Lab 9 Photosynthesis Background Plants, cyanobacteria, and algae convert light energy to chemical energy by the process of photosynthesis. This process involves utilizing light energy to combine water (H 2 O) and carbon dioxide (CO 2 ) to form energy rich carbohydrates. The energy stored in the carbohydrates is then either used by the plant for cellular metabolism or stored as starch. 6CO 2 + 12H 2 O C 6 H 12 O 6 + 6O 2 + 6H 2 O carbon water glucose oxygen water dioxide Some bacteria, such as those found near thermal vents on the ocean floor, are able to produce carbohydrates without light by a process called chemosynthesis. In 1905 F.T. Blackman proposed that photosynthesis has two parts: a light dependent and a light independent reaction. Blackman predicted that the process of photosynthesis would proceed faster in more intense light. His first set of experiments lead him to conclude that increasing light intensity increased photosynthesis but only to a certain level. Increasing light intensity further had no effect. Blackman s second set of experiments involved the effects that temperature and light together had on photosynthesis. He found that increasing the temperature above 40 C lowered the rate of photosynthesis, no matter how much light he provided the plants. Blackman found that increasing the temperature between 0 C and 30 C produced very different effects, depending on how bright the light was. At low light intensity, the increase in temperature had almost no effect on photosynthesis. At high light intensity, the increase in temperature within the range stated above, greatly increased the rate of photosynthesis. Light Absorption Sunlight is generally assumed to be white in color. Actually, white light consists of many different wavelengths combined. Each wavelength by itself exhibits its own color. Light energy is radiated to the earth in packets of energy called photons. Photons are captured by various pigments molecules in photosynthetic organisms. A pigment is a substance that absorbs light of a particular wavelength. A pigment s color depends on the color of light that it reflects. When white light hits a pigmented surface, some of the wavelengths are absorbed and others are reflected. For example, a red object is one which absorbs the blue and green colors of white light and the non-absorbed red is reflected to our eyes. The most important plant pigments in photosynthesis are the chlorophylls. There are two major types, chlorophyll a and chlorophyll b. Green plants contain both types of chlorophyll. In addition to chlorophyll, the leaves of many green plants also contain one or more other pigments, including carotenes, which are orange, and xanthophylls, which are yellow. The presence of these other pigments are masked by the abundance of chlorophyll during most of the year. However, during the fall, when chlorophyll production decreases, the other pigments become more apparent, giving leaves their bright red, orange, and yellow autumn colors. Chromatography To separate the pigments present in a chloroplast we will use a technique known as thin layer chromatography. Chromatography is a technique for separating and identifying substances in a mixture based upon their solubility in a solvent. This process has been used to determine the ingredients that give perfume its scent, analyze environmental pollutants, identify drugs in urine, and even separate proteins. The name chromatography is derived from the Greek words chroma and graph which mean color writing. Chromatography was invented in 1910 by the Russian botanist Mikhail Tswett who used it to separate plant pigments. He filtered a petroleum ether solution of pigments through a simple glass column filled with calcium carbonate. In the 1920 s, two Russians, Ismailov and Shraiber used a layer of alumina spread on a glass plate to separate plant pigments. When a mixture of pigments is placed on a strip of chromatography paper and placed into a solvent solution, the individual pigments in the mixture will Lab 9 Page 1
migrate up the chromatography paper at different rates by capillary action. Paper chromatography allows the pigments to be separated from one another based on their different physical characteristics such as size, solubility, and affinity for the paper surface. For example, some substances that are easily dissolved in the solvent are carried up the chromatography paper, while other pigments that are more strongly attracted to the chromatography paper than to the solvent stop moving and form bands along the paper strip. The final product revealing the separated pigments is called a chromatogram. Following chromatography, each band on the chromatogram can be assigned a Relative Mobility Factor (R f ). Each R f can be associated with a specific substance. The R f is a ratio of the distance the solute (pigment) traveled to the distance the solvent traveled. Distance pigment traveled (D unknown ) R f = Distance solvent traveled (D solvent ) An extract of plant pigments was prepared by using the organic solvents acetone and ethanol to solubilize chloroplast pigments found in spinach leaves. Exercise 9.1 1. Obtain a clear cylindrical developing chamber and cap. 2. Place 25 drops of the developing solvent (iso-octane-acetone) into the developing chamber and place the cap on the top. (The cap is not designed to fit tightly.) This will saturate the atmosphere of the chamber insuring better separation. 3. Obtain one Thin Layer Chromatography (TLC) strip. These strips are made by applying a silica gel absorbent on a plastic strip. Handle carefully by the edges or extreme tip of the strip. The absorbent, on one side, will easily scratch off the backing. 4. Place 3 small spots of pigment along an imaginary horizontal line about 1 cm from the bottom of the TLC strip with a capillary tube. Be sure to keep your finger on the open end of the capillary tube to control the Chromatography Paper size of the drop. The smaller the spots, the better the results. 5. Repeat the spotting in the exact same location as the first spots, 5 more times, or until the spot is dark green or orange. Allow each spot to dry completely before adding the next. 6. Allow the TLC strip to dry completely, then place it into the developing chamber with the pigment line down and replace the cap. 7. Allow the developing spot to migrate until it reaches about 1 cm from the top. 8. Remove the strip. Before the solvent dries use a pencil to lightly mark the solvent front near the top of the TLC strip. Allow the TLC strip to air dry. Drying time is only a few minutes. 9. Measure and calculate the R f for each observable band. Solvent Pigment Line Note: light causes the pigment molecules on the chromatogram to disintegrate. You can keep the colors if you keep the chromatogram in the dark, such as between the pages of a book. Lab 9 Page 2
Spectrophotometry One of the most important techniques of analytical chemistry used by biologists is spectroscopy. It has had a profound effect in the conversion of biology from a descriptive to the quantitative science it is today. Theoretical Background The fact that many molecules of biological importance interact with radiant energy in a predictable fashion is fundamental to spectroscopic measurements. The reflected color of a pigment is qualitative and tells what is happening, but not to what degree (quantitative). A great deal of information can be obtained by quantitating the reflected energy. The human eye is not especially accurate as a quantitative receptor of color, so an instrument known as a colorimeter has been developed. There are three factors which limit the accuracy of the human eye as a colorimeter: 1. The range of radiant energy perceived by the human eye is limited to 380-750 nanometers (the visible range). 2. The human eye cannot discriminate between similar types of light (wave lengths close together). 3. Electrical colorimeters are quantitative (provide a number), while the eye is only semi-quantitative. Spectrophotometers The electric photometer apparatus is called a spectrophotometer. It has replaced the colorimeter in most laboratories. A spectrophotometer consists of a light source which is focused on a prism to separate the light into its separate bands of radiant energy. The different bands (colors) may be then focused through a narrow slit. The narrower the slit the more precise the measurement since the absorption is then more closely related to a specific wave length. The beam of light then passes through the sample to be measured. The sample is usually dissolved in a suitable solvent and contained in a specially selected tube called a cuvette. Most cuvettes have a light path of exactly 1.0 cm. After the selected beam of light traverses the sample, it emerges as transmitted light. The transmitted light is reduced in intensity if the substances in the photocell, including the cuvette, has absorbed some of the transmitted light. If none of the incident light is absorbed, the transmitted light will show the same radiant energy as the original light. The transmitted light then strikes a photoelectric tube which generates an electrical current that is proportional to the intensity of the transmitted light. The photoelectric tube is connected to a galvanometer with a graduated scale that permits measuring the intensity of the transmitted beam. The scale is normally graduated in one of two ways: 1. %T = percent transmittance - an arithmetic scale with equidistant units from 0-100% 2. A = absorbance - a logarithmicscale with non-equal units from 0.0-2.0. Biological molecules are usually dissolved in a solvent prior to measurement. Therefore, the solvent itself may absorb light and be a possible source of error. To assure the light absorption of only the solute is determined, a means of subtracting the absorbance of the solvent is necessary. This is done by first using a blank (solvent) in the apparatus. The scale is manually adjusted to read 100% T or 0.0 A, after which the sample (unknown plus solvent) is inserted. A reading of less than 100% T or more than 0.0 A is considered to be the result of absorbance of the unknown solute. If other solutes (buffers, salts, etc.) are present in the sample other than the unknown solute, they must be included in the blank. Lab 9 Page 3
Using the Spectrophotometer 1. Turn on the instrument by turning knob 1 clockwise. Allow the spec. to warm up for at least 15 min. 2. Make sure the sample compartment is empty the spec is set to Trasmittance mode and adjust to 0 with knob 1. 3. Set the display mode to ABSORBANCE by pressing the MODE control key until the appropriate LED is lit. 4. Fill a cuvette with your blank. 5. Wipe the blank cuvette with a kimwipe to remove liquid droplets, dust, and fingerprints. 6. Place the cuvette in the sample compartment and align the mark on the cuvette with the guide mark on the front of the sample compartment. Press the cuvette firmly into the sample compartment and close the lid. 7. Set the desired wavelength with knob 2. 8. Adjust the meter to 0.0A with knob 3 on the far right side of the instrument. 9. Replace the blank cuvette with your unknown cuvette, aligning the guide marks and close the lid. 10.Record the Absorbance from the meter. If you have another unknown, place it in the spec and record the new absorbance. 11.Remove the cuvette from the sample compartment and repeat steps 6-10 for any remaining sample solutions. 12.When you are finished with all your measurements turn off the spectrophotometer by turning knob 1 counterclockwise until it clicks. Notes: A flashing display indicates that the reading is out of range. When changing wavelength it is important to insert the blank and reset the display to 100%T or 0.0A every time. 1 3 2 Exercise 9.2 Spectroscopic Analysis of Leaf Pigments 1. Collect leaves that have turned a color other than brown and if possible leaves from the same tree that are still green. 2. Keep the green and colored leaves separate at all times. 3. Remove the major veins of the leaves and snip the remaining leaf areas between the veins into very small pieces. 4. Place the leaf pieces in a test tube. 5. Add 5 ml of acetone to the test tube. 6. Mix the test tube contents well. 7. Centrifuge the test tubes to pellet the leaf material. 8. Transfer by decanting some of the colored supernatant to a cuvette. 9. Transfer some of the extract solution to another cuvette to use as a blank. 10.Measure the absorbance of each cuvette at the following wavelengths: Start at 400 and increase by 20 nm up to 700 nm. Remember you must use a blank (extract solution) for each wavelength to zero the spectrophotometer. 11.Record your data. Graph your results, you may use the computer if you want. Use different colors for the different colored leaves. Lab 9 Page 4
Exercise 9.1 Report Name 1. Using colored pencils diagram the chromatogram results. Show the relative position of the colors along the paper, beginning with the color nearest the original pigment line. 2. Calculate the R f values for each band visible starting at the solvent front and ending with the band closest to original pigment line. Band # R f Visible color Pigment 3. What light wavelength (color) would you expect carotene to absorb least? 4. What light wavelength (color) does chlorophyll absorb the least? 5. Why is it beneficial for a plant to have pigments of different colors? Lab 9 Page 5
Exercise 9.2 Report Name Wavelength (nm) Absorbance Absorbance 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 1. How might dust and debris in the air affect photosynthesis? 2. Is there any correlation between the green leaf and the colored leaf spectrograph? If so what? 2.00 1.75 1.50 Absorbance 1.25 1.00 0.75 0.50 520 0.25 0.00 400 420 440 460 480 500 540 560 580 600 620 640 660 680 700 Wavelength (nm) Lab 9 Page 6