1. Lab 5 – Photosynthesis Mmm…spinach.

2. 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.

3. 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)

4. 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 R f values. Actually, this is a bit like gel electrophoresis, isn’t it?

5. 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.

6. 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. Cut/prep the filter paper like shown: Paper Clip Hook Spot

7. 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.

8. 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: R f 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 R f values – that is, closer to 1 – indicate greater solubility in the solvent. More info in your lab instructions.

9. 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.

10. Lab 5 – Photosynthesis Do you remember the photosynthesis equation? 6CO 2 + 6H 2 O  C 6 H 12 O 6 + 6O 2 If you wanted to measure the rate of photosynthesis, what reactant(s) or product(s) do you suppose would be most feasible to track? – O 2 produced. – CO 2 consumed. For this lab, we’re going to measure the production of O 2.

11. 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.

12. 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 sink under water. We’ll then force a solution of sodium bicarbonate (NaHCO 3 – baking soda) into the leaf cells to get them to photosynthesize. – The NaHCO 3 provides the source of carbon in lieu of carbon dioxide.

13. 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.

14. The Procedure Prepare 300 mL of 0.2% sodium bicarbonate solution and pour it into a cup so it’s about 3 cm deep. – This is a solution % by mass equation, so 0.2% of the overall mass should be baking soda. Use the provided weigh boats. Yes, I know, this is technically not correct but that kind of precision is unnecessary. – Label the cup “with CO 2.” Fill another cup to 3 cm deep with water. – Label the cup “without CO 2 ” – 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.

15. 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. – 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 into the other syringe. – Suspend the leaves by tapping on the barrel. – Get rid of the air by pushing the plunger back in, slightly.

16. 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 spring back. No spring? Try again. 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.

17. 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.

18. Your Procedure For Day 2, you’ll be designing your own experiment. Consider what variables could affect photosynthesis: – Disk size. – Solution pH. – Light intensity or color. – Temperature. – Soap amount. – Leaf type. – Other? Write your hypothesis before you conduct the test.

19. 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.

20. Okay then. Questions?