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Chemistry 233L

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Experiment 4: Isolation and Characterization of Natural Products. Isolation of Piperine from Black Pepper. Purpose To isolate and characterize a natural product. Introduction A weakly basic substance that could be extracted from a variety of peppers was isolated and characterized in 1882 and given the name piperine, from the Latin name for pepper (piper). Piperine (I) is a 1,4-disubstituted butadiene; in this experiment we will determine the geometry about those double bonds. Piperine constitutes about 10% of the weight of black pepper, with the remaining 90% being comprised of starches (20-40%), volatile oils (1-3%), and water (8-13%). O O HC

CH

CH CH

O

N

Piperine (I)

Piperine is not responsible for the aroma of black pepper due to its low volatility; however the taste of black pepper is partially attributable to it. Although piperine is tasteless at first, it does ultimately produce a burning sensation and sharp aftertaste, possibly because of its extremely low solubility in water (and therefore unable to penetrate the layer of saliva on the tongue and reach the taste buds). If piperine is wetted with ethanol first, an immediate pepper taste is noted lending credibility to this suggestion. The isolation of piperine can be accomplished simply by extraction of ground pepper with 95% ethanol. The crude extract obtained by heating black pepper in ethanol also contains some acidic, resinous materials that must not be allowed to precipitate with and therefore contaminate the piperine. In order to prevent co-precipitation of piperine and the resin acids, dilute ethanolic potassium hydroxide is added to the concentrated extract to keep acidic materials in solution as their potassium salts. Hydrolysis of piperine. When an attempt to determine the structure of an unknown natural product is being made, it is often useful to cleave the unknown substance into smaller fragments by a chemical reaction; these fragments can generally be more readily identified than the original molecule. Once they have been identified, these smaller molecules represent pieces of a "jigsaw puzzle" which the chemist must put together in a rational way, the goal being, of course, to fit the pieces back together so that the structure of the original substance is duplicated.

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Chemistry 233L

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In the case of piperine, the gross structure is that shown above, however, the stereochemistry about the double bonds is not specified. In the original proof of structure of this substance, it was recognized that the single nitrogen atom present in the molecule was part of an amide linkage so that hydrolysis of this functional group should produce an acid and an amine, both of which might be of known structure. In fact, base-catalyzed hydrolysis followed by appropriate work-up allowed isolation of piperidine, a known cyclic amine, and piperic acid (see Equation 1) allowing one to propose a structure for piperine. O NH

Piperine (I)

+

HC

CH

CH CH Ar

HO

Piperidine

Piperic acid

Equation 1

Assignment of the full structure to piperine requires that the geometry about the double bonds be specified. Given that piperic acid is one of the four geometric isomers shown below and that the melting point of each isomer is that given, isolation of piperic acid and determination of its melting point should allow assignment of the required stereochemistry. It is this task that you will accomplish in this experiment. Ar CO OH

COO H

Ar

mp = 215-217

mp = 134-136 Ar

Ar COO H COO H

mp = 154-156

mp = 200-202

Materials needed • See Experimental Procedure (below) for specifics • 30 g black pepper (whole peppercorns preferred) • 95% ethanol • 2 M ethanolic KOH (KOH(s) in ethanol; should be made just prior to use) • 6 M HCl

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Chemistry 233L

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Procedure Isolation of piperine Place 30 g of FRESH*, finely ground black pepper in a 500 mL sized round-bottomed flask, add ca. 300 mL of 95% ethanol, and gently heat the mixture under reflux for ca. 3 hr (NOTE: The entire reflux need not be done during a single laboratory session). Because there is solid present in the boiling mixture, bumping may occur, particularly if heating is too vigorous. Filter the mixture by suction (vacuum filtration) and concentrate the filtrate to a volume of 20-30 mL by distillation or rotary evaporation. Add 30 mL of warm 2 M ethanolic potassium hydroxide solution to the residue from the distillation, stir and decant (or filter) the warm mixture to remove any insoluble materials. While keeping the solution warm in a hot water bath, add 15-20 mL of water; the solution should become turbid and yellow needles may be seen. Allow the resulting solution to stand until the next laboratory period and then isolate the yellow precipitate of piperine that has formed. Recrystallize the crude piperine from acetone (Be careful! Acetone is flammable!). The resulting crystals should be formed as fine yellow needles. Determine the yield and melting point of the product. The reported melting point of piperine is 129-131°C. Obtain an NMR and IR of your product. Hydrolysis and characterization of piperine (Note: Quantities in the following procedure will be adjusted according to the amount of piperine that is available.) Heat a mixture of 1 g of piperine and 10 mL of 2 N ethanolic potassium hydroxide at reflux for 1.5 hr. Evaporate the ethanolic solution to dryness using a rotary evaporator. Suspend the solid potassium piperate that remains in the flask in about 20 mL of hot water and carefully acidify this suspension with 6 N hydrochloric acid. Collect the precipitate that results, wash it with cold water, and recrystallize the crude piperic acid from absolute ethanol. Determine the yield and melting point of the isolated product. Obtain an NMR and IR of your product. Qualitative detection of piperidine in the distillate can be accomplished by dissolving a few drops of the distillate in a few milliliters of water and determining the pH of the solution. The distillate should also have an odor characteristic of an amine. Note the pH and odor and discard this solution in the "Amine Waste" jar in the hood. Notes General: Nothing of note to add here *Fresh pepper, ground from peppercorns is best as oxidative damage should be at a minimum.

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