biology homework

Isolation and Analysis of Chlorophyll

Content Objectives

1. Determine the absorbance spectra of freshly isolated chlorophyll from spinach leaves, and

different grades of bottled olive oils.

2. Interpret the grade of an unknown olive oil sample based on the absorbance spectra of extra

virgin, regular and light olive oil samples.

Process Objectives

1. Extract chlorophyll with an organic solvent.

2. Obtain an absorption spectrum of chlorophyll using a spectrophotometer.

Plants are primary producers in that they capture sunlight energy and transform it into chemical bond energy of organic molecules. Chlorophyll is the main photosynthetic pigment. It absorbs energy from most of the visible light spectrum.

The transformation of sunlight energy to chemical bond energy occurs in the anabolic

process called photosynthesis, which consists of two separate pathways.

The first pathway of photosynthesis is called the light reactions. In this anabolic

pathway, sunlight energy is transformed to chemical bond energy of ATP and NADPH. These

high energy molecules are then used in the second anabolic pathway called the Calvin Cycle (or

dark reactions) to synthesize the intermediate organic compounds that will used to make glucose.

Chlorophyll appears green because it reflects that part of the visible light spectrum.

There are two types of chlorophyll, a and b. Only chlorophyll a participates directly in the light

reactions, but accessory photosynthetic pigments absorb light and transfer energy to chlorophyll

a. Chlorophyll b has a slightly different structure than chlorophyll a, has a slightly different

absorption spectrum and funnels the energy from these wavelengths to chlorophyll a.

Some accessory pigments absorb the greenish-blue part of the visible light spectrum

(Figure 1). Other accessory pigments (e.g., carotenoids) reflect the green region of the visible

light spectrum. And appear as orange or red, which are often visible in leaves during the fall

when chlorophyll production ceases. An absorption spectrum plots a pigment’s light absorption

versus wavelength, usually shown as nm (Figure 2).

As shown in Figure 2, there are maximum absorbance peaks of chlorophyll and b. The

absorbance spectrum will also show the presence of accessory pigments, with maximum

absorbance in the blue range of the visible light. Collectively, chlorophyll a, b and the accessory

pigments appear green to the human eye.

Figure 1 Figure 2

Chlorophyll molecules absorb light energy and transfer it to a special chlorophyll

molecule, called photosystem II (Figure 3). The photosystem ejects a high energy electron to a

type of an electron transport chain. ATP is produced by chemiosmosis. The electron ejected by

the photosystem II must be replaced and the splitting of water provides the electron to the

photosystem II. Oxygen is produced as a result of the oxidation of water.

Figure 3

Images: Campbell 10/e. Pearson Education, Inc.

This activity will measure the absorbance spectrum of chlorophyll freshly extracted from

young spinach leaves.

Safety Precaution: Acetone is an organic solvent. Students must wear gloves, safety eye

glasses and plastic aprons. Dispose of acetone as instructed! Do not pour it down the sink!

Protocols:

PART I: CHLOROPHYLL ISOLATION

1. Obtain three or four small spinach leaves. Tear into small pieces, and remove the “mid vein.”

2. Place the spinach in a mortar. Add about 2-5 ml of acetone CAUTION— HAZARDOUS CHEMICAL

3. Grind the leaf pieces until all visible tissue is gone. Add additional acetone as needed.

4. Pour solution through cheesecloth into a small beaker. 5. Prepare your sample by filling an empty cuvette ¾ full of ethyl alcohol. 6. Transfer one drop of the filtered chlorophyll solution to the cuvette. 7. The color of the extract should be a pale grass-green. Dilute with ethanol if

needed in a separate tube

PART II: ABSORPTION SPECTRUM

CALIBRATION

1. Connect the Spectrometer to the USB port of your LabQuest Vernier Device. 2. The screen should read what probe is being connected and will show immediately on

the screen.

3. Prepare a blank by filling an empty cuvette ¾ full with ethanol. 4. Choose Calibrate-> Spectrometer form the Experiment menu 5. The lamp will indicate it is warming up (usually 2 min.) after which it will signal it is

complete.

6. Place the blank in the Spectrometer (non-ridged sides facing to the left and right and ridged sides are facing top and bottom).

7. Click Finish Calibration and then click OK.

OBTAINING THE FULL SPECTRUM OF FRESH CHLOROPHYLL EXTRACT

1. Place the cuvette containing the chlorophyll sample into the spectrometer (non-ridged sides facing to the left and right and ridged sides are facing top and bottom).

2. Click collect (green arrow at the left hand bottom of the screen) and a full spectrum graph will be displayed

3. Click on stop (red block at the left hand bottom of the screen) to complete the analysis.

4. Use the stylus to tap on the spectrum line. This will display the absorbance reading from that part of the line.

5. Determine the absorbance at the two peaks of the spectrum and record the values in Table 1.

6. Before moving on, sketch the graph of the absorbance spectrum on the results page (Question 1).

OBTAINING THE FULL SPECTRUM OF OLIVE OIL SAMPLES

1. Fill three separate cuvettes ¾ full with the known olive oil samples (Virgin Olive Oil, Regular Olive Oil, and Light Olive Oil).

2. Fill a separate cuvette ¾ full with the unknown sample of olive oil. 3. One at a time, place the cuvettes containing the olive oil samples into the

spectrometer (non-ridged sides facing to the left and right and ridged sides are facing

top and bottom).

4. Click collect (green arrow at the left hand bottom of the screen) and a full spectrum graph will be displayed

5. Click on stop (red block at the left hand bottom of the screen) to complete the analysis.

6. Use the stylus to tap on the spectrum line. This will display the absorbance reading from that part of the line.

7. Determine the absorbance at the two peaks of the spectrum and record the values in Table 1 for each sample.