Chem 14CL Summer 2006 Isolation of Chlorophyll and Carotenoid Pigments from Spinach Pre-lab report must be written inside your lab notebook. IMPORTANT: Make sure that you always follow the proper laboratory safety protocol BEFORE going to the lab. Note: The Experimental Procedures for this Experiment are provided as a Handout, which will distributed in lab (I) On-line Resources For This Experiment •TLC Procedure on this Orgchem site(II) Pre-lab Report Guideline In preparing for lab set up your notebook with the following information: • Title of the experiment • A reference identifying the procedure handout • A short introduction summarizing the goals of the experiment and the techniques that you will use in the experiment • A flow chart summary of the ENTIRE experimental procedures • MSDS Information (Refer to the MSDS handout for more information) The following chemicals will require you to use the MSDS database on the Web: Hexane; Acetone You should record the following MSDS information in your notebook for the chemicals listed above. (Printouts directly from the Web pages will NOT be accepted!!) (a) Product Name (b) Chemical Formula (c) Formula Weight (d) Melting Point; Boiling Point and Density (e) Health Hazard Data (summarize in your own words) (f) Spill and Disposal procedures (summarize in your own words Chem 14CL Summer 2006 1Isolation of Chlorophyll and Carotenoid Pigments from Spinach Pre-lab report must be written inside your lab notebook. IMPORTANT: Make sure that you always follow the proper laboratory safety protocol (refer to the course syllabus) BEFORE going to the lab. (II) Pre-lab Report Guideline (Continued) DATA TABLES & OBSERVATIONS USE A NEW PAGE TO SET UP THE FOLLOWING BLANK TABLES. • Set up a blank table for recording the column chromatography data. Include a column for the number of the fraction being collected, the time over which the fraction is collected, the concentration of the solvent on the column, the appearance of colors on the column, and the color of the solution in the fraction being collected. • Set up a blank data table for recording the thin-layer chromatography data. The data should include the components of the mobile phase; the identity of the initial spots on the TLC plate; the distance travel by each spot on the TLC plate at the end of the analysis; the distance traveled by the solvent front at the end of the TLC analysis. Keep the original TLC plate in your lab notebook for future reference. Chem 14CL Summer 06 Isolation of Chlorophyll and Carotenoid Pigments from Spinach 2Post-lab Report Guidelines The post-lab report must be written inside your lab notebook (except graphs) THIS IS A GROUP REPORT POST LAB REPORT (I) ABSTRACT – Summarize the goal(s) of the experiment plus a summary of your experimental results (i.e. identities of components and their Rf values etc.) (II) OBSERVATIONS Isolation Rewrite all your observations during the extraction of the pigment and the chromatographic separation in an organized format. In tabular format record the number of the fraction being collected, the time over which the fraction is collected, the composition of the solvent used in the column (i.e. the mobile phase used), the appearance of colors on the column, and the color of the solution in the fraction being collected. Characterization Attach the ORIGINAL TLC plate in your report. In tabular format record the color of each component separated on the TLC plate, and its corresponding Rf value. (III) DATA ANALYSIS Identify the absorption signal(s) in the UV spectra that give rise to the colors of the chlorophyll and beta- carotene solutions (refer to the table listed below for colors of various components) Based on the absorption signals you identified, explain whether the locations of the absorptions make physical sense. Calculate the Rf values for all the components on the TLC plate. Show all your work. Using the guide below, identify as many spots on the TLC plate as you can. Determine which pigments were present in the yellow band and which were present in the green band from your column. Chem 14CL Summer 06 Isolation of Chlorophyll and Carotenoid Pigments from Spinach 3Post-lab Report Guidelines The post-lab report must be written inside your lab notebook (except graphs) THIS IS A GROUP REPORT POST LAB REPORT Pigments in spinach in order of decreasing Rf values: Carotenes (1 spot) (yellow-orange) Pheophytin a (gray, may be nearly as intense as chlorophyll b) Pheophytin b (gray, may not be visible) Chlorophyll a (blue-green, more intense than chlorophyll b) Chlorophyll b (green) Xanthophylls (possibly 3 spots: yellow) (IV) CONCLUSIONS In your conclusion address how well the column separated the various pigments based on the following results. (i) Compare the separation of components on the TLC plate. (ii) Based on the locations of the absorption bands on the UV-Vis spectra Attach the ORIGINAL EXPERIMENTAL TLC plate with this post-lab report and turn in the completed report to your TA on the due date listed on the lab schedule
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