Understanding Photosynthesis: Pigment Separation and Rates
School
CUNY Lehman College**We aren't endorsed by this school
Course
BIO 166
Subject
Biology
Date
Dec 11, 2024
Pages
4
Uploaded by MinisterLlama4966
PhotosynthesisKaram KhamashtaDecember 5th, 2024Lab Report 8IntroductionPhotosynthesis is a complex process in which plants convert light energy into chemical energy, producing glucose and oxygen from carbon dioxide and water. This lab focused on two essential aspects of photosynthesis: separating plant pigments and measuring the photosynthetic rate in isolated chloroplasts. The first part of the lab used chromatography to separate the plant pigments. Chromatography is a method that separates mixtures based on differential partitioning between mobile and stationary phases. In this experiment, the photosynthetic pigments from spinach leaves were separated through paper chromatography. The second part of the lab looked at the light reactions of photosynthesis using a dye-reduction technique. This experiment tested the hypothesis that light and chloroplast enzymes are required for the light reactions. DPIP (Indophenol solution) was substituted for the electron acceptor NADP+, turning from blue to colorless as it was reduced by electrons from the light reactions.Methods and MaterialsThe lab involved two main parts: chromatography of plant pigments and photosynthesis/light reactions. For the chromatography experiment, materials included a chromatography vial, chromatography paper strip, automatic pipette, chromatography solvent (9:1 petroleum ether: acetone), and a spinach leaf. The procedure began by preparing the chromatography vial with solvent and ensuring it was dry. Researchers carefully handled the chromatography strip, marking a 1.5 cm line from one end and creating a pointed bottom. Pigments were extracted by rubbing a coin over a spinach leaf along the pencil line; then, the strip was placed in the solvent-prepared vial, ensuring the pigment line remained above the solvent. For the photosynthesis rate measurement, materials included spectrophotometric cuvettes, pipettes, DPIP solution, phosphatebuffer, isolated chloroplasts (both boiled and unboiled), a spectrophotometer, and a 100W floodlight. Chloroplasts were prepared by blending spinach leaves with ice-cold sucrose solution,filtering through cheesecloth, and separating them into boiled and unboiled samples. The experiment involved setting up four labeled cuvettes with specific combinations of chloroplasts, DPIP, and buffer. Researchers zeroed the spectrophotometer and collected transmittance
measurements at 0, 5, 10, and 15 minutes, with cuvettes exposed to light (except for the dark control), to measure the reduction of DPIP from blue to colorless.ResultsPart A: Chromatography of Plant PigmentsBand numberColor of the bandMigration distance (mm)Rf ValueIdentified pigment1Yellow80.8Carotene2Green/Yellow70.7Xanothonhyll 3Green50.5Chlorophyll A4Pale Green40.4Chlorophyll BPigment size, solubility, and affinity for the stationary phase are all factors that affect separation. Rf values may change when using a different solvent because of variations in the pigment's solubility and interaction with the mobile phase. While auxiliary pigments extend the spectrum and shield chlorophyll, chlorophyll absorbs light energy. Part B: TransmittanceTime (min)Cuvette 2 (unboiled/darkCuvette 3 (unboiled/light)Cuvette 4 (boiled/light)030%30%30%530%35%30%1530%40%30%3030%45%30%DiscussionAs a result of the experiments conducted, the chromatography experiment separated photosynthetic pigments depending on their various chemical properties. Pigments move at different speeds depending on their size and hydrophobicity, which are identified by Rf values.
Chlorophyll a is the primary photosynthetic pigment; accessory pigments increase the range of light wavelengths used for photosynthesis. During the DPIP reduction experiment, DPIP acted asan electron acceptor replacement for NADP+1. These two key factors were realized in this experiment as some requirements for successful photosynthetic reaction-light and functional chloroplast enzymes. Boiling of chloroplast deactivates their enzymatic capabilities, and darkness hinders the light-dependent reactions. Results have shown photosynthesis involves detailed biochemical processes, which critically depend on specific plant cell energy conversion conditions. In addition, further research can study the varying intensity of light or different wavelengths that may impact the photosynthetic rate.
Citations“CUNY Blackboard Resources.” The City University of New York, www.cuny.edu/about/administration/offices/cis/core-functions/cuny-blackboard/.Accessed 5 Dec. 2024.Rajkhowa, Neha. “CUNY Blackboard Resources.” Photosynthesis Slide Show, www.cuny.edu/about/administration/offices/cis/core-functions/cuny-blackboard/.Accessed 5 Dec. 2024.