Photosynthesis. Introduction

Transcription

1 Photosynthesis Learning Objectives: Explain the importance of photosynthetic pigments for transformation of light energy into chemical bond and the advantage of having more than one pigment in the same plant. Describe the physicochemical properties of the photosynthetic pigments that can be used for their separation. Understand and explain the principle of paper chromatography and use the technique to separate photosynthetic pigments from spinach leaves. Understand and explain spectrophotometry and use it to determine the spectral properties of spinach pigments collectively and separately. Introduction Photosynthesis is used by plants, some bacteria, and some protists to convert the carbon dioxide and water using the energy from sunlight into sugar and it results in the release of O 2 as a byproduct. The sugar molecules produced during photosynthesis are used by plant cells as fuel for cellular respiration to produce ATP for cellular work. In Plants, photosynthesis takes place in the chloroplasts within the cells of the mesophyll tissue in leaves. Photosynthesis consists of two cyclic pathways: the light-dependent Light Reactions that takes place in the thylakoids where the light-capturing photosynthetic pigments are located, and the light-independent Calvin Cycle that takes place in the stroma of the chloroplast. During the Light Reactions sunlight energy is converted into enough ATP and NADPH as cellular forms of energy and reducing power for use during the light-independent Calvin Cycle to convert inorganic molecules of CO 2 into glucose.

2 Exercise 1. Identification and isolation of spinach pigments separated by paper chromatography Plants contain organic pigment molecules called photosynthetic pigments: chlorophylls, and carotenoids. Chlorophylls are composed of carbon, hydrogen, oxygen and nitrogen and they contain a porphyrin ring with a Mg atom at its center, which has the potential to gain or lose electrons easily to provide energized electrons to other molecules. There are two chlorophyll pigments that differ slightly in structure, and as such in color: chlorophyll a (blue-green) and chlorophyll b (yellow-green). Carotenoids have hydrocarbon isoprenoid chains, with alternating double and single bonds. There are two classes of carotenoids: carotenes, such as Beta carotene (orange), that are composed purely of carbon and hydrogen, and xanthophylls (yellow), also known as carotenols, which also contain oxygen in the form of hydroxyl or keto groups. Fill-in this following table, based on what you already learned about the chemical structure influencing physical properties: Photosynthetic pigment molecule Number of oxygen atoms Polarity (from 0-3) (0 for non-polar, and 3 for the most polar) 2

3 Photosynthetic pigments can be extracted collectively from leaves using organic solvents such as ethanol or acetone (total pigment). Separation of individual pigments from the total pigment can be achieved by paper chromatography. In paper chromatography, the total pigment is applied to the stationery polar paper and the paper is then dipped in a layer of chromatography solvent (a non-polar mixture of ether and acetone). As the chromatography solvent moves up the paper, the individual pigments will ascend with it at different rates, based on their solubility in the non-polar chromatography solvent. In this way, the individual pigments are separated and can be identified by their color and position on the paper. In this experiment, you will separate individual photosynthetic pigments from spinach leaves using the techniques of paper chromatography, you will identify each by color and by relative migration rate (Rf value), and you will prepare individual isolated pigments extracts. Materials 1 Chromatography paper (square) Spinach extract (pre-prepared) Glass Jar with lid Chromatography solvent: (a mixture of ether and acetone) Glass capillary tube Pipet Ruler Pencil Staple Procedure Exercise 1: Separation and isolation of spinach photosynthetic pigments A. Separation and identification of individual photosynthetic pigments 1. Draw a pencil line across the paper square 10 mm from one end, to define the position of the origin Apply the spinach extract to the origin line across the length of the paper using the glass capillary tube. You will need to fill more than once. Allow to paper to air dry, and repeat the extract application 5-6 times. This number of applications is necessary for obtain enough pigment for use in the next experiment. Allow to completely dry before proceeding. 2. Turn the square paper into a cylinder and staple the top and bottom edges to hold the shape of the cylinder. 3. Pipet 10 ml of the chromatography solvent into the glass jar, without wetting the sides. 4. Gently lower the paper cylinder into the chromatography solvent and let it sit on the bottom. Close the lid and don t move the jar. 5. Observe what happens as the solvent moves up the stationary paper and moves beyond the origin line where the extract was applied. Record your simple observation 6. The solvent will move quickly up the paper and the pigments will be separated in about 15 minutes. When the solvent is about 5-10 mm from the top, remove the paper from the cylinder and pencil-mark the position of the leading edge of each band. Mark the pigments while the paper is still wet. Some colors will fade upon drying. 7. Measure the distance from the pencil line to the leading edge of each clear pigment and work out the Rf value for each one using this formula: Rf = a / b, where a = distance moved by substance from its original position; b = distance moved by solvent from the same position. Enter the distance travelled by the solvent and each of the pigments in the space provided. You will need this information to be presented in your scientific report. Substance Distance travelled by the end Color of chromatography Solvent Beta carotene Chlorophyll a Chlorophyll b Xanthophyll 1 Xanthophyll 2 3

4 Draw a diagram of the paper chromatogram at the end of the separation, which includes: the position of the origin and each of the separated pigments, their individual names and colors. You will need this information to be presented in your scientific report. B. Isolation of individual photosynthetic pigments Individual pigments will be isolated using cut-out paper strips from the paper chromatogram. Pigments will be eluted by soaking the paper strips in acetone, the acetone-soluble pigments will be transferred from the paper into the liquid acetone. 1. After recoding all your observation and measurements, bring your chromatogram to your instructor. 2. Your instructor will cut out chromatograms of all groups and will provide each group with enough strips for extraction. 3. Transfer 8 ml of acetone into a small 25 ml beaker. 4. Soak the cut out strips provided by instructor and allow all pigment to dissolve in acetone 5. Transfer the acetone with the dissolved pigment into a test tube labeled with the isolated pigment you are assigned to use in the next exercise. Exercise 2. Determination of absorption spectra of photosynthetic pigments The first step of photosynthesis is the capture of energy of certain visible light photons through absorption by photosynthetic pigments. The color of all pigments comes from the wavelengths of visible light spectrum that are reflected or transmitted. The ability of a pigment to absorb various wavelengths of light can be determined by spectrophotometry. A spectrophotometer can detect and record both the percentage (%) of the received light that is transmitted by the substance placed in a cuvette in the path of light, as well as its absorbance light. Note that in all spectrophotometers, the absorbance is a log function of the % of the light that could not be transmitted, and it has not units. Also note the position of % transmittance (T) and absorbance (A) displays on your Spectronic 20, the linear scale of transmittance, and the log scale of absorbance. In this exercise, you will determine the absorption spectra of individual isolated photosynthetic pigments and of a diluted sample of the total pigment, prepared by your instructor, by spectrophotometry using the Spectronic 20, shown on the right. Turn on knob A to warm the spectrophotometer for at least 10 minutes before use. Materials Rack with three test tubes Isolated spinach pigments Diluted spinach total pigment, prepared by instructor Spectronic 20 Kim Wipes 4

5 Procedure 1. Follow your instructor explanation of the parts of the spectrophotometer and its proper use. Fill in the name and function in the table. Part Name Function A B C D F 2. Label the tube with 8 ml of the isolated pigment, and the two other tubes. Label one tube with (T) and fill with 8 ml of the diluted total pigment and a second tube with (B) and fill with 8 ml of the solvent to which no pigment was added. Explain the purpose for using tube S. 3. Calibrate the spectrophotometer following you instructor s explanation. Using the cuvette or tube with solvent only, as a blank, measure and record the absorbance of each re-dissolved pigment at 10 nm intervals from Enter the absorbance of the total diluted pigment and your isolated pigment at each of wavelengths in the table. You will obtain the absorbance of the other isolated pigments from other lab groups. Note the position of the filter (F) before you start and switch it over to the appropriate position before recording your readings of the absorbance at 600 nm. Wavelength (nm) Total pigment Chlorophyll a Chlorophyll b Carotenoids (Beta carotene and Xanthophylls)