CONNECTION ALERT! Cellular respiration is the topic of Chapter 10 in your BIOL 120 lecture. Please review your textbook as needed for this lab.
Spectrophotometry is the technique we would have used in lab this week. It is a technique that can be used to identify unknown concentrations in a sample, based on its color! In our lab, samples that undergo a lot of photosynthesis will become a lighter color purple. Solutions that undergo little photosynthesis will stay a darker color purple.
Spectophotometry uses the principles of light and color to help identify concentrations of various components in a solution.
Wavelengths of light are associated with various colors due to their frequency. When light hits an object or solution (like a plant leaf in the figure at right), wavelengths are either absorbed or reflected/transmitted. We see color based on the associated wavelengths that are transmitted or reflected. We do not see the colors associated with the wavelengths that are absorbed.
For example, view the absorption spectrum for typical plant pigments (graph in the sidebar). The graph shows that when light hits a plant, purple, blue, red, and orange wavelengths have high absorbance values, meaning they are mostly absorbed by the plant and we don not see these colors. Green and yellow wavelengths have low absorbance values- meaning they are reflected... this is what gives plants their green color! When a color-sensitive indicator is used the absorbance values are directly proportional to the concentration in the solution.
Recall that during the light reactions of photosynthesis, a molecule called NADP+ is reduced to NADPH (energy is loaded onto it in the form of electrons). So the rate at which NADP+ becomes NADPH approximates the actual rate of photosynthesis. In our lab this week, we will be using something called DIP as a substitute for NADP+. DIP = NADP+ DIPH = NADPH DIP is a dark purple color. As the light reactions of photosynthesis proceed, the DIP becomes lighter and lighter as it is reduced to DIPH (like NADP+ to NADH). BUT! We cannot see the subtle changes in color. Instead, we will use the spec to determine the value of the color, as measured by its absorbance.
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REMEMBER! You designed a research proposal for this lab! You have selected the independent variable you are interested in. Your options included: wavelength of light (with colored lenses), type of light, amount of light, temperature (hot, cold), addition of fertilizer, herbicide or pesticide, or concentration of chloroplasts. The procedure here is what would have been done for you to collect data on the affect of your selected independent variable on the dependent variable of "rate of photosynthesis."
REMINDER: What the heck is DIP? Normally, the light reactions require NADP+. Photons excite electrons and they are used to reduced NADP+ to NADPH (which is then used in the Clavin Cycle). Instead of adding NADP to "feed" our chloroplasts, we are adding DIP as a stand in. This is because DIP is color reactive, meaning we can use color changes to determine how much DIP is reduced to DIPH. That is how we will determine the rate of the light reactions in our differing environments: DIP is a blue/purple color, as it is reduced to DIPH the color intensity is diminished, it gets closer to clear. |
Lab 8: ProtocolIn today's lab you will work with your lab group to review and revise your photosynthesis research proposal, conduct your experiment and analyze your data.
Exercise I. Review/revise your proposal Exercise II. Conduct a virtual lab using spectrophotometry Exercise III. Begin to analyze your data |
Procedure
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Spectrophotometry is an increbly useful technique! This virtual lab is meant to give you sense of what this technique is like and how it can be used. Hopefully you'll get to use this skill later on in other classes.
The solutions you will prepare in step 2 below will closely mirror the possible colors of the DIP solutions we would have used in lab, meaning the model we will create from these solutions to applicable to DIP and will be used in Exercise III.
AnalysisProcedure
Remember: Statistics solve the problem of determining if "more" or "higher" or "different" than is actually enough to be important and biological relevant. Using the principles of probability, they help us parse what we observe from randomness (chance alone) as meaning (a real difference, or a real relationship). Statistics tell us how likely we would be to make the same observations we have made, if chance and randomness were the only drivers. If the probability is very low (<5%), we refer to these patterns as significant.
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The data generator used to create the original photosynthesis data sheet contained an error. If you feel your results are counter intuitive and you'd like to give it another shot, please use these "new data" for analysis.
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Each team member MUST submit your poster individually in blackboard before the due date/time. You can turn in a completed draft for feedback before you final is due is you would like. Final posters will not be accepted late, even if others from your group already turned them in. Currently, your final poster is due the week of Apr 13 - 17. The template on the right has detailed directions for each section.
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