HERMIN HARDYANTI UTAMI (111 304 0202)

CHEMISTRY DEPARTMENT

MATHEMATIC AND SCIENCE FACULTY

STATE UNIVERSITY OF MAKASSAR

2012

APPROVAL SHEET

Complete report of organic chemistry II with the tittle “Piperine Isolation

from Pepper and Chemical Properties of Piperine” was created by :

Name : Hermin Hardyanti Utami

Reg. Number : 1113040202

Group : VII (seventh)

Class : ICP Chemistry “B”

after corrected and consulted by assistant and assistant coordinator, so it was

accepted.

Makassar, December 2012

Assistant Coordinator Assistant

Fandi Ahmad, S.Pd Yanti Madong, S.Si.

Known by

Responsibility Lecturer

Drs. Hj. Ramdani, M.Si.

NIP : 19630221 198803 2 001

A. Title of experiment

Piperine Isolation from Pepper and Chemical Properties of Piperine

B. Purposes of experiment

At the last experiment, student can be understand about :

1. Isolation principle of organic compound from nature material, especially

alkaloid group.

2. Isolation technique in nature material, continue extraction, separating, and

it purification.

3. Knowing chemicals properties of piperine based on the structure and it

degradation result.

C. Background

A natural product is a compound synthesized by a plant or an animal.

Alkalords are natural products that contain one or more nitrogen hetero atoms and

are found in leaves, bark, roots or seeds of plants. Examples include caffeine

(found in tea leaves, coffee beans, and cola nuts) and nicotine (found in tobacco

leaves). Morphine is an alkaloid obtained from opium, the juice derived from a

species of poppy.

Morfphine is so times stronger than aspirin as an analgesic but it is addictive

and suppresses respiration. Heroin is a syntetic compound that is made by a

cetylating morphine. (Bruice, P.Y. 2003:884).

With identities and amounts of amino acids known, the peptide is sequenced

to find out in what order the amount amino acid are linked together. Much peptide

sequencing is now done by mass spectrometry, using either electropspay

ionization (ESI) or matrix-assisted laser desoroption ionization (MALDI) linked

to a time of flight (TOF) mass analyzer as described. Also in common use is a

chemical method of peptide seqencing by called the Edman degradation.

Edman degradation involves treatment of a peptide with phenil isothiocyanale

(PLTC), C6H5-N = C = S, followed by treatmen with trifluoroastic acid. The first

step splits tne N-terminal residue from the peptide chain, yielding an anilinotr

zlinone (ATZ) derivative with shortened peptide. Futher acid catalyzed

rearrangement of the ATZ derivative with aqueous acid converts it into a

phenylthrohydantion (PTH), which is derivative with aquous acid converts it into

by chromatographicaly by comparasion if its solution time with the known elution

times of PTH derivaties of the 20 common amino acids. The chain-shortened

peptide is then automatically resubmitted to anther round of Edman degradation

(Mc Murry, John. 2008 : 1031).

Protein metabolism. Catabolism of proteins: transamination

1. The pathway to amino acid catabolism

a. The amino group is removed by transamination

b. What remains is converted a compound that enters the citric acid cycle.

2. Transamination

a. The NH2 group of an amino acid adds to the aldehyde group of pyridoxal

phospate to form an imine.

b. The imine tautomerizes to different imine.

c. The second imine is hydrolyzed to give an keto acid and an amino

derivative of pyridoxal phosphate.

d. The pyridoxal derivate transfers its amino group to -ketoglutarate, to

regenerate pyridoval phasphate and form glutamate.

3. Deamination

The glutamate from transamination undergoes oxidative deamination to yield

ammonium ion and -ketoglutarate (McMurry, Susan. 2008:796).

Spectrophotometric metods, involving UU absorbance measurements at the

pipenrine absorption maximum near 343 nm measure predominantly piperine but

include some contribution from piperyline, piperettine and any other components

having substantial absorption at this wave lenght. Piperine is extracted from

ground pepper by refluxing 3 hour with ethanol (96% v/v) whilst mogenized

oleoresins are simply dissolve solvent (Wood, A.B. 988:55).

Piperine [(1-5-(1,3) – benzodioxol -5 – yl.) . 1 -0 x 0 – 2, 4 – pentadieny]

piperidine) an alkalord responsible for the pungency of black that pepper and long

pepper. Systemic pharmacological studies on the piperine have revealed that this

compound elicited diverse was pharmacological activities, analgesic, anty-pyretic,

antrinflammatory, anti convulsant and CNS depressant activities. Piperine was

isolated from piper nigium Linn. (Piperaceae) and identified by TLC. Piperine

was fabricated into alginate beads by using sodium alignate. The main aim of this

study was to demonstrate that the sutained release of piperie from alginate beads

by invitro evalution. The drug release studies were showed that the alginate beads

sustained the release of drug with % drug released in hours (Madhavi, B.B.

2009:156).

D. Chemical and Equipment

1. Chemical

a. Pepper sample

b. Aquadest (H2O)

c. Ethanol (C2H5OH) solution

d. Alcoholist potassium hydroxide (KOH alcoholist) solution

e. Potassium Permanganate (KMnO4) solution

f. Concentrated hydrochloric acid (HCl) solution

g. Aqueous hydrochloric acid (HCl) soution

h. Aqueous sodium hydroxy (NaOH) solution

i. Molisch reagent

j. Benedict reagent

k. Aluminium foil

l. Matches

m. Litmus paper

n. Universal indicator

o. Filter paper

p. Whatmann filter paper.

2. Equipment

a. Soxclet equipment (1 set)

b. Analytical scale (1 piece)

c. Buchner funnel (1 piece)

d. Test tube (4 pieces)

e. Test tube rack (1 piece )

f. Stir bar (2 pieces)

g. Dropping pipette (5 pieces)

h. Beaker glass 50 ml and 250 ml (2 pieces)

i. Funnel (2 pieces)

j. Graduated cylinder 10 ml, 25 ml and 100 ml (1 piece)

k. Bunsen burner (1 piece)

l. Triangle (1 piece)

m. Asbestoz Gauze (1 piece)

n. Spray flask (1 piece)

o. Water bath (2 pieces)

p. Round flask 250 ml (2 pieces)

q. Soft cloth and rough cloth (1 piece)

E. Work Procedure

1. Piperine Isolation

a. 20 grams of pepper sample was balanced on analytical scale and was filled

into soxchlet that has covered by filter paper.

b. It was added by ethanol with 3 times of circulation.

c. It was soxcheletd by 15 times circulation.

d. It was let stand and added by 60 ml alcoholist KOH.

e. It was heated on water bath until concentrated and stirred.

f. It was filtered by funnel and divided into 3 part (for piperine isolation,

degradation, and it reaction).

g. It was decreased the volume and let stand on the temperature until 1 day.

h. It was filtered on Buchner funnel and the residue was took.

2. Piperine degradation

a. 1/3 part of isolation result was decreased the volume by reflux it.

b. It was added by HCl 10 M and stirred until dissolve and forming yellow

precipitate.

c. It was let stand on the temperature until 1 day.

d. It was filtered on Buchner funnel and the residue was took.

3. Piperine reaction

a. 3 pieces of test tube was filled by 2 ml of piprine isolation result.

b. It was added by HCl and reacted with :

1)Test tube 1st was reacted by KMnO4

2)Test tube 2nd

was reacted by benedict reagent

3)Test tube 3rd

was reacted by molisch reagent.

c. Each was neutralized the pH and was checked by litmus paper.

F.Observation result

No Activity Result

1.

2.

3.

4.

5.

6.

Isolation of Piperine

Balanced dust paper on the

analitycal scale

Covered with filter paper, filled

to soxlet and added ethanol

The mixture soxlet

1st circulation

2nd

circulation

3rd

circulation

4th

circulation

5th

circulation

6th

circulation

7th

circulation

8th

circulation

9th

circulation

10th circulation

11th circulation

12th circulation

13th circulation

14th circulation

15th circulation

Soxlet mixture heat 10 minutes

Added 60 ml KOH alkoholis

Stir and filtered

25.0124 g

3 times circulation of soxlet

11 minutes

7 minutes

6 minutes

6 minutes

6 minutes

6 minutes

6 minutes

6 minutes

6 minutes

6 minutes

6 minutes

6 minutes

6 minutes

6 minutes

Yellow solution

Yellow solution

Yellow solution

7.

8.

9.

10.

Devided the solution

Vaporated until saturated (b)

Let it stand 1 day

Filtered it with ethanol

a. 6 ml reaction

b. 59 ml for vapor (isolation)

c. 59 ml for reflux (degradation)

Brown solution

Brown solution

Not crystall

1.

2.

3.

4.

Degradation (c)

59 ml solution reflux

Vapored it until 10 minutes

Added 10 M 10 ml HCl and 7

ml H2O

Filtered with ethanol

Dark yellow solution

Yellownish brown solution

Yellownish brown

Not crystall

1.

2

3

Piperin reaaction

2 ml piperin+HCl dilute

Added NaOH

5 drops KMnO4

2 ml piperin+HCl dilute

Added NaOH

5 drops Benedict

2 ml piperin+HCl dilute

Added NaOH

5 drops Molish

Acid solution

Unknown pH

2 layers :

1st :yellow

2nd: turbid (negative)

Acid solution

Unknown pH

Greenish brown (negative)

Acid solution

Unknown pH

Yellow solution (negative)

G. Data Analyzed

1. Isolation of piperine

Known. Weight of sample : 25.0124 g

Mr of Pyperine (C17H19O3N) : 285 g/mol

Mr of pyperidine (C5H10NH) : 85 g/mol

Question=….?

Mass of theory =

=

= 0.2982

2. Degradation of Piperin

Dik : Weight of sample : 25.0124 g

Weight molecule of pyperine (C17H19O3N) : 285 g/mol

Weight molecule of piperidine hydrokloride (C5H10NHCl): 120,5

gram/mol

Weight of theory =

=

= 0,4228

H. Discussion

1. Isolation of piperine

Isolation is seperating process organic compound from nature. In this

experiment would be isolated is pepper that have done smoothly and technique

that we used is continue extraction(soxletation), separation and purity from

another compound that contain in black pepper. The principle soxletation is

pulling chemical compound that done by the dust put on the filtering paper, the

liquid heated in round flask until vapor and condetation by condensor ball become

liquid molecule that drops until in the syfon surface, all of liquid will be down and

back to theflask occured capiler pipes until became circulation. In this experiment

the sample is black pepper that want to get the piperine. Piperine is the alkaloid

responsible for the pungency of black pepper and long pepper, along with

chavicine (an isomer of piperine). Alkaloids are a group of naturally occurring

chemical compounds that contain mostly basic nitrogen atoms.

At this experiment, solvent that used is etanol. Etanol as solvent to solute

all of compound that want in black pepper. Etanol used because has same polarity

is polar until ethanol can solute as the prinsiple like dissolve like. Ethanol used

because volatil properties where in hot condition easy soluble piperin and in cold

condition easy to formed crystall. Then we do extraction continue until 15 times

circulation. At circulation process used boiling stone in flask solvent that purpose

is to keep the pressure and temperature of solution for stable and not occured

explosion.

Process that happen belong circulation is ethanol solvent heated in round

flask until vapor and colded used condensor, until fall become liquid to sample

(black pepper) to soluble active element in sample black pepper and if liquid

solvent have done reached the bottom of syfon then all of liquid from ethanol will

be solute that have in syfon surface will be out occured small pipes to round flask

in this process will occured continue and occured soxletation process, that process

we call the principle of continue extraction. From soxletation process will be

produced 124 ml extraction and in soxletation process occurs 15 circulations

produced transparant yellow extract. After that, extract will dried belong 10

minutes until the solution concentration. It is because to vapor ethanol that contain

in solution. Then added KOH alkoholis 10%. The function is to bond piperine

from solution because occured hydrolysis and to avoid the compotition in

compound other it the function of KOH for get piperine from concentration

extract where in the extract exist another compound when added KOH alkoholis

and caused piperine in extract react become salt acid piperate and to eliminated

other compound because in the extract still contain purity compound. Purity

compound cause piperine compound is alkaloid compound from amida that

sustain hydrolisis reaction. So, alkoholis of KOH for isolated piperine in salt is

piperate acid. Then solution will stir and filter. Filtering process to filtrate can be

seperated from purity.

After that, the filtrate divede 3 part, first part 6ml for piperine reaction,

59ml for piperine isolation and another part for degradation. In isolation of

piperine, the extract will concentrated until saturated and easy formed crystall.

Then, keep it 1 days and not formed crystall. Then crystalitation with ethanol

based on the principle of recriztalitation based the different principle in solubilty

in the solvent and temperature. In the room temperature, piperine compound in the

crystall and polar properties and soluble in etanol as solvent, then piperine in the

filtrate will soluble but purity compound will be left in the residue. In this

process, we didnt get crystall it is because when heated apperantice not attend to

see the extract and the extract exhaused vaporated.

Reaction

N

O

O

O

CH 3CH 2OH+KOH

piperine

ethanol

NH

+O

OH

+ CH 3CH 2OK

. Equation reaction:

1.

N

O O

O

+C 2H 5OH

N+

O O

O

H

+ CH 3CH 2O-

2.

N+

O O

O

H

+ CH 3CH 2O-

NH

+

C+ O + CH 3CH 2O

-

3.

NH

+

C+ O + CH 3CH 2O

-

NH

+

CO

OH

+ CH 3CH 2OK

Piperidine piperate acid

2. Piperine degradation

Degradation is decompotition of compound become a simple compound.

In this thing, piperine degradation to get salt of piperate acid. In this degradation

process, extract will be reacted with KOH 10% (same with piperine isolation) then

concentrated belong 5minutes, produced dark yellow solution. Then, it mixture

reflux belong a hour. This reflux to occurred hydrolysis reaction perfectly. In this

process happen is solvent is KOH filled with piperin in round flask, then heat it.

Vapor of solvent condentation by condenser become solvent molecule until down

to round flask and will solute back piperine that in the flask until surface and all of

by continue. Then suspentition by water to dilute and hydrolysis piperine. Then

add HCl 10M to neutralization because hydrolysis occur in base condition and

must neutralization. Reaction:

O

OHOOC

O

OO-OC

+ H+

Salt piperate acid piperate acid

HCl 10M addition to get proton until formed piperate acid occurred

hydrolysis reaction. Next, solution keep silent belong one day then filtered and

crystallization with ethanol. This process not crystal formed because apperantice

mistaken when reflux and added HCl or water.

3. Reaction of piperine

a. KMnO4 reaction

This experiment we show that piperine easy oxidate because have double

bond. This experiment, alkoholis solution add with HCl dilute to decreasse pH

because solution in base condition to get netral solution. Then reacted with

KMnO4 to oxidate piperine and produce two layers, yellow in uplayer and turbity

in the down layer. It is not same with theory must be purple. It is caused while

netralization not universal indicator and only litmus paper and don’t know the

solution netral or not if only used litmus.

Reaction is

b. Benedict reaction

At this experiment to show that piperine have aldehide group that is

reducing group. Solution that get from isolation added with HCl diluted to

decreasse pH because solution in base condition to get netral solution. Solution

reacted with benedict reagent produce green brown solution. It is not same with

theory must result brick red, green or yellow. It is not same with theory because

piperine still contain purity compound and while netralization not universal

indicator and only litmus paper and don’t know the solution netral or not if only

used litmus.

Reaction :

c.Molish reaction

The purpose of this experiment to identify existence of epoxy group that

sign with furfural ring in piperine. Alkoholis solution from extraction added with

HCl dilute to decreasse pH because solution in base condition to get netral

solution. Then reacted with Molish reaction and formed yellow solution. It is not

same with theory because piperine still contain purity compound and while

netralization not universal indicator and only litmus paper and don’t know the

solution netral or not if only used litmus.

Reaction is

N

O

O

ON

H

HCl + CH3CH3

O

+O

O

piperidine piperanol epoksi

hydrochloride

I.Closing

1. Concusion

a. The principle of organic compound from chemical nature specially alkaloids

extraction alkaloid in acid solution in water and netral or base compound from

mixture to separate with solvent. After this solution base so alkaloid set with

extraction in solvent.

b. Technique isolation in chemical nature is extraction continue, purifying with

method crystalization and purifying step with determine melting point.

c. Piperine reaction with KMnO4 to show that piperine easy oxidezed. Piperine

reaction with benedict to test for carbonyl group in piperine can be reduced by

Cu2+

. Piperine reaction with molish reagent to find a ring that piperine furfural

in epoxy groups.

2.Suggestion

a. Apperantice must be attend when vapor the extraction,

b. Laboran must prepare our reagent before experiment.

BIBLIOGRAPHY

Bruice, P.Y. 2003.Orgnic Chemistry 4th

Edition.New York. Longman Hill.

Madhavi, B.B. 2009. Extraction, Identification, Formulation and Evaluation of

Piperine in Alginate Beads. India. International Journal of Pharmacy

and Pharmaceutical Sciences.

McMurry, John. 2008. Organic Chemistry 7th

edition.USA. Thomson Learning,

Inc.

McMurry, Susan. 2008. Study Guide and Solutions Manual Fundamental of

Organic Chemistry. USA. Thomson Learning, Inc.

Tim Dosen. 2012. Penuntun Organik 2. Makassar.Universitas Negeri Makassar.

Wood, A.B. 1988. Piperine Determination in Pepper (Pipper ningrum L.) and its

oleorensis A reversed phase High-performance Liquid

chromatographic Method. England. Flavour and Fragrance Journal.

Answer Question

1.Using potassium hydroxide to remove resin(said to contain chavicine an isomer

of piperine.

2. Degradation of piperine

O

OHOOC

O

OO-OC

+ H+

Salt piperate acid piperate acid

3. Reaction of Piperine

a. Reaction with KMnO4

b. Reaction with benedict

c.Reaction with Molish

NH

HCl + CH 3CH 3

O

+O

O

piperidine piperanol epoksi

hydrochloride

N

O

O

O