Overview: Chlorophyll-a and Chlorophyll-b are the green pigments that were used in the

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1 Extension: Spectrum just Fluoresce Me The purpose of this activity is to explore more into the topic of wavelengths and reflectance. This will be shown onto different kinds of colored paper, olive oils of different brands, and other vegetation. One will view it by either using a laser with the help of spectrum glasses, or UV black light, and/or a spectrometer. Overview: Chlorophyll-a and Chlorophyll-b are the green pigments that were used in the previous activity. These pigments are usually found in leafy green plants such as spinach. It has the structure as shown below. The only difference between the two groups is that b has a formyl (-CHO) group as oppose to the methyl (designated Y: CH3) group. chlorophyll-a chlorophyll-b Chlorophyll and other pigments (b-carotene, lycopene, or even Vitamin A to name a few) can be found in other organic materials, such as purple cabbage, olive oil, red wine, white wine, tonic water, bluing that is used in laundry detergent, and even tomato paste. From the previous activity one may have noted that the spinach mixture was dark green and translucent. When a UV black light was shined onto the mixture, the material would fluoresce (glow) as a thick murky dark red liquid that one cannot see through it. The reason for not being able to see this under visible light the murky red color is due to the mixture s chlorophyll absorbing some of the wavelengths of visible light of 400 to 500nm (blue region) and 600 to 700 nm (red region). By shining UV light on a mixture, the mixture s atoms will absorbed some of the UV s photons. This will excite an electron of the mixture s atoms to jump to a higher energy level from the ground state. When the excited electron drops to a lower energy level or the

2 ground state, it will emit a photon of light. This emitted photon of light may have a longer wavelength compared to the UV light s photons. By using a UV light source, we will be able to test a variety of mixtures for fluorescence and the color (wavelength/energy) of their photons. Second, using the idea of energy excitation, a laser of different wavelengths will be shined through the mixture to see if the laser s wavelengths will change and note the spectrum of the material using a diffraction grating. Then a spectrometer will be used to see the actual curve of the Absorbance to the wavelength to conclude if the above findings make sense. And finally to view solid material, paper for this activity, repeating what was done in the first and second activity, if certain paper fluoresces and what would it spectrum be. Materials: UV black light, laser pointers (red, green, and blue), RED Tide USB650 spectrometer (attached to a Lab Quest or a LabPro/Lab Quest mini acquisition set), white paper, colored construction paper or pastel sticky notes, and fluorescent paper or neon sticky notes, a group of spectrum glasses one for each person who is doing the activity, olive oil, three small clear beakers, some kind of extract of green leafy vegetation (kale, greens, spinach, get wild and choose what is available in the store), and rubbing alcohol. If one has a limited budget, just get one set of lasers to use for the whole class and have it as a class activity. Also, check to see what the safety regulation is for bringing laser diodes on campus; for an example, in Texas one has to wear laser safety glasses if the lasers are above a Class IIa. That is the reason for the prices on the green and blue lasers being expensive to purchase, but if you do not meet this regulation then a Class IIIb laser should be fine but still follow safety guidelines of making sure that the laser is not pointing into the eyes of another individual. Extraction for Green Leafy Vegetation: Obtain spinach at the fresh produce aisle one can use other leafy greens if desired. Put on goggles and some vinyl gloves to protect the hands because the spinach may stain and be careful around others and your clothing. Pull about 8 fresh leaves (about a gram) without the steams and tear it into little pieces into the mortar. Pour about 20 ml of 91% of rubbing alcohol (Isopropyl Alcohol and one can use 70% but remember which one is being used because one will need some of the alcohol for the calibration of the spectrometer) and a little play sand into the mortar (this is to assist in the process mulching the spinach). Carefully and slowly grind the spinach in the alcohol. Let it stand for about 10 minutes. Fit a filter paper into a funnel and place it into a clean beaker. Pour the mixture into a funnel fitted with the filter paper. The funnel will filter the extract into a clean beaker. Take a pipette and place some of the extract into the cuvette.

3 The beaker will be used during the UV light and laser spectrum activities and the column will be used in the spectrometer activity. UV Black Light Coleparmer.com (EW UVL-4 Handheld Mini Ultraviolet Lamp, 4-watt, 365 nm) $33.00 Spectrometer Vernier.com (VSP-UV Vernier UV-VIS Spectrophotometer, nm ±2.0 nm) $ Cuvettes Vernier.com (CUV Plastic Cuvettes (visible range), 20 lids, 100/pk) $15 Note: The above cuvettes work fine as long as the ribs are not facing the diodes in the spectrometer but one can also purchase fluorometer cuvettes from of the 3.5ml, 9012 or 9014, where the price range from $42.50 to $91.50 for 500/pk and $19.50 for 500 caps. Red Laser pointer Vernier.com (LASER Red-Class 2 <1mW) $19.00 Green Laser Pointer Shop.Z-Bolt.com (BTG-2 <1mW Class II Green Laser Pointers UK, Australia, EU Approved; - one can also purchase through Amazon, 532nm) $68.00 Freemascot.com (1mW-200mW 532nm Green Laser Pointer Pen + Match-Lighting (Black) Choose: Output Power 1mW) $9.99 Carolina.com ( Green-Class II <1mW, 532nm) $ Blue Laser Pointer - Freemascot.com (1mW-50mW 405nm Violet-Blue Laser Pointer Pen (Black) Choose: Output Power 1mW) $12.99 Spectrum glasses one for each student ( D Fireworks Glasses Laser Viewers Buy The Case Special!!, $ for 600 glasses) $0.25 Linear Spectrum glasses one for each student (Diffraction Grating Glasses Linear 1000 line/mm, $ for 1000 glasses) $0.30 Either glasses should work but if one believes that students can get confused by the hatch grating of the spectrum glasses then purchase Linear Spectrum Glasses. Sticky Notes, Fluorescent Colors ( Post-it 1 ½ x 2 Notes, Neon Collection, 100 Sheets/pad, 12/pk) $7.99 Sticky Notes, Pastel Colors ( Post-it 3 x 3 Notes, Pastel Collection, 100 Sheets/Pad, 12/PK) $14.99

4 2 different brands of Olive Oils Grocery Store (example Pompeian Robust Flavor Extra Virgin Olive Oil, first cold press, 8 oz, $5.00 and Badia Extra Virgin First Cold Press Olive Oil, 17 oz, $7). Extra Virgin Light Olive Oil (example Pompeian Mild Flavor Extra Light Olive Oil, 16 oz, $5.00) Tonic Water w/ equine- $1.50 Group Questions to discuss before starting activity two (white-board discussion): 1. What is a wavelength and support the answer with an example? Students should be instructed that they would explain what wavelength should mean and model ways to show different kinds of wavelength. Examples that they may come up with are: 1. Wavelength of an oscillating spring. 2. Wavelength of the string on a guitar or key from a piano and going down the fret or keyboard changes the wavelength. 3. Wavelength of the current on an AC circuit. 4. Wavelength of light (white light would go through between two polaroid s and random pieces of scotch tape between them and different colors would show a kaleidoscope) and it s color associated with the wavelength and it goes through. 2. With just a piece of rope, brainstorm a list of possible ways how one can test ways to affect the wavelength. Students should look at the materials and brainstorm a list of possible factors that may have an effect on wavelength. One is by fixed length and oscillating fast, have about 6 humps to give 3 wavelengths, compared to slow, have it show 2 humps for one wavelength. Another is to change the distance and oscillate to have one wavelength but the length has change. 3. On each of these factors, give an example of the setup you would do to test it. Students should give some examples of a setup of using the lasers to demonstrate wavelength. 4. If mono means one kind or singular and chromatic means produced color, please state what is meant to be monochromatic?. It means one color like all red, all blue, all yellow, etc.

5 Activity One: Viewing mixtures under UV. There are 5 small glass beakers labeled olive oil 1, olive oil 2, olive oil 3, leafy greens, and tonic water. Take the UV and shine it on each of the beakers and note what is seen. Note: Leafy Greens: Spinach it glowed a dense bright red. Olive Oil 1: Pampian Extra Virgin Olive Oil it glowed a dense bright orange. Olive Oil 2: Badia Extra Virgin Olive Oil it glowed a less dense bright orange. Olive Oil 3: Pampian Extra Light Virgin Olive Oil it glowed a dense bright yellowish white. Tonic Water: It glowed a bright bluish white. 1. What mixture gave the most change in color? The leafy greens. 2. Did all the mixtures have the same color? No. 3. Did the olive oils have the same florescence? No. 4. What would account for this? The grade of the olive oil. This is due to the process the manufactures utilize in extracting the oil. Also, when it is light olive oil, they probably did more than one press, LOTS MORE, and the light olive oil could as well just be vegetable oil. Activity Two: Lasers and Spectrums Take a red laser and shine in on the side of the beaker, labeled Leafy Green, where it would go through the mixture to the other side of the beaker. Do this with the other lasers as well. What did you noticed? Did the color of the laser change as it was going through the mixture? Nothing. The laser does not show going through the mixture. This could be due to that the green solution is absorbing the red to prevent it from going through the beaker. Like a colored filtered. Put on the pair of spectrum glasses and view from above. What did you see? What colors are showing when viewing through the spectrum glasses? What is the order after the main color coming from the laser?

6 Reflections of the beaker being hit with laser on the side. Red from the reflection of the laser hitting the side of the beaker. There is just one color, red. Fill in the data into the table below and repeat for the other mixtures. Mixture Leafy Green Spinach Colors Observed for Red Laser glow very little. The red laser does not goes through the mixture. Colors Observed for Green Laser glow. The green laser, as it goes through the mixture and is visible, changes color from a green solid line to a thin bright orange line. Colors Observed for Blue Laser glow a little. The blue laser, as it goes through the mixture, is a short visible, reddish orange color. The laser changed from the blue light to one that does not make it through the beaker. It is just of a bright red dot on the side of the beaker. Olive Oil 1 Pompeian Robust Flavor Extra Virgin Olive Oil surrounding the object are the same as the object but transparent. glow. The red laser, as it goes through the mixture and is visible, solid thick red line. pattern where the bright orange line is replaced with thin green, a thick band of black, and then thick band of bright red where the green line is closer to the object. glow. The green laser, as it goes through the mixture and is visible, changes color from a green solid line to a solid bright orange line. pattern where the reddish orange spot replaced with long vertical line of blue, green, and bright red where the blue is closer to the object. glow a little. The blue laser, as it goes through the mixture, is a short visible, reddish orange color. The laser changed from the blue light to one that barely makes it through the beaker of a bright red spot on the side of the beaker but bigger then the one did for the Leafy Green.

7 Olive Oil 2 Badia Extra Virgin First Cold Press Olive Oil Olive Oil 3 Pompeian Mild Flavor Extra Light Olive Oil surrounding the object are the same as the object but transparent. glow. The red laser, as it goes through the mixture and is visible as a thick dense red line. surrounding the object are the same as the object but transparent. It makes the oil glow and the red laser goes through the oil in a solid thin red line. *This to show that maybe the oil is not as viscous and the previous two oils. surrounding the object are the same as the object but transparent. pattern where the bright red line is replaced with thin green, a thick band of black, and then thick bright red band where the green line is closer to the object. glow. The green laser, as it goes through the mixture and is visible, changes color from a green solid line to a solid orange line. pattern where the orange line is replaced with thin green line, a thick band of black, and then thick dark red line where the green line is closer to the object. It makes the oil glow and the green laser goes through the oil as a visible solid green line. the same as the object but transparent. pattern where the reddish orange spot replaced with thick spots of blue, dark green, and bright red where the blue spot is closer to the object. glow. The blue laser, as it goes through the mixture and is visible, changes color from a blue solid line to a solid white line. pattern where the white line is replaced with thick blue, green, and bright red bands where the blue line is closer to the object. It makes the oil glow to an opaque white with the blue laser changes color to a solid white line going through the solution. pattern where the opaque white line is replaced with blue, green, and then bright red lines where the blue line is closer to the object.

8 Red laser just goes through with a very thin red dashed line and makes it glow bright, bubbly, and transparent. Green laser just goes through with a very thin green dashed line and it makes the tonic glow a bright, bubbly, and transparent It makes the tonic water glow to an opaque bluish white with the blue laser changes color to a solid bluish white line going through the solution. Tonic Water surrounding the object is the same as the object but transparent. surrounding the object is the same as the object but transparent. pattern where the opaque bluish white line is replaced with blue, green, and then dark red lines where the blue line is closer to the object. 1. Which laser and its wavelength shows the most dramatic change on the mixture? What would be reason for this? Blue of 403nm. It has the shortest wavelength to excite the atoms of the mixture to show a more dramatic change. 2. What can you conclude about the olive oil? Are they manufactured the same way? What about the one that is suppose to be Extra Virgin Olive Oil, did it looked the same with the laser when viewed with and without the glasses? No, the Extra Light Olive Oil was different from the other oils. Refer to table. Use the mixtures plus the alcohol used in making the extraction (this is the blank cuvette that will be used for calibration) that are in the sealed columns. They will be placed one at a time into the spectrometer. Connect the spectrometer to the Red Tide and open the Logger Pro software. Place the alcohol into the spectrometer. Go to Experiment -> Calibrate -> Spectrometer 1. Wait for the lamp to warm up and then click Finish Calibration when asked to place the blank cuvette into the column. Then click OK to return to the main screen. Pull out the alcohol and place olive oil 1 into the spectrometer. Click collect and a spectrum graph will appear. When it looks stable click the stop button to stop collecting. Go to Data and Store latest run. Do this for the other cuvettes and be sure to note which run is for what mixture in the table below.

9 Activity Three: Spectrometer and Mixtures Run Mixture Peaks of Wavelength 672.1nm, 612.6nm, 538.1nm, 483.8nm, 1 Olive Oil nm, 419.1nm (the reds, green, and blues region) 2 Olive Oil nm, nm, nm (all blue region) 3 Olive Oil 3 4 Tonic water 5 Spinach (or other leafy substance) 382.5nm (near the infrared) 382.5nm (near the infrared) 652nm, 620nm, 541nm, nm (reds, green, and blue region) 3. Go to Analyze -> Examine and sweep over till you see peaks of which the mixture fall under and view the bottom left of the graph and you will see the coordinates for the (wavelength, Absorbance). On some it will be maxed out so please put a range (width of the peak like for an example you may have nm maximum saturation so put that as one of the peaks for the mixture. Looking at the mixtures, what was the region of wavelengths did each of the mixture falls under of the maximum Absorbance? Did any have more than one maximum peak? If yes which ones? 4. What did you noticed about the olive oil mixtures? Did they all have the same wavelength and intensity (absorbance)? If any, what would account for the differences? The Pampian gave the spectrum in the reds, green, and blues. The Badia gave the spectrum in the blues. The Light Pampian gave the spectrum just in the violet. This makes sense when viewing this with the blue laser pointer and the spectrum glasses, for it gave us the colors that is accounted with the wavelength. They do not all have the same intensity and wavelength. Again it is the way the manufacture is processing the olives. There may be olives that were riper, grown in more enriched soil, and maybe they were heated in the process which can ruin some of the chlorophyll.

10 5. Refer to what you did with the UV light (Activity One) to compare with the findings of the spectrum graphs. Did the color that appeared from the UV agree with what you see on the graphs? Note you are looking at mixtures of color of some of the samples that may give orange, red, yellow, or white. So when looking at the peaks of wavelengths of the mixture and the amount of absorbance, if you mixed them (the wavelength color by the percentage of absorbance) would that give the color that was viewed with the UV? The color of what the UV gave agrees with the spectrum given by the graph. Refer to the graph on the side. If I mixed the color by the amount of absorbance, one should get what was viewed under the UV as well as with the spectrum glasses and laser. The only thing that I question would be the light olive oil for I viewed it as being a less bluish white that is tinted yellow but the graphs shows mostly a long range of blue. If using the spectrometer from Vernier, I may be able to use it to see the fluorescence and then see more details. Use the lasers and the glasses to model wavelength in the following activities by observing if the laser s color is changing or is the spacing of each laser s dots is increasing or decreasing. Activity Four: Lasers and the spectrum glasses on white paper Point the lasers onto a piece of white paper. Put on your spectrum glasses and view the dots on the paper. As you change from one laser to the next, be sure to view from the same distance, as you have done when viewing from the previous laser. 5. What did you notice? The students should see that the color of the dots except for the blue didn t change but for each laser the spacing between the dots changed. From greatest to least on the spacing between each dots of the laser: Red, Green and then Blue.

11 Taken with the fluorescents on: Taken with the fluorescents off: 6. Is the laser s specific color affected by the grating? What given evidence supports this? The color from the main dot didn t change for the red or green except for the blue. It went from a dull blue to a bright blue. And it showed a spectrum on the second dots (second order) from the main dot. (Note: this is the spectrum of the reflected light of the paper.) Activity Five: On colored paper Point the laser on a range of pastel color paper. View it and note any change and then put on the spectrum glasses and view the dots on the paper. 7. What did you notice for each laser? The students should see: Red the color itself did not change.

12 For the Green laser the color was a little dimmer and when viewing through the glasses the images surrounding the dots around the main point dot will show a dash of color. Depending on the paper like yellow paper sticky notes, the students would not see it but if you have colored construction paper then the students would see a tiny bit of spectrum, like if the paper was red construction paper then the spectrum will show a bit of red on the image. For the Blue laser the color will change depending the paper you have given them. Again, an example is that for the yellow paper the blue dot will appear brighter and when viewing through the glasses you will a reflected spectrum due to than the red 8. Is the laser s specific color affected by the grating? What given evidence supports this? The same effect as on number 6. Point the laser on a range of neon colored paper. Put on your spectrum glasses and view the dots on the paper. 9. What did you notice for each laser without the glasses? The students should see a change to the intensity and color change on the green and blue lasers. 10. Is the laser s specific color affected by the grating? What given evidence supports this? No, looking at the images from the main dot, the laser color went back to its original color (red, green, or blue) as shown when using white paper and pastel paper. 11. Does the neon paper affect the laser s specific color? What can you can you state for this effect? On the red laser, one didn t see any change. On the green dot of the first order is different and then from on the images from the main dot it changes back to its original color with a reflectance spectrum due to the neon paper. Where reflectance spectrum is a short thin band where the red band is the longest. This effect happens to the blue laser as well. On the blue dot of the first order is more dramatically different and after the first order the main dot changes back to its original color. With each dot after the first order, each of the images shows a

13 reflectance spectrum due to the neon where the spectrum is thicker and longer then the green but it has a bump somewhere in the range of the yellow to red band starts and it is shorter then shown by the green laser. Note: If you have time, you may want to see about taking a picture and having them compare the bands between the green and blue bands and which band is longer or shorter as well as compare with a different color of neon paper. The reason why the red laser does not reflect the spectrum is due to its frequency being so shorter then the green and blue laser or, since we are looking at wavelength, that it has a larger wavelength then the others. νν = EE h cc λλ = EE h cch λλ = EE *where νν is the frequency, λ is the wavelength, EE is the change in Energy, and h is the Planck constant So, the shorter the wavelength means a greater energy level change where there is greater chance to induce fluorescence. This is the reason for the blue laser being more dramatic then the green laser. So, what is the deal with the white paper? White paper should have all pigment of colors the red, blue, and green, so one would think it should also fluoresce, but because the colors of the laser is already in the paper it would not give a reflected spectrum. But, you may have a white paper that is not exactly white meaning that all pigments are not the same amount of mixture. You may have a spectrum that has more blue pigment then green and red having the smallest amount. As a side note: Laundromats or for those who try to whiten their whites they may use a bluing substance, for example Mrs. Stewart's Bluing Whitener, instead of bleach. This will fake your eyes to seeing the whites are far brighter than it already is by putting a little pigment with the laundry detergent. To even extend the activity, one can also write a vpython program to show the excitation of the electron from the laser onto the paper and change the paper type from white, colored, and fluorescent. Also see if the spectrometer you are using can do florescent vs wavelength and not just Absorbance and Transmittance.