C246 Analysis of Menthol Stereoisomers

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CHEM246 - Fundamentals of Organic Chemistry Laboratory Analysis of Menthol Stereoisomers Reference: Treadwell, E. and Black, T. Journal of Chemical Education , 2005 , vol. 82 , pp. 1046 - 1048. INTRODUCTION Stereoisomers are molecules with identical molecular formulas and connectivity, but different arrangements of groups in space on at least one atom (which is called a stereocenter) . When any two groups attached to a stereocenter are switched with each other, it will result in a different stereoisomer. When a carbon has four unique groups bound to it, this always results in a stereocenter called a chiral-center , such as the compound shown below. The maximum possible number of stereoisomers for a given molecule is determined by the equation 2 n , where n = the number of chiral centers in the molecule. When a molecule has at least one chiral carbon center, there are only two unique ways of orientating the groups in spaces. These are called enantiomers ; they are specific types of stereoisomers that have the same molecular formula and connectivity, but are also non-superimposable mirror images of one another; in other words, they have opposite stereochemical configurations. Since the molecule shown above has only one chiral center (the arrow points to it), a maximum of two stereoisomers is possible for this molecule (2 1 = 2). The two possibilities are shown below: the original molecule ( S ) and its enantiomer ( R ). All chiral molecules, regardless of how many chiral centers they have, will have one (and only one) enantiomer. In molecules with more than one chiral center, the other stereoisomers that are possible are called diastereomers . Diastereomers also have the same molecular formula and connectivity, but the stereochemistry is not completely opposite from one molecule to the other. In order to familiarize you with diastereomers, we will examine the molecule shown below which has two chiral centers, each marked with an arrow. This would suggest that a maximum of 4 stereoisomers possible for this molecule (2 2 = 4). Of these, two are the molecule shown above and its enantiomer (recall that all chiral molecules have one and only one enantiomer). Page 1 of 12

CHEM246 - Fundamentals of Organic Chemistry Laboratory Notice that the two isomers above are not the only possibilities. The two shown have the bromine and the alcohol spatially arranged opposite to one another. Therefore, a molecule with the bromine and alcohol on the same side would be a different isomer, as shown below. Br OH R S But again, since this molecule is chiral, it has an enantiomer: Now that the four different stereoisomers are accounted for, all that remains is to figure out how the two pairs of enantiomers are related to each other. The two molecules on the left side of the page are diastereomers of one another; they have the same molecular formula, the same connectivity, and different absolute stereochemistry, but they are not non-superimposable mirror images of one another. By the same logic, other pairs of diastereomers can be found as shown below. Unlike diastereomers which differ slightly in their physical properties, enantiomers have identical physical properties (melting points, boiling points, solubilities, etc.) except for optical rotation . Page 2 of 12

CHEM246 - Fundamentals of Organic Chemistry Laboratory Optical rotation is measured using a polarimeter, which works by shining plane-polarized light through a sample containing the molecules in question and measuring the degree to which the light rotates while passing through the sample. The optical rotation is read by looking through the measuring field, adjusting, and then reading the scale. To see this in practice, you will directly compare the physical properties of enantiomers and diastereomers. You will analyze different physical properties (polarity and optical rotation) of three stereoisomers of menthol, (+)-menthol, ( − )-menthol, and (+)- neomenthol, in the lab, and find their melting point/boiling point data from the literature. The chemical structures of the three stereoisomers are shown below. OH OH OH (+)-menthol ( − )-menthol (+)-neomenthol ASSIGNMENT 1: Watch the three videos in “ Menthol Stereoisomers Video 1: Theory ” (Canvas Module, Start Here page), and read the stereochemistry section of your organic chemistry textbook to review the concepts. It will prepare you better for the quiz questions and pre-lab/post-lab questions. IMPORTANT TECHNIQUES USED IN THIS LAB: • Thin Layer Chromatography (TLC) Chromatography is one of the most important laboratory techniques used for separating, purifying, and identifying certain chemical compounds. Chromatography literally means "written in color," as it was a technique originally used to separate colored materials, like the pigments in flowers. “Thin layer” chromatography (TLC) is among the most useful tools for answering questions such as “is my reaction done yet?” (by watching the starting material spot disappear and the product spot grow) and “is my compound pure?” (the number of spots that appear can indicate the number of compounds present, and thus the purity of the sample). Like all chromatographic methods, TLC takes advantage of the difference in affinity of the analyte (compound of interest) for the mobile phase (developing solvent) and stationary phase (silica gel) to achieve separation of complex mixtures of organic molecules. In this particular experiment, you are working with three isomers of the same compound; so what differences in them help you separate one from the other on a TLC plate? Think about the spatial orientation of the functional group in the three isomers. Luckily, you have had the opportunity to learn and use the TLC technique in previous module(s), so feel free to review those module(s) as refresher training. The Canvas Module ( Start Here page) has some very useful videos (specifically, Basics of TLC, and TLC Staining), that walk you through the technique. Page 3 of 12

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CHEM246 - Fundamentals of Organic Chemistry Laboratory • Polarimetry Enantiomers, chiral molecules, are molecules that lack an internal plane of symmetry and have a non-superimposable mirror image. Since their physical properties such as melting point, boiling point, solubility, etc. are identical, one way to tell these molecules apart is by polarimetry. It is a technique that allows us to determine the optical activity of a specific chemical compound being investigated, and thereby study various characteristics of it, including the identity. Polarimeter is the instrument that measures the angle of rotation of an optically active compound using polarized light. The polarized light will either rotate clockwise or counter- clockwise in the presence of an optically active compound, and the amount it rotates indicates the angle of rotation. The polarimeter reading of a given sample can be used to calculate the specific rotation , a common standard for comparing optical activity among compounds. The specific rotation is defined as the rotation in degrees observed upon passing polarized light through a path length of 1 decimetre (dm) at a concentration of 1 g/mL. 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑆𝑆𝑟𝑟𝑟𝑟 = 𝑂𝑂𝑂𝑂𝑂𝑂𝑆𝑆𝑟𝑟𝑂𝑂𝑆𝑆𝑂𝑂 𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑆𝑆𝑟𝑟𝑟𝑟 ( 𝑆𝑆𝑟𝑟𝑝𝑝𝑟𝑟𝑟𝑟𝑆𝑆𝑝𝑝𝑆𝑆𝑟𝑟𝑆𝑆𝑟𝑟 𝑟𝑟𝑆𝑆𝑟𝑟𝑂𝑂𝑆𝑆𝑟𝑟𝑟𝑟 ) 𝐶𝐶𝑟𝑟𝑟𝑟𝑆𝑆𝑆𝑆𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑆𝑆𝑟𝑟𝑟𝑟 𝑟𝑟𝑆𝑆 𝑟𝑟ℎ𝑆𝑆 𝑂𝑂𝑟𝑟𝑝𝑝𝑆𝑆𝑝𝑝𝑆𝑆 X 𝑆𝑆𝑟𝑟𝑝𝑝𝑆𝑆𝑝𝑝𝑆𝑆 𝑆𝑆𝑟𝑟𝑟𝑟ℎ 𝑝𝑝𝑆𝑆𝑟𝑟𝑟𝑟𝑟𝑟ℎ This equation will make more sense when you use the polarimetry data to calculate the specific rotation of the menthol stereoisomers in this lab. • Melting Point You already know this technique from the Recrystallization and Melting Point Determination laboratory experiment that was completed early in this course. Page 4 of 12

CHEM246 - Fundamentals of Organic Chemistry Laboratory PRE-LAB QUESTIONS: 1. How many possible stereoisomers does menthol have? 2. Of the molecules you are analyzing by TLC in this lab, do any of them have the same R f ? If so, which ones, and why? If not, why? 3. Assign absolute stereochemistry ( R or S ) to the molecule below. OH Page 5 of 12 3KSYMUQ MGY ± IMOWGQ IKSYKWY YU ]OYM YMK KW[GYOUS ²CS ]K MKY ³ YYKWKUOYUSKWY ´²C±#³µ 3KSYMUQ ¶ GSI SKSYMUQ · ]U[QI MG[K YOSOQGW 7K [GQ[KY YOSIK YMK_ GWK KSGSYOUSKWY¸ 'QYMU[MM YMK_ IGS [GW_ YQOMMYQ_ OY OY IQUYK KSU[MM YMGY YMK_ GWK IUSYOIKWKI YMK YGSK¸ 9MK WKGYUS KUW YMK [GWOGYOUS SG_ GK I[K YU IUSUU[SI UW IMOWGQ U[WOY_¸ 7

CHEM246 - Fundamentals of Organic Chemistry Laboratory 4. A different menthol stereoisomer, (+)-isomenthol is shown below. OH Draw the structure of ( − )-isomenthol. 5. From the list below, which compounds have the same melting point? Which ones have the same optical rotation? Which compound(s) could be separated from others by TLC? (Hint: See how these stereoisomers differ in R/S, cis/trans, or E/Z descriptors) OH OH OH Cl Cl Cl Cl OH I II III IV Page 6 of 12 9GSK SKQYOSM UUOSYY· /¹ ///¹ GSI /;¸ º GSI ± GWK KSGSYOUSKWY UK KGIM UYMKW YU YMK_ MG[K YMK YGSK SKQYOSM UUOSY¸ ± GSI » GWK YMK YGSK IUSUU[SI YU OY YUU ]OQQ MG[K YMK YGSK SKQYOSM UUOSY¸ 9GSK UUYOIGQ WUYGYOUS· /// GSI /;¸ 9MK_ GWK YMK YGSK IUSUU[SI YU YMK_ ]OQQ GK OIKSYOIGQ KUW WUYGYOUS¸ 91) YKUGWGYOUS· // GSI ///¸ 9MKYK YKYY GWK IOGYYKWUSKWY YMKWKKUWK YMK_ IGS GK YKUGWGYKI G_ 91)¸ 9KK GYYGIMKI KOQKY KUW GQQ IWG]OSMY

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CHEM246 - Fundamentals of Organic Chemistry Laboratory TABLE OF PHYSICAL CONSTANTS AND CHEMICAL HAZARDS: Compound name Molecular weight M.P. (or B.P.) o C Chemical Hazards (+)-menthol (− )-menthol (+)-neomenthol Acetone Hexanes Ethyl Acetate Cite your references for the above information. (Not sure how to properly cite a reference? See the single-page document on “How to cite journals websites and books in your lab report” in Canvas module-2) : ASSIGNMENT 2 : Watch Menthol Stereoisomers lab videos (TLC, Staining, and Polarimetry) provided on Canvas ( Start Here page). It explains how to spot samples on a TLC plate, develop it, and stain it to visualize the spots, and how to run a sample in the polarimeter. Page 7 of 12 º¼½¸²¾ M¿SUQ º¼½¸²¾ M¿SUQ º¼½¸²¾ M¿SUQ ·²²) 9QOS¹ K_K¹ GSI WKYUOWGYUW_ KUW GQQ ± OYUSKWY¸ ·À¼ ) YQOS K_K WKYUOWGYUW_ YQOS K_K WKYUOWGYUW_ YQOS K_K WKYUOWGYUW_ ½³¸¾) ¾¾¸º ) ¼³¸Á³ M¿SUQ ³½¸º³ M¿SUQ ³³¸ººM¿SUQ ±½·±³ ) ±½·±³ ) º IS ´ºÁISµ 'QQ YU[WIKI KWUS ]]]¸]OQOUKIOG¸IUS

CHEM246 - Fundamentals of Organic Chemistry Laboratory EXPERIMENTAL PROCEDURE TLC Analysis of Menthol Stereoisomers Solutions of (+)-menthol, ( − )-menthol, and (+)- neomenthol are provided in the chemical hood. Procedure Notes & Observation 1. Obtain a TLC plate from your TA. Obtain ~1 mL (just 2-3 drops is sufficient) of each solution of (+)- menthol, ( − )-menthol, and (+)- neomenthol into separate vials. 2. Using a pencil , carefully and very gently draw two straight lines across the width of the TLC plate; 0.5 cm from the top and 1 cm from the bottom. Mark three X’s, evenly spaced, along the line. Label them (+)-menthol, ( − )-menthol, and (+)- neomenthol. 3. Spot each solution on its matching X using a TLC spotter. Make sure to clean the spotter in between each sample ; do this by dipping the spotter in ~3 mLs of acetone (in a small vial) and draining it on a clean paper towel (repeat the process three times). 4. Measure ~5 mL of 4:1 Hexane–Ethyl acetate mixture (TLC developing solvent) into a TLC beaker; this should result in a solvent depth of about 0.5 cm. 5. Place ½ of a large piece of filter paper in the beaker to act as a wick, then cover the beaker with a watch glass. This saturates the beaker with solvent vapor. 6. Place the TLC plate in the beaker; leaning it so only the top of the plate is touching the side of the beaker (shown in the video guide). T he solvent level should be below the spots. Re-cover the beaker immediately with a watch glass and let the solvent move up the plate until it reaches the top pencil line. 7. Remove the TLC plate and allow the solvent to evaporate from the plate completely. Remove the filter paper, empty the liquid in the TLC beaker into the non-halogenated waste, and return the TLC beaker to the collection box. 8. To visualize the spots, hold the TLC plate with tweezers and briefly dip it into the vanillin stain. Touch the corner of the plate to a paper towel to drain off excess solution. Hold the TLC plate over a hotplate until a blue spot appears for each sample. Sketch of the developed TLC plate (with distances marked): For example, Page 8 of 12 9KK GYYGIMKI KOQK KUW GQQ IWG]OSMY

CHEM246 - Fundamentals of Organic Chemistry Laboratory Optical Rotation Determination There are prepared samples of (+)-menthol, (-)-menthol, and (+)-neomenthol available to analyze in the polarimeter. Your TA will guide you on how to use the polarimeter, and let you know which measurements you need to take. Procedure Notes & Observations 1. Make sure that you write down the concentrations of the samples to be used in the polarimeter; your TA will provide this information. 2. When it is your turn to measure the optical rotation, look through the measuring field magnifying lens. One half of the view field should be darker than the other. Push the L (left) or R (right) button, depending on which side of the circle in the viewfinder is lighter, until both halves are of equal brightness. (If the left side of the circle is brighter, push the L button and vice versa.) 3. Write your observed rotation on the blackboard. 4. Record the average values (from the numbers on the board) for all three stereoisomers. (+)-menthol concentration: ( − )-menthol concentration: (+)-neomenthol concentration: Observed rotation (+)-menthol: Observed rotation ( − )-menthol: Observed rotation (+)-neomenthol: Page 9 of 12 º¸¼± Á¸Áº¼± M¿S1 º¸¼Á º¸¾± M¿ ºÁÁSQ Á¸²¼ ·Á¸¼¼ Á¸½¼ Á¸Áº¾± M¿SQ Á¸Áº¼Á M¿S1

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CHEM246 - Fundamentals of Organic Chemistry Laboratory CALCULATIONS: 1. Calculate the R f for each of the isomers. Show your calculations. R f of (+)-menthol: R f of ( − )-menthol: R f of (+)-neomenthol: 2. Calculate the specific rotation from the observed rotation for (+)-menthol, ( − )-menthol, and (+)-neomenthol. The specific rotation [ α ] λ t is reported along with the temperature, t, and the wavelength of light, λ , used to make that measurement. In our case, λ is designated as D for the sodium D line. Specific rotation is determined by the experimental data using: Specific rotation = [ α ] D t = α c × l where : α = observed rotation in degrees (from polarimeter reading; find this information in Optical Rotation Determination section above) c = concentration in grams per milliliter (g/mL) of solution (concentration of solution; find this information in Optical Rotation Determination section above) l = length of sample tube in decimeters (generally, l = 1 dm, since the cell used in this experiment is 10 cm. Check with your TA to confirm!) Specific rotation of (+)-menthol: Specific rotation of ( − )-menthol: Specific rotation of (+)-neomenthol: Page 10 of 12 »¸¼IS ¿ ¾IS # Á¸½» ±¸¼IS ¿ ¾ IS # Á¸¼Á ±¸¼IS ¿ ¾IS # Á¸¼Á Á¸½¼ ¿ ´Á¸Áº¼± M¿S1 ] º¸Á ISµ# »²¸»³ ·Á¸¼¼ ¿ ´Á¸Áº¼Á M¿S1 ] º¸Á ISµ# ·±½¸½¾ Á¸²¼ ¿ ´Á¸Áº¾± M¿S1 ] º¸Á ISµ# º»¸»¼

CHEM246 - Fundamentals of Organic Chemistry Laboratory WASTE DISPOSAL: Discard all solvent waste into the “NON- HALOGENATED WASTE” bottle. Dispose of the TLC plates and microcapillary tubes in the glass waste. Discard used filter papers and paper towels in the regular trash. ANALYSIS: Complete the table below, using all of the data you have gathered and calculated for (+)-menthol, (-)- menthol, and (+)-neomenthol. Browse scientific literature for the melting points of (+)-menthol and (−) -menthol, and the boiling point of (+)- neomenthol. Be sure to cite your references. Name M.P. (or B.P.) o C from literature R f Specific rotation (+)-menthol ( − )-menthol (+)-neomenthol References used for finding the boiling point/melting point of each compound: Page 11 of 12 ·²²) ±½·±³) ±½·±³) Á¸½» Á¸¼Á Á¸¼Á »²¸»³ ·±½¸½¾ º»¸»¼ 'QQ QOYKWGY[WK [GQ[KY YU[WIKI KWUS ]]]±]OQOUKIOG±IUS

CHEM246 - Fundamentals of Organic Chemistry Laboratory POST-LAB QUESTIONS: 1. What is the relationship between (+)-neomenthol and ( − )-menthol? 2. Assign absolute stereochemistry ( R or S ) to each of the stereocenters in (+)-menthol and ( − )-menthol (Draw the structures here). 3. Draw the structures of all of menthol’s stereoisomers (feel free to add more pages, as needed) . Page 12 of 12 9MK_ GWK YYKWKUOYUSKWY UK KGIM UYMKW 9KK GYYGIMKI KOQK KUW GQQ IWG]OSMY 9KK GYYGIMKI KOQK KUW GQQ IWG]OSMY

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