HARAMAYA UNIVERSITY
COLLEGE OF HEALTH AND MEDICAL SCIENCES
SCHOOL OF PHARMACY
PRACTICAL ORGANIC CHEMISTRY
LABORATORY MANUAL
Compiled by
TESHOME FIKRE (MSc.)
November 2022
Table of Contents
PREFACE ...................................................................................................................................................... iii
LABORATORY SAFETY ............................................................................................................................. v
Experiment 1 ................................................................................................................................................. 11
PREPARATION OF ASPIRIN ................................................................................................................. 11
Experiment 2 ................................................................................................................................................. 13
Recrystallization ........................................................................................................................................ 13
Experiment 3 ................................................................................................................................................. 15
Determination of melting point ..................................................................................................................... 15
EXPERIMENT 4........................................................................................................................................... 17
SURVEY OF SOME FUNCTIONAL GROUPS ..................................................................................... 17
EXPERIMENT 5........................................................................................................................................... 20
PREPARATION OF SOAP ...................................................................................................................... 20
EXPERIMENT 6........................................................................................................................................... 23
CHROMATOGRAPHY ........................................................................................................................... 23
EXPERIMENT 7........................................................................................................................................... 27
QUALITATIVE ANALYSIS OF CARBOHYDRATES ......................................................................... 27
REFERENCE ................................................................................................................................................ 31
APPENDIX I ................................................................................................................................................. 32
Special Reagents ....................................................................................................................................... 32
APPENDIX II ............................................................................................................................................... 34
Physical Constants of some Organic Compounds ..................................................................................... 34
ii
PREFACE
This Manual is intended for use in one semester Practical Organic Chemistry course at the university level.
Such a practical course is usually offered simultaneously with an organic chemistry lecture course to
beginning students of chemistry, medicine, biology and pharmacy.
Students need to know that instructor may announce supplemental to this manual as deemed necessary. In
the laboratory, students shall work in pairs or more at each set-up place. A member of each group cannot
merely be a bystander. Leaving, clean, apparatus, glassware and the work place at the end of each experiment
is so important. Keeping good discipline, having followed safety rules in each experiments help to avoid
accident and is an indispensable condition while working in the laboratory. Your final grade for this
laboratory course will depend on performance at each experiment, how well you keep records during
laboratory session, how well you write laboratory reports. Your skills, as well as continuous assessment by
your instructor, quiz and result in the final examination.
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About your report
You should submit a properly written report on the date announced by your instructor.
The following format summarizes the information to be included in writing report.

Date

Experiment number

Title

Theory

Apparatus and chemicals: list of apparatus and chemicals use in the experiment.

Procedure: write a procedure in such a way that you or someone else can repeat the experiments by
reading what is written here and it should be written passive voice.

Result and Discussion: All data which were obtained from the experimental work are written here.
The discussion section is where you explain experimental observations. It is also important to give
here reason behind some of the major steps followed while conducting the experiment. You must
attempt, for instance the reasons why yield of products you got is good or bad. You should also
compare the actual yield you got with the calculated or theoretical yield.

Answer to Questions:
Remember to write the report neatly, in such a way it is easily understandable and readable
iv
LABORATORY SAFETY
Some laboratory safety and guidelines
In any laboratory course, familiarity with the fundamentals of laboratory safety is critical. Any
chemistry laboratory, particularly an organic chemistry laboratory can be dangerous place in
which to work. Understanding potential hazards will serve well minimizing that danger for you.
We have pointed out specific hazards in the experiments found in this manual. However, it is
ultimately your responsibility, along with the laboratory instructor, to make sure that all
laboratory work is carried out in a safe manner.
Organic solvents: Their hazards
Avoid contact with organic solvents. It is essential to remember that most organic solvents are
flammable and will burn if they exposed to an open flame or match. Remember also that on
repeated or excessive exposure, some may be toxic or carcinogenic or both. If you want to check
the odor of a substance, be careful not to inhale very much of the material. The technique for
smelling flowers is not advisable here; you could inhale dangerous amount of the compound.
NEVER hold your nose over the container and inhale deeply! If reagents are spilled on the skin,
flush immediately with large amount of water. Remove any spilled material from your desk
and try to maintain as clean as possible through pout the laboratory session.
Some laboratory safety and guidelines
1- Safety goggles or eye shields must be worn at all times to guard against the laboratory
accidents.
2- Wear only shoes that shed liquids, sandal, canvas shoes, and high-heeled shoes are not
permitted.
3- Wear non synthetic (cotton) clothing that is not torn or frayed.
4- Clothing should cover the skin from neck to below the knee and at least to the elbow.
5- Gloves are often to be worn to protect the hand when transferring corrosive liquids.
6- To protect outer clothing wear an apron or coat with snap fastener only.
7- Never taste, smell or touch a chemical unless specifically to do so.
8- Never eat or chew gum or smoke in the laboratory.
9- Notify all the accidents, which will occur in your laboratory as soon as possible to your
instructor.
10- For abrasions or cuts flush the affected area with water or submerged into an ice bath.
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11- In case of fire, discharge a fir extinguisher at the base of the flame and move it from one side
to the other side.
12- At the end of the experiment, completely clean all the laboratory bench of equipment with a
damp sponge or paper towel and also clean the sinks, all glassware, used in it.
13- Inquisitiveness and creativeness in the laboratory are encouraged.
Remember that the laboratory is a work place, is not a place to play!
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ABBREVIATIONS
ASA
Acetylsalicylic acid
Atm
Atmosphere
bp
boiling point
CC
column chromatography
conc
Concentrated
GLC
Gas liquid chromatography
HPLC
High performance liquid chromatography
mL
Milliliter
mmp
mixed melting point
mp
melting point
PC
paper chromatography
TLC
thin layer chromatography
vii
GLASSWARE AND UTENSILS
Test Tube Holder
Ring stands
Funnel
Wash Bottle
Test Tube Brushes
Test Tube
Glass Stir Rod
Spatulas
Dropper
Measuring Cylinder
Conical flask
Buchner flask
viii
Test tube racks
Litmus Paper
Beaker
Buchner funnel
Mortar & pistil
Clamp
Burette
Separatory funnel
Beaker Tongs
Filter paper
Florence Flask
Watch Glass
Pipette
Rubber Stoppers
Condenser
Bunsen Burner
Evaporating Dish
Round-bottom Flask
ix
Symbols
DDFFGHjkkll;;;;;;;;okklll
x
EXPERIMENT 1
PREPARATION OF ASPIRIN
Objective: To prepare acetylsalicylic acid, commonly known as “aspirin”
Theory
Aspirin was first synthesized in 1893 by Felix Hofmann, a chemist scientist worked in German
firm of Bayer. Acetylsalicylic acid (ASA), generally called “Aspirin” is the most widely used analgesic at
the present time. It is commonly prescribed alone or with other drugs for the treatment of headache, cold,
influenza, arthritis etc.
Aspirin can be prepared by the acetylation of salicylic acid with acetic anhydride in the presence of catalytic
amounts of mineral acid like sulfuric acid. Aspirin is a white crystalline solid with a melting point of about
135 Co or (138-140Co). Acetylsalicylic acid is stable in dry air, but gradually hydrolyses in contact with
moisture to acetic and salicylic acids.
Chemicals and glassware/apparatus required
Chemicals: salicylic acid, acetic anhydride, sulfuric acid and distilled water.
Glassware/Apparatus
Analytical balance, conical flask, measuring cylinder, pipette, filter paper, water bath, magnetic stirrer,
magnetic bar and suction filtration with pump.
Procedure
1. In a dry conical flask containing (1.5g) of salicylic acid, add (3mL) of acetic anhydride in a small
portion.
2. Add (3-5) drops of conc. sulfuric acid to the solution and stir the mixture well.
3. Enclose the conical flask, then heat the solution in a water bath to about (10) minutes.
4. Add (3ml) of distilled water to the hot solution.
5. In continuous process add (25ml) of distilled water to the solution.
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6. Cool the mixture at room temperature until the white crystals formed inside the solution.
7. Separate the crystals (Aspirin) from the solution by filtration process.
8. Dry the crystals then calculate the percentage yield of the prepared aspirin.
Perform the following test with the product:
1. Solubility test: By taking a small amount of the product on the tip of spatula and placing it in a small test
tube check the solubility of your product in water, ethanol and 10% sodium bicarbonate solution.
2. Test for the phenolic hydroxyl group: Dissolve a few crystals of aspirin in 1 mL of ethanol in a test tube
and add one drop of 1% ferric chloride solution.
12
EXPERIMENT 2
RECRYSTALLIZATION
Objective: To purify a contaminated solid compound by recrystallization
Theory
Recrystallization is a very important purification technique, purifying substances by removing unwanted byproducts. It is also used to manufacture the correct crystal size and shape of a material.
What are the principles behind recrystallization?
The method of purification is based on the principle that the solubility of most solids increases with increased
temperature. This means that as temperature increases, the amount of solute that can be dissolved in a solvent
increase. Recrystallization process is a laboratory technique for purifying different types of solids. The basic
features of this technique are causing a solid to go into solution and then gradually allowing the dissolved
solid to crystallize.
Recrystallization process:

The organic substance is dissolved in a minimum amount of suitable solvent, then the solution heated
near the boiling point of the solvent.

Insoluble (suspended) impurities can be filtered away while the solution is hot.

Cooling the solution till the room temperature, then using an ice bath to form a beautiful crystals.

Separation of the crystals from the solution by filtration process, Finally the crystals should be dried
using an oven.
Chemicals and glassware/apparatus required
Chemicals: as per specific requirement
Depending on the availability of sample any one of the following compound may be used. Salicylic acid,
aspirin, acetanilide, benzoic acid etc.
Solvents1. Polar compounds are soluble in polar solvents such as water, methanol and ethanol.
2. Nonpolar compounds are soluble in non-polar solvents such as hexanes and diethyl ether.
Glassware/Apparatus
Conical flask, Beaker, Measuring cylinder, Hot plate, Filter paper, Thermometer, Glass stirrer Water bath
and Pipette.
13
Procedure

Weight out (1.0) g of crude (impure) sample then put it into a beaker.

Dissolve the sample in (30) mL of solvent (ethanol).

Heat the solution near the boiling point of the solvent.

Cool the solution at room temperature, then using an ice bath (If necessary).

Separate the purified crystals from the solution by the filtration process.

Dry the collected crystals, then calculate the percentage yield of the purified sample.
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EXPERIMENT 3
DETERMINATION OF MELTING POINT
Objective: To determine the melting point of recrystallized sample (aspirin).
Theory
Melting point is a temperature at which the solid organic compound completely converts to the
corresponded liquid form or it is a temperature at which the first crystal starts to melt until the temperature
at which the last crystal disappears.
All the solid organic compounds during the heating gave a range, known as melting range. Pure organic
compounds during heating process characterized by a sharp melting range (1-2) °C, while impure organic
compounds characterized by a broad (wide) melting point range (higher than 2) °C.
Uses of melting point
 Identification of solid organic compounds.
Ex. M.P. of pure Benzoic acid=121-122 °C
 Detecting the impurity inside the solid organic compounds.
Ex. M.P. of impure Benzoic acid=121-124 °C
Impurities lower melting point:

Takes less energy to disrupt crystal lattice when impurities are present

Melting point will be broader (in range)
Melting point apparatus
Generally we have two types of melting point apparatus:1. Classical melting point apparatus (Water bath, Oil bath).
2. Electrical (digital) melting point apparatus.
Chemicals and glassware/apparatus required
Chemicals: Aspirin, paraffin liquid.
Glassware/Apparatus
Capillary tube, water bath, beaker, thermometer, hot plate
Procedure
1. Enclose one ends of the capillary tube by a source of heat.
2. Add a small amount of the aspirin into a capillary tube.
15
3. Place the capillary tube beside a thermometer, using a rubber for such process.
4. Put the (capillary tube + thermometer) inside an oil bath or water bath gently.
5. Heat the oil bath or water bath gently.
6. Record the temperature at which the first crystal converts to the liquid form, until all the crystals
completely converts to the liquid form.
16
EXPERIMENT 4
SURVEY OF SOME FUNCTIONAL GROUPS
Objective: To study the characteristic chemical properties of some functional groups
Theory
A functional group is a unique combination of atoms present in a molecule that is particularly responsible
for the characteristic set of reactions that the molecule exhibits. Some of the most important functional groups
include: hydroxyl, olefin, ketone, aldehyde, amine, carboxylix acid, ester, acid halide, anhydride, nitrile,
ether, halide, aromatic ring etc. It is possible to distinguish by means of simple tests one functional group
from the other. In this experiment attempts will be made to highlight some of the most important tests in
organic chemistry that aid one to tell functional groups apart.
A. Alkanes
Gasoline commonly known as benzene or petrol is one of the most common sources of energy. Similarly
naphta or kerosene is also a commonly used source of energy. Both these fuels are mixtures of hydrocarbons.
They contain mainly alkanes such as isomeric pentanes, hexanes, heptanes and octanes and small amounts
of unsaturated and aromatic hydrocarbons. Petroleum ether is an example of a mixture of alkanes and is
prepared by means of fractional distillation of petroleum by-products. Carry out the following tests in dry
test tubes.
Procedure:
Solubility of Alkanes: Test the solubility of Gasoline in water, ethanol, petroleum ether and concentrated
sulfuric acid (alkanes do not react with concentrated acids!). In each case place 5 drops of gasoline in a small,
dry test tube and add not more than 3 mL of solvent. Observe and record your results in a table.
Repeat the above experiment for kerosene.
Reaction of Alkanes: Alkanes react slowly or not at all with bromine in the dark but in the presence of light
react fairly rapidly. [Write the equation for this light catalyzed reaction and explain why it proceeds so
rapidly].
In three separate test tubes place 1 mL portions of petroleum ether, gasoline and kerosene and add to each
4– 5 drops of a solution of bromine in carbon tetrachloride (5 %). Place the three test tubes in the dark (i.e.
in your cupboard). Observe the color of each test mixture after 15 minutes and note your observation. Blow
your breath gently across the mouth of each tube and observe the result. If HBr is present, it will combine
17
with the moisture of the breath to form a faint cloud. The presence of HBr can also be confirmed by holding
a wet blue litmus paper on the mouth of the tube.
Dump the contents of the three tubes into the provided special container for keeping solvents to be discarded
later. Never throw away chemicals in the sink.
Repeat the above experiment but this time take the three test tubes outside in the sun for 5 minutes or so.
Compare the result with the previous experiment. Is there any difference? Explain the results. Hint: Gasoline
contains sufficient amount of unsaturated hydrocarbons to effect a positive reaction with bromine. How about
kerosene?
Observe the effect of oxidizing agents on the above hydrocarbons as follows: Place 10 drops of each of the
above mentioned three hydrocarbons in three test tubes and add to each 1 ml of dilute (0.5%) potassium
permanganate solution. Shake well, observe the result and record it as either + or – in a table.
B. Alkenes
Solubility of alkenes: Place 0.5 mL (10 drops) of cyclohexene in a dry test tube. Check the solubility of this
compound in concentrated sulfuric acid (3 drops). Repeat this experiment with water and ethyl alcohol.
Explain your results in the Discussion Section of your report.
Reactions of alkenes: With bromine in carbon tetrachloride: To 0.5 mL (10 drops) of the given alkene add
3 drops of bromine/carbon tetrachloride solution. Note your observation.
With aqueous permanganate solution: To 0.5 ml of the given alkene add 1 ml of 0.5% aq. Potassium
permanganate solution. Shake well and record your observation. Explain the result by means of chemical
equations.
C. Alkynes
This simplest member of this class is acetylene and is widely used industrially. Acetylene is readily prepared
according to the following reaction.
CaC2 + 2H2O  H – C  C – H + Ca(OH)2
Generation of Acetylene: Carefully examine the acetylene generator which has been set up by your
instructor in the fume hood. Draw a diagram of the generator in your notebook. Allow water drop by drop
into the calcium carbide so that acetylene is generated gently.
Bromination Test: Bubble acetylene into a test tube that contains 1 mL of a bromine/carbon tetrachloride
solution (0.25%). Observe and record the result.
Baeyer’s Test: Bubble acetylene into a test tube containing 2 mL of dilute (0.5%) potassium permanganate
solution. Observe and record the result.
18
D. Aromatic hydrocarbons
Test for unsaturation: To 6 drops of benzene or toluene add 3 drops of bromine/carbon tetrachloride
solution. Observe if there is any reaction. If there is no reaction explain why aromatic compounds do not
undergo the typical reactions of olefins.
Nitration of benzene or toluene: In a test prepare a nitrating mixture by adding 10 drops of concentrated
sulfuric acid and 3 drops concentrated nitric acid. This mixture is believed to generate the reactive species
known as the nitronium ion which is quite reactive and is capable of attacking the benzene ring. Add to this
4 drops of benzene or toluene. Shake the test tube and let it stand for 15 minutes and then pour the mixture
into a large test tube containing a piece of ice. The heavy oil at the bottom is due to nitrobenzene.
[Nitrobenzene is toxic, avoid contact.] Write the complete equation showing this transformation.
E. Test for ketones
A typical reaction of ketones is their reaction with 2,4-dinitrophenylhydrazine (check the structure and
mechanism of the reaction from a text book). Place 10 drops of the acetone solution in a small test tube. Add
12 drops of the reagent to it. Note the color of the precipitate and explain by chemical equation the reaction
responsible for the formation of this derivative.
F. Test for phenols
Place 20 drops of 10% aq solution of phenol in a test tube. Add to this 3 drops of 2% of neutral ferric chloride
solution. The development of a violet color is characteristic of the phenol functional group.
19
EXPERIMENT 5
PREPARATION OF SOAP
Objective: -To prepare ordinary soap and to examine its properties
Theory
The term "saponification" refers to the hydrolysis of ester under alkaline conditions, and is derived from the
age long practice of making soap by the alkaline hydrolysis of fats. Soaps are sodium salts of long chain fatty
acids, while the natural fats are esters of these acids with glycerol. fats are therefore sometimes nature are
fats from animal and vegetable origin. Animals tallow, lard, palm oil, coconut oil, olive oil, cotton seed oil,
corn oil etc. are but a few of the naturally occurring glycosides.
Natural fats and oils are mixtures of glycosides. For instance, tallow contains myristic, palmitic, stearic, oleic
and linoleic esters of glycerol. The difference between fats and oils is caused by the fact that facts are
composed of glycosides of saturated acids, while oils are made up of unsaturated fatty acids. The extent of
instauration in fat or oil is expressed in terms of its iodine value, which is defined as the number of grams of
iodine, which will add to 100g of fat or oil. Thus a high iodine value as in vegetable oil indicates a high
degree of instaurations.
The hydrolysis of a fat with alkali yields glycerol and three molecules of the salt of the fatty acid
(soap).
The soap molecules possess detergent or cleansing properties because of their ability to form aggregates with
fat soluble materials in which the long, fatty acid chains surround the "dirt" in such a way as to enclose it
within a cluster of soap anions leaving the hydrophilic carboxyl groups as a peripheral water solubilizing
envelope. The resulting "dirt-soap" complex can then be carried away in the water.
Solubility of organic compounds in water is affected by the presence of inorganic salts. Organic compounds
that are miscible in water, decrease its solubility when is added inorganic salts of Na+ or K+ and precipitate.
The acidity of a fat or and eatable oil depends of free fatty acids that there are. Acidity index of fat or eatable
oil is defined as the amount (mg) of NaOH that is necessary to neutralize 1 gm. of fat.
20
This method of
neutralization is based in the titration of fatty acids by a known concentration of a base using phenolphthalein
as indicator.
Materials and reagents required
Round bottom flask
Boiling chips
Condenser
Bunsen burner
Beaker
Suction filtration apparatus
Erlenmeyer
Phenolphthalein
Long stemmed funnel
Detergent
5% MgCl2 solution
Distillated water
5% MgCl2 solution
Beef tallow or oil
20% NaOH solution
Ethanol
Brine solution
ice
5% FeCl3 solution
6M HCl solution
5 % CaCl2 solution
Procedure
In a round bottom flask place 10g of beef tallow, 20 ml of 20% sodium hydroxide solution, 15 mL
95% ethanol and 2 boiling chips. Attach a condenser and reflux the mixture for 1 hr at the end of
which a clear solution should be present. Allow the flask to cool a bit and then pour the warm
solution in to a beaker containing 100 ml of brine solution (saturated salt solution). Cool the
solution in an ice bath, filter the solid using suction filtration and wash it once with 20 mL of ice
21
water. When most of the water has been removed transfer the soap to a small beaker. Heat the soap
to soften. Allow to cool to room temperature and then cool the beaker in an ice bath to harden the
soap. You may take soap with you after conducting the tests described below.
Properties of soap:
1. Dissolve 1 g of the soap you prepared in 50 ml of distilled water, warmed on a water bath
(Test solution 1).
2. Similarly dissolve 0.5 g of ordinary soap in 25 ml of water (Test solution 2) and also 0.5 g
of detergent (Omo or Roll) in 25 ml of water (Test solution 3).
Perform the following experiments on the above three test solutions and note the results in your
report book.
1. Test the alkalinity of each of the above solutions by means of litmus paper.
2. Place 2 mL of test solution 1, into 4 test tubes. Add 3-5 drops of calcium chloride solution
to the first test tube, 5% magnesium chloride to the second, 5% ferric chloride to the third
and 6M hydrochloric acid to the fourth. Shake each test tube well and note your
observations and present the result in a Table.
Repeat this experiment with Test solutions 2 and 3. Explain the different behaviors of the test
solutions in the Discussion section of your report.
22
EXPERIMENT 6
CHROMATOGRAPHY
Objective: To learn the use of chromatographic techniques in the separation and identification of organic
compounds.
Theory
Chromatography is one of the most valuable techniques for the separation, identification and purification of
organic compounds. Although chromatography was first discovered using coloured substances (chroma,
Greek for color), colourless compounds can also be analyzed by this method.
Paper chromatography was first discovered by a British team of scientists) Consden, Gordon, Martin and
Synge, 1944) whose contribution was recognized by the award of the Nobel Prize. The usefulness of the
technique was first appreciated by the ease with which amino acids were separated by paper chromatography.
This further led to the investigation of a variety of other complex substances. Medicine has in particular
benefited from paper chromatography by its application to laboratory examination of body fluids. Other
applications include analysis of poisons, and identification of sugars and other natural metabolites.
Chromatographic separations depend on the differences in the partition coefficients of the components
between two immiscible phases, the mobile and the stationary phases. Thus in the so called partition
chromatography (liquid-liquid chromatography), components partition between two liquids phases i.e. a
mobile and a stationary phase. The following Table shows the major types of chromatography.
The different chromatographic techniques that are often used in laboratories include: paper (PC), thin layer
(TLC), ion exchange, column (CC). In this experiment paper chromatography will be used to separate
components of a mixture while TLC and CC techniques will only be demonstrated.
Technique of Paper Chromatography:
Paper chromatography is an example of liquid – liquid chromatography (LLC). It finds application of small
amounts of materials.
The cellulose, of which ordinary filter paper is composed, has water absorbed on its surface and inside. The
water is called the stationary phase and the cellulose fiber is the solid support. A material applied on the filter
paper when immersed in a solvent moves due to capillary action and partitions between the mobile solvent
and the cellulose bound water. Hence the name liquid- liquid chromatography and this is an excellent
example of partition chromatography.
The technique of paper chromatography is quite simple. It involves the separation and identification of a
chemical substances by a moving solvent on sheets or strips of filter paper. A tiny drop of spot of solution
containing a mixture of the substances to be separated is spotted by means of a capillary tube near one end
of a strip of filter paper. The drop is allowed to dry, leaving a spot of the mixed substances. The end of the
paper nearest the spot is immersed in a suitable solvent system, without immersing the spot itself. The solvent
rises up the paper by means of capillarity.
23
The components of a mixture applied as a spot move at different rates based on the distribution of the
components between the stationary and mobile phases. The developed paper chromatogram is usually
recorded in terms of Rf number. In general polar substances have lower Rf values than less polar substances
(Why).
Rf
distance the compound travelled

dist a n c e the solvent travelled
The Rf value is dependent on factors such as nature of component, type of paper and solvent system.
PROCEDURE
a. Paper chromatography: This involves separation and identification of a mixture of two
indicators by using the method of paper chromatography. In this experiment you will be provided
with a mixture containing any two of the following dyes: malachite green, fluorecein, eosin, and
methyl orange. Your task is to determine the identity of the components of the mixtureby spotting
a sample of the mixture along each side of the pure reference materials.
In the middle of a stripe of Whatman chromatography paper size 6 x 16cm and 2cm from the bottom,
spot by means of a capillary tube a solution of a mixture assigned to your group by your instructor.
Circle with pencil (caution! Not ink or ball point) the spot and write mix at the top of the paper
directly above the circle. To the right and left of this spot and with separation of at least 1cm apply
the reference substance. Mark the identity of each reference substance at the top ofthe paper
using the following abbreviations: malachite green (mg), fluorescein (fl), eosin (Eo)and methyl
orange (Mo).
24
Pour 35 - 40 mL of the developing solvent (n-butanol/ethanol/2N ammonia 2:2:1) in to the
provided chromatographic tank. Close the lid and shake the jar so that the space inside the jar is
saturated with the vapor of the solvent system. Place then your chromatographic paper in the jar
and develop chromatogram by allowing the solvent to gradually rise through the paper. Let this
proceed till the solvent font has risen to approximately 4/5 of the paper height, remove the paper
from the tank and immediately mark the solvent front with pencil. Allow the paper to dry and then
measure the distance from the starting point to the center of each of the oval spots. Calculate the
Rf value of each of the components. Tabulate your results and suggest the identity of the
components in the given mixture.
b. Demonstration of thin layer chromatography: your instructor will demonstrate how this
technique is applied in the separation and identification of organic compounds. Take notes during
the demonstration session and describe the technique in your report book.
25
c. Demonstration of the application of column chromatography: This demonstration is done by using a
tiny column packed with silica gel to which one of the above extracts will be applied. Elution will be done
first with hexane and acetone. This will help the student appreciate the importance of CC to separate natural
products.
26
EXPERIMENT 7
QUALITATIVE ANALYSIS OF CARBOHYDRATES
Objective: To identify the given sample
Theory
A carbohydrate is an organic compound with the general formula Cm(H2O)n, that is, consists only of
carbon, hydrogen and oxygen, with the last two in the 2:1 atom ratio. Carbohydrates make up the bulk of
organic substances on earth and perform numerous roles in living things.
The carbohydrates (saccharides) are divided into four chemical groups: monosaccharides, disaccharides,
oligosaccharides and polysaccharides. Polysaccharides serve for the storage of energy (e.g., starch in plants
and glycogen in animals) and as structural components (e.g., cellulose in plants and chitin in arthropods).
Structural polysaccharides are frequently found in combination with proteins (glycoproteins or
mucoproteins) or lipids (lipopolysaccharides). The 5-carbon monosaccharide ribose is an important
component of coenzymes (e.g., ATP, FAD and NAD) and the backbone of the genetic molecule known as
RNA. The related deoxyribose is a component of DNA. Saccharides and their derivatives include many other
important biomolecules that play key roles in the immune system, fertilization, preventing pathogenesis,
blood clotting and development.
This experiment aims to introduce you with the identification of unknown carbohydrates.
Some important points:
1. Most of the tests and reactions described are not quantitative and volumes are approximate, despite
these facts some tests do not work if quantities greatly in excess of those stated are used.
2. DO NOT place your pipettes in reagent bottles as this leads to contamination.
3. In most tests, it is important to apply a control test using water instead of the solution under
examination. If you are in doubt about the result of a test, perform the reaction with a suitable known
compound.
4. In this experiment, sugar samples are given in their solid state. To perform each procedure, you should
prepare your own sugar solution by taking very small amounts of solid sugars.
5. When you need to boil your sample in a test tube, prepare a hot water in a large beaker and put your
test tube inside the beaker. DO NOT forget to put boiling chips in the beaker.
27
TESTS ON CARBOHYDRATES:
1) Molisch’s Test:
Molisch’s Test is a sensitive chemical test for all carbohydrates, and some compounds containing
carbohydrates in a combined form, based on the dehydration of the carbohydrate by sulfuric acid to produce
an aldehyde, which then condenses with the phenolic structure resulting in a red or purple-colored compound.
Apparatus and materials:
Test tubes, reagents
Procedure:
1. Place 2 mL of a known carbohydrate solution in a test tube, add 1 drop of Molisch’s reagent
(10% α-naphthol in ethanol).
2. Pour 1-2 mL of conc. H2SO4 down the side of the test tube, so that it forms a layer at the bottom
of the tube.
3. Observe the color at the interface between two layers and compare your result with a control
test.
4. If there is a formation of the violet ring then the presence of carbohydrate is confirmed.
2) Carbohydrates as Reducing Sugars:
A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone group. The enolization of
sugars under alkaline conditions is an important consideration in reduction tests. The ability of a sugar to
reduce alkaline test reagents depends on the availability of an aldehyde or keto group for reduction reactions.
A number of sugars especially disaccharides or polysaccharides have glycosidic linkages which involve
bonding a carbohydrate (sugar) molecule to another one, and hence there is no reducing group on the sugar;
like in the case of sucrose, glycogen, starch and dextrin. In the case of reducing sugars, the presence of alkali
causes extensive enolization especially at high pH and temperature. This leads to a higher susceptibility to
oxidation reactions than at neutral or acidic pH. These sugars, therefore, become potential agents capable of
reducing Cu+2 to Cu+, Ag+ to Ag . Most commonly used tests for detection of reducing sugars are Fehling’s
Test, Benedict’s test and Barfoed’s Test.
a) Fehling’s Test:
Fehling’s Solution (deep blue colored) is used to determine the presence of reducing sugars and
aldehydes. Perform this test with fructose, glucose, maltose and sucrose.
Apparatus and materials:
Test tubes, reagents, water bath
Procedure:
1. Take 2 mL of the sugar solution in a clean test tube.
2. Add 1 mL of Fehling’s solution A and 1 mL of Fehling solution B to it.
3. Keep the solution in a boiling water bath for about 10 minutes.
28
4. If there is the formation of red precipitate then the presence of carbohydrate is confirmed.
b) Benedict’s Test:
Apparatus and materials:
Test tubes, reagents, water bath
Procedure:
1.
2.
3.
4.
5.
Take 2 mL of the sugar solution in a clean test tube.
Add 5 mL of Benedict’s reagent to it.
Boil the solution for about 2 minutes.
Cool the solution and observe the solution.
If there is formation of green, red, or yellow precipitate then the presence of carbohydrate is
confirmed.
c) Barfoed’s Test:
Barfoed’s reagent, cupric acetate in acetic acid, is slightly acidic and is balanced so that is can only be reduced
by monosaccharides but not less powerful reducing sugars. Disaccharides may also react with this reagent,
but the reaction is much slower when compared to monosaccharides. Perform this test with glucose, maltose
and sucrose.
Apparatus and materials:
Test tubes, reagents, water bath
Procedure:
1. To 1-2 mL of Barfoed’s reagent, add an equal volume of sugar solution.
2. Boil for 5 min. in a water bath.
3. You will observe a brick-red cuprous oxide precipitate if reduction has taken place.
d) Seliwanoff’s Test:
Seliwanoff’s Test distinguishes between aldose and ketose sugars. Ketoses are distinguished from
aldoses via their ketone/aldehyde functionality. If the sugar contains a ketone group, it is a ketose and
if it contains an aldehyde group, it is an aldose. This test is based on the fact that, when heated, ketoses
are more rapidly dehydrated than aldoses. Perform this test with glucose, fructose, maltose and
sucrose.
Apparatus and materials:
Test tubes, reagents, water bath
Procedure:
1. Heat 1 mL of sugar solution with 3 mL Seliwanoff’s reagent (0.5 g resorcinol per liter 10% HCl)
in boiling water.
2. In less than 30 seconds, a red color must appear for ketoses.
29
3. Upon prolonged heating, glucose will also give an appreciable color.
e) Iodine Test:
Iodine test is an indicator for the presence of starch. Iodine solution (iodine dissolved in an aqueous solution
of potassium iodide) reacts with starch producing a blue-black color. Apply this test to all the
polysaccharides provided.
Apparatus and materials:
Test tubes, reagents, water bath
Procedure:
1.
2.
3.
4.
Take 2 mL of polysaccharide solution.
Add 1-2 drops of iodine solution.
Observe the change in color.
If there is the appearance of blue-black color then the presence of starch is confirmed
30
REFERENCE
Bonner and Castro. Basic Organic Chemistry
Brewster R.Q. Practical Course Organic Chemistry.
Corey, E. J., Angew. Catalytic Enantioselective Diels-Alder reactions: Methods, mechanistic
fundamentals, pathways, and applications. Chem, Int. Ed. Engl., 2002, 41, 1650.
Ermias Dagne. Experiments in organic Chemistry I: Addis Ababa University; 1978
Hassan Bakr Amin, Riyadh. Practical Organic Chemistry: King Saud University, 2007
McMurry: Organic Chemistry: 5th edition
Menger, Goldsmith, Mandell: Organic Chemistry a Concise Approach; 2nd edition Advanced
Organic Chemistry, Part A and B: By Francis A. Carey, 4th and 5th edition
Morrison and Boyd. Organic Chemistry
Neil Isaacs: Physical Organic Chemistry,2nd Edition, University of Reading, England
Organic Chemistry Laboratory Course. University of Camaguey. Cuba.
Organic Chemistry. Practical Manual. Institute of Agricultural Science. Havana. Cuba
P. Sykes; Guide Book to Mechanism in Organic Chemistry, 1982
Pavia Lampman, Kriz Engel. Introduction to Organic Laboratory Techniques. A Microscale
approach. 3rd. ed. Saunders College Publishing. 1999, U.S.A.
R. B. Grossmann, The Art of Writing Reasonable Organic Reaction Mechanism, 2nd Ed.,2003
Richard C. Larock.
Comprehensive Organic Transformations: A Guide to FunctionalGroup
Preparations. 1989
Vogel, A. I.; Furniss, B. S.; Vogel, Arthur Israel. Vogel's Textbook of practical organic
Chemistry; Longman Scientific & Technical; Wiley: London; New York, 1989.
Wendimagegn Mammo. Practical Organic Chemistry II Laboratory manual: Addis Ababa
University; 1996.
31
APPENDIX I
Special Reagents
Tollens reagent
In 500 ml. of distillated water place 30 gm. of AgNO3 and add a NH4OH solution until precipitation of
Ag2O formed at the beginning will complete dissolved. Then the obtained solution will be dissolved
until 1 l.
Fehlling reagent
In 350 ml. of distillated water are dissolved 34.64 gms. of pure CuSO4 and dissolve until 500 ml. This
solution will be solution I or solution A.
Prepare another solution with 173 gms. of Rochelle salt (potassium sodium tart rate) and 65 gms. Of
NaOH in 35O ml. distillated water. The obtained solution will be dissolve until 500 ml. This solution
will be solution II or solution B.
When Fehlling reagent is going to be used, is necessary to take the same volume of solution A and B
and mix.
Benedict reagent
Prepare a solution No. 1 dissolving 173 gms. Sodium Citrate and 100 gms. Of Na2CO3 anhydrous in around
600 ml. of distillated water and dissolving the solution until850 ml.
Prepare a solution No. 2 dissolving 17,3 gms. of CuSO4 in 100 ml. of distillated waterand after that
dissolve until 150 ml. To prepare Benedict reagent we mix solution A and B.
Bayer reagent
Dissolve 2 gms of KMnO4 in 100 ml and after that add 4 gms Na2CO3. Stir and keep it in amber flask.
Molish reagent
In 100 ml. ethanol 95% or chloroform, dissolve 10 gms. of alpha naphtol. Stir and keepit in amber
flask.
32
Schiff reagent
In 350 – 400 ml. of hot distillated water dissolve 1 gm. of fushsine (rose aniline). Cold he solution and
bubble SO2 gas until the solution will be uncolored or mightily yellow. Dilute the solution until 1 l.
Millon reagent
Dissolve 10 gms. of metallic Hg, heating in 20 ml. of concentrated HNO3 and obtained solution dilute
with 30 ml. of distillated water.
Lucas reagent
Cooling externally, dissolve 136 gms. anhydrous ZnCl2 in 105 gms concentrated HCl(90 ml.). Stir
and keep it.
Brady reagent (2,4 – dinitrophenylhydrazine)
In 15 ml. of concentrated H2SO4 dissolve 3 gms. of 2,4 – dinitrophenylhydrazine, the obtained solution
is added with stirring to 70 ml. of ethanol 95%. Stir strongly the mixture and filter. The solution is
used as Brady reagent.
Lugol reagent
In 200 ml. of distillated water dissolve 10 gm. of KI and then 5 gms. of I2. The obtained solution has a
sufficiently concentration to use in iodoform test. Hen you want to use to detect the presence of starch,
dilute 10 or 25 times the volume.
Sellivanov reagent
Dissolve 0,5 gms resorcinol in 100 ml. of concentrated HCl and complete with distillated water until
300 ml. of solution. (This reagent brings positive test with ketoses, but brings negative test with aldoses).
Sulfochromic mixture
Weight 10 gms of K2Cr2O7 and dissolve in 15 ml. of concentrated H2SO4, complete with distillated
water until 100 ml. of solution.
Starch solution as indicator
Weight 1 gm of starch and dissolve in 50 ml. of distillated water at room temperature andafter that add
50 ml. boiling water.
33
APPENDIX II
Physical Constants of some Organic Compounds
M.M molar mass
M.P. melting point
M.M. much soluble
S
S.S. some soluble
inf. infinite soluble
B.P. boiling point
soluble
I
insoluble
W.S. weakly soluble
Name
M.M.
Density M.P.
B.P.
Acetaldehyde
Acetaldoxime
Acetamide
Acetanilide
Acetic acid
Acetic anhyd.
Acetone
Acetoxime
Acetophenyl
hydrazone
Acetyl chloride
Amilic alcohol
Aniline
Benzaldehyde
Benzene
Benzoyl chloride
Biuret
Bromobenzene
Bromo nitrobenzene (o)
Bromo nitrobenzene (m)
Bromo nitrobenzene (p)
Butylacetate
Butylic alcohol (n)
Butylic alcohol (sec)
Butylic alcohol (terc)
Butyl chloride (terc)
Carbon tetrachloride
44,05
59,07
59.07
135.16
60.05
102.09
58.08
73.09
148.20
0.783
0.965
1.159
1.21
1.049
1.082
0.792
0.97
-------
-123,5
47 (13)
81
113-4
16.7
-73.0
-94.6
60-1
26.6
20.02
114-5
222.0
305.0
118.1
139.6
56.5
136.3
163
Solubility
Water ethanol ether
inf
inf
inf
S
inf
inf
S
S
LS.
0.53
21
7
inf
inf
inf
12
inf
inf
inf
inf
inf
m.s.
m.s.
m.s.
S
------------
78.59
88.15
93.12
106.12
78.11
140.57
103.08
157.02
202.02
202.02
202.02
116.16
74.12
74.12
74.12
92.57
153.84
1.105
0.817
1.022
1.046
0.879
1.212
------1.495
1.623
1.704
1.938
0.882
0.810
0.808
0.779
0.847
1.595
-112.0
---------6.2
-26
5.5
-0.5
192.3
-30.6
43.0
56.4
126-7
-76.3
-79.9
-114.7
25.5
-26.5
-22.6
51-2
137.8
184.4
179.0
80.1
197.2.
------156.2
261
257
256
125.1
117.0
99.5
82.9
51-2
76.8
W.S.
2.7
3.6
0.3
0.07
W.S.
1.25
I
I
I
I
0.7
9
12.5
inf.
I
0.097
34
W.S.
inf
inf
inf
inf
W.S.
inf
S
M.S.
S
1.4 c
inf.
inf.
inf.
inf.
inf.
inf.
W.S.
inf
inf
inf
inf
inf.
-----inf.
S
S
S
inf.
inf.
inf.
inf.
inf.
inf.
Chloroaceticacid
Chlorophorm
Dietylamine
Dimetylamine
Dinitrobenzene (o)
Dinitrobenzene (m)
Dinitrobenzene (p)
Metyl benzoate
Nitrobenzene
Propanoic acid
Propylic alcohol
Ribose
Sucrose
Toluene
Urea
2-4 dinitro phenyl
hydrazine
Dietylic ether
Etyl acetate
Etilic alcohol
Formic acid
Fructose
Glucose
Glucose penta-cetate
(alpha)
Glucose penta-acetate
(beta)
Glycerine
Galactose
Lactose
Maltose
Metyl acetate
Xylene (o)
Xylene (m)
Xylene (p)
94.50
119.39
73.14
45.08
168.11
168.11
168.11
136.14
123.11
74.08
60.09
150.13
342.30
92.13
60.06
198.14
1.58
1.489
0.712
0.680
1.59
1.57
1.625
1.087
1.205
0,992
0.804
------1.588
0.868
1.335
--------
61.2
-63.5
-38.9
-96
117-8
89.8
173-4
-12.5
5.6-5.7
-22
-127
87
170
95
132.7
197-8
189.5
61.2
55.5
7.4
319
300-2
299
198-9
210.9
141.1
97.8
-------------110.8
---------------
M.S.
0.82
M.S.
M.S.
0.01
0.3
0.18
I
0.2
inf.
inf.
S
179
I
100
I
S
inf.
S
S
1.9
3.3
0.18
S
M.S.
inf.
inf.
S.S.
0.9
inf.
20
inf.
S
inf.
S
S
5.7
39.5
2.6
S
inf.
inf.
inf.
-----I
inf.
S.S.
inf.
74.12
88.10
46.07
46.03
180.16
180.16
390.34
0.708
0.901
0.789
1.220
1.669
1.544
--------
116.3
-82.4
-112
8.6
95-105
146anh
112-3
34.0
77.1
78.4
100.8
----------------------
7.5
8.5
inf.
inf.
M.S.
82
0.15
inf.
inf.
inf.
inf.
8.5
S.S.
1.3
inf.
inf.
inf.
-----S
I
2.8
131-2
--------
0.1
0.8
2.1
290
---------------------57.1
144
139.3
138.5
inf.
M.S.
17
M.S.
33
I
I
I
inf.
0.6
I
S
inf.
inf.
inf.
S
I
S
I
I
inf.
inf.
inf.
M.S.
390.34 --------
92.09 1.260
17.9
180.16 -------- 169.5
360.31 1.525 202anh
360.31 1.540 --------74.08 0.9240
-98.7
106.16 0.881
-25
106.16 0.867
-47.4
106.16 0.861
13.2
35