Question: "Transmittance is measured in a percentage, which is easy enough, but what are the units for absorbance? I have a Bausch and Lomb Spectronic 20, and corresponding absorbance numbers are below the percentage transmittance. One hundred percent transmittance is 0 absorbance, 50 percent transmittance corresponds to .3, and 25 percent to about .6. What units go after these numbers, and what do the units stand for? I just know a student is going to ask me this."
Answer: "Absorbance is a ratio—there are no units. This is the short answer. I'm sure someone will send a long answer."
—Israel Solon, Greenhill School, Dallas, Texas. 1/27/99
Question: "Why is it essential that the beaker containing the solvent and paper must be covered?"
Answer 1: "You cover or else the chromatography solvent (very volatile pet ether + acetone) will evaporate without your leading edge getting very far. Covering keeps the environment within the test tube 'humid.'"
—Jerry Burke, St. Mary's School, Medford, Oregon. 11/7/99
Answer 2: "I always work with it under my fume hood.... I insist upon it with my students. If you don't have one in your room, make sure that there is plenty of ventilation. I find that it can evaporate out of my chromatography jars, even when I have the lids on!"
—Jo Ann Burman, Andress High School, El Paso, Texas. 11/7/99
Answer 3: "The answer is far closer to the notion of pressure than it is with toxicity. Chromatography on any support, whether paper or thin layer, involves partition between components in the solvent. Consider wanting to separate two amino acids, one with a water soluble R group, and one with an aromatic, relatively water insoluble group. Let's separate them in a solvent that is mixture of phenol and water using paper chromatography. Ask yourself, which of the two components in the solvent will be more attracted to the cellulose that makes up the paper? Paper gets 'wetter' with water than with phenol. (Remember, H-O-H vs. benzene ring-O-H.) When the two are allowed to wet the paper, the water pushes the phenol off where both can attach to the glucose molecules that make up the cellulose in paper. As the solvent wicks either up or down the paper, the solvent front will be enriched with the less water-soluble group, phenol in this case. The mixture of amino acids will also separate depending upon each acid's relative solubility in phenol relative to water. I haven't looked at the lab, but you can easily detect the separation of the two acids by their reaction in ninhydrin.
Now, to the covering of the vessel. If either component of the solvent evaporates, then the composition of the mixture is not a constant, and highly variable results will occur. The Rf values will differ from the standards, and will differ from one student to the next because of differences in their local environment, i.e., sun shining on the setup vs. temperature differences vs. location of drafts, etc. So anything that alters the vapor pressure of the solvents will introduce variability in your results.
What is established on the chromatogram is a gradient of the solvent components, with the more nonpolar components moving toward the front and the more polar components staying behind. That's what separates the two amino acids from each other. Remember the aphorism: Like dissolves like. The amino acids that are more nonpolar, less water-soluble, more hydrophobic will move toward the solvent front while those with the opposite character will stay behind. If the solvent that is toward the solvent front evaporates, the gradient then becomes altered. So, to optimize the reliability of your system, reduce the volume of the chamber, put pads around the inside that are saturated with the solvent, and put a lid on soon after you insert the chromatogram."
—Neal Anderson, Fitchburg State College, Fitchburg, Massachusetts. 11/7/99 and 11/8/99
Equipment and Supply Modifications
Question: "Which brand of spectrophotometers do you suggest purchasing for the AP labs?"
Answer 1: "I have excellent luck with the Spec 20. I bought a newer one from Flinn Scientific and it works well also."
—Robin Groch, San Ramon Valley High School Danville, California. 1/08/01
Answer 2: "If you can afford it, I would recommend that you get at least three or four specs. If you get a professional quality unit, they should be trouble free for years. We use our units both in AP Biology and in AP Chemistry, and we also teach about the technology of the units. You can structure several experiments around these. We have Milton Roy Spectronics (20s and one digital unit) and (knock wood!) they have been flawless for over a decade. They produce reliable, repeatable data."
—Bruce Faitsch, Guilford High School, Guilford, Connecticut. 01/09/01
Answer 3: "I really like to use spectrophotometers because it lets students work with stuff similar to the real-world labs and can be used by the chemists."
—Dave Hall, Martin County High, Stuart, Florida. 1/10/01
Question: "Does anyone have a solution for the coagulation that occurs in the boiled chloroplast cuvette? The results my students get are always disappointing because suspended clumps drift to the bottom, which reads as an increase in transmittance in that cuvette."
Answer 1: " You can avoid this problem by NOT heating the chloroplasts to boiling. Just get them very warm, enough to denature the proteins, but not enough to cause coagulation. A temperature of about 45° C will do nicely. I often check the chloroplast suspension by sticking my finger in the suspension as it is heated in a pan of water on a hot plate. When it gets to the point of being too hot for comfort, the chloroplasts will be nicely denatured, but don't coagulate."
—Jeff Smith, Indiana Academy, Muncie, Indiana. 1/29/99
Answer 2: "Shake well before reading. This should be done with all the cuvettes. The coagulant should remain suspended long enough for a reading, as long as this doesn't take all period."
—Israel Solon, Greenhill School, Dallas, Texas. 1/29/99
Question: "What are the best leaves to use for chromatography?"
Answer 1: "Use Magnolia leaves. They work great! Just have the students place them on the paper, lower epidermis against the paper, and press the pigment onto the paper. I like to use the rounded tips of scissors or the blunt end of forceps to get a nice even line. Another good thing is that it only takes a couple of leaves for the entire class."
—Bobbie Hinson, Providence Day School, Charlotte, North Carolina. 2/8/99
Answer 2: "We had great luck this year using geranium leaves and quarters. We lay a geranium leaf on the chromatography paper about 3 cm from the tip, rub the serrated edge of the quarter across it as many times as we can without shredding the paper, and move the leaf after each 'rub.' We put the chromatography paper in a test tube with petroleum ether solvent, enough so that it wouldn't evaporate too quickly; then cover the test tube. We had spectacular chromatograms this way."
—Eloise Farmer, Torrington High School, Torrington, Connecticut. 2/8/99
Answer 3: " I've used mulberry leaves for this pigment chromatography lab. The darker the leaf, the better. It works best if you get a really dark line."
—Jo Ann Burman, Andress High School, El Paso, Texas. 2/8/99
Tip: "I had dropped the photosynthesis lab when I first started teaching AP Biology out of frustration. When the lab manual first came out (OK, I'm dating myself here) and the lab was included, I sunk into a short-lived funk (we were on shortened periods that day so I didn't have a full prep period). Then I had an epiphany.
The key to the success is in the chloroplast preparation. Follow the instructions in the Edition D teacher's edition to the letter. I put my blender and beaker in the freezer the day before, and the glucose in the fridge. Use only the nice, crisp, green parts of the spinach. Ten seconds of blending, 30 seconds of rest, 10 seconds on, 30 seconds off, 10 seconds on. Filter through the cheesecloth and put on ice in the dark. The biggest problem with preparation is over blending. I have had success with this lab ever since.
The directions themselves for this lab are a little unclear. Have the students make a flowchart before starting, as most of them do not interpret the directions properly, especially since they pertain to putting chloroplasts in the blank before setting 100 percent transmittance and then adding them to all the other tubes at the same time and running them together. My biggest problem has been with the DPIP going from purple to clear too soon—by the first reading—chloroplasts are too hyper!
If you do not already subscribe to the Biology Place (www.biology.com), this would be a good time to try out the two-week free trial offer. The students can use the tutorial and quiz on the photosynthesis lab and will get a better understanding. It was after my trial that I decided the site was definitely worth the money (I receive no commission for this ad)."
—Israel Solon, Greenhill School, Dallas, Texas. 10/17/99
Tip: "Thomas W. Englemann demonstrated in 1883 that in eukaryotic cells photosynthesis occurs in chloroplasts and only in chloroplasts. This was the first case in which a specific physiological or biochemical function could be ascribed to a particular organelle. A number of different plants may be used as sources for chloroplasts; spinach is usually the most readily available and most dependable. Get the freshest spinach available. Frozen spinach will not work. Badly wilted or bruised spinach may not work, or may give such low rates that the reaction will take a long time to complete. It usually helps to soak the spinach in cool or cold water under a light for a few hours. Do not let the spinach leaves get hot while under the light.
NOTES AND COMMENTS: Isolation of chloroplasts
Why use spinach?
- Cheap and available virtually all year round.
- High number of chloroplasts per cell.
- Cells and chloroplasts stay viable for relatively long times in dark and without O2.
- Cells relatively easy to disrupt but chloroplasts are relatively tough.
- Has been used for such experiments for almost 50 years; protocols for chloroplast isolation in part specific for spinach. Soak the spinach in cool water under a light for a few hours. Don't let the spinach get hot. Why? To provide remaining living cells or chloroplasts the energy necessary to repair damaged/deteriorating chloroplasts, perhaps to produce new chloroplasts, and to build up pools of redox intermediates in thylakoids and lamellae.
Why worry about keeping things cold, i.e., in a chilling blender, sucrose solution, etc.? Delays deterioration of chloroplasts and loss of activity. The physical and chemical environment changes as cells are broken. Acids, hydrolytic enzymes, and waste materials are released from broken vacuoles; supplies of metabolites are cut off. All molecules (structural, enzymatic, electron transport) have limited lifetimes. Chilling makes them last a little longer.
Why use 0.5 M sucrose solution as an isolating medium? To prevent osmotic shock and lysis of chloroplasts. Chloroplasts are removed from 0.3 M cytoplasm, into 0.5 M sucrose solution; thus into a hypertonic medium, so chloroplasts shrink slightly rather than swell.
NOTE: Edition D (1997) of the AP Biology Lab Manual contains an important omission. Students are not explicitly told to add unboiled chloroplasts to cuvette #1 (the blank), although this is indicated in Table 4.3 (p. 49). It is VERY IMPORTANT to add unboiled chloroplasts to cuvette #1 before using this blank to set the Spec 20 to 100 percent transmittance. If unboiled chloroplasts are not in the blank cuvette, all the other readings will be nonsense. Set the Spec 20 to 605 nanometers (use the wavelength control knob, on top of the instrument). Some older versions of this exercise suggested using 660 nm, but we have found that 605 nm works better. Adjust the 'zero' setting with no cuvette in the sample holder. Then insert cuvette #1 (containing the mixture shown above, INCLUDING three drops of unboiled chloroplasts) and adjust the dial to indicate 100 percent transmittance. Then insert each of the other tubes to read the absorption of the DPIP."
—C.O. Patterson, Texas A&M University, College Station, Texas. 10/17/99
Question: "It says to set the spectrophotometer to 605 nm. Why that wavelength, and how important is it to be exact? Would it work at other wavelengths as long as you were consistent? Looking in the teachers' guide, I see spinach extract has its lowest absorbance around 600-605nm. So I'm thinking maybe we set it there in order to let as much light as possible initially hit the DPIP? Is this correct? Also, can you calibrate using distilled water, or should you always use ethanol? Any 'light' you can shed on this whole part of the lab would help."
Answer 1: "In choosing the wavelength setting for the spectrophotometer in the photosynthesis lab, you are trying to find a wavelength at which you can most easily see changes in absorbance by DCPIP against a background of absorbance by chlorophyll. So you are not necessarily choosing the wavelength at which DCPIP absorbance is greatest, or the wavelength at which chlorophyll absorbance is lowest. Instead, you are choosing the wavelength at which the 'difference' between the absorbances is greatest. In other words, you compare the entire absorption spectrum of DCPIP with the entire absorption spectrum of chlorophyll, and then choose the wavelength at which the spectra are furthest apart (show the greatest difference). This wavelength is 605nm. Of course, in making this choice, you must pick a wavelength that is strongly absorbed by reduced DCPIP. So it won't work to pick a wavelength down in the blue region of the visible spectrum—DCPIP doesn't absorb blue much at all. Instead it reflects blue wavelengths, which is why it appears blue to our eyes. Other wavelengths will work, but the sensitivity of the measurement is less at these other wavelengths. You are correct—consistency is important, and as long as you use the same wavelength (that is, any wavelength which DCPIP absorbs) throughout the experiment, you should be able to get results. But it just makes the whole procedure so much easier if you choose the best wavelength to begin with. It is not necessary to be 'very exact'—the setting could be 602nm or 607nm but, again, it just makes things easier if you make an effort to set it at the best wavelength when you begin.
I am a little unsure what you mean about calibrating with water versus ethanol. You set the zero on the spec with nothing in the holder—no cuvette, no liquid, nothing. Then you set the 100 percent transmittance with a cuvette containing phosphate buffer, distilled water, and unboiled chloroplasts (reaction tube #1 in the 1997 edition D of the lab manual). Unfortunately, edition D is not very clear on how to set the 100 percent transmittance. It is indicated in Table 4.3 (p. 49) but never explicitly stated in the written step-by-step instructions. It is absolutely necessary to include the correct volume of water, the correct volume of phosphate buffer, and the correct amount (three drops) of unboiled chloroplasts in the cuvette used for setting 100 percent transmittance (setting the blank). I don't see any way that ethanol would work at all. But maybe I am misunderstanding the question."
—C.O. Patterson, Texas A&M University, College Station, Texas. 1/26/99
Answer 2: "You set the wavelength at 605 nm because that is the wavelength for which DPIP has its greatest absorbance (darkest color). As for the filter setting, I have two specs that don't have a filter setting and one that does. Mine has two settings only, 300-600 and 600-900. We set it on the latter (600+). When you are using the Vernier probes and the colorimeter, you use the red setting."
—Bobbie Hinson, Providence Day School, Charlotte, North Carolina. 10/31/00
Tip: "Regarding the photosynthesis lab and the spinach shake, I have found that the amount of sucrose solution is never enough to cover the blades. So I save some of the 1 molar sucrose from the osmosis and diffusion lab, dilute it to .5 molar, and add to the kit's bottle."
—Tricia Glidewell, Marist School, Atlanta, Georgia. 2/1/99
Tip: "Since we only have one Spec 20, I had to get really creative. I had each group of three students do a separate part of the lab and plan an assembly line. One kid would calibrate with the blank cuvette, another would put the next cuvette in, etc. It went quickly and although we did not get large amounts of data, it exposed the kids to the procedure. They felt very confident answering the free-response question on photosynthesis and scored well on it, so I guess it worked."
— Eloise Farmer, Torrington High School, Torrington, Connecticut. 10/17/99
Tips: Here are some useful tips:
- The chloroplasts don't have to be boiled. Heating them to about 40° C will inactivate them without causing the clumping and the problems with the particles interfering with the spec readings.
- If the students don't get a measurable change in the cuvette in the light in the first five minutes, they probably won't. Have them add more drops of chloroplasts, reblank the spec, then start their readings over again. Its better than going the whole 20 minutes and then finding out they don't have any data. The problem is often due to the chloroplast suspension being too weak. Adding more chloroplasts corrects this.
- Most overhead projectors are bright enough to use as your light source. Aim the beam down on the counter top, place the cuvettes in the light, and you won't need a heat trap.
- Have one group isolate the pigments from a leaf that has turned color in the fall. You'll still have the accessory pigments, but the chlorophylls will be gone (which is what we tell the students should be happening, but its nice to confirm it experimentally). " Jeff Smith, Indiana Academy Muncie, Indiana. 10/18/99
Tip: "...For the boiled chloroplasts, you must vigorously re-suspend with pipette (Pasteur or disposable) until the clumps are broken up. Also, if the students don't re-suspend their curvettes/test tubes by tipping back and forth, they seem to get very low readings because the chloroplasts have settled to the bottom. There is a great resource from Flinn Scientific with some labs using the spectrophotometer, Flinn Scientific Spectrophotometer Laboratory Manual, Catalog # AP8685A. The labs included are:
- Analysis of chlorophyll using the spectrophotometer
- How pH affects the growth rate of yeast
- Population growth in E. coli
- DNA extraction and determination
- Relative determination of pigment content in changing leaves"
- Robin Groch, San Ramon Valley High School, Danville, California. 3/9/00 Trouble Shooting and Cleanup
Question: "We just finished the photosynthesis lab and unfortunately it didn't work even though the spec was set right and the kids appeared to measure everything correctly. Is there a trick to making the unboiled chloroplasts in light photosynthesize?"
Answer: "Was your DPIP fresh and how did you prepare the chloroplast suspension? If the sucrose solution isn't absolutely cold, the heat of the blender will deactivate the chloroplasts. I have done this lab with no problems for several years (as long as I can find at least one working Spec 20 in the department). Everything has to be kept as cold as possible until you are ready to use it. Also, did you use a heat sink and activate the spinach with the light for a couple of hours? Even without the Spec 20 results, my students could see that a change was taking place because the chloroplast cuvette kept getting lighter and lighter in color. If you see this happening and it still doesn't register on the Spec 20, there is something wrong with the machine. Those things can get really finicky sometimes. Also, fingerprints on the cuvette and any of a number of other things can get you."
—Jo Ann Burman, Andress High School, El Paso, Texas. 1/28/99
Question: "For the second year in a row measuring the rate of photosynthesis using spinach leaves, DPIP and a Spec 20 have failed to show any activity with spinach chloroplasts. Since I followed the directions exactly for producing a chloroplast solution and knowing my Spec 20s work properly, I am left to wonder about DPIP. I use a 0.1 percent DPIP solution from Carolina Science and Boreal Labs. Should this be diluted further? I am at a loss for this lab."
Answer: "Mine has never failed to work. There is an error in the AP manual I remember and the College Board has it listed on their site (it may be out of date). It involves one of the controls if I remember correctly. I made up my DPIP about 10 years ago according to directions in the original teacher's manual and I store it in a brown bottle and I'm still using it years later. You can see the change occurring easily.... It fades quite dramatically in the 'exposed to light' batch. I put my spinach under the grow light overnight before the lab and blend them in sucrose solution and filter the puree through cheesecloth. I have always had textbook-perfect results unless I had clumps in the boiled ones. Then that one had some erratic points. I have had groups who will reach for the wavelength knob and turn it instead of the knobs on the front of the machine. That will really skew the data if you don't catch it. We usually have to start over if I don't catch that, so now I put a piece of masking tape over that knob after we choose the correct wavelength to prevent inadvertent resetting of the wavelength."
—Charlotte Freeman, Girls Preparatory School, Chattanooga, Tennessee. 11/18/99
Tip: "I use the Spec 20 and it does well even though the groups stack up waiting a turn; I got a second one this year and it helped. One thing I did this year was make my chloroplast suspension too concentrated (I assume). The DPIP was clear by the end of the first five-minute reading and then the graphs flattened out. I assume the rxn was running so fast that it completely reduced all the available DPIP. If one looks at only the first five minutes of data, the results were as predicted. After that, they were all over the place."
—Charlotte Freeman, Girls Preparatory School, Chattanooga, Tennessee. 3/8/00
Question: "How do I dispose of the used petroleum ether/acetone mixture? Any suggestions?"
Answer: "Don't dispose of it, keep it for next year! Tightly cap the bottle and store it with your other organic solvents. I have used the same bottle of solvent for two to three years with excellent results."
—Franklin M. Bell, St. Mary's Hall, San Antonio, Texas. 1/26/99
Conducting Lab Using Probes and Computer/Calculator
Tip: "I have used a modified version of the lab using PASCO CBL. The range of colorimeter is more limited, but the probe is much cheaper (about $2,000 less) than a spectrophotometer (which our school does not have). Students were able to collect decent data."
—Heda O'Brien, The Bullis School, Potomac, Maryland. 3/08/00
Tip: "The Vernier colorimeter and CBL/calculator option works great for the photosynthesis lab. The colorimeters are only about $100, so a whole class can be supplied with them for less than the cost of one Spec 20. The only catch is that it only runs at three wavelengths. That's not a problem for the DPIP. However, you cannot use it for making an absorption spectrum (unless you're satisfied with only seeing the absorption at three wavelengths).
In my class I use the Vernier colorimeters for the DPIP part of the lab—each group can work with their own set up. We also have two Spec 20s that groups take turns using in order to determine the absorption spectrum. As Doug pointed out, when you get to the longer red wavelengths you lose some accuracy. I'm not sure if that is true with the newer Spec 20s."
—Bob Goodman, Hunter College High School, New York City. 3/9/00
Tip: "I used the new oxygen gas probe for a photosynthesis lab and in less than six minutes using three leaves snipped off a house plant I had the neatest curve you have ever seen. I have also used the CO2 probe from Vernier with excellent results in photosynthesis and other labs. The CBL manual from Vernier in biology or the Biology and Computers Lab Manual is excellent."
—Doug Herman, Iowa City West High School, Iowa City, Iowa. 11/02/00
Answer 4: "If you had time to make full use of the spectrophotometers within your department, that might be more worthwhile. However, having enough colorimeters and LabPros to allow smaller student groupings, and maybe even to be able to purchase some other probes as well would make more sense to me. To maximize each student's experience with the equipment and procedure, I like having students work in pairs as much as possible."
—Paula Petterson, Kent Place School, Summit, New Jersey. 01/9/01
Answer 5: "I love spectrophotometers—but, if I was starting out, I'd consider buying colorimeters rather than a spec for biology. I'm glad I have two specs for AP Chemistry and AP Biology, but it's really hard to have a class of 25 use two pieces of equipment. You can buy about 8-10 colorimeters that work with graphing calculators and data collection devices (CBLs, etc.) for the price of one spec. If your school is utilizing these data collection devices, it is a more student friendly way to go! Additionally, if I did decide to buy colorimeters, I'd definitely consult the appendix of a college chemistry book for the condensed lesson on the full-blown spectrophotometer so that my students knew how the 'real deal' worked. The data you collect with the colorimeter will be as (if not more) accurate than the full-blown spectrophotometer."
—Rene McCormick, Carroll High School, Southlake, Texas. 1/5/01
Answer 6: "I have been really pleased with the results I've gotten with the colorimeter that Vernier puts out to go with TI CBL. I have quit using the Spec 20s and gone to doing all my AP Chemistry experiments with these. They are cheap enough that I have 12 of them and can have students working in pairs."
—Louise Mowbray, Career Center, Lewisville, North Carolina. 1/5/01
Answer 7: "I had a spectrophotometer from Fischer that interfaced with the PC. It never worked right. We have eight Spec 20s to share between myself (AP Biology) and the AP Chemistry teacher. Last year I had a great deal of money to start the AP Biology course and I liked the versatility of the Vernier product line. I already had familiarity with their Serial Box interface and the probes are very reliable. This year I purchased six DataLogger interfaces and two colorimeters. I didn't need more since the AP Chemistry teacher had four colorimeters already. I recently finished the photosynthesis lab using the colorimeters and got great results on my first time (three sections of AP Biology). My advice is to stay away from the SCI KIT/Milton Roy products. We had our specs tuned up and the company wouldn't even touch them. The DataLoggers are so versatile that you can use them with PCs, TI8X, or better, and you can even remotely gather data as you would with the CBLs. The PC interface is very intuitive and the support from Vernier is fantastic. The AP Chemistry teacher is now going to return the CBLs she purchased and get the DataLoggers instead. I highly recommend the Vernier line of products. PASCO also puts out a similar line of interfaces and probes."
—Harry Padden, Washington Twp High School, Sewell, New Jersey. 1/6/01
Answer 8: "I would concur with those who have had good data from the Vernier colorimeters. They can be interfaced to a computer with the serial box interface or interfaced to a TI-graphing calculator with the CBL or CBL 2. Even better, with a LabPro they can be interfaced to a computer OR calculator. My class results for the AP photosynthesis lab are beautiful. I do have some Spec 20s, but the Vernier colorimeters give nearly equally reliable data. As was mentioned in an earlier message, at about $100 each, you can buy a class set of Vernier colorimeters for the price of one Spec 20 (or its equivalent). The only disadvantage is that with three wavelengths you cannot do a meaningful absorption spectrum of a sample. I use Vernier colorimeters for some other experiments though, and they are great. I would be wary of some of the Carolina products, which I feel are marked up substantially above their value."
—Bob Goodman, Hunter College High School, New York City. 1/6/01
Answer 9: "I see uses for both pieces of equipment. My vote (IMHO) is to use the spec since I also like to work a little bit with absorption, transmission, and emission ideas. I tie them into light, photosynthesis, and vision; not too much time here, but enough that the spec seems like a natural extension. So, if you might have a grant, that would be my vote if you're thinking of including ancillary topics. Since you can't adjust the 'black box' colorimeter (and peek inside) beyond a few set wavelengths, then it's tougher to get the students to understand it. Now that I've stuck my neck out, it might not surprise you to know that of the specs, I even like the 'older' style with a dial rather than the digital read out. For me (IMHO) there is value to seeing the dial move, and to seeing the inverse relationship between absorbance and transmission labeled on the dial. On the other hand, I certainly see the value of the colorimeter; it might be fine, if your real goal is to get data, or if integrating data into CBLs is important to you and your school. I find that they both give good data (Vernier is the one I have used). The preset wavelengths can be used for a variety of labs, including bacterial growth (by turbidity) that I recently worked on. Since creating a growth curve was my goal here (biology 1), the quick colorimeter was great. Now, one political tip: If after reading all the postings and you're interested in both, you could get the expensive specs now while the money is available, and gradually get the less expensive colorimeters over a period of years if you want them for other uses."
—Elliot Mitchell, Tower Hill School, Wilmington, Delaware. 1/09/01
Tip: "On the photosynthesis lab, the teacher's manual on page 22 (Edition D) gives a satisfactory protocol to prepare a colorimeter in place of using the Spec 20. Eleven tubes are prepared for the students to use as comparison. This has worked well for me when my spectrophotometer quit working. I would not, however, add any food coloring. When we tried this, the results were much harder to determine than it would have been if left alone. The color difference is minimal. In fact, their experimental tubes were more blue than blue-green. On their graphs, most students used the test tube numbers 1 through 11 on the y-axis to show the percent of transmittance, but some related #11 tube to 100 percent transmittance and adjusted the rest accordingly. Its at least worth checking it out and letting the students try it. There are yeast labs to show fermentation (I do this as an additional lab) that are much easier if you have no time to make substitute equipment (aquarium rocks for glass beads, beans instead of peas, soda lime in the respirometers in place of KOH, etc.) for the cell respirometer lab. But there are also protocols for respirometers made from Gatorade bottles, and you can use crickets, or whatever, to measure cell respiration. The first year is hectic to say the least; sometimes graphing and analyzing 'ideal data' from the teacher's manual is an option rather than leaving the lab out. In my opinion, both are quite important labs to address."
—Lynn Cook, Putnam City West High School, Oklahoma City, Oklahoma. 11/12/00
Question: "I have some great new computers and Vernier colorimeters... to do part of the photosynthesis lab. The protocol gives directions for the phosphate buffer but doesn't dilute it to 0.1M as per the instructions of the AP Teacher's Manual. Opinions? Does the molarity of the buffer matter that much?"
Answer: "If you follow the instructions in the lab manual for mixing up the phosphate buffer, you will get a 0.1 M buffer. When mixed with the DPIP and distilled water in a 1:1:3 ratio, the experiment works great. If you choose to use the prepared buffer and DPIP solutions available for order from Carolina, you just need to keep the 1:1:3 ratio of drops or mL."
—Gretchen Stahmer DeMoss, Vernier Software & Technology, Beaverton, Oregon. 12/11/00
Alternative Lab Ideas
Question: "To show that chlorophyll emits red light, I followed the directions suggested as further study for lab 4... but I can't get any redness when the chlorophyll is exposed to light."
Answer 1: "Try extracting the chlorophyll with 95 percent ethanol—that always works well for me. You can just soak the spinach leaves in the ethanol for awhile, or grind some in a mortar and pestle along with some ethanol, then drain through cheesecloth. Be sure to use 95 percent EtOH and not drugstore isopropyl (70 percent), though some drugstores carry 91 percent EtOH, which will work.
"For a brilliant red color, try shining a 'black light' (longwave UV) on the chlorophyll solution while all other lights in the room are turned off. This works great! Putting the chlorophyll in front of the overhead or any other strong white light will also work, but the effect is not as striking as the black-light version.
"It is also instructive to shine the black light on a chloroplast solution (from lab 4, part B) as it will not fluoresce. Challenge the students to explain why one fluoresces and the other does not."
—Robert Dennison, Jersey Village High School, Houston, Texas. 10/7/99
Answer 2: "To show the florescence of chlorophyll, you need a bright light point source such as a halogen bulb flashlight (Mag-light®) and dark room. Shine the bright light into the chlorophyll suspension and view perpendicularly to the axis of the light beam. In other words, if you shine your light along a north to south axis, look for florescence along an east to west axis."
—Brad Williamson, Olathe East High School, Olathe, Kansas. 10/7/99
Answer 3: "I soak green grass in acetone in a beaker overnight and then decant the green solution into a test tube and hold it in front of a projector light. It works!"
—Charlotte Freeman, Girls Preparatory School, Chattanooga, Tennessee. 10/10/99
Answer 4: "An earlier message suggested putting acetone in a blender to do chlorophyll extraction from frozen spinach. PLEASE DO NOT DO THIS!!! Acetone is highly flammable—flash point is -18° C. Acetone is also very volatile, so if you are using acetone, you almost certainly have acetone fumes in the area. I have never seen a blender motor that did not make sparks. Therefore, if you use this suggested technique, you have 100 mL of a volatile, highly flammable (potentially explosive) liquid just a few inches from unshielded electrical contacts. Sure, you may get away with it 100 times... but what about the 101st time, or what about some other classroom teacher who isn't so lucky? Don't do it!!"
—C.O. Patterson, Texas A&M University, College Station, Texas. 2/8/99
Answer 5: "Fluorescence of chlorophyll. Extract the chlorophyll from any leaves. I just dip them in boiling water for about one minute Tear them into pieces and place in methanol or isopropyl or whatever organic solvent is cheap and available. Boil in a water bath. Pour off the chlorophyll solution into a boiling tube. Hold the boiling tube in front of your overhead projector. (Aside: yes, Israel, it's on!) The students directly in front of the projector will claim that the extract is green. Students at about 45 degrees will claim that it is red. If you have students at 90 degrees to the direction of projection of the light, they will claim that the solution is split vertically—one-half is green and the other is red."
—Peter Gardiner, St Michaels University School, Victoria, British Columbia, Canada. 10/17/00
Absorption Spectrum of Pigments
Tip: "Another activity with the Spec 20 and the photosynthesis lab is to cut the bands from the chromatograms. For example, take all of the carotene bands, soak them in a small amount of the solvent, then put the solvent in a test tube and run an absorption spectrum on that particular pigment. Do the same thing with all the other pigment bands. If each student in a class of 20 makes a chromatogram, there is plenty of pigment to extract."
—Bobbie Hinson, Providence Day School, Charlotte, North Carolina. 3/9/00