*O chem lab report

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School

Grand Canyon University *

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231L

Subject

Chemistry

Date

Feb 20, 2024

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docx

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Uploaded by ConstableIronBoar27

Abstract Natural products are materials extracting from organic compounds that can contribute to many medical, scientific, and cultural advancements. This study investigates the extraction and examination of organic compounds found naturally, particularly the chromophores in grape leaves. The main objective revolved around isolating and recognizing specific compounds using methods like column chromatography and UV-Vis Spectrophotometry. The findings pointed to the presence of Chlorophyll A, Chlorophyll B, and Pheophytin A within the grape leaves. To identify these compounds, data was compared with existing literature values within reasonable anaylsis. Recommendations of future experiments highlight the constraints of the procedures and suggests alternatives such as flash chromatography and high-performance liquid chromatography to address error within the lab. By refining extraction methods and broadening comparative studies across various plant species, future experiments could be greatly enhanced. Extracting natural products plays a significant role in scientific exploration and is vital to continue research as modern medicine and science continues to expand.

Introduction Natural products are a diverse group consisting of organic products produced by life which can range from wood and soil, to milk and plants. Natural products can be found in prokaryotes such as bacteria and archaea and eukaryotes such as fungi, plants, and animals. Natural product extraction is the isolation of various compounds such as enzymes, proteins, pigments, terpenes, and antibiotics. The use of extraction can be traced back to beginning civilizations who harnessed the healing and regenerative properties of plants, grains, and soils. Extraction is a key role in agriculture, food, cosmetics, and fragrances. Natural product extraction also plays a key role in medicinal advancements such as cancer research, drug discovery, and preventative medicine. In pharmaceuticals, 49% of the drugs are either natural products or derived from natural products. (Cragg, 2016). Morphine, opium from poppy, quinine from cochine bark, and aspirin from willow bark all are drugs synthesized from natural products as therapeutic agents. Antibiotics such as penicillin from fungus are another example of medicinal extraction. Natural extractions can be used in cancer research such as the compound, vincristine from the Madagascar Periwinkle w;hich can be used to treat leukemia. Taxol from the Pacific Yew Tree has also been used in cancer therapy (Cragg, 2016). A useful organic product from plants are chromophores which give pigmentation and certain chemical properties to the organic compound. Chromophores play a crucial role in the plant's photosynthetic capabilities. During photosynthesis, light energy is converted into glucose, providing energy for metabolic processes, growth, and reproduction. Moreover, photosynthesis releases oxygen and contributes to the regulation of carbon dioxide levels. Chlorophylls, carotenoids, and anthocyanins are

examples of pigments found in various plants. UV-visible spectroscopy is a technique often used to identify and examine the absorption of light by the sample of these chromophores. Isolation of these natural products is a mutli-stage process consisting of extraction and purification. Extraction, purification, structural identification, and synthesis are all processes involving isolation of natural products, Extraction is the first step to separate the desired natural products from the raw materials. This is based on solubility and acid base properties. Purification is based on chromatography and recrystallization. Identification of products can be found through many procedures such as Solvent extraction, thin layer chromatography, ultraviolet visible spectroscopy, infrared spectroscopy, nuclear magnetic resonance, mass spectrometry, atomic spectroscopy, and X-ray spectroscopy (Smith, 2020). Chromophores specifically are best analyzed through the use of column chromatography and UV-Vis spectroscopy. Chromophores are grouped into subgroups such as carotenes, pheophytins, chlorophylls, and xanthophylls. Derivatives of these subgroups involve α-carotene, β-carotene, phenophytin B and chlorophyll B (Smith, 2020). Science utilizes these methods to identify and analyze natural products and compounds. The compounds must be lysed, centrifuged, and analyzed with thin layer column chromatography. The absorption of pigments extracted from the plants is analyzed with UV- spectroscopy. The analysis of these natural compounds derived from organic compounds plays a crucial role in development of medicine, pharmaceuticals, agriculture, and technology. Methods and Results

Week 1: Extraction of a Natural Product Two grams of recently harvested green grape leaves were finely ground using a mortar and pestle provided by the laboratory instructor. Following the addition of 2 mL of acetone, the resulting mixture was carefully transferred to a labeled centrifuge tube (Tube 1). The mortar underwent two additional rinses with acetone, ensuring a cumulative volume of at least 6 mL in Tube 1. After a thorough 2-minute vortexing, Tube 1 underwent centrifugation at 2000 rpm for 2 minutes, employing a counterweight. Subsequently, the tube was delicately handled to avoid disturbing the sediment, and the acetone fraction was poured into a separate labeled tube (Tube 2). Tube 2 was then supplemented with 3 mL each of hexane and deionized water. Following a minute of vortexing and centrifugation at 1000 rpm for 2 minutes, Tube 1 underwent a cleaning process to eliminate plant debris and was dried using compressed air under the fume hood. The acetone/water layer from Tube 2 was then carefully transferred back to Tube 1 utilizing a Pasteur pipette. Meanwhile, the hexane from Tube 2 was reserved, and 2 mL of hexane was introduced to Tube 1, followed by a minute of vortexing and centrifugation at 1000 rpm for 2 minutes. The acetone layer was discarded, and the amalgamated hexane solution from both tubes was prepared for subsequent steps. Figure 2: Sample tube after centrifugation. Sample product assembled at the bottom of the tube. Figure 1: Balanced centrifuge tubes ready for centrifugation.

To establish a drying column, a Pasteur pipette was packed with cotton and sodium sulfate, affixed to a support, with a fresh centrifuge tube positioned below. The hexane layer was meticulously passed through the column. Following this, three TLC plates were readied, each delineated with a line positioned 1 cm above the bottom edge. Extract samples were applied to the plates using glass microtubes: four drops on the first spot, eight drops on the second, and sixteen drops on the third along the marked line. Three distinct jars containing hexane with ratios of 90:10, 80:20, and 70:30 were prepared. Each TLC plate was immersed in one of the jars and retrieved when the solvent had traversed ¾ up the plate. Subsequently, the samples were stored for evaporation, to be utilized in the upcoming week's procedures. Week 2: Separation of Chromophores by Column Chromatography The procedure began with the combination of 4.0 grams of silica gel and 15 mL of hexane in a beaker. The contents for stirred eliminate air from the silica, and the mixture was set aside for later use. A column assembly involved gently compressing a small cotton piece at its base, securing the column to the workbench, and affixing a funnel on top. Approximately 2–4 mm of sand was add to the sand and the column was leveled by tapping it gently with a plastic tool. 5 mL of hexane was slowly added using a Pasteur pipette. 15 mL of hexane was added with a collection beaker placed beneath. Figure 3: TLC plates immersed in hexane filled jar to allow the pigments to travel up the jar.

Silica gel was loaded into the column by pouring the stationary phase solution with a slow, steady flow, tapping the column to level the gel. Maintaining a level solvent layer was crucial to prevent drying and potential damage to the silica gel, which could compromise the separation process. The hexane was run through the column until it nearly reached the top of the silica gel. The column was then filled with hexane, settled, and the process was repeated twice more. Sand was reintroduced, and the hexane level was carefully drained to a few millimeters above the sand. The sample was dissolved in approximately ¾ mL of hexane and added to the column. Additional hexane was added while maintaining the sand's position to maintain the solvent flow. A solution of a 90:10 hexane to acetone mixture was introduced, collecting distinct bands in separate test tubes. As the column progressed, solvent ratios were adjusted, and bands were collected, particularly focusing on the distinct yellow band. The ratios were altered to 80:20 and 70:30, respectively. The column was stopped after collecting the yellow bands using pure acetone. During this process, TLC plates were prepared, marked, and spotted with samples. They were immersed in a 70:30 hexane to acetone solution, and after solvent migration, the test tubes containing bands closest to a single pigment band were photographed, labeled, and stored for the upcoming week's experiment. Figure 4: Separatory column filtering the sample though the silica gel. Figure 5: Separate bands filtered into test tubes after separatory column.

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