Practical Organic Chemistry

Student Manual for

BDC 12061
Organic Chemistry Laboratory

Bachelor of Education (Biological Science) Degree Program

Faculty of Education
Horizon Campus
Malabe, Sri Lanka

0
Contents
1. Introduction ............................................................................................................................ 2
2. Qualitative elemental analysis ............................................................................................... 3
2.1. Preliminary tests .......................................................................................................... 3
2.2. Conformational tests (Sodium fusion / Lassaigne’s test) ............................................ 3
3. Solubility Behaviour .............................................................................................................. 6
4. Tests for Functional Groups................................................................................................... 9
5. Systematic Analysis of an unknown compound .................................................................. 16

1
1. Introduction
The procedure used to identify and characterize the constituents present in the organic compounds is
known as Qualitative Organic Analysis and it is an important part of practical organic chemistry.

All organic compounds most commonly contain carbon (C), hydrogen (H) and oxygen (O). Some
organic compounds may contain other elements such as nitrogen, sulphur, phosphorous, halogen and
metals. Qualitative Organic Analysis is important to determine the presence and nature of these
elements in a given compound. The presence or the absence of any element will provide us information
to classify the given compound according to its functionality.

During this course, you will be able to learn the appropriate methods need to establish the class to which
an organic compound belongs and to identify the functional group present in that compound.
Spectroscopy is probably the most powerful and modern tool available to the chemist for determine the
structure of an unknown compound. It is often possible to determine the structure using only
spectroscopic techniques. Whereas, qualitative organic analysis gives you the chance to learn and
understand the chemistry of different functional groups.

References
1. Vogel, Arthur Israel, (5th Ed.) Vogel’s textbook of practical organic chemistry, Longman
Scientific & Technical Ltd., 1989

2
2. Qualitative elemental analysis
Carbon (C), hydrogen (H), oxygen (O), sulfur (S) and the halogens are elements most commonly found
in organic compounds. Phosphorous may be present to a lesser extent. Detection of the elements present
in the unknown sample is necessary because this provides information on the functional group present
in the compounds and hence is a guide to the types of tests to be carried out for each sample.

E.g.: the absence of nitrogen means the absence of amines, amides, ammonium salts and nitro
compounds and thus tests for those functional groups need not to be performed.

Carbon and hydrogen are assumed to be present in all organic compounds. There is no specific test for
oxygen. Tests given below are used to detect the presence of nitrogen, sulfur, halogens and phosphorous
in an unknown compound. It is also necessary to find out whether those elements present in the
compound are in the ionic or non-ionic forms.

2.1. Preliminary tests

Beilstein test
Compounds containing halogens give a green flame when heated on a small piece of copper gauze. This
is due to volatile copper halides formed on the copper. Some organic compounds containing nitrogen
but not containing halogens. Also give a positive test due to the formation of volatile copper cyanide
under these conditions. Therefore a green flame indicates that halogen is probably present and a
negative result (no green flame) means the definite absence of halogens.
Procedure: Heat a small piece of copper gauze in a Bunsen flame until it oxidized and there is no green
flame. Dip the gauze in the sample and heat it again in the flame and observe the colour. A positive test
is indicated by a green flame.

Ignition test
Compounds giving a sooty yellow flame have a high degree of unsaturation and may be aromatic. These
compounds contain little oxygen, and have a high carbon to hydrogen ratio. They burn at a low
temperature with a yellow flame. E.g.: naphthalene
Much carbon is produced when they are burned. Compounds that contain oxygen generally burns at a
high temperature with a clean blue flame. E.g.: ethanol
Procedure: Place a small amount of the compound on a metal spatula and place it in the flame of Bunsen
burner.

2.2. Conformational tests (Sodium fusion / Lassaigne’s test)

The elements commonly occurring along with carbon and hydrogen are oxygen, nitrogen, sulphur,
chlorine, bromine and iodine. The detection of these elements depends upon converting them to water-
soluble ionic compounds and the application of specific tests.
C, H, O, N, S, X → NaX/ NaCN/ NaCN/ Na2S

Preparation of Lassaigne’s extract

Place about 50 mg of the solid compound (if the compound is a liquid make a paste with anhydrous
Na2CO3) in the bottom of an ignition tube and keep a small piece of metallic sodium in the midway of
the wall of the ignition tube. Now heats the piece of sodium, first gently and then strongly in a Bunsen
flame until the sodium melts, and allow melted sodium to come to the bottom of the tube. The sodium
reacts with the sample and if the reaction is too vigorous, remove the tube from the flame and allow the
reaction to occur. Then heat the tube strongly until it is red hot.

3
Then plunge the red hot tube in to a boiling tube containing 20 mL of distilled water and quickly cover
the boiling tube with a wire mesh. (Care should be taken because un-reacted sodium gives rise to slight
explosions).
Stir well, boil for few minutes and filter the content using filter paper. The resultant solution should be
a clear solution even if the original compound has a colour.

Na + C + N → NaCN (if N is present)

2Na + S → Na2S (if S is present)
Na + S + C + N → NaSCN (if both N & S are present & insufficient amount of Na is used)
Na + X → NaX (If halogens are present)

Test for sulphur

1) Acidify 2 mL of the fusion solution with dil. acetic acid and add a few drops of lead acetate solution.
A black precipitate of lead sulphide indicates the presence of sulphur.
Pb2+ + S2- → PbS
2) To 2 mL of the fusion solution, add 2-3 drops of a freshly prepared dilute solution (0.1%) of
disodium pentacyanonitrosylferrate Na2[Fe(CN)5NO] (Sodium nitroprusside). A purple coloration
indicates sulphur; the coloration slowly fades on standing.
Na2S + Na2[Fe(CN)5NO] → Na4[Fe(CN)5NOS]
Violet colour

Test for nitrogen

To a portion of the fusion extract, add a freshly prepared solution (2-3 mL) of iron (II) sulphate solution.
When sulfur is absent a greyish green precipitate of Fe(OH) 2 is formed. Boil gently to ensure the
formation of ferrocyanide and to oxidize some of the Fe2+ to Fe3+. Cool and acidify with sufficient
amount of dil. sulphuric acid. A Prussian Blue precipitate or coloration (due to the formation of Iron
(III) hexacyanoferrate) indicates the presence of nitrogen in original sample.
FeSO4 + NaOH → Fe(OH)2 + Na2SO4
6NaCN + Fe(OH)2 → Na4[Fe(CN)6]
3Na4[Fe(CN)6] + 4FeCl3 → Fe4[Fe(CN)6]3 + 12NaCl
(Prussian blue)
 Upon boiling the alkaline iron (II) salt solution, some iron III ions are produced by the action of the
air. Dil. sulphuric acid dissolves iron II and iron III hydroxides and hexacyanoferrate(II) reacts with
Iron III salt, producing Iron(III) hexacyanoferrate (Prussian Blue)

4
 If no blue precipitate appears at once, allow to stand for 15 minutes, filter through a small filter
paper and wash the paper with water to remove all traces of colored solution: any Prussian blue
present will then become perceptible in the cone of the filter paper.
 If sulphur is present, a black precipitate of iron (II) sulphide is obtained when the iron (II) sulphate
is dissolve. Boil the mixture for about 30 sec., and acidify with dilute sulphuric acid; the iron (II)
sulphide dissolves and a precipitate of Prussian blue remains if nitrogen is present.

Test for halogens (If nitrogen and sulphur are present)

Cyanide and sulfide ions formed during the sodium fusion will interfere with the test for halides because
AgCN an Ag2S may be precipitated. Therefore, presence of these ions have to be removed first.
Acidify a portion of the sodium fusion extract with dil. nitric acid and boil it for 2 minutes. This will
drive off any HCN or H2S that is formed. When cools, add an excess of silver nitrate (AgNO3) solution.
A precipitate indicates the presence of a halogen. Treat the precipitate with dil. aqueous ammonia
solution. If the precipitate is white and readily soluble in ammonia solution, chlorine is present; if it is
pale yellow and is sparingly soluble, bromine is present; if it is yellow and insoluble, then iodine is
indicated.
NaCl + AgNO3 → AgCl (Pale yellow) + NaNO3
NaBr + AgNO3 → AgBr (Yellow) + NaNO3
NaI + AgNO3 → AgI (Yellow) + NaNO3

Differentiation of chloride, bromide and iodide

 Acidify 1-2 mL of the fusion solution with dil. HNO3 acid and boil for 2 minutes cool the solution
and add 1 mL of chloroform and few drops of chlorine water. Shake the contents of the tube
vigorously and set aside to allow the layers to separate.
 If iodine is present the organic phase first becomes purple colour disappears (owing to oxidation of
iodine to iodate) and, an orange to brown colour in the organic layer indicates bromine. Absence of
a colour indicates chlorine.

Test for Phosphorous

Phosphide (P3-) present in the sodium fusion extract is detected by converting it to phosphate (PO43-).
Boil a portion of the fusion extract with excess conc. HNO3 acid to oxidize Phosphide (P3-) to phosphate
(PO43-), cool and add an equal volume of ammonium molybdate solution and warm the mixture. A
yellow precipitate of ammonium phosphomolybdate indicates the presence of phosphorous.
HPO42- + 3NH4+ + 12MoO42- + 23H+→ (NH4)3PMo12O40 ↓ + 12H2O

Ionic or non-ionic nature of sulfur, halogens and phosphorus;

1. SO42- - To an aqueous solution of the original sample, add dill. HNO3 and dill. BaCl2 solution. A
white precipitate indicates the presence of sulphur as ionic SO42-.
2. PO43- - To the solution of original compound add ammonium molibdate solution and warm. Yellow
precipitate indicates the presence of P as ionic PO43-.
3. Halide - Add excess dil. HNO3 and few drops of AgNO3 solution. A precipitate indicates the
presence of halide ions.

5
3. Solubility Behaviour
The objective of this experiment is to investigate the solubility characteristics of some simple organic
molecules. This knowledge can be used to look at relationships between the solubility properties of an
organic molecule and its structure, and vice versa. This leads to the important idea that structure dictates
function which means that, by knowing the structure of a molecule it is possible to predict its “function”
such as its solubility, acidity or basicity, stability, reactivity etc. Predicting the solubility of an organic
molecule is a useful skill for recrystallization, extraction, designing a new pharmaceutical and
performing reactions to synthesize molecules.
Polar molecules dissolve in polar solvents (e.g. water, alcohols) and non-polar molecules in non-polar
solvents (e.g. hydrocarbon, hexane). This is why ionic compounds like table salt (sodium chloride) or
compounds like sugar dissolve in water but do not dissolve to any great extent in most organic solvents.
It also applies to the separation of oil and water. The polarity of organic molecules is determined by the
presence of polar bonds due to electronegative atoms (e.g. N, O) in polar functional groups such as
amines (-NH2) and alcohols (-OH).
The solubility characteristics of an organic compound can provide experimental evidence for the
presence (or absence) of several important organic functional groups. Most organic molecules are
relatively non-polar and are usually soluble in organic solvents (e.g. diethyl ether, dichloromethane,
chloroform, petroleum ether, hexanes etc.) but not in polar solvents like water. However, some organic
molecules which are more polar could be soluble in water. Compounds that are insoluble in water can
become soluble in an aqueous environment if they form an ionic species when treated with an acid or a
base. This is because the ionic form is much more polar.

6
 H2O
Insoluble
Compound

H2O soluble aq. NaHCO3

Turns red Turns blue Colour of the Soluble Insoluble

litmus into blue litmus into red litmus is Strong acidic Weak acidic
Basic Acidic unchanged E.g. Carboxylic acids E.g. Phenols
Low Mw Low Mw Neutral
Low Mw Nitrophenols
Amines carboxylic neutral
acids

Insoluble Insoluble
aq. NaOH HCl

Soluble Insoluble
Soluble Insoluble
Neutral
Amines Neutral compounds
compounds
Hydrocarbons, Alcohols,
Esters, Amides, Nitro
NH3 evolved Carboxylic acids, Yellow solution - compounds
Amine salts Phenols Nitrophenol

Note - Determine the solubility of an unknown compound in a particular solvent, by using only a
small amount of the sample.

Procedure:
Place about 1 mL of the solvent in a small test tube and few crystals of the unknown solid or one drop
of the unknown liquid directly in to the solvent. Gently tap the test tube to ensure mixing and observe
the disappearance of the liquid or solid, which indicate that solution is taking place. (Do not use a large
amount of the test sample. A mistake is determining the solubility is testing with a quantity of the
unknown too large to dissolve in the amount of solvent taken. Use small amounts.)
Low molecular weight compounds that contain up to 4 carbons and also contain O, N or S are often
soluble in water. Almost any functional group containing these elements will lead to water solubility
for low- molecular weight (C4) compounds. When the ratio of the O, N or S atoms (polar functional
groups) in a compound to the carbon atoms is increased, the solubility of that compound in water
increases.
E.g.: (Glucose) However, as the size of the side chain is increased, the influence of the polar functional
group is less and water solubility decreases.
Branching of the alkyl chain lowers intra molecular forces between molecules and leads to increased
solubility in water. E.g.: t-butyl alcohol is expected to be more soluble in water than n-butyl alcohol.
Compounds soluble in water are usually in all aqueous solvents and reagents. For example, acetic acid
is soluble in water, dilute HCl and dilute NaOH. But if a compound is only slightly soluble in water, it
may be more soluble in another aqueous solvent.

7
E.g.: A carboxylic acid such as butanoic acid may be only slightly soluble in water but very soluble in
diluted bases. Acidic compounds which are insoluble in water, e.g.: phenol and benzoic acid are
insoluble in dil. HCl and are soluble in dil. NaOH solution forming sodium salts. Therefore solubility
in NaOH is an indication of the acidity of these compounds. Acidification of the basic solution with dil.
HCl will re-precipitate the acid.
RCOOH + NaOH → RCOO-Na+ → RCOOH

Aniline is a basic compound which is insoluble in water ad forms a separate layer. It is soluble in dil.
NaOH and soluble in dil. HCl forming aniline hydrochloride. Bacification of the acidic solution will
release the basic compound and form an emulsion or release or separate layer.
NaOH
Ph-NH2 + HCl → Ph-NH3+Cl- → Ph- NH2

Phenols are acidic enough to dissolve in dil. NaOH, but are not acidic enough to liberate CO2 and
dissolve in a solution of either dil. NaHCO3 or Na2CO3. But nitrophenols are highly acidic and dissolve
in both these reagents. Acetone is a neutral organic compound which is soluble in water and is therefore
soluble in dil. NaOH and dil. HCl. But neutral compounds such as ethyl benzoate and acetophenone are
insoluble in water dil. Bases and dil. acids.

8
 4. Tests for Functional Groups
The test for functional groups given below will help you to determine the presence or absence of a
particular functional group. An understanding of these tests is useful to identify the class which an
unknown organic compound belongs. Procedures for carrying out the tests for functional groups have
been described under different classes of organic compounds. Record all observations for each reaction
in your record book immediately.
Preliminary classification
Physical characteristic of the unknown compound should be noted carefully. These includes its physical
state (liquid, solid, crystalline form etc.) its colour and its odor. Many compounds have characteristic
colors or odors. Compounds with a high degree of conjugate are frequently yellow or red. Amines often
have a fish like odor. Esters have a pleasant fruity or floral odor. Acids have a sharp and pungent odor.
(Caution: Many compounds have distinctly unpleasant or nauseating odors. Some have corrosive
vapors. Therefore any unknown substance should be sniffed with the greatest caution)

Tests for Unsaturation

1) Br2/ CCl4 Test
Procedure:
Dissolve few crystals (50 mg) of a solid unknown or two drops of a liquid unknown in CCl 4 or CH2Cl2
(1 mL) and add a solution of Br2 in CCl4/CH2Cl2 drop wise with shaking, until the bromine colour
persists.
The test is positive if more than five drops of the bromine solution are needed so that the colour remains
for one minute. Usually, many drops of the bromine solution will be needed if unsaturation is present.

2) Potassium Permanganate (Bayer Test)

A positive test is given by compounds with double and triple bonds but aromatic rings do not react. The
test depends on the conversion of the purple MnO4- to a brown precipitate of MnO2 following the
oxidation of an unsaturated compound. Other easily oxidizes compounds such as aldehydes, some
alcohols, phenols and aromatic amines also give a positive test with KMnO4.
Procedure:
Dissolve the unknown compound in water or in 95% ethanol and slowly add a 1% aq. solution of
KMnO4 drop by drop with shaking. In a positive test, the purple colour of the reagent is discharged, and
a brown precipitate of manganese dioxide is formed.

Tests for Aldehydes and Ketones

Aldehydes and ketones contain a carbonyl group and often react in a similar way. However, aldehydes
usually react faster than ketones with the same reagent because of lesser crowding around the carbonyl
carbon atom in aldehydes. Aldehydes are also more readily oxidized than ketones. Note any odour,
solubility in water and action on litmus paper.

1) Brady’s Reagent
Aldehydes and ketones condense with hydrazine (NH2-NH2) and substituted hydrazines to form
hydrazones and substituted hydrazones. Even lower aliphatic aldehydes and ketones give orange to red
precipitates with 2, 4- dinitrophenylhydrazine (Brady’s Reagent).

9
Procedure:
Add one drop of the liquid unknown to brady’s reagent (1 mL). If the unknown is a solid, dissolve a
few crystals (10 mg) in a minimum amount of 95% ethanol before adding it to the reagent. Shake the
mixture vigorously. Most carbonyl compounds give yellow to red precipitates immediately. However,
some compounds will require up to 15 minutes, or even gentle heating, to give a precipitate.

2) Tollen’s reagent/ Ammoniacal silver nitrate

Aldehydes are readily oxidized to carboxylic acids and will reduce Tollen's reagent to produce a silver
mirror on the inside of a clean test tube. The reagent must be prepared immediately before use and all
residues disposed of immediately after use. On standing, the reagent tends to form silver fulminate, a
very explosive substance.
Procedure:
Add aqueous sodium hydroxide (NaOH) one drop to silver nitrate (AgNO3) solution (about 2 mL)
contained in a test tube. Then add dilute ammonium hydroxide drop wise until the precipitate (Ag2O)
formed is just dissolved. Add 2 - 3 drops of the liquid aldehyde or few crystals of the solid aldehyde
dissolved in alcohol to this solution. Allow to stand at room temperature. If a silver mirror is not formed,
warm gently on a warm water bath. Aldehydes, reducing sugars and other reducing agents reduce Ag+
to metallic silver, which forms a mirror on the inner wall of the test tube.

3) Fehling’s test
Fehling’s solution is made when required by mixing two Fehling’s solutions, Fehling’s A and Fehling’s
B, in equal proportions.
Procedure:
Mix equal volumes of Fehling's A and Fehling's B solutions and add a few drops or crystal of the
unknown carbonyl compound. Boil the mixture gently on a boiling water bath for 3 - 4 minutes. Note
the time taken to form the reddish brown precipitate of Cu2O. Aliphatic aldehydes reduce Fehling’s
solution rapidly and the reduction is slow with aromatic aldehydes. Reducing sugars also rapidly reduce
Fehling’s solution.
Refer the reactions involve in Fehling’s test

4) Iodoform test
An alkaline solution of sodium hypoiodite (from NaOH and I2) converts acetaldehyde, methyl ketones
and aliphatic alcohols of the general formula, RCH(OH)CH3 into iodoform (CHI3). Iodoform is a yellow
solid with a characteristic smell.

10
Procedure:
Place aqueous NaOH solution (6-8 drops) and an equal volume of I2/KI solution in attest tube. Add the
test compound (3-4 drops) and warm gently in a warm water bath. Shake the test tube vigorously, cool,
dilute with a little water and allow to stand for 15 minutes. A yellow precipitate of iodoform with the
characteristic antiseptic smell will be formed.

5) Chromic acid test (Bordwell-wellman reagent)

Procedure:
Dissolve the liquid unknown (one drop) or the solid unknown (10 mg) in acetone (1 mL). Add several
drops of the chromic acid reagent, a drop at a time with shaking. A positive test is indicated by green
precipitate or a green colour and a loss of the orange colour of the reagent.
3RCHO + H2Cr2O7 + 3H2SO4 → 3 RCOOH + Cr2(SO4)3 + 4H2O
(Orange) (Green)

Primary and secondary alcohols are also oxidized by this reagent. Therefore this test is not useful in
identifying aldehydes unless a positive identification of the carbonyl group has already been made.

Tests for Carboxylic acids

Carboxylic acids are detectable mainly by their solubility characteristics. They are soluble in both dilute
NaOH and NaHCO3 solutions.

1) Test with 5% aq. NaHCO3

Procedure:
Dissolve a small amount of the compound in cold/ hot water and add 5% aqueous NaHCO3 solution.
Observe the solution carefully. If the compound is an acid, bubbles of CO2 will be seen to form.
R-CO2H + NaHCO3 → R-CO2-Na+ + CO2 + H2O
Phenols do not usually liberate CO2 from NaHCO3. However certain phenols are acidic enough to
behave much like carboxylic acids. This is especially true of those substituted with electron-
withdrawing groups at the ortho and para ring positions. Amine salts C6H5NH3+Cl- also liberate CO2
from aqueous NaHCO3

2) Esterification
Procedure:
Warm a small amount of the compound with a few drops of ethanol and one drop of Conc. H2SO4 in a
dry test tube. Cool and pour in to a little water contained in a boiling tube. Note the pleasant fruity smell
of ester.
RCOOH + C2H5OH → RCOOC2H5 + H2O

11
3) Ferric chloride test
Neutral solutions should be used for this test. Excess acid prevents the formation of colour and or
precipitates. Excess alkali gives a reddish brown precipitate of Fe(OH)3.
Procedure:
Dissolve the compound in water or if insoluble, in a few drops of ethanol and add NH4OH drop by drop
to make the solution just alkaline to litmus. Boil gently until the smell of NH3 is completely removed.
Cool this neutralized solution and add neutral ferric chloride. Observe any colorations or precipitates.
Warm the mixture and observe any colour changes. Cool and add dil. H2SO4.
Preparation of neutral ferric chloride solution
Add NH4OH drop by drop in to a solution of FeCl3 until faint but permanent precipitate of Fe(OH)3 is
obtained. Filter and use the filtrate, which should be reddish brown in colour. Neutral FeCl 3 must be
prepared freshly as the neutral solution slowly becomes acidic with time.

Tests for phenols

The pKa of a typical phenol is 10, whereas the pKa of a carboxylic acid is usually near 5. Hence, phenols
are generally not soluble in weakly basic NaHCO3 but do dissolve in NaOH solution, which is more
strongly basic. (More acidic phenols like the nitrophenols dissolve in 10% aq. NaOH solutions forming
colored phenoxide anions that have a high degree of conjugation)
Caution: Phenols have a caustic action on skin and can cause severe burns. Wash with plenty of water
and with a very dilute solution of bromine water if you spill phenol on your skin.

1) FeCl3 test
Procedure:
Add a few drops of an aq. solution of neutral FeCl3 to a dilute aqueous or ethanolic solution of phenol.
Most phenols produce an intense red, blue, purple or green colour. Add dil. HCl and observe any colour
changes.

2) Bromine water test

Procedure:
Add a saturation solution of bromine in water to a 1% aq. solution of the unknown drop by drop with
shaking, until the bromine colour is no longer discharged. A positive test is indicated by the precipitation
of a substitution product (usually turbidity) at the same time that the bromine colour is discharged.
Phenols in alkaline solution form azo dyes with diazotized aniline. Procedure for this test is given under
the tests for amines.

Tests for Amines

Amines are detected best by their solubility behavior and their basicity. If the unknown is soluble in
water, the aqueous solution will have a high pH. Amines dissolve in dil. HCl forming the amine
hydrochloride. Treatment with NaOH solution liberates the free amine, which separates as oily drops
or as a solid from the alkaline solution.
𝐍𝐚𝐎𝐇
Ph-NH2 + HCl → Ph-NH3+Cl- → Ph- NH2

1) Carbylamine test (Isocyanide formation)

Primary amines react with chloroform and alcoholic KOH to form volatile isocyanides or carbylamines
which are recognized by their unpleasant odors.

12
Amphoteric amino compounds (e.g. Amino carboxylic acids and amino phenols) give potassium salts
of the isocyanide, which may not give a smell as they are non- volatile. N- substituted amides like
acetanilide are hydrolyzed by the alkali to primary amines, which will answer the test.

Procedure:
Add a few drops of CHCl3 and alcoholic KOH solution (2 mL) to a small amount of the compound.
Mix well and gently warm for a few minutes. Odor of carbylamine is detected easily when a few drops
are added to a boiling tube containing water.
Caution!
Isocyanides are extremely poisonous. Immediately after this test is completed, cool and carefully
add a few drops of conc. HCl to hydrolyze the isocyanides to the amine, which is odorless.

2) Nitrous acid test

Primary aliphatic amines react with HNO2 liberating N2 and primary aromatic amines from diazonium
salts which are stable at 0-5 ℃ and can be coupled with phenols to give azo dyes. Secondary amines
form nitroso derivatives.
Procedure:
Dissolve the unknown (0.1 g) in water (2 mL) to which conc. H2SO4 (6-8 drops) have been added. Cool
the solution to 0-5 ℃ in an ice bath. Also cool 2 ml of aq. NaNO2 in another test tube. In a 3rd test tube,
prepare solution of 2-napthol (0.1 g) in aq. NaOH (2 mL) and place it in an ice bath to cool. Add the
cold NaNO2 solution, drop by drop with shaking, to the cooled solution of the amine. Look for bubbles
of N2 gas.
Substantial evolution of gas at 0-5 ℃ indicates a primary aliphatic amines, RNH2. The formation of a
yellow oil or a yellow solid usually indicates a secondary amines, R2HH. Tertiary amines do not react.
If little or no gas evolves at 5 ℃, take the half solution and warm it gently to room temperature. Nitrogen
gas bubbles at this elevated temperature indicates that the original compound was a primary aromatic
amine, ArNH2. Add the remaining diazo solution to the solution of β- napthol in alkali. If a red dye
precipitates, the unknown has been conclusively shown to be a primary aromatic amine.

Tests for ammonium salts and amides

Ammonium salts liberate ammonia when treated with aq. NaOH in the cold while amides are
hydrolyzed upon warming with NaOH solution. Ammonia is liberated from primary amides.
Ammonium salts and amides of aromatic carboxylic acids give aromatic carboxylic acids which are
precipitated upon acidification of the solution.
Procedure:
Treat with aq. NaOH solution in the cold and test for ammonia. Warm gently with the NaOH solution
for about 5 minutes and test for the evolution of ammonia. Cool and acidify with conc. HCl. Ammonia
can be identified by its characteristic odor or by using a red litmus paper. A filter paper dipped in
Nessler’s reagent (which will turn brown) or the stopper from the conc. HCl bottle (will form white
fumes of NH4Cl) can also be used.

Tests for Nitro compounds

Nitro phenols usually dissolve in aq. NaOH giving yellow solutions.
1) Reduction to NH2
If the nitro compound already contains an NH2 group, this test cannot be used to detect the presence of
an NO2 group.

13
Procedure:
Add conc. HCl (about 3 mL) and granulated tin (a few pieces) to a few drops of the sample. Warm
gently to complete the reduction. Filter the solution and carry out the carbylamine test with the basified
filtrate.

Tests for Alcohols

1. Chromic acid test
This test is based on the reduction of chromium (VI), which is orange to chromium (III), which is green,
when an alcohol is oxidized by the reagent. Primary alcohols are oxidized to carboxylic acids and
secondary alcohols are oxidized to ketones.
Procedure:
Dissolve the unknown liquid (10 drops) or the unknown solid (10 mg) in acetone (1 mL). Add one drop
of chromic acid reagent and note the result that occurs within 2 seconds. A positive test for a primary
or a secondary alcohol is the appearance of a blue-green colour. Tertiary alcohols do not give the test
within two seconds, and the solution remains orange.

2. Lucas test
This test depends on the appearance of an alkyl chloride as an insoluble second layer when an alcohol
is treated with a mixture of conc. HCl and ZnCl2 (Lucas reagent). This reagent converts alcohols into
the corresponding alkyl chlorides. Zinc chloride (a Lewis acid) increases the reactivity of alcohols
towards acid. The test depends on the rate of reaction of primary, secondary, and tertiary alcohols with
the reagent at room temperature
𝐙𝐧𝐂𝐥𝟐
R-OH + HCl → → R-Cl + H2O

I. RCH2OH → no reaction at room temperature.

II. R2CHOH → R2CHCl + H2O react slowly (1 hour or maybe longer)
III. R3COH → R3CCl + H2O (immediately) benzylic and allylic alcohols react instantly.

This test works only for alcohols that are soluble in the reagent. This often means that alcohols with
more than six carbon atoms cannot be tested.
Procedure:
Cool 1 mL of conc. HCl in an ice bath and dissolve 1 g of ZnCl2 in it. Add 2-3 drops of the unknown
compound to this solution and shake vigorously. Tertiary, benzylic and allylic alcohols give an
immediate cloudiness in the solution as the insoluble alkyl halide separates from the aqueous solution.
After a short time, the immiscible alkyl halide will form a separate layer. Secondary alcohols produce
a cloudiness after 2-5 minutes. Primary alcohols dissolve in the reagent giving a clear solution.

Tests for Esters

Note the characteristic pleasant fruity odour.
1. Basic hydrolysis
Procedure:
Boil a little of the unknown with 1-2 mL of dilute aq. NaOH for 5-10 minutes. (Add boiling stones)
stop heating and observe the solution to determine whether the oily ester layer has disappeared or
whether the pleasant odor of the ester has disappeared. Acidify with conc. HCl to see whether any
precipitate is formed. Precipitate indicates an aromatic acid.

14
2. Ferric hydroxamate test
On being heated with NH2OH, esters (and lactones) are converted to corresponding hydroxamic acids
(N-hydroxyamides) which form strong, colored complexes with Fe3+ ions in acidic medium.
Procedure:
Dissolve 2-3 drops of the liquid unknown, or about 40 mg of a solid unknown, in a mixture of 0.5 mL
of 25% hydroxylamine hydrochloride solution and 1 mL of aq. NaOH. Heat the mixture to boiling for
a few minutes. Cool the solution and then acidify with dilute HCl. If the solution becomes cloudy, add
2 mL of 95% ethanol to clarify it. Add a drop of FeCl3 solution and note whether a colour produced. If
the colour fades, continue to add ferric chloride until the colour persists. A positive test should give a
deep red or violet colour.

15
5. Systematic Analysis of an unknown compound
Functional groups in an unknown organic compound are identified by carrying out the following tests.
A confirmatory test is then carried out for each functional group detected. Treat the unknown compound
with the following reagents and record all your observations and inferences.
1. Check the solubility in water.
2. Treat with 5% aq. HCl. Observe solubility if it was insoluble in water.
3. Treat with 5% aq. NaOH solution. Observe solubility and the colour of the solution. If N is present,
see if ammonia is evolved. Heat the solution and observe any changes.
4. Treat with 5% aq. NaHCO3 solution. Observe solubility and test for the evolution of CO2.
5. Heat with excess soda lime. Test for any gaseous products (NH3)
6. Collect the volatile products (phenols, anilines etc.) in a test tube containing alcohol. Carry out the
test for phenols and amines on the condensate.
7. Treat with the Brady’s reagent. If positive, test for aldehydes using Fehling’s test, Tollen’s test or
chromic test.
8. Add neutral FeCl3 to a solution of a sample.
9. Carry out the ferric hydroxamate test for esters.
10. Test with Br2/CCl4 or with Br2/water.
11. If N present, carry out the carbylamine test.
12. Lucas test.

16