Experiment 4_ Extraction of Acidic and Neutral Compounds

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Megan Cousins Chem-341-11 Experiment 4: Extraction of Acidic and Neutral Compounds Objective The objective of this lab is to separate all of the different pigmented compounds in spinach leaves through a liquid-liquid extraction. Each one of these compounds has a different polarity in which they can be removed from each other using their distinct polarities. To confirm that we had extracted all of the compounds, the Rf values were given but calculations had to be done to confirm their presence and identity. Rf values are calculated by determining how far the compound has traveled in relation to the distance that it has been allowed to travel, and this can be controlled even further by measuring the distance that the compounds will be allowed to travel through marking the TLC plate previous to placing in the solution. There was a crude extraction done through extracting the pigments and placing the extract onto a TLC place which was submerged into a solution of hexane that has a polarity of 0.5 and acetone that has a polarity of 5.1. These differing polarities will help pull different compounds present in the extract to different heights through the stationary phase of silica. A full column chromatography was done to extract each band into singular test tubes also using solutions with different polarities to extract each particular band. The three different solutions are made up of 8 mL of hexane to 2 mL of ethyl acetate, which has a polarity of 4.4. As well as 7 mL of hexane to 3 mL of ethyl acetate and 6 mL of hexane to 4 mL of ethyl acetate. Each different color band that was collected

was individually placed onto another, new TLC place to complete another identity confirmation using Rf. Procedure 1. Weigh out approximately 0.5 g of fresh spinach leaves a. Remove stems b. record the mass 2. Tear the leaves into confetti sized pieces and place them into a mortar a. Add about 1 mL hexanes b. Add about 1 mL acetone c. Add a small amount of sand d. grind the leaves with a mortar and pestle until the liquid turns a bright, deep green 3. Using a 5” Pasteur pipet, transfer the liquid to a centrifuge tube 4. Rinse the mortar and pestle with another 0.5 mL of acetone and transfer this to the centrifuge tube also 5. If there have more than 1.5 mL of acetone/hexanes in the centrifuge tube at this point, reduce the volume via evaporation in a warm water bath 6. Add another 1.0 mL of hexanes and 2.0 mL of distilled water to the extract in the centrifuge tube 7. Using a Pasteur pipet, thoroughly mix the two layers by gently drawing the mixture in and out of the centrifuge tube a. Allow the tube to stand a few minutes undisturbed to maximize separation of these layers

b. Add a small amount of saturated NaCl if there is an emulsion layer or do not observe clear separation 8. Using a 9” Pasteur pipet, carefully draw off the aqueous layer and transfer it to a small beaker a. Add another 2.0 mL of distilled water to the centrifuge tube as a wash 9. Repeat step 8 10. Dry the organic extract in the centrifuge tube with anhydrous Na 2 SO 4 11. Filter the Drying Agent a. Prepare a filter pipet by inserting a small cotton plug into a 5” Pasteur pipet b. Using a micro clamp, secure the pipet on a ring stand c. Place a clean collection vial below pipet system d. Transfer the extract via pipet to the filter pipet e. Rinse the centrifuge tube containing the drying agent with another 1.0 mL of hexanes f. Filter remaining liquid 12. Obtain a TLC plate a. Mark the TLC plate 1 cm from the top and bottom b. Spot it with the extract i. Spot the plate around 9 times to make sure it is concentrated enough 13. Develop the plate in a solution of hexanes:acetone (7:3) a. Place a strip of paper towel in the developing chamber and allow the chamber contents to equilibrate before inserting the plate b. Set a watch glass over the beaker to prevent evaporation c. Remove the TLC plate when the liquid has risen up to the top line (1 cm from the top) 14. Use a UV lamp to note all possible spots

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a. Circle each spot with a pencil 15. Determine Rf values for all spots a. and try to match them to the compounds shown below (listed in order of decreasing Rf values) b. Include a drawing of the TLC plate and RF values in the lab report i. carotenes (1-2 yellow-orange spots) ii. pheophytin A (gray, intense) iii. pheophytin B (gray, may only be visible under UV) iv. chlorophyll A (blue-green, intense) v. chlorophyll B (green) vi. xanthophylls (as many as 3 yellow spots) Column Chromatography Procedure 1. Prepare 3 Test Tubes of ethyl acetate and hexane mix a. Test tube A: 2 mL ethyl acetate/8 mL hexane b. Test tube B: 3 mL ethyl acetate/7 mL hexane c. Test tube C: 4 mL ethyl acetate/6 mL hexane 2. Keep extra hexane and ethyl acetate on the side to work with during the chromatography process 3. Obtain a microcolumn and insert a cotton plug a. Clamp the column on a ring stand and place a small beaker beneath the system to collect excess solvent during the column preparation b. Place a small layer of sand on the cotton plug c. Wet the cotton and sand with hexane 4. Weigh out about 2.0 g of silica gel in a small beaker

a. break off the tip of a pipet to widen the diameter b. Add hexane to create a slurry c. From here on BE SURE that the column does not run dry, keep the solvent level above the level of the silica d. Begin pipetting the slurry into the column using a 5” Pasteur pipet e. Tap the side of the column gently i. Continue to add slurry until the column packing is about 2 inches from the top of the glass column f. Add a small amount of sand to the top of the column 5. Using a 5” Pasteur pipet, load the sample onto the prepared column a. Allow the extract to travel into the sand layer and then complete the transfer of the sample by adding another 0.5 mL of hexanes to the holding container b. Load that on the column 6. Immediately begin the chromatography process with pure hexanes a. A tight yellow band should be beginning to move down the column while the green layer remains at the top b. If the yellow band is sluggish and doesn’t begin to move down the column within 10 minutes, add a drop or two of Solution A to the pure hexane c. When the yellow band is approximately 3/4 of the way down the column, begin collecting fractions in numbered test tubes 7. Begin to increase the polarity of the solvent system a. gradually include more of Solution A b. If the more polar bands aren’t moving, a drop or two of solution B can be added i. gradually include more of Solution B c. If the more polar bands aren’t moving, a drop or two of solution C can be added d. gradually include more of Solution C

e. If the more polar bands aren’t moving, a drop or two of ethyl acetate can be added f. gradually include more ethyl acetate 8. Collect each different band into a different test tube a. Keep track of which order these bands emerged 9. Obtain one of the larger TLCplates available and number it along the baseline with the number of fractions collected a. multiple spottings for each fraction to visualize them after development are needed i. Mark in pencil where the fraction will be spotted ii. Spot the fraction and allow a few seconds for it to dry before spotting again in the same place iii. Repeat spotting 10 or 12 times for each fraction iv. After one fraction has been spotted multiple times, clean the capillary tube by dipping it in acetone and touching it to some paper towel to draw out the acetone wash v. Clean it with acetone 2-3 times between fractions 2. Develop the plate a. Calculate Rf values and make identifications. b. Be sure to comment on how well the column effected separation in the final lab report c. Show a clear understand of the concept of chromatography d. If the results are anomalous, is there an explanation? e. Include TLC drawing and Rf factors in the lab report

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Observations Results Calculating Rf values on the crude TLC plate Y distance - distance between the placement of the extract and where the system was stopped Y distance = 3.1 cm X distance - distance between the placement of the extract and where the compound stopped on the TLC plate Rf equation Rf = x y From Bottom Up First mark = 0.15 cm Appearance: Very pale yellow Assumed Identity: xanthophylls Rf value = 0.04 cm Rf = 0.15 3.1 Second mark = 0.30 cm Appearance: pale yellow

Assumed Identity: xanthophylls Rf value = 0.097 cm Rf = 0.30 3.1 Third mark = 0.50 cm Appearance: light yellow Assumed Identity: xanthophylls Rf value = 0.161 cm Rf = 0.50 3.1 Fourth mark = 0.80 cm Appearance: Bright lime green Assumed Identity: chlorophyll B Rf value = 0.258 cm Rf = 0.80 3.1 Fifth mark = 0.90 cm Appearance: Bright bluish green Assumed Identity: chlorophyll A Rf value = 0.290 cm Rf = 0.80 3.1

Sixth mark = 1.35 cm Appearance: Pale olive green Assumed Identity: pheophytin B Rf value = 0.435 cm Rf = 1.35 3.1 Seventh mark = 2.50 cm Appearance: Yellow Assumed Identity: carotenes Rf value = 0.806 cm Rf = 2.5 3.1 Calculating Rf values on the individual TLC plate Y distance = 3.3 cm 1st mark = 2nd Test Tube = 0.8 cm Appearance: Light true green Assumed Identity: chlorophyll B Rf value = 0.242 cm Rf = 0.8 3.3 2nd mark = 2nd Test Tube = 1.6 cm Appearance: Pale olive green

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Assumed Identity: pheophytin B Rf value = 0.485 cm Rf = 1.6 3.3 3rd mark = 3rd Test Tube = 0.65 cm Appearance: Light green Assumed Identity: xanthophylls Rf value = 0.197 cm Rf = 0.65 3.3 4th mark = 4th Test Tube = 0.5 cm Appearance: Pale yellow Assumed Identity: xanthophylls Rf value = 0.151 cm Rf = 0.5 3.3 Discussion and Conclusion The objective of this lab was to separate all of the different pigmented compounds in spinach leaves through a liquid-liquid extraction. Each one of these compounds has a different polarity in which they can be removed from each other using their distinct polarities. To confirm that we had extracted all of the compounds, the Rf values were given but calculations had to be done to confirm their presence and identity. Rf values are calculated by determining how far the

compound has traveled in relation to the distance that it has been allowed to travel, and this can be controlled even further by measuring the distance that the compounds will be allowed to travel through marking the TLC plate previous to placing in the solution. The process of TCL or Thin Layer Chromatography is utilized to extract and separate different chemical compounds based off of how fast they travel up the stationary phase using the solution that the plate is placed in. In this solution, hexane is being used and has a polarity of 0.5, the other liquid is acetone which has a polarity of 5.1. These differing polarities will help pull different compounds present in the extract to different heights through the stationary phase of silica. This showed 7 different marks of varying color. Based on the color, and the known range based solely on polarity, they can be identified as from bottom to top, xanthophylls, chlorophyll B, chlorophyll A, pheophytin B, and carotenes. After the crude TLC was performed, a full column chromatography was then done to extract each band into singular test tubes. This process also utilized solutions with different polarities to extract each particular band. The three different solutions are made up of 8 mL of hexane to 2 mL of ethyl acetate, which has a polarity of 4.4. As well as 7 mL of hexane to 3 mL of ethyl acetate and 6 mL of hexane to 4 mL of ethyl acetate. Each different color band that was collected was individually placed onto another, new TLC place to complete another identity organic compound is exposed to infrared radiation over a range of different frequencies (called wavenumber), it will be absorbed differently by each bond in the molecule that can be graphed in order to identify which types of bonds and functional groups are present in a sample. For this specific instance, the IR spectroscopy is being utilized to try and test the identity of the samples. The main way that these compounds can be separately identified is through the two different compounds is through their skeletal structure. With Benzoic acid, the main functional group is carboxylic acid which has a very identifiable peak that is around the 1750 cm -1 mark which

should not at all be present in the 1,4-dimethoxybenzene sample, of which it isn't. The first, and main thing that must be done to separate the two compounds is to use their confirmation using Rf. This produced only 4 bands, and comparing their coloring and Rf values to that of the pure extract, they appeared to be xanthophylls, chlorophyll B, and pheophytin B. A reason why the second time through there were significantly less bands and could have more errors in general is due to how diluted the sample became since there had to be constant upkeep on the liquid in the system. This dilution most likely could have been the reason that not many of the pigments from the compounds showed through, and since not many are UV reactive either, they could not have been noted even if they were present. The only way that one could tell that the compound was in the separate test tubes is by the color of the solution itself. This lab was not dependent on the quantity of the compound collected, but the fact that it was actually collected. Post Lab Questions

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