Writing Assignment: Optical Spectra of Bean Leaf Pigments Rev. 1/18

Writing Assignment: Optical Spectra of Bean Leaf Pigments rev. 1/18 For your next writing assignment you will learn to make a graph with two y-axes as these figures are important to many projects in biology. To generate the data needed, you will use a spectrophotometer. You will use a Bausch and Lomb Spectronic 20™ spectrophotometer which is just one particular model of spectrophotometer. This instrument focuses a beam of light of a specific wavelength and known intensity through a 13x100 culture tube (cuvette) and measures the amount of light lost through light absorption by the contents of the tube. Of course it determines this by measuring the percentage of light being transmitted through the tube and hitting a light sensor. The amount of light blocked by the contents of the tube is absorbance. Other brands and models of spectrophotometer may us other kinds of cuvettes, and have other specific operating procedures, but we will learn the procedures for the “Spec 20” as it is commonly known. Plug in and turn on your spectrophotometer. To turn the instrument on, rotate the front left knob clockwise until it clicks on. Observing how a spectrophotometer works o Fill a 13x100 mm cuvette to half-full with water from the wash bottle. o Add a pinch of corn starch from the small jar at your station and mix by inversion. o Insert this cuvette of starch slurry into the cuvette holder but leave the door open. o Set the wavelength control (top knob) to 400 nm and set the range shift lever to the left. o The room lights will be dimmed or you can look through a cardboard tube to restrict room light. o Rotate the front knobs clockwise multiple turns to increase the light intensity while looking down into the cuvette until you can see a violet/purple color of light reflecting off the starch particles in suspension. If you and your partner both see NOTHING, ask the TA or instructor for help. o Using the top wavelength control, increase the wavelength from 400 nm toward 600 nm while looking down into the cuvette and record the colors you see. At 600 nm, move the range shift lever to the right, and continue your observations of color up to 700 nm. 400 425 450 475 500 525 550 575 600 625 650 675 700

 Did you shift the lever here? You have just documented the colors of the human visible spectrum. You should observe the same sequence of colors across the width of a rainbow. Of course artists sometimes modify reality in their art, but as a scientist you know the difference! As soon as possible, remove your cuvette from the instrument, close the lid, move the range shift lever back to the left, set the wavelength to 400 nm (green light), and rotate the front knobs counter-clockwise until the needle is somewhere in mid-scale. Allow your Bausch and Lomb Spectronic 20™ spectrophotometer to recover from the abuse we have given it for at least 5 minutes. We need it stabilized for making precise measurement of the amount of light passing through a cuvette. As you wait, proceed to the next steps: Extracting leaf pigments  Use a hand punch to cut ten 8-mm diameter discs of bean (Phaseolus vulgaris ‘Roman’) primary leaf blade tissue into a 13x100 mm culture tube.  Add 5 mL of ethanol using a 3 mL transfer pipette.  Use the test tube holder and heat the alcohol in a hot water bath (~90°C) to extract the leaf pigments. Watch the alcohol carefully as ethanol boils at a lower temperature than water. One-minute of simmering is all that you need.  Decant the green alcohol extract into a clean 13x100 mm cuvette. The leaf discs should look white and the extract should be a medium green color. Discard the cuvette with the now very pale leaf discs.  Check to be sure you still have a cuvette that is at least half-full of green leaf extract. If it is not, then add more alcohol to match the depth of 5 mL of alcohol in the calibration (aka blank) tube! It is absolutely critical that the cuvette contain enough liquid in it. The light is shone by the instrument Page 2 through the cuvette and is measured at a level maybe 3 centimeters above the bottom! Our instrument CANNOT measure volumes less than 4 mL. This fact is critical to remember for future projects.  Use a Kim-wipe to clean any finger prints from the lower two-thirds of the calibration (blank) and the extract cuvettes. You can set these tubes into a test-tube rack for a moment. Measuring the optical properties of the leaf pigment extract  1. On your spectrophotometer, set the range lever and the wavelength using the top knob to the value indicated in the chart below.  2. With the chamber empty and closed, set the needle to read 0% transmittance at the left end of the top scale by adjusting the left knob on the front of the instrument. Do not use other knobs at this time. This step is called “zeroing” the instrument; you are adjusting the electronics in the instrument so that it indicates no light. In adjusting the needle to match the 0% mark, move your head to the left or right as needed to make the real needle and its reflection in the mirror of the scale superimpose at the 0% mark. This tells the instrument what we consider to be NO light of the set wavelength being measured.  3. Insert the cleaned calibration (aka blank) tube (5 mL ethanol), close the cover, and calibrate the instrument to 100%T at the right end of the scale by adjusting the right knob on the front of the instrument. Do not touch any other knobs at this time; if you accidentally adjusted another knob, start this protocol over at step #1 above! For accuracy, remember to superimpose the real and reflected needle at the 100%T mark. This step is tells the instrument what we consider to be the MAXIMUM possible light of the set wavelength that could be measured.  4. Touching NO knobs, insert the cuvette of leaf pigment extract, close the cover, and record the Transmittance (top scale) and Absorbance (bottom scale) measurements accurately. Obviously this cuvette has some intermediate level of light coming through; the needle should be between 0 and 100%T. Some light at the set wavelength was passed through the extract and appears as %T. But some of the light at the set wavelength was absorbed by the extract and appears as the Absorbance. Be very careful in recording the two values; the Absorbance scale is backwards (increasing from right to left) and the subdivisions on the scale change across its range! This is further complicated by the absorbance scales having been printed without leading zeroes! Notice how there is NOT a tick mark that is correctly 1.9 OD (optical density is the unit of absorbance); there IS a tick mark at 1 (without being printed 1.0) and there IS a tick mark at 0.9 (but the leading zero is absent!). You know better about leading zeroes; this is why it is important! Both partners should check the measurements and come to agreement on them before recording them! Be sure to use the full precision of each scale, and use leading zeroes correctly in recording the absorbance data. Repeat all of the numbered steps (1-4) above for each wavelength. Be sure the filter lever is to the left! Move the filter lever to the right! Wavelength (nm) %T and Abs. Wavelength (nm) %T and Abs. 400 — 600 — 420 — 620 — 440 — 640 — 460 — 660 — 480 — 680 — 500 — 700 — 520 — 540 — 560 — Don’t discard your extract 580 — yet! Read and carry on! Page 3 Observing fluorescence of the extracted leaf pigments When a pigment, such as green chlorophyll, absorbs light, that light energy is driving some working process. In photosynthesis the light energy is transferred from chlorophyll to members of an electron transport system (usually called the light reactions). The absorbed energy splits water into oxygen, electrons, and protons. So when we want to measure the presence of a pigment, the absorbance data are more important than the percent transmittance. In other words green (transmitted) light that we see does not drive photosynthesis, blue and red light are strongly absorbed and those wavelengths of light DO drive photosynthesis. The light energy ultimately ends up in carbon-carbon bonds in carbohydrates. But here in the cuvette we have extracted pigments, they are no longer in association with chloroplasts and their biochemistry that includes the membrane transport proteins, C-3 (Calvin) cycle enzyems, etc. So the absorbed energy in this cuvette cannot be passed into photosynthesis. What happens to the absorbed light? When a chlorophyll molecule is excited by light, an electron of a Magnesium ion that is articulated in the hydrophilic ring system of chlorophyll is lifted out of the electron cloud of Mg and into an electron cloud formed by the resonating double-bonds (shown as alternating single and double bonds) of the chlorophyll ring system. This absorbed light energy is captured in this electron movement. But with nowhere to go (no ETS!), the electron eventually falls back to its place in the Magnesium electron cloud. The energy is now lost from the chlorophyll molecule and is released as light energy. Light is emitted and this emission of some light is called fluorescence. In this project we will shine Blue/UV light from a penlight into our leaf pigment extract. Will the pigment inside release Blue/UV light? You may recall from Chemistry or Physics class that there is a “Second Law of Thermodynamics” which must obeyed in the real universe. It basically demands that a system processing energy must always lose some energy as heat in all exchanges. So while chlorophyll will absorb and be excited by Blue/UV light as you demonstrated in the spectrophotometer, it will obey the 2nd Law and lose energy in fluorescence. So instead of getting the higher energy Blue/UV light back we used for excitation, our fluorescence light will be of lower energy and longer wavelength. In other words it will be a color of light that is toward the red end of the spectrum. Use the Blue/UV penlight to excite the extracted pigments in the cuvette, what do you observe as fluorescence?

______As a check (not a control), shine the Blue/UV penlight on an intact leaf on the bean (Phaseolus vulgaris ‘Roman’) plant. What do you observe about the excitation light or any fluorescence?

______Useful vocabulary for your responses include incident light, absorbed light, reflected light, excitation light, fluorescent light and the color words you used on the first page to describe colors of light. You have completed the work needed to do in laboratory today! Follow any cleanup procedures given by your instructor!

Constructing Double-Y Plots Previously you have learned how to use Microsoft Excel™ to make figures of publishable quality. In this exercise, you will apply this knowledge to a new kind of graph: one which has two y-axes. This is a writing assignment; completion of this assignment is required of all. Your mission is to use Microsoft Excel™ to produce a single figure to clearly show the relationships between three variables: wavelength, absorbance, and transmittance of the extracted leaf pigments (page 2). The kind of chart you need to create is a scatter plot with two different y-axes. Which of the variables should be on the x-axis? Obviously, it is the independent variable…the one you manipulated…you set the spectrophotometer to different wavelengths of light. The other two variables are dependent variables; their values measured are clearly dependent upon the wavelength of light that you set. The two dependent variables need to be on the y-axes [sic]. The scales for these two variables are quite different…by about 100X. Therefore you would not be able to observe the relationship between both variables if they were plotted on just one axis scale (as we did for the hand width data earlier in the semester). So you need to create a double-y axis plot; there will be two y-axes in the final figure. Page 4 In Microsoft Excel™ enter all sixteen (16) of your wavelengths in one column, and the corresponding %T and A values in two adjacent columns. Select the three columns of data, and create a Scatter plot through the usual steps (remember: NEVER use the line-graph plot). The “default” chart that the software creates will probably have the correct variable on the x-axis, but will probably have the two dependent variables shown on the same y-axis. One of the data series (absorbance) is crunched down to the x-axis. This is the reason a second y-axis is needed! Double-click the transmittance symbols to Format the Data Series. In the side palette, click 3-bars icon — SERIES OPTIONS — Plot Series On — Secondary Axis. If needed (but it should be already done), set the absorbance series to the primary axis. Your figure should now show a second axis scale on the right for transmittance and the absorbance data markers should now be spread properly on the left-axis to reveal the relationships between all three variables. The absorbance scale should be on the left axis. In this figure, neither the absorbance nor the % transmittance data appear to fall on any regular line, so we are NOT doing any kind of regression (i.e. we are not adding a “trendline”). Select the symbols of each series and, on the line option, set a smoothed line (checkbox at bottom of dialog) to connect the data points to show the absorption and transmission spectra for your alcohol leaf pigment extract. Maybe one should have a dashed line to go with its symbols (markers). Hints:  set  Marker Border Join type to miter, add outside tick marks to axis scales;  turn off any marker shadows. You might notice that these curves are symmetrical reflections, with an axis of symmetry above and parallel to the x-axis. The smoothed curves should especially show this relationship, and also reveal any errors in data recording (with an asymmetrical blip in one spectrum not reflected in the other spectrum). As careful scientists in the laboratory, we chose to record both absorbance and transmittance…why? Recording both parameters in the lab provides the scientist with a good cross- check of the data later in analysis. If needed, you can correct an erroneous value by converting its associated correct value by applying one of two relationship formulae in Excel™ (you are not being asked to do this unless you do have erroneous values!): A=2-log(T) and/or T=100*10^(-A) For example, imagine that you had recorded, at one wavelength, a %T of 95 and an absorbance of 0.22 and this resulted in a huge jump in the absorbance spectrum that you did not find reflected as a huge dip in the transmittance spectrum. You are suspicious of the value for absorbance because of its hard- to-read scale and the missing leading zero on the instrument. To diagnose the recording error, you would use the formula =2-log(T) in Excel and plug 95 into it as the value of T. In this case Excel returns an absorbance of 0.022. A yes, there is the error that both lab partners failed to record the decimal values correctly! In real science we would have to start over and do it again but without human measurement errors. In this case, we cannot go back to lab, so you can use the Excel-calculated value for absorbance. Bringing your Double-Y Plot to biology legibility standards Based on your previous experience in this course, you should know what to do next. You need to remember your model of Figure 4 from the Figuring Biological Data exercise. There are many quirks in the default graphics produced by Excel™ that must be corrected to make a figure that meets the specifications of professional journals in biology. You should now be a very good critical inspector of these features. You have prior rubrics to guide you, and the FBD checklists for Tables and Figures. So be sure to make the necessary corrections to remove “cute” 3D and color effects, remove shading and gridlines, and use bold lines, larger sans-serif fonts and standard symbols. Absorbance blocks light, so maybe a black square () is an intuitive data marker for that variable. Transmittance implies light goes through, so an open circle () might be a good choice for the marker? As was true in the Seed Germination writing assignment, format the Percent Transmittance axis scale to have fewer “tick marks” and numbers; recall that our target is 4-8 of these and you likely have Page 5 10 here by default. Set the maximum to 110, the major unit to 20, and the minimum to 0. The maximum tick mark should now show 100; the value of 120 previously shown was irrational (except on “reality” TV shows)! After clicking OK you should see that this axis scale now meets our specifications. The Media Browser helps insert graphics font (Wingdings) symbols (in black fill or no-fill outline) in the axis titles instead of adding apparent outlier point on your scatter plot with a “legend” or “key” inside the plot area, and also not in the caption for the figure. Remember too that a numbered figure caption to make the figure free-standing is required. Make sure your caption has all the features of its model(s) too! Hint: what is that “(s)” all about? Is there a caption sentence that is NOT required for this project? How is our title different from the model? Writing Materials and Methods and Results As you prepared your seed germination writing assignment, read the pertinent sections of Knisely [or Pechenik] about the Materials and Methods and Results sections of a standard laboratory report. Using the description and the guidelines in the seed germination worksheet, prepare a draft of these sections of a laboratory report. You need to follow those suggestions completely, but we reiterate here just a few that are commonly missed by freshmen. The Materials and Methods section does not start with a list of the materials that are needed for the project. The Materials and Methods section does not instruct the reader in what they should do, but instead informs the reader of what you did. So do not use imperative mood (do not give commands!)! [that sentence and its parenthetical are examples of imperative mood…the text inside this bracket is in indicative mood]. Moreover, you write in PAST tense (as opposed to present or future tenses). Passive voice (the indirect object was manipulated by the direct object) is commonly used but active voice (the direct variable influenced the indirect variable) is increasingly preferred. The use of pronouns (I or we) is acceptable, but should not be over-used; science should be objective rather than subjective. The scientist should not be the critical factor most of the time. A Materials and Methods section in a complex project can be subdivided with subheadings to allow modular references to “subroutines.” In this case, the project is so simple, just a couple of paragraphs can likely suffice. One paragraph would be on growing the plants and extracting the leaf pigments. The second would deal with the spectrophotometric measurements. There was no statistical analysis so you do not need that part. Don’t forget a paragraph about the use of the UV/blue penlight! Whether subheadings are used or not, multiple paragraphs are expected. Remember that essential details are included, but standard procedures such as labeling of tubes, pipetting techniques, the specific cuvette used, the calibrating procedure for instruments are assumed to be known by a scientist reader. What you did use in the cuvette for calibrating would be a critical mention. The results text should not include numerical values; use color words for the wavelengths, and use relative (maxiumum, peak, trough, minimum) word values rather than numerical %T or A values. The Results section does NOT rehash the methods. This text section is where you get to write about what you, the author, observed about the plant, the discs, and the extract in laboratory, what you observed in the data (Fig. 1). It is critical that you write about any other observations you made that are NOT in the figure. You get to write the story of what you observed in this project in the Results text section. The project is over so it should all be written in past tense! Because these are your observations, they are subjective, and personal pronouns are appropriate and recommended. This text in a complex project can be divided under subheadings, but for our simple project a few paragraphs are likely all you need. Do not forget to include your observations from the use of the UV penlight. The results section includes any figure(s) and table(s) with your data, but showing only figures and tables in this section is not acceptable. In the figure/table caption you tell (concisely!) what was done and how the data were analyzed only. Some of you know that you do NOT tell what you observe in the Page 6 figure in the figure caption; you let the reader interpret the graph for her/himself. In a complete lab report, your interpretations would go into the discussion section; the discussion section will be taught to you in the sophomore core courses. What to Hand In For this project you should hand in a computer-printed stack of pages neatly stapled together near the upper left corner. The body text printing should be double-spaced in 12 point serif font (Times), with at least 1” (2.5 cm) margins all around. The first page should have a suitable title centered at the top. A model for a title based on an experiment would be: The effect of independent variable on dependent variable in Taxon binomial. Please do not type this unaltered model into your document...until you have substituted the variable you manipulated, the variable(s) you measured, and the scientific name of the organism into this model! The font size should be the largest and boldest in this paper! Did our project here represent an experiment? If not, then perhaps the model should be modified accordingly. Beneath this line should be a line with your name first and then your lab partners in alphabetical order by last name. You have learned their names, right?! These lines should be centered in normal size font. The next line should include your contact information: department address and email address, centered in normal font size. The next line should have the major heading Materials and Methods centered in a larger, bolder font (but a size smaller than the title). The next line should be any subheading you are choosing to use at the left margin in the first paragraph of the section. The subheading can be normal size font, but made bold to stand out. At the end of the Materials and Methods section, you can continue on the same sheet with the heading Results centered on its own line in the same larger, bolder font used for Materials and Methods. This is followed by any subheadings and the paragraphs of the Results text section. To ensure that the Results heading or a subheading does not appear on the last line of a sheet, remember to use the Insert-Page-Break function in Microsoft Word™. Your double-y plot (Fig. 1) can be inserted between or after the paragraphs of this section in the Microsoft Word™ document. Alternatively the figure can be on a separate sheet at the end of the Results section (i.e. the last page!). Be sure your Figure still has its font as sans-serif (Arial); Word and Excel do not play well together unless you specify the font you need! Its caption should be in the usual 12 point serif font (Times) properly aligned with 2 cm (0.75”) left- and right- margin indents from the page margins. Since there is just one figure in this paper, the title in its caption might match the paper title! You will not need to have the usual last sentence on statistical analysis (we did none). Double check that you have all of the pages correctly sequenced, that your figure meets the specifications of previous grading rubrics and the checklists in Figuring Biological Data. Make sure you leave enough time to complete this assignment while you can still get any help you need to make the software or text do what you need it to do. Hand your stapled stack of papers into the instructor at the beginning of class on the due date. This writing assignment “worksheet” is not to be handed in. Please staple your document together yourself before the due date...do not bring a stack of loose sheets to class to hand in. Please never attempt to keep your papers together by folding down their corners; this always fails and pages of your precious work could be lost or credited to someone else! There are staplers all over campus if you do not have one yourself. If you do not have one, I know what you should ask for as your next birthday present!