week 7-8 asa lab finsihed

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Column Chromatography 10/12/2023 and 10/19/2023 Asael Garcia Abisai Garcia Chem 2023 1L0 FL23 Sarita Sitaula

Purpose Carry out the separation and purification of solids and liquids through column chromatography. Part A of this lab the preparation of the sample Lycopene and  -Carotene is extracted from tomato paste through a 50:50 ratio mixture of Acetone: Petroleum ether. Part B of this lab the column chromatography separation of Lycopene and  -Carotene is conducted through three different steps. 1. Column packing (slurry method) (silica + hexane), 2.Sample preparation and loading, 3.Column running (elution). Background Column Chromatography is excellent at removing impurities and organic chemists obtain new compounds by synthesizing or isolating natural products that have been biosynthesized by microbes, plants, or animals. The most common adsorbent for column chromatography, silica gel and alumina, are the same stationary phase as seen in Thin Layer Chorography. The eluents, instead of flowing up flows down through the column filled with the absorbent. Three mutual interactions must be considered: the activity of the stationary absorbent phase, the polarity of the eluting mobile solvent phase, and the polarity of the compounds in the mixture being chromatographed. Table of Physical Constants Substance Mol Formula Mol Weight m.p (C) b.p. (C) Density Lycopene C 40 H 56 536.888 g/mol 177 660.9 0.8895 g/cm 3  -Carotene C 40 H 56 536.873 g/mol 180 633 1.00 g/cm 3 Acetone C 3 H 6 O 58.08 g/mol -95 56 0.791 g/mL Petroleum Ether C 6 H 14 86.178 g/mol -40 60-90 0.650 g/mL Sodium Chloride NaCl 58.44 g/mol 801 1465 2.16 g/cm 3 Magnesium sulfate MgSO 4 120.366 g/mol 1124 330 2.66 g/cm 3 Silica Gel SiO 2 60.99 g/mol 1200 n/a 0.1 g/cm 3 Hexane C 6 H 14 86.18 g/mol -95.3 68.73 0.6606 g/mL Potassium Carbonate K 2 CO 3 138.205 g/mol 891 Decomposes 2.43 g/cm 3 Hazards Lycopene is a general fire hazard.  -Carotene can cause skin and eye irritation and is hazardous if inhaled or ingested. Exposure to acetone can irritate the eyes, nose, and throat. Petroleum ether is harful if inhaled o ingested causing dizziness and drowsiness which can lead to loss of

consciousness. Sodium chloride may cause skin or eye irritation. Silica gel can cause redness and irritation when in contact with skin and eyes. Hexane contact can cause skin rash, dryness, and redness. Potassium Carbonate is toxic to mucous membranes, eyes, and skin. Gloves, goggles, and proper lab attire covering all exposed skin must be always worn. Proper care and caution should be followed when handling lycopene. Procedure • Set up a gravity filtration system. • Weigh about 4.0 g of tomato paste into a small beaker. • Add 10 mL of the extraction solvent (a 50:50 mixture of acetone and petroleum ether). • Stir the mixture vigorously with a glass rod. (The solvent turns red-yellow). • Decant the liquid onto the filter paper leaving the solid residue behind. • Repeat this process 3 more times. • After the 4 th extraction, transfer the residue to the filter and wash it with 5 mL of extraction solvent. • Press the residue in the funnel with a spatula to squeeze out as much liquid as possible. • Transfer the combined liquid extract to a separatory funnel.Add 20 mL of 10% aqueous K2CO3 solution to the separatory funnel, stopper and shake it vigorously. • From time to time release the pressure built up inside the funnel by opening the stopcock. • When the two layers have separated, drain the lower aqueous layer into a beaker. Discard the aqueous layer. • Repeat the process with 20 mL of 10% NaCl solution. • Pour the top organic layer into a clean Erlenmeyer flask and add a small amount of anhydrous MgSO4 (drying agent). • Decant the clear solution to another clean and dry flask making sure that the MgSO4 stays behind. • Label the flask and store it in a cool and dark place for next week. Part II: Chromatographic Separation of Lycopene and  -Carotene A. Packing the Column (Slurry method) • Fill the chromatography column two-thirds with dry silica gel. • Pour this out into a small beaker. • Add about 8 mL hexane to the beaker. • Stir the mixture vigorously to remove air bubbles.

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• Swirl the mixture to get the silica gel suspended and immediately pour the entire slurry into the funnel. • Open the stopcock, drain some solvent into the same beaker and finish transferring the remaining slurry into the column. • Place an empty flask under the column and allow the solvent to drain to about 5 mm above the top surface of the silica gel. • Tap the column with a pencil until the column settles to a minimum height. • Pack the column as evenly as possible. • Cracks, air bubbles, and channels will lead to a poor separation. B. Column Chromatography • Dissolve the pigment sample obtained from tomato paste in the least possible amount of dichloromethane. • Immediately transfer the pigment extract to the top of the column with a pipette, using a few drops of hexane to rinse any remaining extract onto the column. • When the extract surface just disappears into the sand, fill the column nearly to the top with hexane and continue to add hexane to keep its level more or less constant. • When the yellow  -carotene band begins to drain out of the column, replace the beaker with a clean, dry, and pre-weighed flask and elute the yellow  -carotene band completely. • Then fill the column with the second eluent, 10% acetone in hexanes, and continue to replenish the eluent as before. • When the orange-red lycopene band begins to leave the column, replace the beaker with a clean, dry, and pre-weighed flask and elute the orangered lycopene band completely. • Label the flasks and store it in a cool and dark place for next week when you weigh the flasks and calculate the percent yields. Observations • The tomato paste was bright red and thick. • With 50:50 mixture added it became bright orange-red color with a liquid consistency. • Extract can be seen separated into 2 different layers with the organic layer on top and water on the bottom. • The substance after a week was dried out and was a red-orange-yellow color of dried out substance that was stuck to the bottom of a flask. • The column became fully one long orange color without any separation. • Once separation began, bands can be seen within the column. • A yellow and orange separation of color can be seen within the column. • A small red band portion can be seen towards the upper portion of the column.

Clean Up Dismantle gravity filtration system. Discard left over solids and liquids into their designated waste containers. Discard used pipettes into their designated waste container. Clean all beakers and flasks used with acetone. Return all equipment to their designated locations in the lab. Data % of Extract = (mass of extract / mass of tomato) x 100 2.5% = (.1g / 4g) x 100 % of  -Carotene = (mass of  -Carotene / mass of extract) x 100 3% = (0.0030 / .1g) x 100 % of Lycopene = (mass of Lycopene / mass of extract) x 100 2.5 % = (0.0025 / .1g) x 100 Discussion During the first part of the lab when preparing the substance, some of it was spilled. This error was due to using faulty equipment, which was a cap that wouldn’t fully close. Then during the separation portion of the lab some of the substance was poured onto the funnel. This led to cross contamination and poor separation happening. This is why during one portion of the lab, instead of the compound being separated in the column it was all bunched together in one orange color. Conclusion Column Chromatography is the separation and purification of solids and liquids through 2 different parts. Part A of this lab the preparation of the sample Lycopene and  -Carotene and part B is the separation of Lycopene and  -Carotene. The percent yield calculated at the end of the lab were 2.5 % of Extract, and 3% of  -Carotene, and 2.5 % of Lycopene.

References Beta-Carotene | C40H56 | CID 5280489 . (n.d.). PubChem. Retrieved October 26, 2023, from https://pubchem.ncbi.nlm.nih.gov/compound/Beta-Carotene Hexane . (n.d.). Fisher Scientific. Retrieved October 26, 2023, from https://www.fishersci.com/us/en/scientific-products/selection-guides/chemicals/hexane.html magnesium sulfate, 7487-88-9 . (n.d.). The Good Scents Company. Retrieved October 26, 2023, from http://www.thegoodscentscompany.com/data/rw1256141.html Petroleum ether . (n.d.). Wikipedia. Retrieved October 26, 2023, from https://en.wikipedia.org/wiki/Petroleum_ether Potassium carbonate . (n.d.). Wikipedia. Retrieved October 26, 2023, from https://en.wikipedia.org/wiki/Potassium_carbonate Salek, R., & Neveu, V. (n.d.). Lycopene | C40H56 | CID 446925 . PubChem. Retrieved October 26, 2023, from https://pubchem.ncbi.nlm.nih.gov/compound/Lycopene Silica gel | 112926-00-8 . (2023, September 7). ChemicalBook. Retrieved October 26, 2023, from https://www.chemicalbook.com/ChemicalProductProperty_EN_CB1199389.htm Slobodnik, J. (n.d.). Acetone | CH3COCH3 | CID 180 . PubChem. Retrieved October 26, 2023, from https://pubchem.ncbi.nlm.nih.gov/compound/Acetone Sodium Chloride - Properties, Uses, Occurrence and Preparation . (n.d.). Vedantu. Retrieved October 26, 2023, from https://www.vedantu.com/chemistry/sodium-chloride

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Post Lab 1.Predict the order of elution of a mixture of triphenylmethanol, biphenyl, benzoic acid, and methyl benzoate from an alumina column. This mixture of molecules would elute in the following order: biphenyl, methyl benzoate, triphenylmethanol, and benzoic acid. 2.What would be the effect of collecting larger fractions when carrying out the experiments in this chapter? When doing experiments, gathering bigger fractions will result in a decrease in the chemicals' purity. The substances that are being eluted will contaminate one another while collecting bigger fractions. Generally, the existence of several chemicals in the fraction is one of the probable results. 3.What would have been the result if a large quantity of petroleum ether alone were used as the eluent in either Experiment 1 or Experiment 2? Not all of the chemicals would elute if a lot of petroleum ether were utilized. This is so that the column will only elute the non-polar compounds because petroleum ether is a non-polar solvent. preventing the polar chemicals from eluting. 4.Once the chromatographic column has been prepared, why is it important to allow the level of the liquid in the column to drop to the level of the alumina before applying the solution of the compound to be separated? Before adding the compound solution, it is crucial to level the liquid in the column so that it drops to the alumina's level; otherwise, a bigger band will form. In addition, the experiment's goal is to find a distinct thin band for data collection. This is due to the fact that the compound combination will dilute with the solvent if the solvent level is not lowered to the level of alumina. As the chromatography process progresses, the band will get bigger since there is more liquid. 5.A chemist started to carry out column chromatography on a Friday afternoon, reached the point at which the two compounds being separated were about three-fourths of the way down the column, and then returned on Monday to find that the compounds came off the column as a mixture. Speculate the reason for this. The column had not run dry over the weekend. The compounds were simply left in the column for too long during the weekend, which is why they were discovered to have come off the column as a combination. The column's compounds travel at various speeds. But over time, as long as the compounds are left in the column, they will start to mix together and diffuse into one another.