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Light is composed of photons with quantized or specific wavelengths and energies. The longer the
wavelength, the lower the energy. Types of light are categorized as: gamma, x-ray, ultraviolet (UV),
visible (vis), infrared (IR), microwave, and radio waves, depending on the wavelength of the
photon. The light that our eyes can detect, conveniently referred to as the visible region, is a very
small section of the light spectrum. Spectrophotometry is the study of the transmission or
absorbance of light through a substance. This technology is crucial in research and industry, as it is
used to identify unknown compounds and measure their concentration, be it an illicit drug found at
a crime scene, a newly synthesized drug created in the laboratory, or a food product ready for
market. Some molecules absorb UV light, others visible light, and even others IR light.
Transmittance is a measure of the amount of light passing through a substance; absorbance is the
amount of light that was captured by a substance. A clear colorless piece of glass has close to 100%
transmittance and 0% absorbance of visible light. In colored liquids, for example, the color we see
is a result of the different wavelengths absorbed and total amount of light absorbed.

Olive oil is made by pressing and extracting the rich oil from the olive fruit by various methods.
There are various grades of olive oil; three common grades are: extra virgin, regular, and light.
Extra Virgin olive oil is considered the highest quality. It is the first pressing from freshly prepared
olives. It has a greenish-yellow tint and a distinctively fruity aroma because of the high levels of
chlorophyll and other pigments (such as carotenoid) and volatile materials extracted from the fruit.
Regular olive oil is collected with the help of a warm water slurry to increase yield. It is pale yellow
in color, with a slight aroma, because it contains fewer volatile compounds. Light olive oil is very
light in color and has no aroma because it has been processed under pressure to remove the
chlorophyll and volatile compounds. Light olive oil is commonly used for frying because it does not
affect the taste of fried foods and it is relatively inexpensive.

The absorbance spectrum, in the visible light range, of chlorophyll gives interesting results. The
chemistry of chlorophyll (there are two essential chlorophyll molecules – chlorophyll A and B)
displays optimal absorbances in the visible spectrum that optimize plant photosynthesis. The
combination of these wavelengths is green to the human eye, but different sources of chlorophylls
will have different ratios of these peaks, which create various shades of green. Thus, in the world of
chlorophyll, all greens are not the same. Each type of chlorophyll, therefore, has a spectral
fingerprint or set of absorbances that is unique to that molecule. All of the millions of molecules
known to man, in fact, have a unique spectral fingerprint.

Beer's law states that the spectral absorbance of a sample is directly proportional to the
concentration of the sample. Meaning, if a substance becomes more concentrated, it will absorb
more of a particular frequency of light. Plants contain chlorophyll, a green pigment responsible for
photosynthesis. Chlorophyll absorbs light most strongly in the blue spectrum. Therefore, the
greater the concentration of chlorophyll in a plant leaf, the greater the amount of blue light
absorbed. If you plot absorbance versus concentration, the resulting graph yields a straight line.
The equation for the straight line (termed regression line) can be used to determine the
concentration of an unknown solution. Beer’s law is an invaluable technique in accurately
measuring concentrations of new pharmaceutical drugs being produced for industry, and in
breathalyzers, measuring the concentration of blood alcohol in suspects.
First, you will analyze a sample of the three grades of olive oil to determine the absorbance peaks
that are present. You will use a Vernier Spectrovis to measure the absorbance of the olive oil
samples over the visible-near infrared (NIR) light spectrum (380 – 850 nm). Second, you will test
an unknown sample of olive oil and grade it as extra virgin, regular, or light, as well as match it to a
commercial brand. Third, you will determine the concentration of an unknown sample of olive oil
using Beer’s law, given known standards of olive oil. Finally, you will do a spectral analysis
comparing and contrasting the spectral fingerprint of olive oil and extracted chlorophyll.


      To create an absorbance spectrum for three samples of olive oil
      To identify an unknown sample of olive oil as extra virgin, regular, or light
      To use Beers’ law to determine an unknown concentration of olive oil
      To identify similarities and differences in spectral graphs of olive oil and pure extracted


Computer                       Spectrovis                             blank cuvettes
8 samples olive oil            distilled water


   1. Obtain and wear goggles. Obtain 5 samples:

          Sample A         Sample B          Sample C        Sample D              Unknown Sample

   2. Use a USB cable to connect a SpectroVis to a computer.
   3. Start Logger Pro 3 (version 3.6 or newer) on your computer.
   4. Open the file marked “Spectrometer Lab” on the desktop. To calibrate your Spectrovis:
          a. Prepare a blank by filling an empty cuvette ¾ full with distilled water. Place the
              blank cuvette in the spectrometer.
          b. Select Calibrate Spectrometer from the Experiment menu. The calibration dialog box
              will display the message: “Waiting for lamp to warm up”. The minimum warm up
              time is one minute. Follow the instructions in the dialog box to complete the
   5. To create a spectral fingerprint graph:
          a. Empty the blank cuvette and rinse it twice with small amounts of the first olive oil
              solution. Fill the cuvette ¾ full with the solution and place it in the spectrometer.
              Make sure the cuvette sides have been cleanly wiped with a kimwipe.
          b. Click “Collect”. A full spectrum graph of the solution will be displayed, showing
              wavelengths of high and low absorbances. This is your spectral fingerprint. Note
                that some areas of the graph contain peak absorbances, and others that contain
                minima that characterize this substance.
            c. Repeat steps a through b for each type of known olive oil, including your unknown
                solution. Identify your unknown olive oil based on this analysis. Double click on
                the graph, and select “Point Protectors” and “Legend”. You must rename each
                data table according to the type of olive oil it represents by clicking on each of
                the data table headers and renaming them. All four olive oil graphs and the
                unknown graph will be on one graph together!
            d. Click “Stop” to complete the analysis. Select “Save As” from the File menu and
                print a copy of this spectrum graph. Make sure the graph is labeled and titled
            e. Click the Configure Spectrometer Data Collection icon on the toolbar. It is the
                rainbow icon. A dialog box will appear.
            f. Select Absorbance vs. Concentration under Set Collection Mode. The wavelength of
                peak absorbance (λ max) should be automatically selected.
   6.   Most olive oils have a maximum absorption at 410.6 nm, which is in the blue spectrum.
        This makes sense, considering olive oil contains a high concentration of chlorophyll. A
        Beer’s Law curve was prepared for you by the instructor, by preparing 4 cuvettes containing
        100.0%, 50.0%, 25.0%, and 12.5%. The absorbance of each was measured at 410.6 nm.
        Open this graph by finding it in the folder marked, “spectrometer lab”.
   7.   Create a line of best fit. Make sure the statistics are shown for your line of best fit. Make
        sure the graph is titled, and both axes are labeled and have appropriate units.
   8.   Select “Save As” from the File menu and print a copy of this absorbance vs. concentration
        graph. Make sure the graph is labeled and titled properly. This is your Beer’s Law graph.
        Print it out for your records and for analysis.
   9.   A 5th cuvette was prepared containing one of your olive oil samples, as it has an unknown
        chlorophyll concentration. It was placed into a cuvette and into the spectrometer, the
        absorbance was read as “.422”. You will use this value to calculate its concentration of


Data:          As taken in class

Graphs:        Include two graphs in your lab report – 1 spectral fingerprint graph, and one Beer’s
               Law graph. Be sure that labels and titles are correct. Be sure to do a line of best fit
               for the last graph, ensuring that the information for the line (y-intercept, slope) are
               shown on the graph so you can determine your unknown.

Calculations: Include any and all relevant calculations, including concentration calculations, if
              applicable, and any calculations required below for the conclusion.


   1. Provide a brief explanation as to how a spectrometer works and why they are of practical
      importance to the scientific community.
   2. Match the olive oil samples with their respective commercial grade. Provide rationale for
      your answer!
3. From your spectral fingerprint analysis, which type of olive oil was your unknown sample?
   Provide clear and scientific evidence that supports this.
4. At what wavelengths did your olive oil have maximum absorbance? At what wavelengths
   did your olive oil have minimum absorbance? Discuss how these results are consistent with
   what the human eye sees visually when looking at olive oil or chlorophyll A/B.

5. Below is the spectral analysis for a sample of pigment extracted from spinach leaf:

       a. Using this graph, label each spectral fingerprint graph as accurately as possible with
           respect to chlorophyll A and B.
       b. Did each olive oil sample contain both chlorophyll A and B? Explain!
       c. Did all samples contain the same proportion or amount of chlorophyll A and B?
       d. It is theorized that Vitamin K, essential for protein function and blood coagulation, is
           coupled with chlorophyll in green leafy vegetables and plant oils. Which olive oil,
           then, would be the greatest source of vitamin K? Explain!
6. Using the statistics from your Beer’s Law graph, calculate the concentration of chlorophyll
   in your unknown olive oil sample in units of %.
7. What would be the concentration of chlorophyll in an olive oil sample that has an absorption
   of .45? What would be the absorbance of a solution that has a concentration of 35.5%?
   These should be found and demonstrated through calculation!
8. Why would it be crucial to obtain concentrations of chemicals, drugs, or solutions accurately
   through a Beer’s Law technique in research or industry?

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