Evaluating Olive Oil Quality through Absorbance and Fluorescence Spectrometry

Transcription

1 Olive_Oil_Spectrometry_v1c.docx Evaluating Olive Oil Quality through Absorbance and Fluorescence Spectrometry A spectrometric evaluation of olive oils. * 1.1 EXPERIMENTAL GOAL 1 OBJECTIVES In this experiment, students will use absorbance and fluorescence spectrometry to evaluate the quality of olive oils. 1.2 PREREQUISITE SKILLS AND KNOWLEDGE Students should have experience using the Vernier Spectro-Vis Spectrophotometer, or a similar spectrometer, and have completed the lab Extraction and Separation of Plant Pigments. 1.3 RESEARCH SKILLS After this lab, students will have had practice in: following laboratory protocols using a laboratory notebook following hazardous waste guidelines using the proper personal protection equipment choosing an appropriate size of micropipette using micropipettes using a transfer pipette pigment extraction using a mini-centrifuge using a spectrophotometer to measure absorbance using a spectrophotometer to measure fluorescence 1.4 LEARNING OBJECTIVES After this lab, students will be able to: use a spectrometer to evaluate the grade and freshness of olive oil * This experiment was adapted from the evaluation copy of the lab Determination of Chlorophyll in Olive Oil in Advanced Biology with Vernier from Vernier Software, accessed November 18, 2016.

2 2 Olive Oil Spectrometry 2 PRE-EXPERIMENT ASSIGNMENT Olive Oil Quality Olive oil is one of the oldest food oils and has had an immense influence on much of European history and culture. Currently, the following grades of olive oil are recognized by the International Olive Council: Extra-virgin olive oil (EVOO): produced by the use of mechanical means only, and is also judged to be of highest quality in terms of taste. Virgin olive oil: produced by the use of mechanical means only, but not as high quality in taste. Refined olive oil: refined from either virgin or extra-virgin olive oil. The refining process uses charcoal or other filters to produce an oil without taste or color. A small amount of extra-virgin olive oil is often added to the refined oil to give it a hint of flavor. Refined olive oil is often just described on a label as olive oil or pure olive oil. Olive pomace oil is made from oil extracted from the paste left over after pressing the oil for extra-virgin or virgin olive oils. This extraction uses solvents or other treatments not sanctioned for the above grades, but is still considered fit for consumption. Olive pomace oil is preferred in some uses because it has a higher smoke point than other olive oils Chlorophyll in Olive Oils Chlorophyll is a component of most virgin olive oils. Other components include other plant pigments such as carotenoids; beta-carotene is one. The amount of chlorophyll in any one kind of olive oil depends on the chlorophyll content of the olive. In higher plants, chlorophyll molecules embedded in the thylakoid membrane of chloroplasts act as light-harvesting antennae to absorb sunlight as the initial step in photosynthesis. The absorbed photon prepares the chlorophyll molecule to donate an electron to the electron transport chain. When the chlorophyll is isolated from the chloroplasts, the energy absorbed by the chlorophyll molecule is instead eventually given off as emission of a lower energy photon. This emission is called fluorescence. The structure of chlorophyll a is shown below. The absorbance spectrum of chlorophyll is shown below. Much of this information was excerpted from Wikipedia s interesting discussion of olive oil.

3 Olive Oil Spectrometry Oxidative Degradation of Olive Oils Olive oils are at their best when they are fresh. With age (or more quickly with exposure to heat, or light) olive oil will lose its green color and fresh, grassy smell. These changes occur when the chlorophyll molecule loses the central magnesium metal ion (Mg 2+ ) and becomes pheophytin. The structure of pheophytin a is shown below. Pheophytins are important parts of the electron transfer pathway in photosystem II, but in olive oil they are merely the first products of chlorophyll degradation. In very little time, the pheophytins break down to pyropheophytins. The structure of pyropheophytin a is shown below. Pheophytins and pyropheophytins have absorbance spectra very similar to chlorophylls, but despite these similarities, you will be able to spectrometrically distinguish between fresh extra-virgin olive oil, refined olive oil, and degraded extra virgin olive oil.

4 4 Olive Oil Spectrometry 2.2 PREPARE FOR THIS EXPERIMENT Read through the entire lab procedure and prepare your lab notebook. When you feel ready for the lab, test your preparation with the Pre-Experiment Quiz on e-learning.

5 Olive Oil Spectrometry MATERIALS CHECK OFF LIST Each group of (2-3) students will have: laptop computer with LoggerPro software Vernier SpectroVis Plus Spectrophotometer 1 glass micropestle 1 conical 1.5 ml microcentrifuge tube 7 disposable cuvettes with four clear sides 7 disposable plastic pipettes cuvette and tube stand Each large group of (1-2) small groups will share: 3 LABORATORY MANUAL spinach leaves bottle containing 2-propanol or ethanol jar of grinding beads mini-centrifuge tubes with 8 ml of the following olive oils o Publix brand olive oil (not virgin) (or equivalent) o Publix brand light olive oil (or equivalent) o Pompeian Robust Extra Virgin Olive Oil (or equivalent) o Eleon Extra Virgin Olive Oil (or similar prestige olive oil) o Saporito Arborsana Extra Virgin Olive Oil (or similar prestige olive oil) o one or more of the above EVOOs heated to 180 C for 30 minutes o one or more of the above EVOOs heated to 180 C for 120 minutes 3.2 SAFETY AND WASTE DISPOSAL PROTOCOLS Goggles must be worn while the spectrometer is in use. Isopropanol-containing solutions should be placed in the labeled waste bottle. Used olive oil may be washed down the drain when no longer needed. Used cuvettes, tubes, and disposable pipettes should be put into the benchtop or classroom lab waste containers. 3.3 EXPERIMENTAL PROCEDURE Collect a Spectrum of Chlorophylls Use 2-propanol (or ethanol) to extract the pigments from a small amount of spinach leaves. Refer to your lab notebook from the early part of this semester for more details on the extraction procedure. Collect an absorbance spectrum of the spinach leaf pigments. Make sure your absorbance is not saturated you may need to dilute the pigment extract with more of the solvent. Q1. Briefly describe how you will extract the pigments from the spinach leaves. Q2. Insert a snip of your publication-ready spectrum here. Set aside your chlorophyll-containing cuvette for the upcoming fluorescence work. Compare your spectrum to the chlorophyll absorbance spectrum from Vernier (in Pre-Experiment). Q3. Account for any differences. Q4. Which spectrum do you think will be more useful for identifying chlorophyll in your olive oil samples? Explain.

6 6 Olive Oil Spectrometry Grades of Olive Oils In this part of the experiment you will compare three different grades of olive oil: light, regular, and extra virgin. 1. Label three cuvettes appropriately and fill each ¾ full with one of the three different grades of olive oil. 2. Prepare a fourth cuvette with DI water for the reference. 3. Calibrate the spectrometer using DI water for the reference. 4. Collect a full spectrum for each olive oil sample. 5. Compare the olive oil absorbance spectra to each other and also to one of the reference chlorophyll spectra. Q5. Describe the spectrum of each olive oil, and the differences between them, in terms of the absorbance peaks and other distinguishing features. Q6. Which grades of olive oil appear to contain chlorophyll? What spectrometric evidence support your answer? Chlorophyll Content Assay Now, use the light grade of olive oil as the reference to recalibrate the spectrometer, so that you can compare the chlorophyll content of the other olive oils. Q7. Explain the purpose for this new calibration. Q8. What makes the light olive oil appropriate as a calibration reference? Collect new spectra of the two different olive oil grades (regular and extra virgin) and compare them, in terms of absorbance peaks and features, to the spectra you collected previously. Q9. How are these spectra of the same olive oils different from the spectra you collected in the previous section? Use your absorbance spectra to estimate how much more chlorophyll one grade contains compared to the other. NOTE: If your absorbance shows signs of saturation, you may need to dilute your olive oil in a known amount of the reference oil. Don t forget to include the dilution in your calculations. Q10. Describe how you used your absorbance spectra to determine the relative ratios of chlorophyll in the two grades of olive oil. Q11. What difficulties, if any, did you encounter, and how did you resolve them? Obtain a sample of a prestige olive oil and use the above assay to determine the relative chlorophyll content. Q12. Describe your results Chlorophyll Fluorescence Chlorophyll will fluoresce under the right wavelength of light. Use the absorbance spectra you recorded to predict which of the two fluorescence wavelengths available on the SpectroVis will be most effective with chlorophyll. Q13. Describe your choice and the reasoning behind it. Confirm your prediction by collecting a fluorescence spectrum using the cuvette containing your plant pigment extraction at each excitation wavelength. You should see a peak from chlorophyll at approximately 675 nm.

7 Olive Oil Spectrometry 7 Q14. Which excitation wavelength was most effective? Chlorophyll Fluorescence Assay Optimize sample time To evaluate the relative fluorescence of each of your assigned olive oils, you will first need to optimize the sample time. Find the olive oil with the greatest fluorescence at the ~675 nm chlorophyll peak. You will use this sample to optimize the sample time. Follow these instructions to optimize the sample time to produce a peak intensity between 0.8 and 1.0: 1. Choose Set up Sensors Spectrometer from the Experiment menu and change the sample time to 150 ms. 2. Collect a spectrum. Observe the height of the peak at ~675 nm. 3. Adjust the sample time up or down until the amplitude of the peak is within the desired range. Q15. Record the optimized sample time in your lab notebook and here Collect and compare fluorescence spectra Use the same optimized sample time to collect and store a full spectrum of each olive oil. Q16. Why is it important to use the same sample time for each oil sample? Use the spectra you collected to calculate the relative chlorophyll content of each olive oil. Q17. Show your calculations and results here. Q18. Compare your results using fluorescence spectrometry to those using absorbance spectrometry. Do they agree? Q19. Is one method better than the other? Explain your reasoning Absorbance and Fluorescence Evaluation of Degraded Olive Oils Three different EVOO s were given the heat treatments described below to accelerate chlorophyll demetalization. You will evaluate the oils for indications of chlorophyll loss, and formation of degradation products pheophytins and pyropheophytins. Heat treatments: 1. The oil was heated to 180C and held at that temperature for 30 minutes. It was then allowed to return to ambient temperature and stored in a jar. 2. The oil was heated to 180C and held at that temperature for 120 minutes. It was then allowed to return to ambient temperature and stored in a jar. Complete the following assays with each of the heat-treated oils, and the controls: 1. Collect and compare the absorbance spectra. 2. Collect and compare the fluorescence spectra. Note any new peaks and shifts in peaks, both of which could be the result of degradation products. 3.4 POST-LAB ASSIGNMENT Prepare an abstract describing just the final experiment: the examination of the heat-treated EVOOs.