EXPERIMENT 1

PREPARATION OF DIBENZALACETONE

1.1 OBJECTIVE

Upon completion of this experiment, student should be able to:

1. carry out a base catalyzed aldol reaction between benzaldehyde and acetone.

1.2 INTRODUCTION

The reaction of an aldehyde with a ketone employing sodium hydroxide as the base is an example
of a mixed aldol condensation reaction, the Claisen-Schmidt reaction. Dibenzalacetone is
prepared by condensation of acetone with two equivalent of benzaldehyde. The aldehyde
carbonyl is more reactive than that of the ketone and therefore reacts rapidly with the anion of the
the ketone to give a β-hydroxyketone, which easily undergoes base catalyzed dehydration.

Chemicals
Acetone
Benzaldehyde
Ethanol 95%
Sodium hydroxide, NaOH 12%
Distilled water

Apparatus
Erlenmeyer flask 250 mL
Aluminium foil
Parafilm
Buchner funnel
Filter paper for Buchner funnel
Ice-water bath

1.3 PROCEDURE

1. Dissolve 5 mL of benzaldehyde and 2 mL of acetone with 50 mL ethanol in a 250 mL

Erlenmeyer flask.
2. Dilute 12 mL NaOH 12% with 38 mL of distilled water in a beaker. Add this base solution
into the Erlenmeyer flask (Procedure 1).
3. Close the Erlenmeyer flask with aluminium foil and seal it with parafilm.
4. Shake it strongly in 10 min.
5. Put it aside, and shakes frequently for another 30 min.
6. Place the Erlenmeyer flask in an ice-water bath, and cool it for a few minutes.
7. Filter your product using Buchner funnel.
8. Rinse with cold distilled water to remove the base, NaOH.
9. Air dry your product.
10. Place them in a low temperature oven until a constant mass is obtained.
11. Record the mass of product.
12. Calculate its percentage yield.
13. Determine the melting point of your product.
14. Record the IR spectrum and determine the important functional groups.

1.4 QUESTIONS

1. Draw the synthesis of the reactions including compound structures and reagents involved.
2. Write the equation of the reaction of the reaction to show the difference of aldol
condensation between benzaldehyde and acetaldehyde.
3. Compare the theoretical and experimental melting point of dibenzalacetone.

1.5 REFERENCE

1. Fieser, L. F., Williamson, K.L. (1998). Organic Experiments; 8th Ed. Houghton Mifflin
Company: Boston, MA.
1.6 WORKSHEET

Observation

Mass of product

Percentage yield

Melting point of dibenzalacetone

(place your product here)

IR spectra MUST be included in the laboratory report

IR spectrum of compound A Completed Yes / No

Observation

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EXPERIMENT 2
SAPONIFICATION OF AN ESTER

4.1 OBJECTIVE

Upon completion of this experiment, students should be able to:

1. explain saponification process

4.2 INTRODUCTION

An ester can be separated into its components of carboxylic acid and alcohol using hydrolysis
reaction in the presence of strong base. This reaction, called saponification, results in the
formation of an alcohol and a carboxylate salt.

Chemicals
Ethyl benzoate
Hydrochloric acid
NaOH
Boiling chips
Litmus paper

Apparatus
Droppers
Hot plate
Reflux condenser
Round bottomed flask
Beaker
Buchner funnel
Filter paper

4.3 PROCEDURE

1. In a round bottom flask, mix 5 mL of ethyl benzoate with a solution of 3 g of NaOH in 15

mL of water.
CAUTION: Sodium hydroxide is a caustic substance. Do not touch this substance. Rinse your
skin with water immediately if it has a soapy feeling. Sodium hydroxide will cause
irreversible eye damage and blindness. Clean up any spilled material at once.
2. Fit the flask with a reflux condenser.
3. Add few boiling chips in the flask to prevent bumping.
4. Boil the mixture gently for at least 15 min or until ester layer has disappear.
5. Cool the flask at room temperature.
6. Dilute the product with 50 mL of water.
7. Separate the aqueous layer from the oily layer using a separating funnel.
8. While stirring, add dilute hydrochloride acid to the aqueous layer until the solution is acidic
to litmus paper.
9. Filter the precipitate using Buchner funnel.
10. Record your observation
11. Write chemical equation for the saponification of ethyl benzoate.

4.4 QUESTION

1. State two uses of saponification and explain.

4.5 REFERENCE

1. Plummer, D. T., (1987). An Introduction to Practical Biochemistry, 3rd edition, McGraw-Hill

Book Company (U.K.) Ltd., London.
4.6 WORKSHEET

Observations

Equations

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EXPERIMENT 3
THIN LAYER CHROMATOGRAPHY

5.1 OBJECTIVES

Upon completion of this experiment, students should be able to:

1. distinguish polar and non-polar solvents.
2. perform analysis technique using thin layer chromatography.
3. differentiate the retention factor, Rf for different compounds.

5.2 INTRODUCTION

Thin layer chromatography (TLC) is a chromatographic technique that is useful for separating
organic compounds. TLC consists of a stationary phase immobilized on a glass or plastic plate,
and an organic solvent as mobile phase. The sample dissolved in a volatile solvent is deposited
as a spot on the stationary phase. Different component in the mixture moves with the different
rates by capillary action, due to the differences of behaviour between the mobile phase and the
stationary phase.

The retention factor, Rf is define as the distance travelled by the compound divided by the distance
travelled by the solvent.

Distance travelled by the compound a

Rf = =
Distance travelled by the solvent b

Figure 5.1: Thin layer chromatogram

Chemicals
Acetone
Chloroform
o-Nitroaniline
p-Nitroaniline
Unknown sample
TLC Silica gel F254 Aluminium sheets 20×20cm

Apparatus
Forcep
UV Lamp
TLC Spotter
TLC Glass Container
TLC Plate Cutter

5.3 PROCEDURE

1. Draw the origin (0.5 cm from the bottom) and solvent front line (0.5 cm from the top) of the
TLC plate (3x5 cm) using pencil.
Note: Use only pencil with blunt end (2B/HB). Do not use mechanical pencil.
2. Place all three samples using TLC spotter on the TLC plate on the origin line with correct
labelling.
3. Allow the sample to dry and place the TLC plate into the TLC container containing
chloroform (5 mL).
Note: Samples spotted have to be above the solvent level.
4. When the solvent reach the solvent front, remove the plate from the container with forceps.
5. Leave it to dry in room temperature.
6. Place the chromatogram under UV lamp and mark the spots using pencil.
6. Calculate the Rf value for each sample.
7. Record and discuss the result.

5.4 QUESTION

1. Discuss the identification of unknown compound from their distinguished Rf values.

5.5 REFERENCE

1. Skoog, D.A., Holler, F.J., Nieman, T.A. (2005). Principles of Instrumental Analysis. 5 th
Edition, Thomson Asia Pte Ltd., Singapore.
5.6 WORKSHEET

Samples o-nitroaniline p-nitroaniline Unknown sample

Distance travelled
by the compound (a)

Distance travelled
by the solvent (b)

Retention factor, Rf

Thin Layer Chromatogram

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EXPERIMENT 4
EXTRACTION AND IDENTIFICATION OF ESSENTIAL OIL USING GAS CHROMATOGRAPHY
AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY ANALYSIS

4.1 OBJECTIVES

Upon completion of this experiment, student should be able to:

1. assemble the hydrodistillation setup.
2. extract the essential oil from plant sample using hydrodistillation technique.
2. identify chemical components using GC and GC-MS analysis.

4.2 INTRODUCTION

Essential oils are the collection of hydrophobic secondary metabolites that can be extracted from
plants and are used in perfumes, flavourings and alternative medicine techniques such as
aromatherapy. While a single compound may have a distinct smell, most essential oils are actually
hundreds of compounds that, when combined, create the smell associated with that particular
plant.

The essential oil can be extracted from the leaves, bark, flowers, roots, seeds and fruits of the
plant. In the following experiments, hydrodistillation is used to extract the essential oils.
Hydrodistillation is a technique used to distill immiscible liquids, for which steam provides one of
the immiscible phases. Hydrodistillation is commonly used to extract perfume and flavour oils
from natural sources.

Chemicals
Plant sample, Zingiber officinale (200 g)
Water
Boiling chips
Diethyl ether
Magnesium sulphate anhydrous.

Apparatus
Heating mantle
Round bottom flask (2 L)
Clavenger type
Condenser
Rubber tube
Erlenmeyer flask (150 mL)
Filter paper
Screw cap vials
4.3 PROCEDURE

1. Cut the plant sample into small pieces.

2. Place the material into a 2 L round bottom flask.
3. Add water until the plant sample was covered and begin the distillation for 4 hours.
4. Collect the essential oil into the Erlenmeyer flask.
5. Extract with 5 mL of diethyl ether for 3 times.
6. Dry the ether layer with anhydrous magnesium sulphate
7. Filter the solution using filter paper.
8. Wash the filter paper with 3 times of 5 mL diethyl ether.
9. Leave the sample at room temperature about 10 minutes to evaporate the ether.
10. Weight and record the yield of essential oil.
11. Run the essential oil using GC and GC-MS analysis.
12. Discuss the results.

4.4 QUESTIONS

1. Explain the characteristics of essential oil.

2. Discuss the industrial application of essential oil.

4.5 REFERENCE

1. Pino, J. A., Marbot, R., Rosado, A., Batista, A. (2004). Chemical composition of the
essential oil of Zingiber officinale Roscoe L. from Cuba. Journal of Essential Oil Research.
16(3): 186-188
4.6 WORKSHEET

Observation

Mass of product

Sample name and location

GC and GC-MS spectra MUST be included in the laboratory report

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EXPERIMENT 5
ISOLATION OF PIPERINE FROM BLACK PEPPER

5.1 OBJECTIVES

Upon completion of this experiment, student should be able to:

1. assemble the Soxhlet extraction apparatus
2. obtain crude extract from black pepper.
3. isolate piperine from black pepper.

5.2 INTRODUCTION

Some of the organic compounds found in nature have specific smell or odour and they are usually
known, in general, as aromatic compound. In early days, aromatic compounds meant then that
the compound had a low hydrogen/carbon ratio and it was fragrant. The parent compound of the
aromatics is benzene. Today, not all aromatic compounds are fragrant but they are called
aromatic compounds because of their structure.

Vanilin is an example of aromatic compound with specific smell. It is obtained from vanilla and is
widely used as deodorant, food flavour and one of the ingredients in pharmaceutical products.
One of the spices which is widely known is black pepper or Piper nigrum. It has a rather hot and
sharp taste. This is due to the presence of a compound called piperine.

Chemicals
Black pepper
Ethanol 95%
Sodium sulphate anhydrous
Alcoholic potassium hydroxide 10%

Apparatus
Soxhlet extractor
Rotary evaporator
Volumetric flask
Bunchner funnel
Aluminium foil
Parafilm
Preparative Thin Layer Chromatography
TLC Silica gel F254 Aluminium sheets 20×20cm

5.3 PROCEDURE

1. Obtain 20 g of the grounded black pepper

2. Extract the sample with 150 mL 95% ethanol in a Soxhlet extractor for 2 hours.
3. Filter the mixture using filter funnel.
4. Add sodium sulphate anhydrous.
5. Concentrate using vacuum using rotary evaporator (until dry).
6. Add 10 mL alcoholic potassium hydroxide 10% to the concentrated filtrate to dissolve the
crude extract.
7. Separate the solution from the undissolved solid filtrate by slanting the flask.
8. Close the flask with aluminium foil then seal it with parafilm.
9. Leave the alcoholic solution for a night.
10. Piperine will be obtained as yellow needles. Collect the crystals using Buchner funnel.
11. Calculate the percentage yield of the crude extract
12. Dissolve a small amount of piperine in acetone and blotch it on the thin layer
chromatography plate.
13. Place the plate in a chromatography tank and develop it using toluene-ethyl acetate
solution (2:1).
14. Place the plate under UV light and take note of your observation and the sample.

5.4 QUESTIONS

1. Why was 10% potassium hydroxide solution added to the filtrate?

5.5 REFERENCE

1. Epstein, W. W., Netz, D. F., Seidel, J. L (1993). Isolation of piperine from black pepper.
Journal of Chemical Education. 70 (7): 598-599
5.6 WORKSHEET

Observation

Mass of product

Sample name and location

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EXPERIMENT 6
SPECTROSCOPY ANALYSIS OF ORGANIC COMPOUND

6.1 OBJECTIVES

Upon completion of this experiment, student should be able to:

1. elucidate structure of organic compound.
2. distinguish character of functional groups in organic compound.

6.2 INTRODUCTION

One of the most common tasks in chemistry today is the ability to identify an unknown compound
isolated from natural products or synthesized in laboratory. In using the latest analytical
instruments we will be able to deduce the complete structure of an unknown. Infrared (IR)
spectroscopy, Ultraviolet-Visible spectroscopy (UV-Vis) and Mass Spectrometry (MS) are
significant in giving us data about the structure present in an unknown compound. Nuclear
Magnetic Resonance (NMR) is the key to elucidating a compound's structure.

Chemicals
Compound A
Deuterated methanol
Deuterated chloroform

Apparatus
Vials
NMR tubes
Droppers

6.3 PROCEDURE

1. Compound A will be provided in the laboratory.

2. Record the IR spectrum of compound A.
3. Record the UV-Visible spectrum of compound A.
4. Analyse compound A using 1H and 13C NMR Spectroscopy.
5. Analyse compound A using GC-MS.
6. Compile all spectra from procedure 2 to 6.
7. Predict the structure of compound A.

6.5 REFERENCES

1. Solomon, T. W. G., Fryhle, C. B., Snyder, S. A. (2018). Organic Chemistry. 12nd Ed., New
York, John Wiley & Sons.
2. Ouellette, R. J., Rawn, J. D. (2015). Principles of Organic Chemistry. 3rd Ed., Elsevier.
6.6 WORKSHEET

All spectra MUST be included in the laboratory report

IR spectrum of compound A Completed Yes / No

Observation

UV-Visible spectrum of compound A Completed Yes / No

Observation

1
H NMR spectrum of compound A Completed Yes / No

Observation

13
C NMR spectrum of compound A Completed Yes / No

Observation

GC-MS spectrum of compound A Completed Yes / No

Observation

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