Lab 5 – Photosynthesis
Mmm…spinach.
Lab Structure
• The first thing to know is that we have a dual-lab
structure here.
– The main lab (Lab 5, straight outta your lab manual).
– The mini-lab (Lab 4, from the old lab manual you
don’t have).
• We’ll start with Lab 4 for our first day, then
continue with Lab 5 over the following two days.
Mini-Lab 4 Information
• Use the photocopied packet for this one.
– Cross off #2 in the OVERVIEW section. You’re not doing that.
– Once you answer question #3, you’re done. Ignore the rest.
• The other objectives are worth noting.
• In this mini-lab, you’ll be doing the following:
– Separating plant pigments using chromatography.
– Identifying the pigments you separate.
– The three pigments you’ll be separating:
• Beta carotene (Vitamin A; makes carrots orange)
• Xanthophyll
• Chlorophyll(s)
Da Procedurez
• To perform this paper chromatography analysis, you’re
going to do the following, in general terms:
– Cut a piece of filter paper and mark a starting line.
– Crush a spinach leaf piece onto the line using a quarter.
• Make it a thick layer and as even a line as possible.
– Put the filter paper into a solvent in a graduated cylinder.
– Watch as the solvent soaks up into the paper, dissolving the
pigments and carrying them up the paper.
– Mark the ending point of the solvent as it soaks up.
– Mark the bands of pigment where they stopped.
– Perform some calculations to determine Rf values.
• Actually, this is a bit like gel electrophoresis, isn’t it?
Video!
• Chromatography!
Da Detailz
• I’ll pour the solvent into the bottom of the graduated cylinder.
– It is quite volatile, which means evaporates easily and isn’t so
great to inhale. Keep it stoppered.
• It’s acetone and petroleum ether. Can you say “nerve damage?”
• When you put the filter paper in, the solvent will begin
soaking up the filter paper.
– Make sure the solvent does not immediately contact the
pigment.
• As the solvent rises, it will carry different pigments from the
leaf at different rates.
• Remove the filter paper when the solvent nears the top.
– Use a pencil to mark all the pigments and solvent before they
evaporate.
Paper Clip Hook Spot
Filter Paper Prep
• Use an empty 100 mL graduated cylinder and
measure out some filter paper that’s long
enough so that:
– You can suspend it from the side of the cylinder
with a paper clip bent into a hook shape.
– Once suspended, its bottom tip hits around the 5
mL mark.
• We have lots of filter paper so test it out.
• Mark a starting line where your pigment will
be about 3 cm away from the bottom.
– Use pencil for all your marks. Ink will separate.
• Cut/prep the filter paper like shown:
Cautions
• Use only glass graduated cylinders.
– Ask if you need one.
• Make sure the filter paper is only a little bit into the
solvent.
– DO NOT let the solvent touch the pigment directly.
• Need I remind you about the dangers of the solvent?
– Hang the paper clip into the “spout” area so the stopper is
most effective.
– Try to avoid inhaling the vapor.
• Clean up by rinsing the graduated cylinder out and
putting the filter paper in the “disposal bag.”
• Don’t steal my quarters.
Calculations
• Like for the electrophoresis lab, you’ll measure the
distances each pigment travels.
– Some bands will travel almost as far as the solvent.
– Some bands will barely move.
• Key: Rf is the constant that relates the distance
traveled by the pigment to the distance traveled by
the solvent.
– Formula is: (distance of pigment) / (distance of solvent)
• Use mm for distance.
• Key: Higher Rf values – that is, closer to 1 – indicate
greater solubility in the solvent.
• More info in your lab instructions.
Onward!
• That’s all you need to do for Day 1 of the lab.
• Spend your time setting up your lab notebook
for both days.
– Refer to your lab manual for more info on the next
lab.
– Lab 5 begins on Page S61.
Lab 5 – Photosynthesis
• Do you remember the photosynthesis equation?
• 6CO2 + 6H2O  C6H12O6 + 6O2
• If you wanted to measure the rate of
photosynthesis, what reactant(s) or product(s) do
you suppose would be most feasible to track?
– O2 produced.
– CO2 consumed.
• For this lab, we’re going to measure the
production of O2.
The Lab
• We’re going to conduct this portion of the lab over
two days.
• The first day, you’re going to get familiar with the
procedure and run a practice lab.
• The second day, you’re going to select a variable you
think will affect photosynthesis, design the
experiment, and run it.
– Then everything gets compiled in a FORMAL lab report.
• Note: You can submit the mini-Lab 4 part separately as
individuals.
The Concept
• We’re going to use a hole punch to create disks of
spinach.
– Regular, grocery store variety spinach.
– In case you’ve never tested it, spinach leaves will
generally float under water.
• We’ll then force a solution of sodium bicarbonate
(NaHCO3 – baking soda) into the leaf cells to get
them to photosynthesize.
– The NaHCO3 provides the source of carbon in lieu of
carbon dioxide.
The Concept
• In the sodium bicarbonate solution, the leaves will
first sink.
• However, as photosynthesis begins, oxygen will
build up around the leaf, causing it to become
buoyant and float.
– Sound familiar?
• You’ll run this for 10 disks and create a graph at
the end showing the number of disks floating as a
function of time.
The Procedure
• Prepare a sodium bicarbonate solution and pour it into
a cup so it’s about 3 cm deep.
– It’s 1 gram of baking soda (NaHCO3) in 100 mL water.
• THIS IS DIFFERENT FROM YOUR LAB MANUAL!
– Label the cup “with CO2.”
• Fill another cup to 3 cm deep with water.
– Label the cup “without CO2” – it’s your control.
• For each cup, add a drop of dilute liquid soap and swirl.
– DO NOT generate suds. That’s too much.
– The soap will help us get the sodium bicarbonate solution
into the leaf.
• The soap is in the plastic stock bottle.
The Procedure
• Punch at least 10 disks of spinach for each cup.
– Be careful to avoid thick veins in the leaf.
• Your lab manual has details on how to get a good disk.
• Use a syringe to remove gases from the leaves and
replace it with bicarbonate solution.
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Remove the plungers and put the leaves inside.
Push the plunger in almost all the way.
Pull in 5 mL of sodium bicarbonate/soap into one syringe.
Pull in 5 mL of water/soap into the other syringe.
Suspend the leaves by tapping on the barrel.
Get rid of the air by pushing the plunger back in, slightly.
The Procedure
• Now to create a vacuum:
– Put your finger over the end of the syringe.
– Pull the plunger back (suction, anyone?) and hold for 10
seconds.
– While holding, swirl the syringe/leaf disks.
– Let the plunger slowly spring back.
• No spring? Try again.
– With your finger still over the end, push in a little,
“compressing” the liquid and leaves.
• Disks not sinking? Try again till they do.
– The disks should sink once this is done, since you
replaced their air spaces with solution or water.
Creating a Vacuum
You’re
replacing
the air
spaces here.
The Procedure
• Pour the whole syringe solution into the cups.
• Put the cups under a light source and start a timer.
– Record the number of floating disks after each minute.
• DON’T record the time it takes for each to float.
• Swirl disks that may stick to the sides of the cup.
– Continue till all of the disks float, unless it gets, like,
ridiculously long.
– Determine the median amount of time for half the disks
to float.
• Record this info in your notebook.
Your Procedure
• For Day 2, you’ll be designing your own experiment.
• Consider what variables could affect photosynthesis:
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Disk size.
Solution pH.
Light intensity or color.
Temperature.
Soap amount.
Leaf type.
Other?
• Write your hypothesis before you conduct the test.
Lab Reports and Graphs
• Make a graph of your average number of disks
floating at a certain time (for each treatment).
• To be clear:
– The mini-lab can be submitted as its own packet.
• It should not be mentioned in the formal lab report.
– The main lab (Lab 5) should be submitted as a
single formal lab report from your entire group.
• Use the standard formal lab report rubric.
Okay then.
• Questions?