Lab 8 REPORT FORM_ CHEKERDJIAN

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Purdue University *

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11500

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Chemistry

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Feb 20, 2024

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Lab 8: Use Chromatography to Separate Pigments REPORT FORM Answer the following discussion questions in complete sentences.. Support your claim using evidence, i.e., experimental data and/or observations that support the claim. Cite specific quantitative results. Connect your evidence (data and/or observations) to your claim using reasoning that explains why your evidence supports your claim. Reasoning should be based on a scientific rule, law, principle or definition. Question #1. Brief description of your leaf samples (color, texture, shape, type of plant collected from (if known), etc.). What solvent did you use for TLC development (identities and volumes)? Question #2. List your sample spotting scheme. Insert the pictures of your TLC plate, including baseline, sample labels, solvent front, and spots that you circled. For each sample, label each component with a number. The two samples we used were from the same plant however one was a flower petal and the other was a leaf. The leaf was a dark green with the underside being light green. There was a visible line in the middle with the leaf’s vein also being clear, the texture was smooth. The flower was of a light purple color, soft and smooth and the sample we used was made up off five petals. For the TLC development we used a mixture of 6 mL of Hexane and 4 mL of Acetone. Hash mark Sample name or # 1 st 2 2 nd 2 3 rd 1 & 2 4 th 1 & 2 5 th 1 & 2

Viewed under visible light Viewed under UV light Question #3. Fill the below tables based on your TLC plate. Table 1. Components of One Spinach Reference Standard Component # Color Identity of Pigment Distance component traveled (mm) Distance solvent front traveled (mm) R f 1 Lime green Chlorophyll b 20 40 0.5 2 Dark green Chlorophyll a 23 40 0.575 3 Yellow Beta - carotene 39 40 0.975

Table 2. Components in Sample 1 Component # Color Distance component traveled (mm) Distance solvent front traveled (mm) R f Possible identity of component Nothing showed for our purple petal Table 3. Components in Sample 2 Component # Color Distance component traveled (mm) Distance solvent front traveled (mm) R f Possible identity of component 1 Pale yellow 90 40 22.5 Xanthophylls 2 Pale yellow 14 40 35 Xanthophylls 3 Bright lime green 18 40 45 Chlorophyll b 4 lime green 19 40 47.5 Chlorophyll b 5 Light green 22 40 55 Chlorophyll a 6 Yellow 39 40 97.5 Beta - carotene

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Question #4. List the following pigments in order of most polar to least polar (non-polar): chlorophyll a, chlorophyll b, xanthophyll, beta-carotene. Justify or explain the basis of your answer. Question #5. Did you see any spots in Samples 1 or 2 that did not appear in your spinach standard? If so, where would the unknown compound (or compounds) fall in the order of polarity you determined in question 4? Support your answer with data (i.e. R f values) from your experiment. Question #6. Explain how you identified the components of your samples. Give at least one specific example. Xanthophyll > Chlorophyll b > chlorophyll a > Beta-carotene Xanthophyll are the most polar and beta-carotene are the least. Beta-carotene is purley hydrocarbons which makes them the least polar. Chlorophyll is more polar then beta- carotene because it has carboxylic acid and ester groups which make it polar. Chlorophyll b is more polar then a because it has most polar functional groups. Yes our 2 nd sample showed 2 more values then the other 2 samples. The unknown compound were beta-carotene as they were yellow and the distance they traveled was 0.9mm and 1.4mm which cause them to have Rf values of 0.225mm and 0.35mm making them the least polar as they didn’t travel far. I used the spinach standard in the intro to figure out the components We need to run a TLC of the spinach instead of just using the picture of the results for several reasons. First off there is always a possibility of human error and the results the professor or TA got would not be the same as mine. Secondly it would give us more accurate results as it makes sure that all phases are correct, and we are doing the experiment like we should.

Question #7. Why do you need to run a TLC of the spinach instead of just using the picture of the spinach TLC results that you see in the lab manual? Question #8. What factors influence how far the bands move up the TLC plate in this experiment? Why does beta-carotene travel farther than chlorophyll? Be specific in your answer. Question #9. What do you think would happen if you run this experiment again using only hexane as the solvent? Explain using detailed predictions. Question #10. Explain how you would i) separate and ii) identify one sample containing two types of large molecules. One of the molecules is very polar and negatively charged while the second molecule is less polar and neutral. The sample is not colored. (Hint: refer back to the section: Introduction .) The factors that influence how far the bands move up the TLC plate in this experiment is the solvent polarity, adsorbent material on the TLC plate, and the concentration of the sample. Beta-carotene travels further than chlorophyll because it is a nonpolar molecule that interacts strongly with the adsorbent. Beta-carotene will also have a weaker interaction with the polar stationary phase causing beta-carotene to travel further up than chlorophyll. If you run this experiment using only hexane as the solvent, it would take a lot more for the process to happen. I say this because we know that polarity of the solvent has a direct effect on the speed of the process, so if we decrease the polarity by using a solvent which is less polar (hexane is less polar then acetone) the components will move much slower. To separate the molecules: 1. Place small spots of the mixture on the baseline. 2. Place the chromatogram into a chamber 3. The solvent will rise and will carry the components of the mixture 4. As the solvents moves it will separate into two groups a fast-moving sport and a slower moving spot To identify: Nonpolar molecules will be carried with the solvent front and move quickly. Polar molecules will be slow. After the solvent has reached ½ of the plate remove it from the beaker and use UV light to visualize the separated molecules. Then determine the Rf value and compare it to the Rf value of a known compound.

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