Experiment #5:
`Resolution of (R,S)–1–Phenylethylamine via
`Diastereoisomer formation with (2R),(3R)–Tartaric Acid
`
`Introduction
`
`A separation of the enantiomers of phenylethylamine can be achieved by exploiting
`the very different solubilities in methanol of their diastereomeric complexes with a
`single diastereomer of tartaric acid.
`Recall that enantiomers have identical physical properties, while those of
`diastereomers differ. When a hot solution of racemic (R,S)–1–phenylethylamine
`and an equivalent amount of (R,R)–tartaric acid in methanol is allowed to cool, the
`less soluble (S)–amine (R,R)–tartrate complex crystallizes out of solution (see
`scheme below). Isolation of this crystalline material and treatment with excess
`aqueous sodium hydroxide allows liberation of the (S)–amine from the tartrate. Just
`as with the extraction experiment you have already done, we now have a situation
`where we can separate a relatively non–polar organic material (the amine) from a
`polar one (the tartrate salt). After extraction the pure (S)–amine is obtained by
`evaporating the methanol solvent on a rotary evaporator.
`In theory, the more soluble diastereomeric complex that remains dissolved in the
`filtrate can also be isolated by treating the filtrate with sodium hydroxide solution,
`extracting the liberated amine into an organic solvent to separate it from tartrate,
`and evaporating the methanol solvent. The structures of the materials used and
`complexes formed are as follows. A flowchart (overleaf) details the steps involved
`in this resolution experiment.
`
`Organic Chemistry I Laboratory Manual (prepared by D. G. Hamilton, October 2006)
`
`Liquidia's Exhibit 1031
`IPR2020-00770
`Page 1
`
`

`

`
`
`
`
`Organic Chemistry I Laboratory Manual (prepared by D. G. Hamilton, October 2006)
`
`
`
`Liquidia's Exhibit 1031
`IPR2020-00770
`Page 2
`
`

`

`In practice, it is very rare for a single crystallization of this kind to prove sufficient to
`fully separate two enantiomers. Invariably, the complex containing the (S)
`enantiomer crystallizes along with some of the complex containing the (R)
`enantiomer, and not all of the (S) enantiomer complex crystallizes. Thus we are left
`with a mixture of some of the (S) containing complex and most of the (R)
`containing complex in solution. If it was required to achieve absolute and complete
`separation of a pair of enantiomers the crystallization experiment would be
`performed several times, in sequence, gradually enriching the percentage of pure
`single enantiomer.
`What you will do
`Work in pairs throughout this experiment. Though the first part of this two week
`experiment is rather short the best results are obtained by being careful at this
`stage and performing the simple operations cautiously.
`
`Organic Chemistry I Laboratory Manual (prepared by D. G. Hamilton, October 2006)
`
`Liquidia's Exhibit 1031
`IPR2020-00770
`Page 3
`
`

`

`1st week
`Care! The racemic amine mixture has a strong and irritating odor. Dispense this
`material and perform all your manipulations in the hood.
`Weigh 6.25g (41.6mmol) of (R,R)–tartaric acid into a 250mL Erlenmeyer flask, add
`100mL of methanol, and heat the mixture gently on a hotplate for a few minutes.
`To
`the warm solution slowly add 5g
`(5.3mL, 41.2mmol) of
`(R,S)–1–
`phenylethylamine. Care! too rapid addition will cause the mixture to boil over. Once
`the addition is complete let the solution boil very gently on the hotplate for at least
`15 minutes (use a boiling stick). Let the solution cool to room temperature, seal the
`flask with parafilm, label it with your names and laboratory section, and place in the
`location indicated by your TA or instructor. The solution must now be left to
`slowly crystallize until next week.
`
`2nd week
`Care! The sodium hydroxide solution you will use will cause serious burns if it
`comes into contact with your skin. Wear gloves and eye protection at all times
`when handling this material.
`Collect the crystalline product by suction filtration and wash the crystals on the filter
`with a little methanol. Dry the crystals on a filter paper and record the yield. DO
`NOT DISCARD THE FILTRATE.
`Partially dissolve the product in around 50mL of water and add around 4.5mL of
`50% aq. NaOH solution. Ensure that the solution is basic by testing with pH paper.
`Transfer the aqueous solution to a separatory funnel, add 30mL of ether, and
`thoroughly mix the two layers using the technique you learned in a previous
`experiment. Carefully return the aqueous layer to the separatory funnel and repeat
`the extraction with a second 30mL portion of ether. Collect the second portion of
`ether, combine with the ether extracts from the first extraction, and dry over
`anhydrous Na2SO4 for a few minutes. Decant the dried extracts into a round
`bottomed flask and remove the solvent using a rotary evaporator. Transfer the
`residue to a screw top vial and record the yield.
`If so directed by your TA or instructor transfer the methanolic filtrate from the
`crystallization process to a round bottomed flask and evaporate the solvent on the
`rotary evaporator. Next, add 50mL of water to the residue. You are now going to
`follow the extract procedure from above, thus: add 4.5mL of 50% aq. NaOH
`solution. Ensure that the solution is basic by testing with pH paper. Transfer the
`aqueous solution to a separatory funnel, add 30mL of ether, and extract. Repeat
`the extraction with a second portion of ether (30mL), combine the ether extracts,
`
`Organic Chemistry I Laboratory Manual (prepared by D. G. Hamilton, October 2006)
`
`Liquidia's Exhibit 1031
`IPR2020-00770
`Page 4
`
`

`

`and dry them over anhydrous Na2SO4 for a few minutes. Decant the dried extracts
`into a round bottomed flask and remove the solvent using a rotary evaporator.
`Transfer the residue to a screw top vial and record the yield.
`With the assistance of your instructor record the optical rotation of your two
`samples using the polarimeter in the central instrument room. In the same manner,
`also record the optical rotation of the orginal racemic mixture, (R,S)–1–
`phenylethylamine.
`Instructions on how
`to prepare
`the samples
`for
`this
`measurement will be provided when you are ready.
`Cleaning Up
`Your purified amines, methanol filtrate, and any ether left over from the extraction
`should be disposed of in the non–chlorinated waste solvent container. You basic
`layers from the extraction should be placed in the aqueous waste container.
`Your Report
`Record the dried weight of the crystals you obtain, and the weights of the two
`samples of amine you isolate. Record the optical rotations of the two samples and
`indicate clearly which figure belongs to which enantiomer, R or S.
`In your discussion section calculate a percentage recovery for the crystalline
`material you isolate and comment on the efficiency of this process. With reference
`to the optical rotations you recorded on the polarimeter discuss how effective was
`your attempt to separate the two amine enantiomers (optical rotations for the pure
`single enantiomers, in other words the theoretical values, are: (S) –38.2° and (R)
`+38.2°).
`Questions
`1. The (S) enantiomer is isolated via a crystalline salt and, as crystals grow in a
`“pure” form, this enantiomer can potentially be isolated in a very pure state.
`Isolating the (R) enantiomer from the residue left in solution inevitably involves
`isolation of that portion of the (S) amine that did not crystallize. The presence
`of this (S) amine has an effect on the optical rotatory power of the sample
`isolated in this part of the experiment. What effect?
`It is possible to separate the two enantiomers comprising a racemic mixture
`using the various forms of chromatography you have met (TLC, column, GC,
`HPLC). These techniques share a common operational basis, but what
`modification is required if separation of enantiomers is to be achieved?
`
`2.
`
`Organic Chemistry I Laboratory Manual (prepared by D. G. Hamilton, October 2006)
`
`Liquidia's Exhibit 1031
`IPR2020-00770
`Page 5
`
`

`

`Answers to Questions
`
`
`
`2.
`
`1. The (S) enantiomer is isolated via a crystalline salt and, as crystals grow in a
`“pure” form, this enantiomer can potentially be isolated in a very pure state.
`Isolating the (R) enantiomer from the residue left in solution inevitably involves
`isolation of that portion of the (S) amine that did not crystallize. The presence
`of this (S) amine has an effect on the optical rotatory power of the sample
`isolated in this part of the experiment. What effect?
`For the sake of argument let’s assume that 90% of the (S) amine crystallized
`as it’s tartrate salt. The remaining 10% of the (S) amine must therefore still be
`in solution along with all of the (R). The optical rotatory power of the material
`left in solution is compromised by the presence of this 10%, i.e. for every (S)
`amine molecule left behind we are canceling the effect of one (R). The optical
`rotation recorded for the material left in solution must therefore be less than the
`theoretical maximum.
`It is possible to separate the two enantiomers comprising a racemic mixture
`using the various forms of chromatography you have met (TLC, column, GC,
`HPLC). These techniques share a common operational basis, but what
`modification is required if separation of enantiomers is to be achieved?
`All of the chromatographic techniques they’ve met rely on polarity differences
`to achieve separation. Enantiomers have identical physical properties so some
`change must be made to the stationary or mobile phases in order to achieve
`separation in this case. They’ve just learned that the ONLY way to discriminate
`between enantiomers is to have them interact with a single enantiomer of a
`second optically active material. Thus, a column packed with a chiral stationary
`phase would be employed in GC and HPLC, and there are (very expensive)
`forms of silica that contain embedded chiral molecules that can be used for
`TLC and column chromatography. The deal here is exactly the same as the
`experiment
`they have
`just done—formation, albeit
`temporarily—of
`diastereomeric complexes with different physical properties (polarity in this
`case).
`
`
`
`Organic Chemistry I Laboratory Manual (prepared by D. G. Hamilton, October 2006)
`
`Liquidia's Exhibit 1031
`IPR2020-00770
`Page 6
`
`

`

`Preparatory Guide
`
`To check
`
`Polarimeter (operation and calibration).
`A balance in one of the dispensing hoods.
`
`Reagents required
`
`(R, R) Tartaric acid
`(R), (S)-Phenylethylamine
`50% NaOH solution
`Methanol
`Diethyl ether
`Anhydrous sodium sulfate
`
`Supplies
`
`pH Paper.
`Screw top sample vials
`Pipettes
`
`Waste
`
`Basic aqueous waste containers. Non–chlorinated waste containers –a few of
`each.
`
`
`
`Organic Chemistry I Laboratory Manual (prepared by D. G. Hamilton, October 2006)
`
`Liquidia's Exhibit 1031
`IPR2020-00770
`Page 7
`
`