Exercise7 10

Laboratory Exercises in Pharmaceutical Organic Chemistry
EXERCISE NO. 7
PROPERTIES AND REACTIONS OF ORGANIC COMPOUNDS:

HYDROCARBONS
I. Intended Learning Outcomes:

At the end of the exercise, the student should be able to:
1. Investigate the properties and reactions of hydrocarbons.
2. Differentiate the types of hydrocarbon compounds.
3. Perform chemical tests to identify hydrocarbons.
4. Distinguish the properties that differentiate hydrocarbons from other organic
compounds.
II. Discussion:
Hydrocarbons are organic compounds consisting completely of carbon and

hydrogen only. These compounds are naturally occurring in natural gas and petroleum
deposits, hence, the major source of petroleum products. Petroleum is a complex
mixture of hydrocarbons that must be separated through fractional distillation and
refined before it can be used. Hydrocarbons have a special property to catenate forming
seemingly endless chain of carbon. This unique property of carbon makes it possible to
form millions of different organic compounds.
Based on the chemical structure types, hydrocarbons are generally classified into
two: the aliphatic and aromatic hydrocarbons. Aliphatic hydrocarbons are compounds
containing carbon and hydrogen bonded together in either straight, branched or non-
aromatic chains. Aliphatic compounds could be saturated, meaning it possess only one
sigma bond such as alkanes and cycloalkanes, or it could be unsaturated, meaning it
possess double or triple bonds such as alkenes, alkynes, cycloalkenes, and
cycloalkynes, all of which contains pi bonds. Aromatic hydrocarbons are cyclic
hydrocarbons with delocalized pi electrons between carbon atoms of ring. Their natural
characteristics are described as aromaticity – and there are certain criteria for a
hydrocarbon to be classified as an aromatic. These compounds should be flat or planar
in configuration, cyclic, must contain conjugated double bonds (alternating double
bonds), and must follow the Hückel’s rule of aromaticity.
Hydrocarbons ignites spontaneously as it reacts with oxygen upon ignition.

Combustion reaction occurs when hydrocarbon reacts with oxygen to produce carbon
dioxide, water, and heat. The general formula for combustion is shown below:
( ) ( ) ( )
When a fuel (e.g. gasoline, kerosene) burns in plenty of air, it receives enough
oxygen for complete combustion, thereby producing non-luminous flame. When a fuel
Centro Escolar University – School of Pharmacy 46

burns in a limited space (e.g. car engine), there is no enough oxygen to completely
oxidize the fuel, thereby producing black smoke/soot. Incomplete combustion reaction
occurs when hydrocarbons react with insufficient amount of oxygen, instead of
producing carbon dioxide, produces carbon monoxide, water and carbon as products.
With the properties of hydrocarbons, saturated hydrocarbons are generally inert,

which means unreactive, with inorganic acids such as hydrochloric and sulfuric acids as
well as with oxidizing agents such as potassium permanganate and sodium dichromate
at room temperature. This inert property is attributed to the strong sigma bond, a single
bond which holds the atoms of saturated hydrocarbons at a relatively stable nature.
Rigorous conditions are required to make these compounds (generally alkanes) to react
through substitution reactions. Meanwhile, unsaturated hydrocarbons react
characteristically on their double bonds. A double bond is composed of one strong
sigma bond and one weak pi bond. The breaking of the weak pi bond is primarily the
reason for the characteristic reactions of unsaturated hydrocarbons. A very typical
reaction of the double bond is shown below:
This addition reaction shows that the pi bond is broken and forms a sigma bond,
placing the substituent group on the location the double bond. Unsaturation of
hydrocarbons can be detected through bromine in dichloromethane, using the reaction
of double bonds as mentioned before. Bromine is decolorized upon reaction with an
unsaturated hydrocarbon.
Another method to detect unsaturation is Baeyer’s test. Alkenes and alkynes are
oxidized through reaction with potassium permanganate, decolorizing the purple-
colored solution of potassium permanganate while producing a brown precipitate of the
manganese dioxide.
Aromatic hydrocarbons contain conjugated pi bonds, that is alternating double

bonds. This special arrangement of pi bonds exhibits resonance which causes the
conjugative stability of the aromatic compounds. Benzene, being the most common
example of an aromatic compound, generally undergo electrophilic aromatic substitution
reactions. Benzene undergoes substitution reaction wherein the ring system is not
destroyed and therefore resist addition of substituent groups within the pi bonds. This

explains the reason why aromatic compounds resembles reactions of saturated rather
than unsaturated hydrocarbons.
Test for aromaticity using nitration reaction involves a benzene nitrated using a
nitrating acid (HNO3 + H2SO4) to produce nitrobenzene. This nitration test predicts the
presence of aromatic ring through the formation of yellow colored layer in solution after
warming the test sample. Only aromatic compounds will proceed through electrophilic
aromatic substitution. Nitration of benzene firstly involves the formation of a very
powerful electrophile, the nitronium ion, which is linear. This occurs following the
interaction of sulfuric and nitric acid. Sulfuric acid is stronger, and it protonates the nitric
acid on the hydroxyl group so that a molecule of water can leave. Benzene attacks the
positively charged nitrogen atom of the electrophile, where one of the N=O bonds is
broken at the same time. This is followed by rapid loss of a proton to regenerate the
aromaticity.
III. Materials, Apparatus and Reagents Needed:
Test Samples Materials and Apparatus Reagents

benzene 1-mL pipet 0.1% potassium permanganate
cyclohexane 5-mL pipet 1% sodium hydroxide
gasoline bulb aspirator 5% bromine in dichloromethane
kerosene cork stoppers concentrated nitric acid
n-hexane evaporating dish concentrated sulfuric acid
glass droppers distilled water
glass rod
porcelain spatula
test tube holder
test tubes
watch glass

IV. Procedures:
1. Ignition Test
a. In a dry watch glass, place 5 drops of benzene then ignite it using a lighted
matchstick.
b. Observe whether the flame produced is luminous or non-luminous (luminous
flame is the one that burns brightly while a non-luminous flame is the one that
burns blue).
c. Then place the bottom of a dry evaporating dish over the flame produced by the
sample and observe if soot is formed.
d. Repeat the procedure with n-hexane, cyclohexane, kerosene, and gasoline, in
place of the benzene. Record the results. (Note: Wipe the bottom of the
evaporating dish immediately before proceeding with the next sample.)
2. Baeyer’s Test for Unsaturation

a. In a dry big test tube, place 5 mL of 0.1% KMnO 4 solution then add 1 drop of 1%
NaOH solution to render it alkaline.
b. Pipet out 1 mL of the prepared alkaline KMnO4 solution and place it in a small
test tube. Label the tube as standard solution.
c. Into five separate small test tubes, label the following samples, and place the
corresponding volume:
 10 drops of benzene
 10 drops of n-hexane
 10 drops of cyclohexane
 10 drops of kerosene
 10 drops of gasoline
d. To each of the five samples, add 15 drops of the alkaline KMnO4 solution.
Compare the color of each test sample with the standard solution.
e. Record the results. (Note: Avoid exposing the test samples to strong light and
heat. Present the results to the instructor immediately.)
3. Bromine in Dichloromethane Test

a. In a dry big test tube, place 5 mL of 5% bromine in dichloromethane. Pipet out 1
mL of the prepared solution and place it in a small test tube. Label the tube as
standard solution. (Caution: Bromine gas is toxic. Ensure that the bottle is tightly
closed after pipetting out the solution.)
b. Into five separate small test tubes, label the following samples, and place the
c. To each of the five samples, add 15 drops of the bromine in dichloromethane
solution. Compare the color of each test sample with the standard solution.

d. Record the results. (Note: Avoid exposing the test samples to strong light and
heat. Present the results to the instructor immediately.)
4. Test for Aromatic Hydrocarbons

a. In a dry beaker, place 2 mL of concentrated nitric acid. Then carefully and slowly
add 2 mL of concentrated sulfuric acid to the beaker. (Caution: Concentrated
nitric and sulfuric acids are highly corrosive and can cause burns.)
b. Into five separate small test tubes, label the following samples, and place the
c. To each of the five samples, add 16 drops of the nitrating acid. Then dilute each
solution with 20 drops of distilled water.
d. Warm the solutions in hot water bath for 10 minutes. (Note: Remember to warm
only and not to boil the samples.)
e. Observe any changes in the solution. Record the results.

Name: __________________________________ Date: _____________________

Year & Section: __________ Group No.: ______ Rating: ___________________
EXERCISE NO. 7

HYDROCARBONS
V. Results and Observations:
Ignition Test
Test Sample Flame Presence of soot
benzene
n-hexane
cyclohexane
kerosene
gasoline
Baeyer’s Test for Unsaturation

Test Sample Color Color of the standard solution
benzene
n-hexane
cyclohexane
kerosene
gasoline
Bromine in Dichloromethane Test

Test Sample Color Color of the standard solution
benzene
n-hexane
cyclohexane
kerosene
gasoline
Test for Aromatic Hydrocarbons

Test Sample Color
benzene
n-hexane
cyclohexane
kerosene
gasoline

VI. Conclusion:
VII. Questions:
1. How do the reactions in the ignition test of hydrocarbon samples compare? What
conclusion can be drawn from the results?
2. How do you account for the observed reactions of the test samples in Baeyer’s test?
What generalizations can be given regarding their unsaturation?
3. What is the reaction involved between the hydrocarbon samples and bromine?
4. Briefly explain what happens to a compound that undergoes electrophilic aromatic

substitution.

EXERCISE NO. 8

ALCOHOLS

1. Investigate the properties and reactions of alcohols.
2. Differentiate the classification of alcohols.
3. Perform chemical tests to identify alcohols.
4. Distinguish the properties that differentiate alcohols from other organic
compounds.
II. Discussion:
Alcohols are organic compounds composed of an aliphatic carbon atom with a

hydroxyl (-OH) functional group. The hydroxyl group is a functional group of all alcohols,
hence the general formula of ROH, where R is the alkyl group. Alcohols are very
common and may exist freely in nature. Ethanol (ethyl alcohol), which is the active
component of alcoholic beverages, is just one and most common among the large
family alcohols. Alcohols exhibit some unique set of physicochemical properties mainly
due to the presence of the hydroxyl functional group. Some of the properties of alcohols
depend on the length of the alkyl chain attached to the hydroxyl group. Alcohols can be
grouped into three classifications based on this premise.
A primary (1°) alcohol has a carbon atom with the hydroxyl group attached to one
other carbon atom. The general formula for primary alcohol is RCH 2OH. A secondary
(2°) alcohol has a carbon atom with the hydroxyl group attached to two other carbon
atoms. The general formula for secondary alcohol is R2CHOH. A tertiary (3°) alcohol
has a carbon atom with the hydroxyl group attached to three other carbon atoms. The
general formula for tertiary alcohol is R3COH.
Alcohols generally have higher boiling points as compared to hydrocarbons

having the same molecular masses. This is mainly due to the intermolecular hydrogen
bonding in between the hydroxyl groups. Moreover, the boiling point of alcohols
increases as the number of carbon atoms in the aliphatic chain increases. However, the

boiling point of alcohols decreases with an increase in branching in aliphatic carbon

chain.
The solubility of alcohols is technically governed by its ability to perform

intermolecular hydrogen bonding with polar solvents. Hydrogen bonds are usually
formed in between the hydroxyl group of alcohol and water, which renders the former to
be soluble to the latter. However, this polarity is affected by the presence of the alkyl
chain attached to the hydroxyl group. The longer the alkyl chain is, the lesser the
alcohol becomes soluble with water.
Alcohols are acidic in nature because of their ability to react with active metals
(e.g. sodium, potassium, etc.) forming the corresponding alkoxide. This acidic nature is
due to the polarity of the hydroxyl bond. The acidity of alcohols decreases when an
electron donating group is attached to the hydroxyl group as the electron density
increases on the oxygen atom. Therefore, primary alcohols are more acidic than
secondary and tertiary alcohols.
Alcohols exhibit a range of spontaneous chemical reactions due to the cleavage

of C-O and O-H bonds. Primary alcohols undergo oxidation in the presence of an
oxidizing agent to produce aldehydes which upon further oxidation give carboxylic
acids. Secondary alcohols are oxidized to ketones, but no further oxidation can occur
due to lack of the reactive C-H bond found in aldehydes. Tertiary alcohols cannot be
oxidized due to lack of the reactive C-H bond also.
These chemical properties of alcohols can be analyzed using qualitative tests.

Primary and secondary alcohols can be distinguished from tertiary alcohol using

chromic acid test or Jones test. As previously mentioned, primary alcohols are oxidized
to aldehydes while secondary alcohols are oxidized to ketones. The solution of
chromium oxide (CrO3) in sulfuric acid (H2SO4) can detect polar functional groups that
can be oxidized. Tertiary alcohols give a negative result with this test because of the
absence of alpha hydrogen. The orange hexavalent chromium (Cr 6+) reagent is
converted to a blue-green trivalent chromium (Cr3+) species, which often precipitates in
acetone.
To further distinguish the classification of alcohols, Lucas test can be employed.

Tertiary alcohols immediately react with Lucas reagent (concentrated HCl and ZnCl 2)
through an SN1 mechanism to produce alkyl chloride that is insoluble in the aqueous
solution which appears as a white precipitate or cloudiness of the solution. This test
cannot be used for water-insoluble alcohols (generally >5 carbon atoms) as they may
immediately produce cloudiness or a second layer, which may impart a false-positive
result. A secondary alcohol reacts slower (approximately 5 minutes) while a primary
alcohol will not react at all. Benzyl alcohol (phenyl–CH2–OH), allylic alcohols (C=C–
CH2–OH) and propargylic alcohols (C≡C–CH2–OH) often give immediate results like
tertiary alcohols.
Alcohols may also react with carboxylic acids when heated in the presence of an
acid catalyst to form an ester. This reaction is known as Fischer esterification. The
reaction is both slow and reversible. A prominent sign that an ester is formed is through
the production of a characteristic odor. Esters are known for their odors, and this is the
reason why they are commonly employed as flavouring agents.

A test used to demonstrate the oxidation of alcohol is through partial oxidation

and dehydrogenation using hot catalyst such as a heated copper or platinum to speed
up chemical reaction. Methanol could be used as a sample to be oxidized to methanal
(formaldehyde). After which, to detect if methanol was oxidized, resorcinol is added to
the solution and a concentrated organic acid (sulfuric acid) is added dropwise. A red-
violet ring at the junction of the two layers with dark red flocculent precipitate is noted as
the positive result, indicating the presence of methanal.
Another test, iodoform test, can be used to identify methyl ketones or secondary
alcohols adjacent to a methyl group. A solution of iodine in potassium iodide (I 2 in KI) in
sodium hydroxide reacts only for compounds with the structure RCH(OH)CH3 or
RC=OCH3 to form a positive result of canary yellow precipitate. This test is very specific
test and it does not work for all alcohols or ketones and does not work also for water-
insoluble compounds.
Lastly, alcohols can also be classified based on the number of hydroxyl groups
present in the alcohol, whether monohydroxy or polyhydroxy alcohol. One of the most
important polyhydroxy alcohol is glycerol, a triol obtained as a by-product from the
manufacture of soap used as a moisturizing agent in cosmetic formulations. The
presence of glycerol is detected using acrolein test. When heated with potassium
bisulfate (KHSO4), glycerol is dehydrated to form propenal (acrolein), which has a
characteristic pungent odor.
The following selected qualitative tests will help each student understand more
the properties and reactions of alcohols.


benzyl alcohol 1-mL pipet 0.5% resorcinol
ethanol 5-mL vial 5% potassium dichromate
glycerol beaker 5% sulfuric acid
methanol bulb aspirator concentrated sulfuric acid
n-butyl alcohol Bunsen burner distilled water
sec-butyl alcohol copper wire iodine solution
tert-butyl alcohol cork stoppers potassium bisulfate
crucible tongs salicylic acid solution
glass droppers
glass rod
hotplate
porcelain spatula
test tubes
IV. Procedures:
1. Jones Test
a. Into three separate small test tubes, label the following alcohols, and place the
 20 drops of n-butyl alcohol
 20 drops of sec-butyl alcohol
 20 drops of tert-butyl alcohol
b. To each of the three samples, add 10 drops of 5% K 2Cr2O7 and 10 drops of 5%
H2SO4.
c. Place all the test tubes in a boiling water bath at the same time.
d. Record the time for each of the sample to change color. Note for the change in
color and odor of the product produced.
2. Lucas Test
a. Into four separate small test tubes, label the following alcohols, and place the
 20 drops of tert-butyl alcohol
 20 drops of sec-butyl alcohol
 20 drops of n-butyl alcohol
 20 drops of benzyl alcohol
b. To each of the four samples, add 20 drops of freshly prepared Lucas reagent.
Shake only once and let it stand.
c. Record the time for each of the sample to form an insoluble layer or emulsion
indicating the production of the alkyl halide.

3. Esterification Reaction
a. Place 20 drops of methanol in a big test tube. Add 10 drops of salicylic acid and
5 drops of concentrated H2SO4.
b. Note the odor of the product formed and the time for it to develop.
4. Test for Methanol

a. In a 5-mL vial, place 1 mL of methanol then add 1 mL of distilled water.
b. Get a stretch of copper wire with reasonable thickness and make a spiral of
copper wire by winding it around a glass rod.
c. Hold the copper spiral using a crucible tongs and heat it to red heat over Bunsen
flame.
d. Dip the heated copper spiral in to the methanol-water mixture in the vial. Repeat
steps c to d for 10 times.
e. Transfer the mixture from the vial to a small test tube, then add 5 drops of 0.5%
resorcinol.
f. Add 20 drops of concentrated H2SO4 to the solution by allowing it to flow down
along the sides of the tilted test tube. Do not shake the solution.
g. Observe and note the color produced at the junction of the two layers.
5. Iodoform Test
a. Into three separate big test tubes, label the following alcohols and place the
 4 drops of ethanol
 4 drops of isopropyl alcohol
 4 drops of methanol
b. To each of the three samples, add 4 mL of iodine solution, then add 10% NaOH
solution dropwise until the solutions are decolorized.
c. Place all the test tubes in a water bath (60°C).
d. Observe and note the odor of the vapors and the color of the precipitates formed.
6. Acrolein Test
a. In a small test tube, place 5 drops of glycerol and add a pinch of KHSO4.
b. Heat the sample over a Bunsen flame.
c. Note and record the odor produced.

Name: __________________________________ Date: _____________________

EXERCISE NO. 8

ALCOHOLS
Jones Test
Alcohol Color Odor Time
n-butyl alcohol
sec-butyl alcohol
tert-butyl alcohol
Lucas Test
Alcohol Changes in the Solution Time
tert-butyl alcohol
sec-butyl alcohol
n-butyl alcohol
benzyl alcohol
Esterification Reaction
Reactant Odor Time
methanol + salicylic
acid
Test for Methanol

Color
Iodoform Test
Alcohol Color Odor
ethanol
isopropyl alcohol
methanol
Acrolein Test Odor

glycerol
VI. Conclusion:

VII. Questions:
1. Compare the relative ease of oxidation of primary, secondary and tertiary butyl
alcohols toward acid dichromate based on the time required for solution to
change in color.
2. What is the significance of the Lucas test? Account for the reaction of tertiary,
secondary and benzyl alcohol?
3. What is the significance of the iodoform test? What result indicates the formation
of iodoform?
4. What is the significance of the Acrolein test?

EXERCISE NO. 9

PHENOLS

1. Investigate the properties and reactions of phenol.
2. Differentiate the derivatives of phenol.
3. Perform chemical tests to identify phenols.
4. Distinguish the properties that differentiate phenols from other organic
compounds.
II. Discussion:
Phenols are organic aromatic compounds wherein the hydroxyl group is attached
to a benzene ring. Phenols and its derivatives can be considered as alcohols due to
their hydroxyl group, however, differ in their chemical behavior so it is sensible enough
to treat these compounds as characteristically distinct group. Their difference with
alcohols technically is due to the phenyl ring attached to the hydroxyl group.
Phenol, also known as hydroxybenzene or carbolic acid, is a toxic, colorless

crystalline solid with sweet tarry odor. It is usually employed as an antiseptic and
disinfectant. Phenol is also used in cosmetic formulations and manufacture of some
drugs (precursor).
Phenols generally have higher boiling points compared to other hydrocarbons

having the same molecular masses. The intermolecular hydrogen bonding between the
hydroxyl groups of phenol molecules affects this property. The boiling point of phenols
increases with an increase in the number of carbon atoms. The phenol’s solubility is
governed by the hydroxyl group on its structure, forming intermolecular hydrogen
bonding. The hydrogen bonds formed in between phenol and water make it possible for

the former to be slightly soluble to the latter. However, an increase in the size of the aryl
group attached to the hydroxyl group decreases it solubility in water. Phenols are acidic
in nature due to its reactivity with active metals (e.g. sodium, potassium, etc.) to form
corresponding phenoxides. The sp2 hybridized carbon of the benzene ring attached
directly to the hydroxyl group functions as an electron withdrawing group, hence,
decreases the electron density on the oxygen. Due to the delocalization of the electrons
in the benzene ring, phenoxide ions are more stable. This indicates that phenols are
more acidic than alcohols. In substituted phenols, its acidity decreases if an electron
donating group is attached to the ring while acidity increases in case of electron
withdrawing group.
The synthesis of phenols can be achieved through diazotization of primary

aromatic amines. When a primary aromatic amine with nitrous acid (NaNO 2 + HCl) at
~5°C, benzene diazonium chloride is formed. These diazonium salts are highly reactive
and upon warming with water, will be hydrolyzed in to phenol.
Phenols react with dilute aqueous sodium hydroxide to form sodium phenoxide.
Sodium phenoxide are soluble with water in contrast with phenol which is only slightly
soluble due to the phenyl ring.
Ferric chloride reacts with several organic compounds such as phenol forming
colored complexes which may vary from compound to compound. Particularly, phenols
produce a purple colored compound upon reaction with ferric chloride. This is due to the

formation of ferric phenoxide that absorbs visible light to give an excited state in which
electrons are delocalized over both the iron atom and the conjugated organic system.
Bromine water reacts with phenols, forming a decolorized solution with white
precipitate. The bromination of phenol results to 2,4,6-tribromophenol product which
appears as white precipitate while the side product of hydrobromic acid results to
decolorization of the solution with an odor of an antiseptic.
A test used to determine the presence of phenolic compounds in amino acids is

known as Millon’s test. Amino acids and proteins with phenol radicals react with Millon’s
reagent [HgNO3 and Hg(NO3)2 in HNO3] to form red colored complexes. Tyrosine is the
only amino acid with phenol in its structure, hence this test is specific for tyrosine and
proteins containing tyrosine.
Phenol can also be used as precursor in the synthesis of several organic

compounds. One of which is an organic indicator phenolphthalein, used in acid-base
titration. It turns colorless in acidic solutions while it turns pink to magenta in basic
solutions. In the synthesis of phenolphthalein, phenol is reacted with phthalic anhydride
under an acid catalyst using an electrophilic aromatic substitution reaction. The resulting
solution can be further tested for its indicator property using acidic and basic solutions.


1% α-naphthol 1-mL pipet 1% ferric chloride
1% catechol 5-mL pipet 5% hydrochloric acid
1% phenol beaker 5% sodium hydroxide
1% thymol bulb aspirator bromine water
5% albumin Bunsen burner concentrated sulfuric acid
concentrated phenol cork stoppers distilled water
salicylic acid solution glass droppers Millon’s reagent
glass rod phthalic anhydride
hotplate
porcelain spatula
test tubes
IV. Procedures:
1. Solubility Test
a. Place 3 mL of 5% NaOH solution in a small test tube.
b. Add a pinch of phenol.
c. Shake and observe its solubility.
2. Reaction with Ferric chloride

a. Into four separate small test tubes, label the following test samples, and place
the corresponding volume:
 1 mL of 1% phenol
 1 mL of 1% catechol
 1 mL of 1% thymol
 1 mL of 1% α-naphthol
b. To each of the four samples, add 1 drop of 1% FeCl3 solution.
c. Observe and note for the color produced.
3. Bromine Water Test

a. Into two separate test tubes, label the following test samples, and place the
 1 mL salicylic acid

b. To each of the two test tubes, add bromine water dropwise until precipitate is
formed.
c. Observe, record and compare the results.
4. Formation of Phenolphthalein
a. In a big test tube, mix 2 drops of phenol and a pinch of phthalic anhydride, then
add 3 drops of concentrated H2SO4.
b. Heat over a low flame until all substance is melted. Set aside to cool.
c. Add 5 mL of distilled water and shake well.
d. Into two separate big test tubes, label the following samples and place the
 2 mL of 5% sodium hydroxide
 2 mL of 5% hydrochloric acid
e. To each of the two test tubes, add 2 drops of the solution from procedure c.
f. Observe and note the color produced in each test tube.
5. Millon’s Test
a. Into two separate big test tubes, label the following test samples, and place the
 1 mL of 5% albumin
b. To each of the two test tubes, add 6 drops of Millon’s reagent. Shake well.
c. Boil both test tubes in a water bath for 1 minute.
d. Observe and note the color produced.

Name: __________________________________ Date: _____________________

EXERCISE NO. 9

PHENOLS
Solubility Test
Test Sample Solubility
phenol in 5%
NaOH
Reaction with Ferric chloride

Test Sample Color
1% phenol
1% catechol
1% thymol
1% α-naphthol
Bromine water Test

Test Sample Result
1% phenol
salicylic acid
Formation of Phenolphthalein
Test Reagent Color
5% NaOH
5% HCl
Millon’s Test
Test Sample Color
1% phenol
5% albumin
VI. Conclusion:

VII. Questions:
1. What type of reaction explains the solubility of phenol in sodium hydroxide? What
is the organic product formed?
2. How do the reactions of the four phenolic samples with ferric chloride compare?
What is the organic product formed upon the reaction of phenol with ferric
chloride?
3. What compound is the precipitate formed in the bromine water test? What is the
type of reaction involved in the formation of the product?
4. Write the chemical reaction involved in the formation of phenolphthalein. What is

the function group responsible for its indicator property?
5. What is the significance of Millon’s test?

EXERCISE NO. 10

ALDEHYDES AND KETONES

1. Investigate the properties and reactions of aldehydes and ketones.
2. Compare and contrast aldehydes and ketones.
3. Perform chemical tests to identify aldehydes and ketones.
4. Distinguish the properties that differentiate aldehydes and ketones from other
organic compounds.
II. Discussion:
Aldehydes and ketones are organic compounds both containing a carbonyl

functional group (C=O). The carbon atom of this group has two more bonds that may be
occupied by hydrogen or alkyl or aryl substituents. If one of the substituents is
hydrogen, the compound is an aldehyde, while if both substituents are neither
hydrogen, the compound is a ketone.
The carbonyl group of both aldehyde and ketone is largely responsible some of
the shared physicochemical properties of these compounds. The carbonyl group is a
highly polarized group because of the greater electronegativity of the oxygen. As a
result of this polarization, the carbonyl group is susceptible to nucleophilic attack.
Aldehydes and ketones are naturally occurring organic compounds, often combined
with other functional groups. Some of these compounds are found in plants, animals,
and even microorganisms.

Aldehydes and ketones are commonly synthesized in the laboratory through

oxidation reaction of alcohols. As previously discussed on alcohols, primary alcohols
are oxidized to aldehydes while secondary alcohols are oxidized to ketones, while these
reactions are under controlled conditions. Aldehydes and ketones may chemically
behave in similar fashion however, specific reactions still occur between the two organic
compounds which distinguishes them from one another.
Tests for Carbonyl Group
2,4-dinitrophenylhydrazine (2,4-DNPH) reacts with aldehydes and ketones

through condensation reaction. When aldehyde reacts with 2,4-DNPH, it forms an
aldehyde 2,4-dinitrophenylhydrazone which appears as yellow to orange precipitate
while when a ketone reacts with the said reagent, it forms a ketone 2,4-
dinitrophenylhydrazone which may appear as red-orange precipitate. This test yields a
positive result with compounds having carbonyl group, which is present in both
aldehydes and ketones.
Sodium bisulfite test is used to determine if a compound is an aldehyde or an

aliphatic methyl ketone. A positive result for the two compounds will yield white
precipitate. The aldehyde or the methyl ketone reacts with sodium bisulfite by breaking
the pi bond of the carbonyl group through nucleophilic attack. The bisulfite ion then

attaches to the original carbonyl group to form the bisulfite addition compound. With
ketones, only methyl ketones will yield positive result since ketones with bulky groups
sterically hinders the bisulfite ion in attaching to the carbonyl group.
Tests for Aldehydes
Qualitative tests that can be used to differentiate aldehydes from ketones are
Schiff’s, Tollens’, and Fehling’s tests. These tests are all based from the oxidation
reaction of aldehydes to form carboxylic acids. The C-H bond of aldehydes is more
readily oxidized compared to the C-C bonds of ketones.
The Schiff’s reagent (sodium bisulfite with the organic dye para-rosaniline
hydrochloride) reacts with aldehyde to produce a positive result of magenta red color of
the solution. The reaction involved is the same as explained in the sodium bisulfite test,
however, an organic dye is added to produce visual results, distinguishing aldehydes
from ketones. Some ketones may also produce a color change but not a magenta red
color.
The Tollens’ reagent (silver nitrate with sodium hydroxide and excess ammonium
hydroxide to render solution colorless) reacts with aldehyde to produce a positive result
of “silver mirror” precipitate. The ammoniacal silver nitrate is reduced to silver which
appears as a silver mirror adhering to the glass walls of the test tube, while producing
the carboxylic acid-ammonium salt product. Tollens’ test can detect both aliphatic and
aromatic aldehydes.
The Fehling’s reagent (a solution of Fehling’s A prepared by mixing concentrated

sulfuric acid with aqueous solution of copper sulfate pentahydrate and Fehling’s B
prepared by mixing sodium hydroxide with potassium tartrate) reacts with aldehyde to
produce a positive result of brick-red precipitate. The aldehyde reduces the cupric ions
in complex with tartrate ions to cuprous ions; forming cuprous oxide which is the reason
for yielding brick-red precipitate. Fehling’s test is very specific to produce the positive
result with an aliphatic aldehyde; aromatic aldehydes and ketones give a negative result
with this test.

Tests for Methyl Ketones
Two specific tests identify methyl ketones: the sodium nitroprusside and iodoform
tests. These test gives only their respective positive results in the presence of an acetyl
group (CH3C=O), hence methyl ketones or compounds that can be oxidized to an acetyl
group such as isopropyl alcohol and acetaldehyde can yield positive result with these
two tests.
Sodium nitroprusside reacts with methyl ketones to produce a wine-red color

solution due to the formation of acetone-nitroprusside ion complex. In presence of alkali,
a methyl ketone such as acetone is converted to CH3C=OCH2- ion which reacts with
nitroprusside ion [Fe(CN)5NO]2- to give highly colored ion [Fe(CN)5NOCH2C=OCH3]2-.
Sodium nitroprusside test is also used in clinical tests to detect the presence of acetone
in urine.
Another test, iodoform test, can be used to identify methyl ketones or secondary
alcohols adjacent to a methyl group. A solution of iodine in potassium iodide (I 2 in KI) in
sodium hydroxide reacts only for compounds with the structure RCH(OH)CH3 or
RC=OCH3 to form a positive result of canary yellow precipitate. This test is very specific
test and it does not work for all ketones and does not work also for water-insoluble
compounds.
Test for Saccharides
Two tests were selected to identify saccharides or sugar compounds: Molisch

test and Bial’s test. Saccharides are classified based on the number of sugar moieties
found in one molecule: monosaccharides, disaccharides, oligosaccaharides, and
polysaccharides. Most of the shared properties of carbohydrates are due to their
functional groups: an aldehyde (for aldoses) or ketone (for ketoses) and hydroxyl
groups.

Molisch test is a general test for all saccharides. Molisch reagent (10% α-
naphthol in 95% ethanol) dehydrates pentoses to furfural derivatives and hexoses to 5-
hydroxymethyl furfural derivatives. The furfural compounds further react with α-naphthol
in Molisch reagent producing a purple-colored product. Monosaccharides yield rapid
results while disaccharides react slower.
Bial’s test is a specific test for pentoses (or carbohydrates with five carbon
chain). Bial’s reagent (orcinol, hydrochloric acid and ferric chloride) dehydrates
pentoses to form furfural. The furfural further reacts with orcinol and the ferric ion
present in Bial’s reagent producing a bluish product.

1% glucose 1-mL pipet 10% sodium hydroxide
1% ribose 5-mL pipet 5% sodium hydroxide
5% glucose beaker 5% sodium nitroprusside
5% starch bulb aspirator Bial’s reagent
acetaldehyde cork stoppers Brady’s reagent
acetone evaporating dish concentrated sulfuric acid
acetophenone glass droppers distilled water
benzaldehyde glass rod Fehling’s A
ethyl acetate hotplate Fehling’s B

ethyl methyl ketone test tube holder glacial acetic acid

formaldehyde test tubes Molisch’s reagent
Schiff’s reagent
sodium bisulfite solution
Tollens’ reagent
Wagner’s reagent
IV. Procedures:
1. Test for Carbonyl Group

1.1. Brady’s Test
a. Into four separate big test tubes, label the following test samples, and place
 1 mL of acetophenone
 1 mL of benzaldehyde
 1 mL of formaldehyde
 1 mL of acetone
b. To each of the test tubes, add 10 drops of Brady’s reagent (2,4-DNPH
reagent).
c. Shake the solutions vigorously.
d. Observe and note for the extent and the time for the precipitates to form.
1.2. Sodium bisulfite Test

a. Into two separate small test tubes, label the following test samples, and place
 1 mL of acetaldehyde
 1 mL of acetone
b. To each of the two test tubes, add 1 mL of NaHSO3 solution.
c. Shake the test tubes and observe for the formation of precipitate.
d. Note and record the extent of precipitates formed. Compare the results.
2. Test for Aldehydes

2.1. Schiff’s Test
a. Prepare four small test tubes and place 1 mL of Schiff’s reagent on each test
tube.
b. Label and add 2 drops of the following test samples on the four test tubes,
individually:
 formaldehyde
 benzaldehyde
 acetone
 acetophenone
c. Shake and observe for changes in the color of the solution.
d. Note and record the results.

2.2. Tollens’ Test

a. Prepare four small test tubes and place 1 mL of Tollens’ reagent on each test
tube.
individually:
 formaldehyde
 benzaldehyde
 acetone
 5% glucose
c. Place all the test tubes in hot water bath for 2 minutes and observe changes
in the solution.
d. Note and record the results. (Note: After documenting the results, clean the
test tubes immediately.)
2.3. Fehling’s Test

a. Prepare four small test tubes and place 10 drops of Fehling’s A and 10 drops
of Fehling’s B on each test tube.
individually:
 formaldehyde
 benzaldehyde
 acetone
 5% glucose
c. Place all the test tubes in hot water bath for 2 minutes and observe for the
formation and color of the precipitate.
d. Note and record the results.
3. Test for Methyl Ketones

3.1. Sodium nitroprusside Test
a. Prepare two evaporating dishes then place 2 drops of distilled water, 3 drops
of 5% sodium nitroprusside and 3 drops of 5% NaOH solution on both
evaporating dishes.
b. Label then add 2 drops of the following test samples on the two evaporating
dishes, individually:
 acetone
 ethyl methyl ketone
c. Then on both evaporating dishes, add 3 drops of glacial acetic acid.
d. Observe and note for the change in color of the solution.
3.2. Iodoform Test

a. Prepare three big test tubes and place 4 mL of Wagner’s reagent on each test
tube.
b. Label and add 5 drops of the following test samples on the three test tubes,
individually:

 acetone
 ethyl methyl ketone
 ethyl acetate
c. Then to each test tube, add 10% NaOH solution dropwise until the color of
the solution disappears.
d. Place all the test tubes in warm water bath (60°C). Observe the color of the
vapors and the color of the precipitate formed.
e. Note and record the results.
4. Test for Saccharides

4.1. Molisch Test
a. Into three separate big test tubes, label the following test samples, and place
 1 mL of 5% glucose
 1 mL of 5% starch
 1 mL of benzaldehyde
b. To each of the test tubes, add 2 drops of Molisch reagent then mix
thoroughly.
c. Then again to each test tube, add 1 mL of concentrated H 2SO4 by letting it
flow down the side of the inclined test tube.
d. Observe and note for the color in the interface of the two layers of liquid.
4.2. Bial’s Test

a. Prepare two big test tubes and place 3 mL of Bial’s reagent on each test tube.
b. Label and add 3 drops of the following test samples on the two test tubes,
individually:
 1% glucose
 1% ribose
c. Place both test tubes in hot water bath until a change of color in the solution
is observed.
d. Note and record the color of the solution and the time for it to form.

Name: __________________________________ Date: _____________________

EXERCISE NO. 10

ALDEHYDES AND KETONES
Test for Carbonyl Group

Brady’s Test
Test Sample Extent of Precipitation Time Formation
acetophenone
benzaldehyde
formaldehyde
acetone
Sodium bisulfite Test

acetaldehyde
acetone
Test for Aldehydes

Schiff’s Test
Test Sample Result
formaldehyde
benzaldehyde
acetone
acetophenone
Tollens’ Test
formaldehyde
benzaldehyde
acetone
5% glucose
Fehling’s Test
formaldehyde
benzaldehyde
acetone
5% glucose

Test for Methyl Ketones

Sodium nitroprusside Test
Test Sample Result
acetone
ethyl methyl ketone
Iodoform Test
acetone
ethyl methyl ketone
ethyl acetate
Tests for Saccharides

Molisch Test
Test Sample Result
5% glucose
5% starch
benzaldehyde
Bial’s Test
Test Sample Color Time Formation
1% glucose
1% ribose
VI. Conclusion:

VII. Questions:
1. Identify the type of reaction involved in: (a) Brady’s test, (b) sodium bisulfite test,
(c) Tollens’ test, and (d) Fehling’s test.
2. Why does glucose yield positive result with Fehling’s test?
3. What is the clinical significance of sodium nitroprusside test?
4. What is the basis of Molisch test employed to differentiate aldehydes and

ketones from carbohydrates?

EXERCISE NO. 11

CARBOXYLIC ACIDS AND THEIR DERIVATIVES

1. Investigate the properties and reactions of carboxylic acids, esters, amides and
acid anhydrides.
2. Differentiate carboxylic acids, esters, amides and acid anhydrides.
3. Perform chemical tests to identify carboxylic acids, esters, amides and acid
anhydrides.
4. Distinguish the properties that differentiate carboxylic acids, esters, amides and
acid anhydrides from other organic compounds.
II. Discussion:
Carboxylic acids are organic compounds containing a carboxyl (R-COOH)

functional group. Carboxylic acids occur widely in nature, often combined with other
functional groups, just like in fats, oils, and waxes. These organic compounds are
important components of many, food, and medicines. Many of these substances are
best known in their common names based on Latin and Greek words describing their
sources.
Carboxylic acids are compounds that also contains and acyl group (RCO–) with a
hydroxyl group attached to it. On the other hand, other functional groups could be
attached to the acyl group through nucleophilic substitution reaction, that is, replacing
the hydroxyl group with substituents such as halo, alkoxyl, amino, and acyloxy. These
compounds are therefore considered as carboxylic acid derivatives, and their properties
substantially change. Shown below are some examples of carboxylic acid derivatives.
As previously mentioned in alcohol, the most common reaction to synthesize

carboxylic acids is through the oxidation of primary alcohols. In the presence of an
oxidizing agent (such as potassium dichromate or potassium permanganate), the
primary alcohol is oxidized to an aldehyde, which is then further oxidized in to a
carboxylic acid. This chemical process also occurs biologically in the liver when ethanol

is metabolized into acetaldehyde through an enzyme known as alcohol dehydrogenase

(this process can also occur through CYP450 enzyme), and then acetaldehyde is
further metabolized into acetic acid through an enzyme known as aldehyde
dehydrogenase. The acetate metabolite is a more excretable form; this process of
ethanol metabolism therefore prevents the accumulation of the toxic acetaldehyde
metabolite.
As indicated in its name, carboxylic acids exhibit acidic property in aqueous

solution. The reaction involves the release of protons from the carboxyl group and the
stability of the conjugate base. Acidity of carboxylic acids can be detected through pH
measurement, the use of pH paper or pH indicators.
Another common chemical reaction of carboxylic acids is neutralization, wherein

they react with strong bases (such as sodium hydroxide or potassium hydroxide)
forming water-soluble salts as products.
However, when carboxylic acids react with weak bases (such as sodium
bicarbonate), a water-soluble salt is still formed but together with a by-product of carbon
dioxide liberated as gas. The reaction is accompanied by the formation of
effervescence, indicating the release of carbon dioxide from the solution. This chemical
reaction is used to differentiate carboxylic acids from other weak acids such as phenols.
A very essential carboxylic acid derivative ester is synthesized through mixing

primary or secondary alcohols with a carboxylic acid in the presence of an acid catalyst.

This reaction is known as Fischer esterification. During the synthesis, the acid catalyst
promotes the protonation of the carbonyl oxygen which makes the carbonyl carbon
electrophilic. The alcohol then acts as the nucleophile that attacks the electrophilic
carbonyl carbon. This would in turn produce a by-product of water and a protonated
ester; the ester would then be deprotonated. It is noted that the chemical reaction is in
equilibrium. The production of an ester is indicated by the presence of characteristic
fruity-like odor because esters are known to exhibit such property.
Carboxylic acids can also be detected by reacting the sample with neutral ferric
chloride, producing a brick red precipitate. The carboxylic acid is neutralized using
ammonium hydroxide to produce ammonium carboxylate while excess ammonia gas is
released through heating. The ammonium carboxylate is then reacted with ferric
chloride producing ferric carboxylate which gives a brick red precipitate. A reaction of
acetic acid through this test is given below.
CH3COOH + NH4OH → CH3COONH4 + H2O

acetic acid ammonium hydroxide ammonium acetate water
CH3COONH4 + FeCl3 → (CH3COO)3Fe + NH4Cl

ammonium acetate ferric chloride ferric acetate ammonium chloride
The presence of carboxyl group could be more than one in a compound, the
classic examples for these compounds are citric acid and tartaric acid. Citric acid is
tricarboxylic acid and has no enantiomer form while tartaric acid is dicarboxylic acid that
has two enantiomer forms: D-tartaric acid and L-tartaric acid. Special test for
identification citric acid and tartaric acid was established by Chapman and Smith
wherein they found out that when citric acid is boiled with a very alkaline solution of
potassium permanganate, it will yield a green solution of alkaline manganate. Whereas
when tartaric acid is treated with the same solution, brown precipitate of manganese
dioxide will be produced.
An essential derivative of carboxylic acid are esters. Esters are organic

compounds with the general formula of R1C=OOR2, where R1 may be hydrogen, alkyl or
aryl groups and R2 may be an alkyl or aryl group but must not be a hydrogen (the
compound will be a carboxylic acid if R2 is a hydrogen atom). Esters are very common

in nature and are known to have characteristic pleasant odors unlike carboxylic acids.
This property enables esters to be utilized as perfuming or flavoring agents, aside from
them being used as organic solvents. An important test to identify esters is known as
ferric hydroxamate test. Esters when heated with hydroxylamine produce hydroxamic
acids that reacts with ferric ion to form intensely colored complexes, commonly dark
maroon in color. This test is related to the phenol test wherein compounds with high
enolic character gives colored complexes with ferric ion. Therefore, a preliminary test
must be performed to check if the carbonyl compound being tested produces enough
enol to form colored complexes with ferric ion, which could lead to a false positive
result.
Other derivatives of carboxylic acid include acid anhydride and amide. Acid
anhydrides are organic compounds with two acyl groups (R-C=O) joined together by an
oxygen atom, while amides are organic compounds with general structure of a nitrogen
atom bonded to the carbonyl carbon atom (RC=ONH2). These two carboxylic acid
derivatives are well known to undergo hydrolysis reaction. For example, acetic
anhydride can be hydrolyzed into two molecules acetic acid whilst acetamide produces
carboxylate salt and free ammonia. The pH of the products formed can be tested to
confirm their presence after the hydrolysis reaction.


5% citric acid 1-mL pipet 0.1% potassium permanganate
5% tartaric acid 5-mL pipet 0.5M hydroxylamine
hydrochloride
acetamide beaker 10% sodium hydroxide
acetic acid blue litmus paper 1N hydrochloric acid
acetic anhydride bulb aspirator 5% ferric chloride
benzoic acid glass droppers 5% sodium carbonate
ethyl acetate glass rod 5% sodium hydroxide
stearic acid graduated cylinder ammonium hydroxide solution
hotplate concentrated sulfuric acid
porcelain spatula distilled water
red litmus paper methyl orange
test tube holder n-propanol
test tubes
IV. Procedures:
1. Test for Carboxylic acids

1.1. Reaction with Water and Indicators
a. Into two separate big test tubes, label the following test samples, and place
the corresponding volume and weight:
 1 mL of acetic acid
 0.5 g of stearic acid
b. To each of the test tubes, add 3 mL of distilled water. Shake and note for the
solubility.
c. Warm the mixtures over hot water bath then note for any changes in the
solubility.
d. Test the solution with blue litmus paper then with methyl orange TS. Note for
changes in the color.
e. Record the results.
1.2. Reaction with Sodium hydroxide

 0.5 g of stearic acid
b. To each of the test tubes, add 3 mL of 5% NaOH. Shake and note for the
solubility.
c. Record the results.
1.3. Reaction with Sodium carbonate

a. In a dry big test tube, place 1 mL of acetic acid then gradually add 5 mL of 5%
Na2CO3.

b. Note for any formation of effervescence. Then warm the solution gently over
warm water bath.
c. Observe for any changes in the solution. Record the results.
1.4. Esterification Reaction

 20 drops of acetic acid
 0.05 g of benzoic acid
b. To each of the test tubes, add 10 drops of n-propanol and 5 drops of
concentrated H2SO4.
c. Note for the odor produced. Compare and record the results.
1.5. Reaction with Neutral Ferric chloride

 1 mL of 5% tartaric acid
b. To each of the test tubes, add sufficient quantity of ammonium hydroxide
solution until the solution turns red litmus paper to blue. Then boil the solution
over hot water bath to remove excess ammonia.
c. In a separate big test tube, place 5 mL of FeCl3 then gradually add 5%
Na2CO3 until faint precipitate of ferric hydroxide is produced. This is the
neutral FeCl3 solution.
d. Add the neutral FeCl3 solution dropwise to the test samples until no further
change in color occurs.
e. Note for the colors of the solution and the precipitate produced. Record the
results.
2. Special Test for Tartaric acid and Citric acid

a. Into two separate test tubes, label the following test samples, and place the
 1 mL of 5% tartaric acid
 1 mL of 5% citric acid
b. To each of the test tubes, add 10 drops of 5% NaOH to render the solution
alkaline. Test the Ph with red litmus paper until it turns to blue.
c. Then again to each test tube, add 5 drops of 0.1% KMnO 4. Note for the color of
the solution. Boil the solution in water bath.
d. Observe and note for the color of the precipitate formed. Record the results.
3. Ferric hydroxamate Test

3.1. Preliminary Test
a. In a small test tube, place 1 mL of 95% ethanol then add 1 drop of ethyl
acetate.
b. Add 1 mL of 1N HCl then add 3 drops of 5% FeCl3.
c. Note for the color produced and record the result.

3.2. Final Test

a. In a small test tube, place 1 mL of saturated alcoholic solution of
hydroxylamine hydrochloride and add 1 drop of ethyl acetate.
b. Add 0.2 mL of 6N NaOH to the solution then heat in a boiling water bath.
c. Cool slightly then add 2 mL of 1N HCl and 3 drops of 5% FeCl3.
d. Note for the color of the solution and compare it to the preliminary test result.
Record the results.
4. Hydrolysis Reaction
a. In a small test tube, mix 1 mL of acetic anhydride and 3 mL of distilled water.
(Caution: Acetic anhydride is a lachrymator. Do this step at the fume hood.)
b. Note the solubility in cold water. Then shake the test tube and observe for any
evolution of heat.
c. Test the solution with blue litmus paper.
d. On another small test tube, place 0.5 g of acetamide and add 5 mL of 10%
NaOH. Heat the solution over boiling water bath.
e. Observe for any evolution of gas. Then test the gas with moist red litmus paper.

Name: __________________________________ Date: _____________________

EXERCISE NO. 11

CARBOXYLIC ACIDS AND THEIR DERIVATIVES
Test for Carboxylic acids

Reaction with Water and Indicators
Test Sample Change in Litmus Paper Change in Color with Methyl
orange
acetic acid
stearic acid
Reaction with Sodium hydroxide

Test Sample Results (Solubility)
acetic acid
stearic acid
Reaction with Sodium carbonate

Test Sample Formation of Effervescence Results (Changes in Solution)
acetic acid
Esterification Reaction
Test Sample Odor
acetic acid
benzoic acid
Reaction with Neutral Ferric chloride

Test Sample Color of the Solution Precipitate Formed
acetic acid
tartaric acid
Special Test for Tartaric acid and Citric acid

Test Sample Results (Changes in Color and Precipitate Formed)
tartaric acid
citric acid

Ferric hydroxamate Test

Preliminary Test
Test Sample Result (Color of the Solution)
ethyl acetate
Final Test
ethyl acetate
Hydrolysis Reaction
Test Sample Change in Litmus Paper Result (Changes in the
Solution)
acetic
anhydride
acetamide
VI. Conclusion:

VII. Questions:
1. How do the acetic acid and stearic acid compare in the reaction with sodium
hydroxide?
2. What may be inferred as the cause of the observed reaction of sodium carbonate
with acetic acid?
3. Write the balanced chemical equations for the following: (a) esterification of
acetic acid, (b) reaction of acetic acid with neutral ferric chloride, (c) hydrolysis of
acetic anhydride, and (d) hydrolysis of acetamide.
4. Give at least five groups of carboxylic acid derivatives and their general formulas.

EXERCISE NO. 12

AMINES AND OTHER NITROGENOUS COMPOUNDS

1. Investigate the properties and reactions of amines and other nitrogenous
compounds.
2. Differentiate the types of amines and other nitrogenous compounds.
3. Perform chemical tests to identify amines and other nitrogenous compounds.
4. Distinguish the properties that differentiate amines and other nitrogenous
compounds from other organic compounds.
II. Discussion:
Amines are organic compounds composed of an amino group (–NH2) and are
derivatives of ammonia in which one or more of the hydrogens has been replaced by an
alkyl or aryl group. These group of compounds are classified as primary, secondary or
tertiary amines depending on the number of alkyl or aryl group directly bonded to the
nitrogen atom.
Amines are commonly prepared through reduction reactions of nitro, nitrites and
amino compounds. Primary amines are usually prepared by direct alkylation of
ammonia by alkyl halides, although alternative procedures are more preferred. The
general strategy is to first form a carbon-nitrogen bond by reacting a nitrogen
nucleophile with a carbon electrophile. Then extraneous nitrogen substituents that may
have facilitated this bonding are removed, thereby producing the amine product.
Reaction of Amines
Most amines are Brønsted-Lewis bases, but their alkaline strength largely
depends on the substituents attached to the amino compound. Examples include aniline
and p-nitroaniline, which are weaker bases due to the delocalization of the nitrogen non-
bonding electron pair into the aromatic ring. Moreover, conjugated amine groups

influence the alkalinity of an amine. Amines react with acids, accepting protons from the
latter to form salt product.
Amines may also react with bromine water, producing a decolorized solution with
white precipitate. The reaction is the same with phenol, however the product formed
results to 2,4,6-tribromophenylamine which appears as white precipitate with a side
product of hydrobromic acid which results to decoloration of the solution. The reaction
involved is an electrophilic aromatic substitution. An example given below is the reaction
of aniline with bromine water.
The Hinsberg’s test is a useful test to differentiate primary, secondary and tertiary
amines. Primary and secondary amines react with benzene sulfonyl chloride to give
solid sulfonamide derivatives whereas, tertiary amines do not give any product other
than the starting amine. The sulfonamides from primary amines are soluble in water and
re-precipitating with dilute hydrochloric acid while those from secondary amines are
insoluble in water and dilute hydrochloric acid. This test is conducted in aqueous base
(sodium hydroxide or potassium hydroxide) and the benzene sulfonyl chloride reagent
appears as an insoluble oil. Due to the heterogenous nature of this system, the
hydrolysis of benzene sulfonyl chloride to its sulfonate salt in the absence of amines is
relatively slow. The amine dissolves in the reagent and immediately reacts producing
the results mentioned.

Another useful test to differentiate the classes of amines is the reaction with
nitrous acid. Primary amines react with nitrous acid producing bubbles from a clear
solution due to the formation of nitrogen gas (with an alcohol as products). Secondary
amines react with nitrous acid producing an oily, yellowish, water-insoluble layer due to
the formation of N-nitrosamine. Tertiary amines react with nitrous acid producing a clear
solution due to the formation of water-soluble ammonium salts.
Reaction of Nitro Compounds
Specific nitrogenous compounds known as nitro compounds are group of organic

chemicals usually composed of benzene and its homologues with an attached nitro
group (–NO2). Most notable compounds under this group includes nitrobenzene, picric
acid (trinitrophenol) and TNT (trinitrotoluene). The nitrogen of this compound has a
formal positive charge, which is a strong electrophile. When the nitro group is attached
to an aromatic ring, the positive nitrogen drains the electron density of the ortho and
para positions in the benzene ring, which renders these positions unreactive. Therefore,
electrophiles will prefer to attack the meta position.

A very common test used to identify nitro compounds is ferrous hydroxide test.
Nitro compounds oxidize ferrous hydroxide forming a reddish-brown precipitate of ferric
hydroxide, while the nitro compound is reduced to a primary amine. A negative result is
indicated by a greenish precipitate. In most cases, all nitro compounds immediately
yield a positive result (while the rate of chemical reaction is dependent on the solubility
of the nitro compound).
R–NO2 + 6 Fe(OH)2 + 4 H2O → R–NH2 + 6 Fe(OH)3
During the late 19th century, Janovsky discovered that when nitro compounds are
treated with acetone in an alkaline environment, these compounds will yield colored
complexes due to the formation of Meisenheimer complexes. Mono-nitro substituted
benzene will give light yellow solution, di-nitro substituted benzene will give a bluish-
purple solution while tri-nitro substituted benzene will give a blood red solution.
Reaction of Amino acids and Peptides
Other essential nitrogenous compounds include a class of compounds known as

amino acids. Amino acids are known as the building blocks of important
macromolecules known as proteins. The structure of an amino acid is composed of a
carboxyl group, an amino group, and substituent groups all attached to an α-carbon.
These amino acids form polymers through a nucleophilic attack to the carbonyl carbon
of the carboxyl group of one amino acid by the amino group of another amino acid. This
reaction forms an amide bond in between the two linked amino acids; the resulting
structure is known as a peptide. At some point, when the link of amino acids is long
enough, it produces the macromolecule protein.

A general test for amino acids known as ninhydrin test is employed to identify the
presence of an amino acid. The test relies on the reaction between the amino group of
the amino acid and ninhydrin. Ninhydrin, which appears as a yellow solution, is a very
strong oxidizing agent. It reacts with the amino acid producing an intermediate product
hydrindantin, which further reacts with ninhydrin producing the ultimate product which is
a Ruhemann’s complex, appearing as a bluish to purple colored solution. However,
imino acid proline produces a bright yellow color while asparagine yields a brown-
colored product.
Biuret test is another general test for compounds with peptide bonds. Biuret is a
compound produced when urea is heated at 180°C. When this compound is treated with
dilute cupric sulfate in alkaline condition, a purple-colored product is formed. This
chemical reaction is widely employed to detect the presence of proteins and peptides
(especially in medical diagnostic testing). The purple color produced is due to the
formation of coordination complex of cupric ions with the unshared electron pairs of
peptide nitrogen and the oxygen in water.

Reaction of Alkaloids
Alkaloids are naturally occurring organic compounds containing basic nitrogen

atoms. These are very diverse biomolecules, but all are secondary metabolites derived
from amino acids or from transamination process. Alkaloids commonly have medicinal
properties such as local anesthetics, stimulants and even as an antimalarial, to name a
few. These compounds are also known to have bitter taste, mostly colorless, crystalline
in structure. Alkaloids can be classified into several ways: biological and ecological
activity, chemical structures and biosynthetic pathways. The three main types of
alkaloids are true alkaloids (derived from amino acids and they share a heterocyclic ring
with nitrogen), protoalkaloids (compounds in which the nitrogen derived from an amino
acid is not a part of the heterocyclic ring), and pseudoalkaloids (their basic carbon
skeletons are not derived from amino acids).
Qualitative chemical tests can be employed to detect the presence of alkaloids

through the formation of precipitates using precipitant reagents. Most of these color
reagents consist of dehydrating, oxidizing, or a combination of the two agents.
Summarized in the table below are the color reagents used to detect the presence of
alkaloids in a compound, together with their expected results.

Table 12.1 Reagents Used for Detecting Alkaloids

Reagent Component Positive Result
Dragendorff’s Potassium bismuth iodide solution orange precipitate
reagent
Mayer’s reagent Potassium mercuric iodide cream precipitate
solution
Wagner’s reagent Iodine in potassium iodide solution reddish brown precipitate
Valser’s reagent Mercuric iodide solution white precipitate
Hager’s reagent Saturated picric acid solution yellow precipitate

1% albumin 1-mL pipet 0.1% ninhydrin
1% glycine 5-mL pipet 1% cupric sulfate
1% tyrosine beaker 10% potassium hydroxide
2,4-dinitrobenzene bulb aspirator 10% sodium hydroxide
aniline Bunsen burner 10% sodium nitrite
brewed coffee cork stoppers 2M hydrochloric acid
powder
N,N-dimethyl aniline glass droppers 2N potassium hydroxide in
methanol
N-methyl aniline glass rod 5% ferrous ammonium sulfate
nitrobenzene hotplate 5% hydrochloric acid
picric acid ice bath 6N sulfuric acid
urea test tube holder acetone
test tubes benzene sulfonyl chloride
watch glass bromine water
concentrated hydrochloric acid
distilled water
Dragendorff’s reagent
Hager’s reagent
Mayer’s reagent
sodium chloride
Valser’s reagent
Wagner’s reagent

IV. Procedures:
1. Reactions of Amines
1.1. Reaction with Concentrated Hydrochloric acid
a. In a dry watch glass, place 3 drops of aniline then add 1 drop of
concentrated HCl.
b. Observe for any changes. Record the result.
1.2. Reaction with Bromine water

a. In a small test tube, place 1 mL of aniline then add dropwise sufficient
quantity of bromine water until precipitate is formed.
b. Remember to shake the test tube in each addition of bromine water.
Note for the color of the precipitate. Record the result.
1.3. Hinsberg’s Test

a. Into three separate big test tubes, place 5 drops of the following test
sample:
 aniline
 N-methyl aniline
 N,N-dimethyl aniline
b. Then on each of the test tube, add 5 mL of 10% NaOH solution and 7
drops of benzene sulfonyl chloride. Put a cork stopper on the test tube.
(Caution: Benzene sulfonyl chloride is a lachrymator. Do this step at
the fume hood.)
c. While holding the cork stopper on the test tube, shake the test tubes
vigorously for five minutes.
d. Separate the oil or precipitate formed through decantation. Then test
for its solubility with distilled water and with 5% HCl. If it is soluble in
distilled water, add 3 drops of concentrated HCl. Observe any changes
in the solution.
e. Record the results.
1.4. Reaction with Nitrous acid

a. In a beaker, place 1 mL of aniline then add 3 mL of concentrated HCl
in 5 mL distilled water.
b. Cool the solution in an ice bath. After the solution cools, gradually add
5 mL of 10% NaNO2 solution.
c. Pipet out 5 mL from the previous mixture into a big test tube and warm
over hot water bath.
d. Note for the evolution of gas. Record the result.
e. Repeat the test with 1% glycine in place of aniline.

2. Reactions of Nitro Compounds

2.1. Ferrous hydroxide Test
a. In a small test tube, place 3 drops of nitrobenzene then add 30 drops
of freshly prepared 5% ferrous ammonium sulfate solution.
b. To the mixture, add 1 drop of 6N H2SO4 solution and 20 drops of 2N
KOH in methanol.
c. Cover the test tube then shake.
d. Note for the positive result of blue precipitate turning to rust-brown
within a minute.
e. Record the result.
2.2. Janovsky’s Test

a. In three small test tube, label the following test samples and place the
 2 drops of nitrobenzene in 1 mL of acetone
 2 drops of picric acid solution in 1 mL of acetone
 2 drops of 2,4-dinitrobenzene in 1 mL of acetone
b. To each of the test sample, add 10 drops of 10% KOH solution.
c. Shake the test tubes and observe the color produced in the solution.
d. Record the results.
3. Reactions of Amino acids and Peptides

3.1. Ninhydrin Test
a. Into two separate big test tubes, label and place 2 mL of the following
test samples:
 1% glycine
 aniline
b. To each of the test tube, add 10 drops of 0.1% ninhydrin solution.
c. Boil in a water bath and note for the color produced in the solution.
d. Record the result.
3.2. Biuret Test

a. In a dry big test tube, heat a pinch of urea over a low flame until it
melts. (Note: Do not overheat and burn the sample). Note for the odor
of the fumes.
b. Cool the test tube then add 20 drops of distilled water until the urea
dissolves.
c. Then add 5 drops of 10% NaOH solution and 3 drops of 1% CuSO4.
d. Note for the color of the solution. Record the result.
e. Into three separate test tubes, label and place 3 drops of the following
test samples:
 1% albumin
 1% tyrosine
 1% glycine

f. To each test tube, add 5 drops of 10% NaOH solution and 3 drops of
1% CuSO4.
g. Shake the test tube and note for the color of the solution formed.
Record the results.
4. Reactions of Alkaloid
4.1. Preparation of Sample
a. In a beaker, place 2 grams of brewed coffee powder, then add 10 mL
of 2M HCl.
b. Heat the solution whilst stirring in a water bath for 10 minutes then set
aside to cool.
c. Add 1 gram of NaCl, stir and filter.
d. Discard the residue and then equally divide the filtrate into 5 portions.
4.2. Dragendorff’s Test

a. Add 20 drops of Dragendorff’s reagent (potassium bismuth iodide TS)
to the filtrate from coffee.
b. Note for the positive result of orange-colored precipitate. Record the
results.
4.3. Mayer’s Test

a. Add 20 drops of Mayer’s reagent (potassium mercuric iodide TS) to the
filtrate from coffee.
b. Note for the positive result of cream-colored precipitate. Record the
results.
4.4. Wagner’s Test

a. Add 20 drops of Wagner’s reagent (iodine in potassium iodide TS) to
the filtrate from coffee.
b. Note for the positive result of reddish brown-colored precipitate.
Record the results.
4.5. Valser’s Test

a. Add 20 drops of Valser’s reagent (mercuric iodide TS) to the filtrate
from coffee.
b. Note for the positive result of white-colored precipitate. Record the
results.
4.6. Hager’s Test

a. Add 20 drops of Hager’s reagent (saturated picric acid TS) to the
filtrate from coffee.
b. Note for the positive result of yellow-colored precipitate. Record the
results.

Name: __________________________________ Date: _____________________

EXERCISE NO. 12
AMINES AND OTHER NITROGENOUS COMPOUNDS
Reactions of Amines
Reaction with Concentrated Hydrochloric acid
Test Sample Result (Changes in the Sample)
aniline
Reaction with Bromine water

Test Sample Precipitate Formed
aniline
Hinsberg’s Test
Solubility Changes upon
Test Sample
5% HCl Distilled water addition of HCl
aniline
N-methyl aniline
N,N-dimethyl aniline
Reaction with Nitrous acid

Test Sample Result (Evolution of Gas)
aniline
1% glycine
Reactions of Nitro Compounds

Ferrous hydroxide Test
Test Sample Result (Formation of Precipitate)
nitrobenzene
Janovsky’s Test
nitrobenzene
picric acid
2,4-dinitrobenzene

Reactions of Amino acids and Peptides

Ninhydrin Test
1% glycine
aniline
Biuret Test
Test Sample Odor upon Heating
urea

urea
1% albumin
1% tyrosine
1% glycine
Reactions of Alkaloids
Test Result (Formation of Precipitate)
Dragendorff’s Test
Mayer’s Test
Wagner’s Test
Valser’s Test
Hager’s Test
VI. Conclusion:

VII. Questions:
1. What is the precipitate formed in the bromination of aniline? Account for the
reaction.
2. Write the chemical reactions involved in (a) ferrous hydroxide test and (b)
Janovsky’s test which resulted to the changes in color observed with each
respective test.
3. Write the chemical reactions involved in (a) Ninhydrin test and (b) Biuret test which
resulted to the changes in color observed with each respective test.
4. Summarize the compounds found on each reagent and its corresponding positive
result with amines: (a) Dragendorff’s test, (b) Mayer’s test, (c) Wagner’s test, (d)
Valser’s test, and € Hager’s test.

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Laboratory Exercises in Pharmaceutical Organic Chemistry

PROPERTIES AND REACTIONS OF ORGANIC COMPOUNDS:

I. Intended Learning Outcomes:

Hydrocarbons are organic compounds consisting completely of carbon and

Hydrocarbons ignites spontaneously as it reacts with oxygen upon ignition.

Centro Escolar University – School of Pharmacy 46

With the properties of hydrocarbons, saturated hydrocarbons are generally inert,

Aromatic hydrocarbons contain conjugated pi bonds, that is alternating double

Centro Escolar University – School of Pharmacy 47

III. Materials, Apparatus and Reagents Needed:

Test Samples Materials and Apparatus Reagents

Centro Escolar University – School of Pharmacy 48

2. Baeyer’s Test for Unsaturation

3. Bromine in Dichloromethane Test

Centro Escolar University – School of Pharmacy 49

4. Test for Aromatic Hydrocarbons

Centro Escolar University – School of Pharmacy 50

Name: __________________________________ Date: _____________________

PROPERTIES AND REACTIONS OF ORGANIC COMPOUNDS:

V. Results and Observations:

Baeyer’s Test for Unsaturation

Bromine in Dichloromethane Test

Test for Aromatic Hydrocarbons

Centro Escolar University – School of Pharmacy 51

4. Briefly explain what happens to a compound that undergoes electrophilic aromatic

Centro Escolar University – School of Pharmacy 52

PROPERTIES AND REACTIONS OF ORGANIC COMPOUNDS:

I. Intended Learning Outcomes:

Alcohols are organic compounds composed of an aliphatic carbon atom with a

Alcohols generally have higher boiling points as compared to hydrocarbons

Centro Escolar University – School of Pharmacy 53

boiling point of alcohols decreases with an increase in branching in aliphatic carbon

The solubility of alcohols is technically governed by its ability to perform

Alcohols exhibit a range of spontaneous chemical reactions due to the cleavage

These chemical properties of alcohols can be analyzed using qualitative tests.

Centro Escolar University – School of Pharmacy 54

To further distinguish the classification of alcohols, Lucas test can be employed.

Centro Escolar University – School of Pharmacy 55

A test used to demonstrate the oxidation of alcohol is through partial oxidation

Centro Escolar University – School of Pharmacy 56

III. Materials, Apparatus and Reagents Needed:

Test Samples Materials and Apparatus Reagents

Centro Escolar University – School of Pharmacy 57

4. Test for Methanol

Centro Escolar University – School of Pharmacy 58

Name: __________________________________ Date: _____________________

PROPERTIES AND REACTIONS OF ORGANIC COMPOUNDS:

V. Results and Observations:

Test for Methanol

Acrolein Test Odor

Centro Escolar University – School of Pharmacy 59

4. What is the significance of the Acrolein test?

Centro Escolar University – School of Pharmacy 60

PROPERTIES AND REACTIONS OF ORGANIC COMPOUNDS:

I. Intended Learning Outcomes:

Phenol, also known as hydroxybenzene or carbolic acid, is a toxic, colorless

Phenols generally have higher boiling points compared to other hydrocarbons

Centro Escolar University – School of Pharmacy 61

The synthesis of phenols can be achieved through diazotization of primary

Centro Escolar University – School of Pharmacy 62

A test used to determine the presence of phenolic compounds in amino acids is

Phenol can also be used as precursor in the synthesis of several organic

Centro Escolar University – School of Pharmacy 63

Name: ______________ Date: _

Name: ______________ Date: _

Name: ______________ Date: _

Name: ______________ Date: _