CAS #
`299-42-3
`64-17-5
`1634-04-4
`7647-01-0
`7487-88-9
`90-64-2
`
`Hazard
`Irritant, hygroscopic
`Flammable, irritant
`Flammable, irritant
`Corrosive, highly toxic
`Hygroscopic
`Light-sensitive
`
`Supplemental Materials
`
`Compound
`(1R, 2S)-(-)-ephedrine
`95% or absolute ethanol
`tert-butyl methyl ether
`6M HCl
`MgSO4
`d,l-mandelic acid
`
`Instructor Notes:
`1. Ephedrine is a controlled substance, so the stockroom measured the reagent for distribution.
`
`2. We checked all student lockers to see if crystallization had occurred before the students returned. If
`no diastereomeric salt crystals had formed overnight, we either scratched the bottom of the Erlenmeyer
`flask, or removed a drop of the solution on a glass rod and allowed the alcohol to evaporate leaving
`crystals. Swirling the tip of the glass rod containing these crystals in the solution seeded the rest.
`
`3. The students were not required to recover the other diastereomeric salt from the filtrate. However, in
`developing this experiment, the (1R,2S)-(-)-ephedrine-(S)-(+)- mandelate salt was recovered from a
`concentrate of the initial filtrate in lower yield (32% crude, 8% recrystallized). Its recrystallization and
`neutralization yielded resolved (S)-(+)-mandelic acid with an optical purity of 77% and mp of 124.0-
`128.50C.
`
`4. The optical rotation of the resolved mandelic acid was performed using a ~ 0.50 g dissolved in a 10.0
`mL of absolute ethanol. The solution was then placed in a 1-dm polarimetry tube and optical rotations
`between –6 to -70 were obtained. If students had more than 0.5 g, they removed a melting point sample
`and committed the rest to polarimetry. If their yield was low, they used their entire sample for
`polarimetry, then recovered the product for melting point by rotary evaporation to remove the ethanol.
`
`5. We found excellent correlation between melting point and % optical purity of the resolved (-)-
`mandelic acid. If the student had a poor melting point indicating contamination by the other enantiomer,
`the optical purity was also low. Occasionally a student had an excellent melting point, but obtained a
`lower optical purity. We believe this was due to student error in preparing the solution for the
`polarimeter. Many removed too much sample for melting point and utilized the initial mass for their
`specific rotation calculation. We now have the students reweigh after removing a sample for melting
`point determination.
`
`6. The optical rotation in water is also known (Aldrich), but we found that a small amount of the
`resolved mandelic acid did not dissolve, so we preferentially used the absolute ethanol for clearer
`solutions.
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`Liquidia's Exhibit 1028
`IPR2020-00770
`Page 1
`
`

`

` ENANTIOMERIC RESOLUTION of
`(+)-Mandelic Acid by (1R,2S)-(-)-Ephedrine
`
`This experiment was adapted from Jarowski, C. and Hartung, W.H., J. Org. Chem., 8, 564 (1943);
`Manske, R.H. and Johnson, T.B., J. Am. Chem. Soc., 51, 1909 (1929) by Andrea Cerrone, Class of ’02.
`
`Objectives:
` To review 2 classes of stereoisomers: enantiomers & diastereomers.
` To use acid/base chemistry and varying salt solubilities to effect a physical separation.
` To provide experience with the purification techniques of recrystallization, extraction, drying,
` rotary evaporation and the analytical techniques of melting point and polarimetry.
`
`Theory:
` (R)-(-)-mandelic acid and (S)-(+)-mandelic acid are enantiomers, stereoisomers that are non-
`superimposable mirror images which differ only by the internal spatial relationship of their atoms.
`Except for the difference in the way they rotate PPL (optical activity), enantiomers are identical in
`physical and chemical properties. Only another chiral agent can recognize the difference between
`enantiomers.
` There is an acidic proton in mandelic acid that if reacted with a chiral organic base in a neutralization
`reaction will produce salts that are diastereomers. Diastereomers are stereoisomers that also differ by
`their internal spatial arrangement of atoms, but they are not mirror images, nor superimposable.
`Diastereomers have different physical and chemical properties which can be exploited to effect a
`separation.
`
`
`OH
`
`HO
`
`H3C
`H
`
`Ph
`
`CH3HN
`
`H
`OH
`(1R,2S)-(-)-ephedrine
`an organic base (R2NH)
`
`
`
`HP
`
`h
`
`C
`
`Ph
`
`C
`
`CO2H
`
`HO2C
`H
`(S)-(+)-mandelic acid
`(R)-(-)-mandelic acid
`(boldfaced acidic proton)
`
`
` In this experiment you will separate (resolve) a racemic (50:50) mixture of the two mandelic acids by
`converting them into diastereomeric ammonium salts using the chiral base, (1R,2S)-(-)-ephedrine (also
`known as (1R,2S)-(-)-α-(1-methylaminomethyl)benzyl alcohol). The resultant diastereomeric salts have
`different physical properties, particularly their solubility in 95% ethanol. The (1R,2S)-(-)-ephedrine-
`(R)-(-)-mandelate preferentially crystallizes from ethanol. A suction filtration will allow us to collect
`this salt, leaving the (1R,2S)-(-)-ephedrine-(S)-(+)-ephedrine mandelate in solution.
`
`ACID/BASE NEUTRALIZATION REACTION FORMING DIASTEREOMERIC SALTS:
`
`
`NHCH3
`
`CH3
`
`H
`
`H
`
`OH
`
`Ph
`(1R,2S)-(-)-
`ephedrine
`
`OH
`
`+
`
`Ph
`
`CH CO2H
`*
`R,S-mandelic acid
`
`
`
`
`+NH2CH3
`
`CH3
`H
`
`H
`
`H
`
`OH
`
`+NH2CH3
`
`H
`
`OH
`
`and
`
`HO
`
`CO2
`
`H
`
`CH3
`OH
`
`H
`
`Ph
`Ph
`(1R,2S)-(-)-ephedrine
`-(R)-(-)-mandelate
`
`CO2
`
`Ph
`
`Ph
`(1R,2S)-(-)-ephedrine
`-(S)-(+)-mandelate
`
`
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`Liquidia's Exhibit 1028
`IPR2020-00770
`Page 2
`
`

`

`-2-
`
` The resolved (R)-(-)-mandelic acid can be freed from its diastereomeric salt by neutralization with
`HCl. HCl is a stronger acid that substitutes for the mandelic acid within the salt by replacing the proton
`on the carboxylate group. The free, reprotonated, resolved (R)-(-)-mandelic acid is then isolated by
`ether extraction followed by rotatory evaporation of the ether.
`
`NEUTRALIZATION OF (1R,2S)-(-)-EPHEDRINE-R-(-)-MANDELATE SALT:
`+NH2CH3
`+NH2CH3
`
`Cl
`
`CO2
`
`H
`
`CH3
`OH
`
`H
`
`H
`
`OH
`
`+ HCl
`
`Ph
`Ph
`(1R,2S)-(-)-ephedrine
`-(R)-(-)-mandelate
`
`CO2H
`
`H
`
`OH
`
`and
`
`Ph
`reprotonated
`free, resolved
`(R)-(-)-mandelic acid
`
`CH3
`
`H
`
`H
`
`OH
`
`Ph
`new ephedrine
`hydrochloride salt
`
`
`
`
` You will then analyze the resolved (R)-(-)-mandelic acid by measuring its mass, optical rotation and
`melting point. The success of your effort will be judged by melting point and the calculated % optical
`purity based on your specific rotation.
`
`Experimental Procedure:
`
` week #1 - Preparation of Diastereomeric Salt Solution
`
`SAFETY:
`1. Ethanol is flammable. No open flames. Your heat source is a hotplate on a low setting.
`2. Look up the hazards associated with the amine, (1R,2S)-(-)-ephedrine, and (+)-mandelic
` acid to understand why it is important to wear gloves when handling them.
`
`Preparation of diastereomeric salts:
`
` Obtain from the stockroom a packet containing 3.00 g + 0.05 g of (-)-ephedrine and empty the
`contents into your jointed 125 mL Erlenmeyer flask (E.flask). In the hood, set 22 mL of 95% ethanol in
`a non-jointed 125 mL E. flask to warm on a hotplate set to 1. Weigh 3.00 g + 0.05 g of d,l-mandelic
`acid in a 50 mL beaker. Add 10 mL of warm ethanol to each container and stir to dissolve both solids.
`Keep all containers on the hotplate through dissolution. Once both solids have dissolved, pour the
`solution containing the d,l-mandelic acid into the E. flask containing the (-)-ephedrine. Remove the 125
`mL E. flask from the hot plate and allow it to stand and cool. Scratch the bottom of the E. flask with a
`glass rod to promote crystallization over the week. Then stopper the E. flask and wrap the neck with
`parafilm, label it with your name and contents, and store the flask in your locker until next period.
`
`week #2: Recrystallization, Neutralization of Diastereomeric Salt and
` Extraction of Free Resolved (R)-(-)-Mandelic Acid
`
`SAFETY:
`1. 6M HClaq solution is very corrosive. Wear gloves when handling.
`2. Ethanol and ether are flammable, no open flames.
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`Liquidia's Exhibit 1028
`IPR2020-00770
`Page 3
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`

`

`-3-
`
`Recrystallization of (1R,2S)-(-)-Ephedrine-(R)-(-)-Mandelate
`
` Collect the ephedrine mandelate crystals that have formed over the week by vacuum filtration
`(suction flask and Büchner funnel can be obtained from wall cabinet). (See note 1 if no crystals formed.)
`The ethanol filtrate should be placed in the appropriately labeled waste container located in the hood.
`Before proceeding remove a small sample of this crude salt and store it in a small, labeled test tube
`(uncapped) for future melting point analysis.
` The salt crystals are contaminated with a small amount of the other diastereomeric salt, so they need
`purification. You will recrystallize them from 95% ethanol. Transfer the crystals to a 50 mL beaker,
`add 25 mL of ethanol and heat on a hotplate (setting 1) to dissolve the crystals. Once the crystals have
`dissolved, remove the beaker from the hotplate and allow it to cool slowly to room temperature on the
`benchtop. Utilize this cooling period to clean and set-up your suction assembly for a collection of the
`purified salt crystals. Also prepare an ice-water bath and chill 10 mL of 95% ethanol to be used later for
`a rinse. Once the solution has cooled to RT, place the beaker in the ice/water bath and cool to 00C with
`occasional stirring. Collect the purified crystals by suction filtration and rinse with the chilled ethanol.
`Again pour the ethanol filtrate into the waste container.
` Remove a small portion of the recrystallized salt and store them in a small, labeled test tube
`(uncapped) for a future melting point analysis. (See note 2.)
`
`An aside: The ethanol filtrates which are discarded contain more of the (1R, 2S)-(-)- ephedrine-(S)-(+)-
`mandelate. We could rotary evaporate the ethanol to recover this “other” diastereomeric salt, also treat
`it with HCl to effect a neutralization, extract and distill to isolate the resolved (S)-(+)-mandelic acid.
`But we won’t.
`
`Neutralization of (1R,2S)-(-)-Ephedrine-(R)-(-)-Mandelate
`
` Transfer the recrystallized diastereomeric salt crystals into a 125 mL E. flask and add 4 mL of 6M
`HCl and swirl to effect the neutralization reaction. (If the new ephedrine hydrochloride salt does not
`completely dissolve, add 2 mL of water.) Test the solution with litmus paper to ensure acidity.
`
`
`Purification of Resolved R-(-)-Mandelic Acid:
`
` Transfer the acidic solution to the 60 mL separatory funnel (obtain from tub). MAKE SURE THE
`STOPCOCK IS CLOSED. Extract the free, resolved mandelic acid from this solution by using three
`10 mL portions of tert-butyl methyl ether (TBME). The instructor will demonstrate technique. Dry the
`combined ether layers with anhydrous magnesium sulfate for about 10 minutes. Gravity filter the dried
`ether solution into your PRE-WEIGHED 50 mL pear shaped flask. (What solvent will you use to
`moisten the filter paper?) The aqueous layer may be poured down the sink.
` If time permits, rotary evaporate the ether to a constant mass. Instructor will demonstrate the use of
`the rotary evaporator. There will be time next period for rotary evaporation, if necessary.
`
`week #3: Analysis of Free Resolved (R)-(-)-Mandelic Acid
` Optical Rotation and Melting Point Analyses
`
`As part of your pre-lab for week #3, read the Melting Point handout and assigned pages from Zubrick’s
`“The Organic Chem Lab Survival Manual”.
`
`Experimental: Obtain a 10.0 mL volumetric flask from the stockroom.
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`Liquidia's Exhibit 1028
`IPR2020-00770
`Page 4
`
`

`

`
`
`-4-
`
` Mass the resolved mandelic acid, then remove a small sample for a melting point, store as usual in a
`labeled test tube. Remass the sample and use it all for the polarimetry sample; ~0.50 g provides a
`meaningful optical rotation. If you must use your entire sample for the optical rotation, then rotary
`evaporate off the ethanol to reclaim the solid for melting point analysis.
` Add 8 mL of absolute ethanol to the pear shaped flask containing the resolved mandelic acid. Swirl
`to dissolve the solid mandelic acid. Using a pipette, carefully transfer this solution to the 10 mL
`volumetric flask. Use 2 mL of abs. ethanol to rinse the pear flask and take the solvent level to the 10 mL
`mark line in the volumetric flask. Cap and gently invert the volumetric to make the solution
`homogenous.
` Using a fresh pipette, carefully transfer the solution to the polarimeter tube, note its volume (1-dm)
`and then measure the optical rotation. (Will you rotate the dial clockwise or counterclockwise?) Use the
`above value to calculate the specific rotation and then the % optical purity. (See note 3 below.) If you
`had sufficient mandelic acid for both the polarimetry and melting point analyses, you may discard the
`polarimetry solution in the labeled waste container.
`
`
`Notes:
` 1: If no crystals have formed, try scratching the bottom of the E. flask again, or remove a drop of the
`solution on a glass rod and allow the alcohol to evaporate leaving crystals. Swirl the tip of the glass rod
`containing these seed crystals in the solution.
`
` 2. The solid diastereomeric salt sample that you saved will be melted in two weeks in order to verify
`its identity and purity. A typical melting point reported for this (1R,2S)-(-)-ephedrine-(R)-(-)-
`mandelate salt was 168-1700C. The (1R,2S)-(-)-ephedrine(S)-(+)- mandelate salt melts approximately
`200C lower.
`
` 3. Compare your specific rotation to that obtained by Andrea on a resolved sample and commercial
`(R)-(-)-mandelic acid: -1600.
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`Liquidia's Exhibit 1028
`IPR2020-00770
`Page 5
`
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