10 mL graduated cylinder and
bring them to the same temperature as the potato filtrate.
7.​ Measure with a thermometer and record the temperatures of all three set-ups.
8.​ Get the pressure probe and lab quest ready to record pressure differences every
second for 30 seconds.
9.​ For each of the temperature set-ups: invert the hydrogen peroxide into the test tube
containing the potato filtrate and IMMEDIATELY add the pressure probe stopper and
press start. Record the initial pressure and final pressure after 30 s.
10.​Monitor pH by measuring with pH paper (or probe) all three set-ups. Record.
11.​(If time had allowed: for the best experiment, we would choose at least two
more temperatures and do at least 5 trials of each temperature).

To hand in:
ai) What are the manipulated, responding, and controlled variables for the above
experiment?
MV:
RV:
CV’s:
aii) A data table or data tables that show data (with reasonable uncertainties) that is
requested to be recorded. Insert the table(s) here (on the word doc on D2L).
b) Design an idealized (if time is not an issue to collect sufficient data to do stats)
procedure to investigate the effect of pH on the enzyme catalase. You may copy and
modify relevant items from this procedure you performed (make sure you reference it!).

c) Safety & Environment: Comment on some precautions that you took for experimenter
safety and how you minimized materials and disposed of them safely.
Station 2 – diluting, determining the isotonic point of a potato, calculating % change

Materials
1 potato 1.0​M sucrose solution
knife & potato corer 6 small test tubes
plastic ruler test tube rack
balance plastic wrap or parafilm
paper towel for blotting tweezers for removing potato

Procedure
1) Take a 1.00 M sucrose solution and dilute it to obtain 5.0 mL of the following sucrose
concentrations: 1.00 M, 0.80 M, 0.60 M, 0.40 M, 0.20 M, 0 M by using a 10 mL graduated
cylinder and distilled water (for example – 0.80 M is 4.0 mL of 1.00 M sucrose and 1.0 mL of
distilled water).
2) Use a potato corer or cut an approximately 1.0 cm wide strip of potato from the middle.
Use a ruler to slice 6 approximately 1.0 x 1.0 cm cubes of potato. Blot each potato cube
lightly, mass it and record its initial mass before placing it in a marked test tube full of a
designated sucrose concentration.
3) Cover the 6 test tubes with plastic wrap or Parafilm and place them in a safe location
overnight.
4) The next day – remove each potato chunk one at a time. Blot them lightly and measure
and record their final mass.

To hand in:
a)​ Data and Analysis combined table with initial masses, final masses, and % change in
mass

b)​ Get % change data from four other groups so you can calculate means & standard
deviations. Make a table of the 5 trials, the mean, and the standard deviation.

c)​ Graph of sucrose concentration vs. average % change in mass of a potato with
isotonic point determined graphically and shown – add in standard deviation
error bars.

Station 3 - Comparing cellular respiration of germinating and non-germinating seeds

in a respirometer

Materials
Carbon dioxide or oxygen probe, 250 mL plastic bottle for probe and Vernier lab quest
25 Germinating and 25 non-germinating seeds of the same species

Procedure
1)​ Program the lab quest so that it will take carbon dioxide concentration (or oxygen)
measurements every 30 seconds for 5 minutes.
 2)​ Choose 25 undamaged dry seeds of _________________________ species and add them to
the dry plastic bottle.
3)​ Press start and make a data table recording the CO2 concentrations every 30 s for 5
minutes (no need to record this table).
4)​ Use the range in answers for number 3 to determine the uncertainty in the carbon
dioxide (or oxygen) probe for the data table.
5)​ Choose 25 undamaged germinating seeds to add to an empty plastic bottle and
record the carbon dioxide (or oxygen) concentration every 30 s for 5 minutes. Make
a simplified data table that records initial and final concentrations.

To hand in
a)​ Data table with uncertainties, Calculate rate of cellular respiration per seed (show
calculations and units).

b)​ Discuss 2 sources of error or limitations and 2 improvements or extensions (list one
strength too!)

Station 4 – Measuring the Rates of Photosynthesis

4a) Leaf Disk Assay with spinach, with and without bicarbonate
(lots of youtube.com videos to help as it is an AP Biology Lab)
Procedure modified from http://www.elbiology.com/labtools/Leafdisk.html

Mini Background Information:

Leaf disks float, normally. When the air spaces are infiltrated with solution the overall density of
the leaf disk increases and the disk sinks. The infiltration solution includes a small amount of
sodium bicarbonate. Bicarbonate ion serves as the carbon source for photosynthesis. As
photosynthesis proceeds oxygen is released into the interior of the leaf which changes the
buoyancy--causing the disks to rise. Since cellular respiration is taking place at the same time,
consuming oxygen, the rate that the disks rise is an indirect measurement of the net rate of
photosynthesis.

Materials:

●​ 0.5 % sodium bicarbonate with a ●​ Hole punch​ ​ ​

drop of liquid soap added ●​ 100 mL graduated cylinder
●​ Liquid Soap ●​ Timer
●​ Plastic syringe (10 cc or larger) ●​ Light source
●​ Leaf material (Spinach) ●​ pH probes or pH paper
Procedure:

●​ Use approximately 50 mL of 0.5% bicarbonate solution with soap

o​ The soap wets the hydrophobic surface of the leaf allowing the solution to be
drawn into the leaf. It’s difficult to quantify this since liquid soaps vary in
concentration. Avoid suds. If your solution generates suds then dilute it with
more bicarbonate solution.

●​ Cut 10 or more uniform leaf disks for each trial using a hole punch.

o​ Single hole punches work well for this but stout plastic straws will work as well.
o​ Choice of the leaf material is perhaps the most critical aspect of this procedure.
The leaf surface should be smooth and not too thick; avoid major veins.

●​ Infiltrate the leaf disks with sodium bicarbonate solution.

o​ Remove the piston or plunger and place the leaf disks into the syringe barrel.
Replace the plunger being careful not to crush the leaf disks. Push on the
plunger until only a small volume of air remains in the barrel (< 10%).

o​ Pull a small volume of sodium bicarbonate solution into the syringe. Invert,
shake and tap the syringe to suspend the leaf disks in the solution.
o​ Holding a finger over the syringe opening, draw back on the plunger to create a
vacuum. Hold this vacuum for about 10 seconds. While holding the vacuum,
swirl the leaf disks to suspend them in the solution. Let off the vacuum. The
bicarbonate solution will infiltrate the air spaces in the leaf causing the disks to
sink. You will probably have to repeat this procedure 2-3 times in order to get the
disks to sink.
o​ Pour the disks and solution into a clear graduated cylinder. Add bicarbonate
solution to a depth of about 3 centimeters. Use the same depth for each trial.

●​ For a control infiltrate leaf disks with a solution of only water with a drop of soap--no
bicarbonate.

●​ Place under the light source and start the timer. At the end of each minute, record the
number of floating disks (there can be an uncertainty in counts!!). Then swirl the disks to
dislodge any that are stuck against the sides of the graduated cylinder. Continue until all
of the disks are floating.​

To Hand in:
a)​ We had a control experiment at this station. Explain what a “control” means and
how it is different from controlled variables

Station 5 – Separating pigments in Spinach Using Paper Chromatography

Materials (experiment to be conducted under fumehood)

Spinach leaves Filter paper
Test tube Petroleum Ether Solvent (and waste
Cork with paper clip hook bottle)

1.​ Pick a few fresh leaves of spinach.

2.​ Place a filter paper strip with a tapering notch (triangle) towards one end of the strip.
3.​ Horizontally trace a line with a pencil that is ~2 cm from the notch’s tip (at the base of
the triangle).
4.​ Crush the spinach leaves onto the midsection of the line.
5.​ In the fume hood, pour the ether acetone solvent into the test tube so that it will just
cover the tip of the filter paper.
6.​ Suspend the strip in the test tube from the hook on the cork.
7.​ The loading spot should remain about 1 cm above the level of the solvent.
8.​ Allow the solvent front to move up the filter paper, bringing the pigment with it.
9.​ Once the solvent gets close to the hook it is suspended from, carefully take the strip off.
10.​ Use a pencil to mark a line at the limit of travel for each pigment and the solvent front.
11.​ Measure the distances traveled by each pigment and the solvent front.

To Hand in:

a)​ Chromatograph and measurements recorded in a data table (use uncertainties)

b)​ Rf calculations which can be compared with theoretical using % error.
c)​ Safety and Environment Section

Theoretical Rf’s

You will find different Rf values on different websites. They vary depending on the method and the solvent
used. I found these ones
from: https://www.depts.ttu.edu/ciser/science.../plants/Plant_Pigment_Chromatography.pdf .
They said they used 90% petroleum ether and 10% acetone which sounds like what we use.
β-carotene - 0.99
chlorophyll a - 0.30
chlorophyll b - 0.13
violaxanthin - 0.40
lutein - 0.68
(violaxanthin, and lutein are carotenoids)