I.

INTRODUCTION

In organic chemistry, a “natural product” is one that is produced by a living organism. In order to
discover the compounds, or the natural products, that form one’s surrounding environment, it is
necessary to isolate compounds from their natural sources and to determine their structures (Roberts
and Caserio 1977).

The process of isolating a natural product can be completed with many different methods, which
include, and are not limited to: solvent extraction, distillation, chromatographic separation, liquid phase
extraction, and so on. Two key steps in the isolation process are purification and characterization, which
ensure eradication of contaminants that may preside in the substance to be isolated and assist in
confirming the substance’s identity, respectively.

However, in this exercise, the distillation and reflux methods will be utilized, as well as recrystallization
for purification. Distillation is a separation process that can be used to separate a pure liquid from a
mixture of liquids. Reflux is an energy-supplication technique involving the condensation of vapors and
the return of said condensate to the system from which it originated, and the setup allows the reaction
to be carried out at the boiling point of the solvent and yet it prevents loss of solvent or reagent due to
evaporation (University of Calgary 2012). The setup for these methods can be found in Figure 2.3. and
2.4. under Setups. Recrystallization, on the other hand, involves dissolution of the solid in an appropriate
solvent at an elevated temperature and the subsequent re-formation of the crystals upon cooling, so
that any impurities remain in solution, and is most effective with the least amount of impurity in the
substance to be purified (University of Toronto 2008).

In this exercise, the natural product to be isolated using these methods is piperine, an alkaloid derived
from black peppercorns or Piper nigrum L. (see Figure 2.1.). It is responsible for the pungency of black
pepper and is tasteless, though imparts a burning aftertaste. Alkaloids, also known as vegetable alkali,
are naturally occurring chemical compounds containing basic nitrogen atoms (amines); they are known
to have bitter taste and physiological activity.

Figure 2.1. Skeletal structure of piperine.

II. OBJECTIVES

III. MATERIALS AND METHODS

A. SCHEMATIC
B. SETUPS
Figure 2.2. A suction filtration setup.
Figure 2.3. A reflux setup.
Figure 2.4. A basic distillation setup.
C. LIST OF NECESSARY CHEMICALS

IV. DATA

Table 2.1. Isolation of crude piperine.

 ACTIONS OBSERVATIONS

Freshly ground black peppercorns

- addition of 95% ethanol

After reflux and suction filtration

- residue

- filtrate

After distillation of filtrate

- distillate

- residue

- addition of 10% ethanolic KOH

- thorough mixing for 5 minutes

After filtering the mixture through suction (if there is formation of any solids)

- residue

- filtrate

- addition of 350 mL water

After standing overnight

After filtering the mixture through suction

- filtrate

- residue (crude piperine)

Table 2.2. Data on isolation of crude piperine.

MASS, g PERCENTAGE YIELD, %

Freshly ground black peppercorns

- sample + vial X
 - sample – vial X

- sample X

Crude piperine

- empty watch glass X

- unused filter paper X

- sample + watch glass + X

filter paper

- sample

Table 2.3. Recrystallization of piperine.

ACTION OBSERVATIONS

Dissolution of crude piperine in acetone

Heating gently in water bath

After storing in refrigerator

After filtering through suction

- filtrate

- residue

Table 2.4. Data on recrystallization of piperine.

MASS, g PERCENTAGE YIELD, %

Purified piperine X

- empty watch glass X

- unused filter paper X

- sample + filter paper + X

watch glass
 - sample

Table 2.5. Melting point determination: oil bath method and Fisher-Johns

ACTION OBSERVATIONS

Piperine crystals

Oil Bath Method

- liquefied piperine

Fisher-Johns Point Apparatus

- crystals after turning on the apparatus

- liquefied piperine

Table 2.6. Data for melting point determination.

TEMPERATURE, ˚C

Oil Bath Method

- recorded melting point

Fisher-Johns Melting Point Apparatus

- initial temperature

- final temperature

- melting point range

Table 2.7. Reaction with concentrated acids.

ACTION OBSERVATIONS

Piperine crystals

Addition of 3-4 drops of concentrated HCl

Addition of 3-4 drops of concentrated H2SO4

Table 2.8. Selecting a solvent for the recrystallization of benzoic acid.

ACTION In water In dichloromethane In ethanol

Observations Sol’n (+/-) Observations Sol’n (+/-) Observations Sol’n (+/-)

Addition of benzoic
acid

After heating mixture

*** + means soluble, - means insoluble, +/- means slightly soluble

Table 2.9. The recrystallization process of benzoic acid with chosen solvent.

ACTION OBSERVATIONS

Crude benzoic acid

- dissolution in hot solvent

Filtering with stemless funnel

- residue

- filtrate

Cool filtrate to room temperature

After immersion in ice bath

After suction filtration

- filtrate

- residue

Table 2.10. Data on recrystallization of benzoic acid.

MASS, g PERCENTAGE YIELD, %

Crude benzoic acid X

Purified benzoic acid

 - empty watch glass X

- unused filter paper X

- sample + filter paper + X

watch glass

- sample X

- percentage yield X

VI. Discussion

A natural product such as piperine can be isolated from its natural resource, which, in this case, are
black peppercorns. It is necessary to use them when freshly ground because stored ground black
peppercorns are known to lose flavor over time, which therefore means that piperine loses its potency,
as it is the reason for the pungency and flavor of the spice. Piperine is a major alkaloid in black pepper,
and aside from its culinary and preservative benefits, it is a herbal bioenhancer; it is known to increase
the bioavailability, or the proportion of a substance that enters human circulation to have an active
effect, of many drugs through specific mechanisms, such as inhibition of specific enzymes that support
biotransformation, and non-specific mechanisms, such as reduction of the stomach’s acidic content to
prevent drug degradation (Turmeric for Health 2016).

The isolation process is not complete unless finished with purification, which is recrystallization in this
exercise, and characterization. The simple distillation used for the isolation of piperine was preceded by
a two-hour reflux of a pepper-ethanol mixture. Due to many organic reactions taking an extended
period of time to achieve, heating is often utilized to increase the rate of reaction. However, many
organic compounds have low boiling points and will vaporize upon exposure to high heat which prevents
the reaction from preceding in full (Chemistry Stack Exchange 2015). In relation to this, reflux refers to
heating a solution with an attached condenser to prevent reagents from escaping in vapor form. The
95% ethanol used in this exercise has a boiling point of 78.3degC, and therefore it is necessary for the
pepper-ethanol mixture to undergo reflux in order to prevent losses, at the same time, the organic
reaction length is sped up.

Following reflux, simple distillation, commonly used for separation of ethanol and water, was used to
separate any excess ethanol from the pepper-ethanol mixture. This is usually possible when the two
substances have different boiling points; ethanol has a lower boiling point (78.3degC) than piperine
(130degC), and thus evaporates and condenses first before exiting into the receiver. The drop rate of 1-2
drops per second was maintained for optimal and uniform separation, considering the apparatus used
(Ahluwalia, Bhagat and Aggarwal 2010). The temperature of the pot residue was measured through the
use of a thermometer positioned an approximate 10mm below the arm so that the resulting
temperature read would be the nearest to the true boiling point value of the solvent. As mentioned in
Table 2.1., the distillate is transparent and colorless, following the appearance of ethanol.
Classically, piperine is isolated by a 95% ethanol extraction of ground pepper followed by an overnight
precipitation from 10% ethanolic potassium hydroxide or KOH (Ikan 1991), which is used in this exercise;
though an alternative method involving extraction into dichloromethane and precipitation via trituration
of the crude oils with diethyl ether was developed (Epstein, Netz and Seidel 1993). After distillation, 10%
ethanolic KOH is added to the pot residue and mixed thoroughly, before water is added with constant
stirring; it is important to add water in order to form aqueous KOH. The hydroxide ion acts as a
nucleophile, wherein reactions carried out in alcohol tend to be elimination reactions, and reactions
carried out in water tend to be substitution reactions (Chemistry Stack Exchange 2014); using water as a
solvent in an elimination reaction involving KOH would cause the equilibrium to shift toward the
reactants, which in this case would be crude piperine. The chemical reaction for the isolation of crude
piperine can be seen in Figure 2.5. below.

[Figure 2.5.]

Piperine can usually be isolated only in 2-4% yield from its natural resource, the data in Table 2.2.
following this with a 3.21% yield before an occurrence of a personal error causing the yield to drop
greatly to 0.53% after a recrystallization attempt, and due to its low percentage yield, several synthetic
routes have been created in order to receive a greater product (Epstein, Netz and Seidel 1993).

Recrystallization, as a purification process, involves dissolution of the solid in an appropriate solvent at

an elevated temperature, followed by the subsequent reformation of the crystals upon cooling, so that
any impurities remain in solution (University of Toronto 2008), which was mentioned in the
introduction. Deciding what solvent to use is of utmost importance, as recrystallization will only work
with the proper solvent. A proper solvent is characterized by the following: (1) the solute must be
relatively insoluble in the solvent at room temperature, (2) the solute will be more soluble in the solvent
at a higher temperature, and (3) impurities present in the solute should either be soluble in the solvent
at room temperature or insoluble at high temperature (Yoder 2017). It would also be appropriate to use
a solvent that has slightly different polarity from that of the solute, as if they are similar the solute will
already be slightly soluble at room temperature, following “like dissolves like”.

With piperine, the recrystallizing solvent used was acetone. The crude piperine isolated in the first
portion of the exercise was made to dissolve in acetone, gently heated, and then refrigerated to
decrease the solubility of piperine in water so that crystals may form.

Based on the recrystallization of benzoic acid, the potential solvents were water, ethanol and
dichloromethane. Based on the data given by Table 2.8., water was chosen as the recrystallizing solvent
due to benzoic acid being insoluble at room temperature and later soluble upon heating, and it was
observed in Table 2.9. that some impurities were insoluble at higher temperature, thus easily filtered
out. Gravity filtration is mostly used for hot solutions because this ensures that the product remains
heated as it is filtered, as compared to using suction filtration, which would cause the solution to cool
rapidly due to the presence of a vacuum (Woodgate 2002). The use of fluted filter paper with the hot
gravity filtration ensures that there is a greater surface area for the solution to be exposed to as it
drains, as doing so speeds up the rate at which the solution transfers through the paper. A stemless
funnel is also necessary, as a slower solution transfer rate could cause the solution to cool, which would
result in premature crystallization in the funnel stem.

In order to obtain the crystals, the filtrate from which they appear is made to stand in room
temperature so that crystallization occurs slowly, as a slower rate of crystal formation ensures greater
purity; this ensures that no impurities are co-crystallized along with the desired product. The ice-bath
may follow after enough crystals have formed, or in the case of piperine, refrigeration; rate of
crystallization slows as temperature decreases so cooling with an ice bath should only be used until
crystals begin to form (Yoder 2017). The purified crystals can then be obtained by use of suction
filtration, which is more appropriate as the rapid cooling will hinder formation of any impure crystals as
the solution is disturbed for the filtration.

There are instances, however, wherein crystals may not form due to possible personal errors. One
instance, which may have occurred in the exercise, is using an overabundance of solvent; this can only
be amended by removing a portion of solvent, done by heating the solution for a period of time to
encourage evaporation of solvent (Yoder 2017). There were little to no crystals seen before and after
suction filtration, most possibly due to the large amount of solvent used in the dissolution crude
piperine, which resulted in a sudden drop of the yield to 0.53% after recrystallization, seen in Table 2.4.
In contrast, the recrystallized benzoic acid yielded a relatively large 30.7%, as seen in Table 2.10.

Other instances include the crystals obtained by suction filtration being allowed to dry without first
being washed with cold solvent, which will result in a negative error due to any possible unformed
crystals in the residue being lost; conversely, if these same crystals are washed with hot solvent instead,
a negative error will occur as the crystals may dissolve and become part of the filtrate.

Decolorizing carbon was added

Characterization is a process wherein the obtained natural product will undergo a series of chemical
tests to determine its melting point and its reaction to certain acids; this is necessary in determining
whether or not the resulting product is indeed the pure substance itself and not greatly contaminated.

The melting point of piperine was determine using a Fisher-Johns Melting Point Apparatus, which is a
device that uses small glass coverslips to hold sample crystals, and incrementally increases the
temperature; the temperature reading is also shown on a mounted thermometer, though the true
melting point value must be calculated using Equation 2.1. The obtained melting point for piperine
ranged from 133-134degC, and the true melting point value was computed to be 145-146degC, which is
higher than the melting point of pure piperine at 130-131degC. Following the principle of boiling point
elevation, this means that the crystallized piperine recovered contained contaminants, therefore was
not completely purified in the recrystallization process or may have been improperly

[EQN 2.1.]

The purified piperine was also made to react with hydrochloric acid (HCl), nitric acid (HNO3), and sulfuric
acid (H2SO4) in a standard color reaction. According to the data in Table 2.7., the reaction between
piperine and HCl yielded an observed yellow color, while HNO3 yielded a yellow-orange color, and
H2SO4 yielded an orange color. Acidified piperine generally yields an unstable yellow color which
changes to an unstable red color upon a succeeding addition of an alkali (Graham 1965); the reactions
piperine exhibited with each of the acids stayed within the suspected range of color, the slight tinge of
orange in HNO3 and H2SO4 due to the earlier presence of KOH with piperine.

VIII. References (ONLY THOSE CITED)

A. Books
AHLUWALIA V.K., BHAGAT P., AGGARWAL R. 2010. Laboratory Techniques in Organic Chemistry.
New Delhi, IN: I.K. International Publishing House Pvt. Ltd.
IKAN R. 1991. Natural Products: A Laboratory Guide. 2nd Edition. New York, USA: Academic
Press.
ROBERTS J.D., CASERIO M.J. 1977. Basic Principles of Organic Chemistry. 2nd Edition. Menlo
Park, CA: W.A. Benjamin, Inc.
B. Journals
EPSTEIN W.W., NETZ D.F., SEIDEL J.L. 1993. Journal of Chemical Education 70(7): 598-599.
GRAHAM H.D. 1965. Journal of Pharmaceutical Sciences 54(2): 319-321.
C. Web Sources
BANDIK G.C. 1999. Decolorizing Carbon. Retrieved September 17, 2017, from:
http://www.pitt.edu/~bandik/organicweb/decolorcarbon.html
CHEMISTRY STACK EXCHANGE. 2014. Why does alcoholic KOH prefer elimination whereas
aqueous KOH prefers substitution? Retrieved September 17, 2017, from:
https://chemistry.stackexchange.com/questions/15728/why-does-alcoholic-koh-prefer-
elimination-whereas-aqueous-koh-prefers-substituti?noredirect=1&lq=1

CHEMISTRY STACK EXCHANGE. 2015. What is “heating under reflux”? Retrieved September 16,
2017, from: https://chemistry.stackexchange.com/questions/22364/what-is-heating-under-
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TURMERIC FOR HEALTH. 2016. Black Pepper or Oil: Which is Better for Turmeric’s Absorption?
Retrieved September 17, 2017, from: https://www.turmericforhealth.com/turmeric-
queries/pepper-or-oil-which-improves-curcumins-absorption
UNIVERSITY OF CALGARY. 2012. Organic Laboratory Techniques: Reflux and Reflux Condenser.
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UNIVERSITY OF TORONTO. 2008. Recrystallization. Retrieved August 29, 2017, from:
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