Practical Organic

Identification
4th-Stage

PhD. Salam G.Taher

M.Sc. Khdir A. Othman

Flammable Corrosive Acute Toxic Health Hazard Irritant Env.Hazard

2022 – 2023
Department of Chemistry, Faculty of science and health, Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Content

1st week:
2 Qualitative Organic Analysis
3 Physical characterization
3 Physical constants
4 Solubility
4 Ignition test
5 Analysis for Elements (To detect extra elements in the given organic compound.)
2nd week:
8 Functional group tests:
9 Unsaturation
11 Alcoholic
15 Aldehyde and ketones
19 Carboxylic acids
21 Ester and anhydride
24 Phenols
26 Amines
30 Ethers
31 Carbohydrates
34 Amino acids

1
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Qualitative Analysis of Organic Compounds

Organic chemists regularly need to identify the compounds that are formed in chemical
reactions or isolated from natural sources. In simple words the identification of unknown organic
compound means the establishing of its molecular and structural formula.
An analyte is considered to be exactly identified, when its Physical, chemical, and biological
properties are Identical to the original compound. This can be confirmed either by Instrumental
methods (IR, NMR, MS, UV, HPLC, etc.…) or via manual methods bases on physical observations
(state, melting point, boiling point, solubility, odor, color, etc.), elemental analysis, and tests for
functional groups.

Chemical Analyses are generally divided into two broad categories:

▪ Qualitative analysis: Deals with the determination of what is present i.e., N, Cl, S…etc.
▪ Quantitative analysis: Deals with the determination of how much is present.

How to start?
Physical Characteristics

Solid Physical Constants Liquid

Melting point Boling point

Solubility tests

General Scheme of Analysis: Ignition tests

Elemental Analysis (Na fusion)

Functional group classification tests

Spectral Analysis IR, NMR,

Derivative(s) synthesis

2
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Step 1. Physical Characteristics:

Record a physical state of the Unknown whether it is Solid or Liquid. Usually, small
Organic molecules are liquid such as Low molecular wight of Alcohols, Aldehydes,
Physical state Ketones, Carboxylic acids, and Esters, etc. Large Organic molecules are solid, such
as high molecular wight of Carboxylic acids, Phenols, Carbohydrates, and Amino
Acids etc.

Common colored compounds include Nitro and Nitroso compounds (YELLOW),

a-diketones (YELLOW), Quinones (YELLOW to RED), Azo compounds
Note the Color (YELLOW to RED), and polyconjugate olefins and Ketones (YELLOW to RED).
Phenols and Amines are often BROWN to dark-PURPLE because of traces of air
oxidation products.

Some liquid and solid Amines are recognizable by their fishy odors; Esters are often
pleasantly fragrant. Alcohols, Ketones, Aromatic hydrocarbons, and Aliphatic
Note the Odor olefins have characteristic odors. On the unpleasant side are Thiols, Isonitriles, and
low-molecular weight Carboxylic acids.
Note: it is wise to sniff them with caution. Some compounds can have corrosive
vapors or make you feel nauseous.

Step 2. Physical Constants:

When you are asked to identify an unknown organic sample the first thing to do is
to check the purity of the Sample, impurities in your compound will make it
extremely difficult to identify. Thus, before you do anything else, you will need to
make sure your unknown compound is pure. (Normally we give you a pure sample).

How to check the purity of your sample?

▪ Melting Point for solid sample.
▪ Boiling Point for liquid sample

3
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Step 3. Solubility Tests:

The solubility of the unknown in the following reagents provides very useful
information. In general, about 1 mL of the Solvent is used with approximately 0.01
g or 0.2 mL (2-3 drops) of the unknown compound.

1N

1N

1N

Conc
.

Step 4. Action of Heat (Ignition):

Place some organic compounds (0.1g) on nickel spatula, heat until ignition occurs:

▪ Yellow, sooty flame is indicative of an aromatic or a highly unsaturated aliphatic

Note: This test should
be done in a hood.
compound.
▪ Yellow-Blue but non-sooty flame is characteristic of simple saturated aliphatic
hydrocarbons. The oxygen content of a substance makes its flame more colorless
or blue. Also, Halogens.
▪ Burnt sugar smell and charring: Carbohydrates(sugars), polyhydroxy
compounds.
▪ Some compounds sublime with heat e.g.: benzoic acid naphthalene.
▪ The presence of residue show that it contains metal atom.

4
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Step 5.
Sodium fusion
test
(Lassaigne’s test)
CAUTION:
Manipulate Sodium
with a knife and
forceps; never touch it
with the fingers. Wipe
it free of kerosene with
a dry towel or filter
paper; return scraps to
the bottle or destroy
scraps with methyl or
ethyl Alcohol, never
with water. Safety
glasses! Hood!
Do not use CHCl3 or
CCl4 as samples in
Sodium fusion. They
react extremely
violently.
Sodium can cause
serious burns and the
Sodium may react
violently with some
substances. Wear
gloves, avoid contact,
and keep the Sodium-‐
‑lead alloy away from
other chemicals.
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
Analysis for Elements:
In organic compounds 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.
How To Perform Experiment:
The experiment consists of two stages:
1. Preparation of sodium fusion extract
2. To test for Nitrogen, Sulphur, and Halogens
▪ Preparation of Sodium Fusion Extract
C, H, O, N, S, X NaX
NaCN
Na2S
NaCNS
Procedure:
1. In the fume hood or under a snorkel, take a small piece of freshly cut Sodium
metal and dry it between the folds of a filter paper. Take it in an ignition tube.
2. Hold the ignition tube with a pair of tongs and heat it till the Sodium melts into
a shining glob. ↺
3. Using a Pasteur pipet, add 2 drops of the unknown (or 10 mg of a solid) directly
onto the molten Sodium, so that it does not touch the sides of the tube and
continue heating gently until the contents of the tube are solid.
4. Heat the ignition tube at the tip of the flame till it becomes red hot.
5. Put the red-hot ignition tube in a beaker containing 10 mL distilled water and
cover it immediately with a wire gauze. Break the bottom of the ignition tube
with the help of a glass rod, if it does not break itself.
(*CAUTION: The tube shatters)
6. Boil for (3-4 min) and filter hot solution through a filter paper.
7. The 'fusion' filtrate which should be clear and colourless. Then divide into
THREE parts for the Specific Tests Described Below:
5
 Part I. Because the Sodium in the extract combines with the excess water to generate
Test for Sodium hydroxide, the Sodium fusion extract is alkaline. To keep the solution
Nitrogen: alkaline, Sodium hydroxide is sometimes added.
Note: This test is not Sodium cyanide (NaCN) reacts with Iron(II)sulfate to produce hexacyanoferrate (II)
given by compounds
sodium Na4[Fe(CN)6], which combines with iron(III) salts (i.e., Fe2(SO4)3),
containing N but not C
inevitably formed by air oxidation in the alkaline solution, to give insoluble Prussian
atoms. For example,
NH2NH2 does not
BLUE, Fe4+3[Fe+2(CN)6]3.
answer this test despite
having an N atom.
This is because both C
and N are required to
form CN– ion.
This test is not given
by diazonium salts as
they decompose to
give nitrogen gas on
heating.
Procedure:
1. Add 0.5 ml of fusion solution to a clean micro test tube.
2. Add 3 drops of 2% FeSO4. xH2O solution.
3. Warm it
4. Add 3 drops of 1N H2SO4
5. Dark BLUE colour indicates that the original substance contains Nitrogen.

Part II. A- Sodium Nitroprusside Test

Tests for Sulphur 1. Add 2 drops of Fusion Extract to a well plate.
2. Add 2 drops of 0.5% Sodium nitroprusside solution
3. The formation of PURPLE colour indicates that the original substance contains
Sulphur.

6
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 B- Lead Acetate Test
1 Add 3 drops of fusion solution to a well plate.
2 Add 2 drops of conc. Acetic acid.
3 Add 2 drops of 0.5N Lead acetate [(CH3COO)2Pb].
4 The formation of BLACK precipitate confirms the presence of Sulphur.

Part III. Procedure.

Tests for 1. Add 5 drops of fusion solution to a clean micro test tube.
Halogens 2. Acidify with 1N Nitric acid (HNO3). Follow the acidification with litmus paper.
3. Boil the solution gently for 2-3 min. to expel any Hydrogen sulfide or cyanide
that may be present. (Sulfide and Cyanide, if present, will interfere with the test
CAUTION:
H2S and HCN are for the halogens, how?).
highly poisonous 4. Cool the solution and add 3 drops of 0.3 N aqueous Silver nitrate (AgNO3)
gases, perform this solution.
experiment inside the
5. A heavy precipitate of Silver halide indicates the presence of Chlorine, Bromine,
fume hood and make
or Iodine in the original organic compound.
sure the hood is
working and the hood If only one halogen is present, it may be identified by decanting the mother liquor,
slide is closed washing the precipitate with distilled water, and treating the precipitate with 1N
completely. aqueous Ammonia.
A faint turbidity
6. If the precipitate is WHITE and dissolves readily in 1N NH3 and reappears on
should not be
acidification of the solution with 1N HNO3 – Chlorine is indicated.
interpreted as a
positive test. 7. If the precipitate is pale YELLOW and partially soluble in 1N NH3- Bromine is
indicated.
8. If the precipitate is YELLOW and insoluble in NH₃(aq) – Iodine is indicated.

AgCl: WHITE ppt., Soluble in Ammonia solution.

AgBr: pale YELLOW ppt., Soluble with Excess amount of Ammonia solution.
AgI: YELLOW ppt., Insoluble in Ammonia solution.

7
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Step 6. Functional group tests
Functional Chemical Test No. Functional groups respond
Group
Potassium permanganate test (Baeyer’s
1.1 Alkene, Alkyne, Alcohols,
Unsaturated test)

Bromine test 1.2 Alkene, Alkyne, Phenols & aromatic Amines

Chromic acid (Jones’ reagent) 2.1 1° Alcohols, 2°Alcohols, & Aldehydes
Sodium metal test 2.2 Alcohols, Phenols, enols, acids
Alcohols Ceric ammonium nitrate test 2.3 Alcohols, Phenols
Lucas’s test 2.4 Used to classify Alcohols as 1°, 2°, or 3° Alcohols
Acetyl chloride 2.5 Alcohols, Phenols, Amines (1ry, 2ry)
2,4-Dinitrophenylhydrazine 3.1 Aldehydes & Ketones
Aldehydes Sodium bisulfite test 3.2 Aldehydes (some Ketones)
& Tollens's (Silver mirror) test 3.3 Aldehydes, Reducing sugars, hydrazo-compounds
Ketones
Sodium nitroprusside test 3.4 Ketones
Iodoform test 3.5 Tests for –CH(OH)CH3 and –COCH3 groupings
Sodium bicarbonate test (NaHCO3) 4.1 Carboxylic acids
Carboxylic Ester test 4.2 Alcohols, acids
Acids
Fluorescein test 4.3 Dicarboxylic acid
Esters Alkaline hydrolysis (Saponification) 5.1 Amides & Esters
& Ferric hydroxamate (Feigl Test)
Acetic
5.2 Esters (amides, acid chlorides, acids, Alcohols)

anhydride Aniline Test 5.3 Acetic anhydride

Ferric chloride test 6.1 Phenols, enols (Amines, acids)

Phenols
Phthalein dye test 6.2 Phenols
Carbylamine test 7.1 Primary Amines
Liebermann's nitroso reaction (Nitrous
acid test), Nitrosamines, Phenols (some)
Amines 7.2
Aromatic Amines (primary), Phenols
Azo dye test
Hinsberg’s test 7.3 Used to classify Amines as 1°, 2°, or 3°
Zeisel test 8.1 Esters or Ethers
Ether
Acidic Hydrolysis 8.2
Molisch's test 9.1 Carbohydrates

Carbohydrates Benedict’s test 9.2 Aldehydes, Reducing sugars

Iodine test 9.3

Ninhydrin test 10.1 Amino acids

Amino acid Copper Complex: (Biuret Test) 10.2 Amino acids

Xanthoproteic test 10.3 Aromatic amino acids, tyrosine, and tryptophan.

8
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 1 . UNSATURATION

Unsaturated hydrocarbons are hydrocarbons that have double or triple covalent bonds
THEORY:
between adjacent carbon atoms.

Unsaturated hydrocarbons of ethylene series have multiple C=C bond which is found
on the following reactions:
1.1. Potassium permanganate test (Baeyer’s test).
1.2. Bromine test.
Unsaturated hydrocarbons of acetylene series have multiple C≡C bond:

TESTS Properties are close to compounds with double bond. Therefore, the same reactions of
accession of Bromine and oxidation of permanganate of Potassium which, proceed with a
smaller speed are suitable for their detection.
To distinguish aromatic hydrocarbons from aliphatic we can use
▪ Alkylation with Chloroform in the presence of aluminum chloride.
▪ Sulfonation and Nitration Experiment

9
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 1.1 . Potassium permanganate (Baeyer’s) test:

In this test the VIOLET colour Potassium permanganate (KMnO4) disappears when an
alkaline Potassium permanganate is added to an unsaturated hydrocarbon. The
disappearance of VIOLET colour may take place with or without the formation of
BROWN precipitate of Manganese oxide. The chemical reaction is given below.

Materials Required, ✓ 1% Alkaline KMnO4: Dissolve about 1 g of KMnO4 in 100 ml of D.W. To that solution add
Preparation: 10 g of anhydrous Sodium carbonate (Na2CO3).

Precautions: ❖ KMnO4 is strong an oxidizing reagent.

Procedure: 1. Dissolve 1 drop (0.01 g) of the unknown in 0.5 ml of Acetone in a clean micro test tube.
2. Add 2 drops of 1% alkaline KMnO4 Solution. Shake the mixture.

Observations: Decolourization of VIOLET colour may take place with or without the formation of BROWN
precipitate, indicates unsaturation.

Questions How Alkynes react with KMnO4? Draw the general reaction.

1.2 . Bromine test:

In this test when Bromine solution is added to the unsaturated hydrocarbon the
Note: ORANGE-RED colour of Bromine solution disappears if the hydrocarbon is unsaturated.
At effect of Bromine on
aromatic Amines and Bromine forms an addition product with the unsaturated hydrocarbon. The chemical
Phenols also there is a
replacement reaction. reaction is given below.
Replacement reaction, as
well as accession reaction,
proceeds with Bromine
discoloration.

Materials Required: ✓ Bromine water solution: Take 5 ml of Bromine add 100 ml of distilled water and shake
Preparation of well. Decant off the clear liquid.
Reagents: ✓ Bromine in CCl4 or CHCl3: Take 5 ml of liquid Bromine in 100 ml of Carbon tetrachloride
or Chloroform, shake it to dissolve properly.
✓ Carbon tetrachloride or Chloroform
✓ Conc. Ammonia

Precautions: ❖ The Bromine solution should be handled carefully.

❖ Do not inhale the vapors of Bromine and Chloroform.
❖ The test should be carried out at room temperature.
❖ Avoid Bromine solution in contact with skin.

10
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Procedure: (I) Water-soluble Compounds:
1. Dissolve 1 drop (0.01 g) of the unknown in 0.5 ml of distilled water in a clean micro
test tube.
2. Add 2 drops of Bromine water with constant shaking.
(II) Water insoluble Compounds:
3. Dissolve 1 drop (0.01 g) of the unknown in 0.5 ml of Carbon tetrachloride in a clean
micro test tube.
4. Add 2 drops of Bromine in CCl4 with constant shaking.
5. Disappearance of ORANGE-RED colour indicates unsaturation.
6. Continue the drop wise addition of Bromine with constant shaking until the BROWN
colour persists.
7. Blow across the mouth of the test tube or bring a rod dipped in Ammonium hydroxide.
Absence of white fumes confirms unsaturation.
(III) Gaseous Compounds:
8. Add 0.5 ml of Bromine solution into a gaseous compound taken in a jar. Shake the
mixture well.

Observations: Decolourization of ORANGE-RED colour of Bromine solution indicates unsaturation.

Questions: Why does Phenol decolorize Bromine water? Draw the reaction.
How Ethers have undergone halogenation ?

2 . ALCOHOLS

The organic compound which has -OH functional group are called Alcohols. The general
THEORY
formula for Alcohol is R-OH. Where R is an alkyl group.

The following test can be carried out to detect the Alcoholic functional group.
2.1. Chromic acid Test
2.2. Sodium metal test
TESTS
2.3. Ceric ammonium nitrate test
2.4. Lucas’s test
2.5. Acetyl chloride test

2.1 . Chromic Acid Test (Jones’ reagent):

Note:
Aldehydes give positive result.
Tertiary Alcohols are not
oxidized by Chromic acid, but
gives Chromic acid Esters
colored in YELLOW or RED
wine.
Alcohols undergo several types of chemical reactions. The most important reaction
Alcohols undergo is oxidation to carbonyl compounds. Primary Alcohols are oxidized
to Aldehydes, which themselves are sensitive to oxidation to Carboxylic acids. The
following reactions show the expected products for different Alcohols with the Jones
reagent, a solution of chromic acid (H2Cr2O7) in concentrated H2SO4. The chromic acid
serves as the oxidizing agent as it changes from an oxidation number of +6 to +3.

11
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Materials Required, ✓ Chromic acid reagent: (0.15 M) (1 N) Dissolve 5 g of the K2Cr2O7 in D.W., add 2.5 ml
Preparation: of Conc.H2SO4 and make up the volume to 100 ml.

Precautions: ❖ The reagents should be freshly prepared to perform the test.

❖ Not to heat the reaction mixture directly on the flame.
❖ Chrome (VI) is a known carcinogen.
❖ Sulfuric acid is highly corrosive.

Procedure: 1. Dissolve 1 drop (0.01 g) of the unknown in 0.5 ml of Acetone in a clean micro test
tube.
2. Add 2 drops of Chromic acid reagent
3. Shake the test tube with gentle warming (40 oC).

Observations: A reaction within 2‑3 seconds indicates a primary or secondary alcohol. With aliphatic
aldehydes, the solution turns cloudy in about 5 seconds and the GREEN suspension forms
within 30 seconds. Aromatic aldehydes require 30-‐‑90 seconds or longer to form the
suspension. Note that the formation of another dark color (particularly with the color of the
liquid remains orange) should be considered a negative test.

Questions Write the reaction between Chromic acid reagent and simple Aldehyde.

2.2 . Sodium Metal Test:

It is based on the appearance of brisk effervescence due to the liberation of Hydrogen gas
when Alcohol reacts with active metals like Sodium. Alcohols and Phenols quite easily
react with metallic Sodium with hydrogen evolution and formation of alkoxides and
phenoxides. This test is favorable if phenyl or carboxyl groups are absent.

Materials Required: ✓ Sodium metal

✓ Calcium sulfate (CaSO4) Anhydrous

12
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Precautions: ❖ The Alcohol to be tested should be dry because water also reacts with Sodium.
❖ Sodium should be handled carefully; unreacted Sodium should be destroyed by adding excess
Alcohol.

Procedure: 1. Add 0.5 ml of the Unknown to a clean micro test tube.

2. Add 0.5 g of Anhydrous CaSO4 and shake well.
3. Decant the solution to another clean test tube.
4. Add a small piece of Sodium metal.
Observations: Evolution of Hydrogen gas cause a brisk effervescence indicates an Alcoholic group.

Questions Why Anhydrous CaSO4 is added to the sample in sodium metal test of alcohols?

2.3 . Ceric ammonium nitrate test:

Note: Phenols react with ceric Alcohols react with Ceric ammonium nitrate to form a RED colour precipitate due to the
ammonium nitrate.
formation of a complex compound and Nitrate. The chemical reaction is given below

Materials Required, ✓ 10% Ceric ammonium nitrate reagent: Take 10 gm of YELLOW crystals of Ceric
Preparation: ammonium nitrate in 100 ml of warm dilute 1N ~ (6.4%) Nitric acid.

Procedure: 1. Dissolve 1 drop (0.01 g) of the unknown in 0.5 mL of Water in a clean micro test tube.
2. Add 2 drops of 10% Ceric ammonium nitrate reagent
3. Shake the solution well.

Observations: wine RED precipitate shows the presence of Alcoholic group.

Questions Draw the reaction between ceric ammonium nitrate and phenol. With an observation.

2.4 . Luca's Reagent:

Note:
This test cannot be used for
solid alcohols or alcohols
that are not soluble in the
Lucas reagent (typically
alcohols that have boiling
points higher than ~140
°C).
The mixture of Zinc chloride and concentrated Hydrochloric acid is called Luca's reagent.
It reacts with primary, secondary and tertiary Alcohols at different rates. This reagent
forms a cloudiness on reacting with Alcohols. Tertiary Alcohols react immediately and
give cloudiness, secondary Alcohols react slowly and gives cloudiness after 5 to 10
minutes and there is no reaction with primary Alcohols.
The chemical reactions are given below.

13
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Materials Required, ✓ Luca's reagent: Dissolve 135 g of anhydrous Zinc chloride (ZnCl2) in 100 ml of
Preparation: concentrated Hhydrochloric acid. (Cool if necessary to avoid the loss of HCl)

Precautions: ❖ The Luca's reagent can cause serious burns, and is harmful

Procedure: 1. Dissolve 1 drop (0.01 g) of the unknown in 0.5 mL of Water in a clean micro test tube.
2. Add 3 drops of Luca's reagent
3. Shake and observe the changes

Observations: Primary Alcohol: Here no visible reaction is observed and the solution remains colorless e.g., 1-
Pentanol
Secondary Alcohol: Here the solution turns turbid or cloudy in 5-20 minutes with slight heating
e.g., 2-Pentanol
Tertiary Alcohol: Here the solution turns turbid or cloudy rapidly with the formation of two
separate layers at room temperature

Questions

2.5 . Acetyl Chloride Test:

Alcohol reacts with Acetyl chloride results in the formation of Ester and Hydrogen
chloride. The resulting Hydrogen chloride on contact with Ammonium hydroxide forms
a white fumes of Ammonium chloride and water. The chemical equation is given below.

Materials Required: ✓ CaSO4 Anhydrous

✓ Acetyl chloride
✓ Ammonium hydroxide (conc. Ammonia solution).
Precautions: ❖ Acetyl chloride is very corrosive and its vapors are irritating and toxic. It reacts violently with
water and some alcohols. Work in a fume hood or under a snorkel and keep away from water.
❖ Ammonia is harmful if inhaled or allowed to contact the skin. Work under in a fume hood or
under a snorkel

14
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Procedure: 1. Add 0.5 ml of the Unknown to a clean micro test tube.
2. Add 0.5 gm of anhydrous Calcium sulfate and shake well.
3. Filter the solution.
4. To the filtrate add 2 drops of Acetyl chloride and shake well.
5. Take a glass rod dipped in Ammonium hydroxide solution.
6. Bring the glass rod near the mouth of the test tube.
Observations: Formation of WHITE fumes indicates the presence of Alcohol

Questions How this test could be performed, when you have acetyl halides as an Unknown?

3 . ALDEHYDES or KETONES

Aldehydes and Ketones of low molecular weights are volatile compounds. Identification
of Aldehydes and Ketones is based on two types of reactions, addition reaction to the
double bond and oxidation reaction.
In Aldehydes, the carbonyl group is attached to a hydrogen atom and an aliphatic or
THEORY:
aromatic radical. Formaldehyde is an exceptional case in which the carbonyl present in
Formaldehyde is attached to two hydrogen atoms. In Ketones, the carbonyl group is
attached to two aliphatic or aromatic group.

The following tests are used to identify the presence of Aldehydes and Ketones.

3.1. 2,4-dinitrophenyl hydrazine test

3.2. Sodium bisulfite test

The difference between Ketone and Aldehyde is the carbonyl group present in Aldehydes

TESTS can be easily oxidised to Carboxylic acids whereas the carbonyl group in Ketones are not
oxidised easily. This difference in reactivity is the basis for the distinction of Aldehydes
and Ketones. They are generally distinguished by the following tests.

3.3. Tollen’s test

3.4. Sodium nitroprusside test
3.5. Iodoform test (Alcohols)

3.1 . 2,4-dinitrophenyl hydrazine test:

15
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Aldehydes and Ketones react with 2,4-dinitrophenylhydrazine give a YELLOW to
ORANGE precipitate. The chemical reaction is given below.

Materials Required, ✓ 2,4-DNPH reagent: Reagent: 0.5 g of the 2,4-dinitrophenyl hydrazine is dissolved in 20 ml
Preparation: conc. HCl also dilute with water to 100 ml.

Precautions: ❖ 2,4‑Dinitrophenylhydrazine (2,4-DNPH) is harmful if absorbed through the skin. Wear gloves
and avoid contact.

Procedure: 1. Dissolve 1 drop (0.01 g) of the unknown in 0.5 mL of Ethanol in a clean micro test
tube.
2. Add 3 drops of 2,4-DNPH reagent.
3. Shake the mixture well.

Observations: Formation of a crystalline ORANGE-YELLOW precipitate indicates an aldehyde or ketone. The

color of the precipitate may give a clue to the structure of the carbonyl compound (unconjugated
aliphatic aldehydes and ketones usually yield a YELLOW precipitate, while aromatic and α, β-
unsaturated aldehydes and ketones yield an ORANGE-RED precipitate).

Questions

3.2 . Sodium Bisulfite (NaHSO3) Test:

Aldehydes and Ketones combine with Sodium bisulfite to for well-crystallized water-
Note:
Acetophenone and soluble products known as “Aldehyde bisulfite” and “Ketone bisulfite". The chemical
Benzophenone do not reaction is given below.
give this test.

16
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Materials Required: ✓ 10% Sodium bisulfite (NaHSO3)
Precautions: ❖ The reagents should be freshly prepared to perform the test.
Procedure: 1. Dissolve 1 drop (0.01 g) of the unknown in 0.5 mL of Ethanol in a clean micro test
tube.
2. Add 3 drops of 10% Sodium bisulfite solution.
3. Shake well and leave it for 15-20 minutes.
Observations: Formation WHITE precipitate confirms the presence of Carbonyl group.

Questions

3.3 . Tollen’s Test (Silver Mirror Test): (Aldehydes)

Tollen's reagent consists of Silver Ammonia complex in Ammonia solution. Aldehydes

reacts with Tollen's reagent gives a GREY-BLACK precipitate or a Silver mirror.
Aldehydes are oxidised to the corresponding acid and Silver in Tollen's reagent is reduced
from +1 oxidation state to its elemental form. Generally, Ketones do not respond to this
test.

Materials Required: ✓ Tollen's reagent: (Transfer 10 drops of 5% AgNO3 and 2 drops of 1N NaOH to a test tube. A
Preparation of BROWN–BLACK precipitate should form. With very thorough stirring add one drop of 10%
Reagents: NH3(aq.). The BROWN precipitate should dissolve, if not add another drop of 10% NH3(aq.).
and stir thoroughly. No more than 3 drops of 10% NH3(aq.). should be necessary to dissolve
the BROWN precipitate. Avoid adding an excess of concentrated NH3(aq.).

Precautions: ❖ Silver nitrate is corrosive and toxic. Wear gloves and avoid contact.
❖ The reagents should be freshly prepared to perform the test.
❖ Not to heat the reaction mixture directly on the flame.
❖ After performing the Tollen’s test wash the test tube with Nitric acid to destroy the Silver
mirror, because (highly explosive when dry, may form if Tollen’s reagent is stored or
improperly disposed.

Procedure: 1. Add 5 drops of freshly prepared Tollen’s reagent to a clean micro test tube
2. Add 2 drops (0.01 g) of the Unknown.
3. Heat the mixture in a water bath gently for about 10 minutes.
Observations: Formation of a Silver mirror or separation of dark BROWN-BLACK precipitate indicates the
presence of Aldehyde.

Questions This test is given by reducing sugars. Why?

17
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 3.4 . Sodium Nitroprusside Test: (Ketones)

Ketone reacts with alkali forms an anion further it reacts with Sodium nitroprusside forms
a colored complex ion. Aldehydes do not respond to this test. The chemical reaction is
given below.

Materials Required, ✓ 0.5% Sodium nitroprusside reagent: Dissolve 0.5 g of sodium nitroprusside
Preparation: Na2[Fe(CN)5(NO)].2H2O in 40 ml of Water and add 10 ml of 1N NaOH. dilute with water to
100 ml.

Precautions: ❖

Procedure: 1. Dissolve 1 drop (0.01 g) of the unknown in 0.5 mL of Water in a clean micro test tube.
2. Add 2 drops of 0.5 % freshly prepared alkaline solution of Sodium nitroprusside
3. Shake well
Observations: RED colouration confirms the presence of Ketone.

Questions

3.5 . Iodoform test: (Ketones)

The iodoform test indicates the presence of an Aldehyde or Ketone in which one of the
groups directly attached to the carbonyl carbon is a methyl group. Such a Ketone is called
a methyl Ketone.

Disadvantages: Some compounds that can be easily oxidized to methyl Ketones give also positive results,
The Iodoform reagent reacts with methyl Alcohols such as 2-propanol, 2-butanol and Ethanol which are
oxidized in the presence of the Iodoform reagent to methyl Ketones.

Materials Required, ✓ Iodine solution: (Dissolve 3 g of Potassium iodide (KI) in 40 ml of Water. To that solution
Preparation: add 1g of solid Iodine (I2) and dilute the solution to 100 ml).
✓ 1N NaOH
Precautions: ❖ Iodine should not be inhaled as it can cause irritation to nose.
❖ The iodine solution color is brown and should be stored in a dark place.

18
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Procedure: 1. Add 1 drop (0.01 g) of the Unknown to a clean micro test tube.
2. Add 2 drops of 1N NaOH (aq.), shake the contents.
3. Add the Iodine solution drop wise until the DARK Iodine color persists. If a
precipitate is not present within 3 to 5 minutes, warm the solution slightly (~60 °C);
if the color fades, add more iodoform reagent until the color remains for 1-2 minutes.
Then add 1-2 drops 1N NaOH to remove excess Iodine (become colorless) and dilute
the mixture with cold water.

Observations: Formation YELLOW precipitate confirms the presence of Ketone.

Questions

4 . CARBOXYLIC ACIDS

Carboxylic acids are organic compounds containing carboxyl functional group. It

is of two types aliphatic and aromatic. Aliphatic acids are soluble in water where
as aromatic acids are sparingly soluble in water. Formic acid and acetic acid are
the simplest aliphatic acid and benzoic acid is the simplest aromatic acid. Formic
THEORY
acid and acetic acid are liquids. Carboxylic acids such as benzoic acid, oxalic acid,
phthalic acid, tartaric acid etc. are colourless crystalline solids.
Carboxylic acids tend to donate protons and act as acids. It is this property which
is helpful in the identification of a -COOH group.

Any of the following test can be carried out to detect the Carboxylic functional
group.

4.1. Sodium bicarbonate test (NaHCO3)

TESTS 4.2. Ester test
4.3. Fluorescein test

Note: Litmus test: If the colour of the universal litmus paper changes to RED then
Carboxylic group is present. Phenol also gives this test.

4.1 . Sodium Bicarbonate Test:

19
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 When Carboxylic acid reacts with Sodium bicarbonate solution carbon dioxide is evolved
with a brisk effervescence along with Sodium acetate is formed. The chemical reaction is
given below.

This test is used to distinguish between Carboxylic acid from Phenol. Phenol does not answer to
this test.

Materials Required: ✓ 5% NaHCO3

Precautions: ❖ In Sodium bicarbonate test use acid free Alcohol so that it should not interfere with the
functional group of the given organic compound.
❖ The addition of Sodium bicarbonate should be carried out slowly so that the effervescence is
visible clearly.

Procedure: 1. Dissolve 2 drop (0.01 g) of the unknown in 0.5 mL of Ethanol in a clean micro test
tube.
2. Add a few drops of 5% NaHCO3,
Observations: Evolution of brisk effervescence confirms the presence of Carboxylic acid.

Questions What causes brisk effervescence in Sodium bicarbonate test?

4.2 . Ester Test:

Carboxylic acid reacts with Alcohol in the presence of concentrated Sulfuric acid forms a
pleasant-smelling Ester. This reaction is known as Esterification. The chemical reaction
is given below.

Materials Required: ✓ Ethanol

✓ Conc. H2SO4
✓ 1N NaOH
Precautions: ❖

Procedure: 1. Add 2 drops (0.01 g) of the Unknown to a clean micro test tube.
2. Add 5 drops of Ethanol and 1 drop of conc. H2SO4
3. Heat the mixture in a water bath gently for about 2 minutes.
4. Pour the mixture into another test tube carefully containing 0.5 ml of Water
5. Add a few drops of 1N NaOH.

20
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Note: The resultant solution should be Neutralize.

Observations: Formation of a sweet-smelling compound indicates the presence of Carboxylic group.

Questions How can manage this test to identify Alcohols?

4.3 . Fluorescein Test:

Note: Dicarboxylic acids
like Oxalic acid,
Terephthalic acid and
Isophthalic acid do not give
fluorescein test.
This test should be performed only if the compound gives positive results in litmus test
and Sodium bicarbonate test. This test is given by Dicarboxylic acid. Dicarboxylic acid
on heating gives Acid anhydride. When this Anhydride is treated with Resorcinol in the
presence of concentrated Sulfuric acid a fluorescent dye is formed and so this reaction is
called Fluorescein test.

Materials Required: ✓ Conc. H2SO4

✓ Resorcinol
✓ 1N NaOH

Precautions: ❖

Procedure: 1. Add 2 drops (0.01 g) of the Unknown to a clean micro test tube.
2. Add 0.01 g of Resorcinol and 2 drops of H2SO4
3. Heat the mixture in a water bath gently for about 2 minutes.
4. Pour the mixture into another test tube carefully containing 0.5 ml of 1N NaOH.
Note: The resultant solution should be alkaline

Observations: GREEN colour fluorescent solution confirms the presence of acid group.

Questions

5 . ESTERS and ANHYDRIDES

An Ester is a chemical compound derived from an acid (organic or inorganic) in

which at least one –OH hydroxyl group is replaced by an –O– alkyl (alkoxy) group,
THEORY as in the substitution reaction of a Carboxylic acid and an Alcohol.
An acid anhydride is a compound that has two acyl groups bonded to the same
oxygen atom.

21
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 The following test can be carried out to detect the Esters functional group.

TESTS 1.1. Alkaline Hydrolysis (Saponification).

1.2. Ferric Hydroxamate (Feigl Test)
1.3. Aniline Test: (Anhydrides)

5.1 . Alkaline hydrolysis (Saponification):

Saponification is the hydrolysis of an Ester under basic conditions. The direct

products are a Carboxylic acid salt and an Alcohol.

Esters with boiling points higher than 200 °C may be unreactive

Materials Required, ✓ 1N NaOH

Preparation: ✓ ph.ph Indicator: Dissolve 1 g of the solid in 100 mL of Ethanol.
Precautions: ❖ Sodium hydroxide is toxic and corrosive, causing severe damage to skin, eyes, and mucous
membranes. Wear gloves and avoid contact.

Procedure: 1. Mix 3 drops of 1N NaOH and 2 drops of ph.ph Indicator in a clean test tube. The
solution became PINK.
2. Add 2 drops (0.01 g) of the Unknown.
3. Heat the mixture in a water bath gently for about 2 minutes.

Observations: Disappearance of PINK color indicates the presence of Ester.

Questions Why the Pink color is disappeared?

5.2 . Ferric hydroxamate (Feigl Test):

22
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 This test used for Acid Anhydride, Acyl Halide, and Ester. Primary and secondary nitro
compounds, imides, some amides, most nitriles, and Aldehydes (with no a-hydrogens give
a positive test).

Materials Required, ✓ 0.5M Hydroxylamine hydrochloride (NH2OH·HCl): Dissolve 3.5 g Hydroxylamine

Preparation: hydrochloride in 100 mL of Ethanol. (Strong reducing agent).
✓ 1N NaOH
✓ 1N HCl
✓ 2% FeCl3

Precautions: ❖ Hydroxylamine hydrochloride is toxic and mutagenic and it can cause a form of anemia. Avoid
contact with its solution. Sodium hydroxide is toxic and corrosive, causing severe damage to
skin, eyes, and mucous membranes. Wear gloves and avoid contact with the NaOH solution.

Procedure: 1. Dissolve 1 drop (0.01 g) of the unknown in 0.5 mL of Water in a clean micro test tube.
2. Add 3 drops of 0.5 M hydroxylamine hydrochloride and 2 drops of 1N NaOH. Then
heat the solution in a water bath gently for about 2 minutes.
3. After the solution has cooled slightly, cautiously add 2 drops of 1N HCl.
4. Add 2 drops of 2% FeCl3 and Observe the color produced. If the color does not persist,
continue to add dropwise.

Observations: VIOLET/WINE/MAGENTA coloration of the Ferric hydroxamate complex is a positive test for
Esters.

Questions

5.3 . Aniline Test: (Anhydrides)

When Aniline reacts with Acetic anhydride by using an acetylation reaction then
Acetanilide and Acetic acid are formed. Acetamides are usually crystalline solids which
can be helpful during the observation.

23
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Materials Required: ✓ Benzene or Chloroform (CHCl3)
✓ Aniline
✓ Conc. Acetic acid.

Precautions: ❖ Aniline, benzene, and Chloroform are harmful.

Procedure: 1. Add 2 drops (0.01 g) of the Unknown to a clean micro test tube.
2. Add 5 drops of Benzene or Chloroform
3. Add 2 drops of Aniline, followed by addition 1 drop of conc. Acetic acid.
4. Heat the mixture in a water bath for 1-2 minutes.
5. Cool the Mixture in a cold water
Observations: Precipitate is obtained (if no ppt. is formed, add 2 drops of cold water or few chunks of ice).

Questions Design another test for anhydrides.

6 . PHENOLS

Phenol is a hydroxyl group (-OH) on an aromatic ring or simply the hydroxy

derivatives of aromatic compounds are known as Phenols. Phenols are weaker
THEORY:
acids than Carboxylic acids. It undergoes substitution reaction easily. Phenol is
one of the most versatile and important industrial organic chemicals.

The following test can be carried out to detect the Phenolic functional group.

6.1. Ferric chloride test

6.2. Phthalein dye test

TESTS Note:
Litmus test: If the colour of the universal litmus paper changes to RED then Phenol
is present. Carboxylic group also gives this test. Compare to Carboxylic acid
Phenol is weakly acidic and it does not give an effervescence with aqueous Sodium
carbonate.

24
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 6.1 . Ferric Chloride Test:

Aqueous solution of Phenol reacts with freshly prepared Ferric chloride solution gives
coloured complex. Most Phenols give DARK coloured solution. The chemical reaction is
given below.

Materials Required: ✓ 2% FeCl3

Precautions: ❖ 2% FeCl3 solution used should be freshly prepared should be neutral and very dilute.
❖ Phenol should be handled with care because it is toxic and corrosive nature.

Procedure: 1. Dissolve 1 drop (0.01 g) of the unknown in 0.5 mL of Water in a clean micro test
tube.
2. Add neutral solution of 2% FeCl3 slowly dropwise.
3. Observe the change in colour.
The colours produced by simple Phenolic compounds with ferric chloride solution is listed below.

Observations: Phenol, Resorcinol, o-Cresol, p-Cresol,

Catechol Pyrogallol
Hydroquinone

VIOLET or BLUE colouration GREEN colouration RED- BROWN

Questions

6.2 . Phthalein Dye Test:

Phenol on heating with Phthalic anhydride in the presence of concentrated Sulfuric acid
forms a colourless condensation compound called Phenolphthalein. On further reaction
with dilute Sodium hydroxide solution gives a PINK colour Fluorescent compound called
Fluorescein.

25
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Materials Required: ✓ Conc. H2SO4
✓ 1N NaOH
✓ Phthalic anhydride

Precautions: ❖ Phenol should be handled with care because it is toxic and corrosive nature.
❖ Concentrated acids should be handled with care.

Procedure: 1. Add 2 drops (0.01 g) of Unknown sample to a clean micro test tube.
The colours produced by 2. Add 0.01 g of Phthalic anhydride to it.
different Phenolic
3. Add 1 drops of Conc. H2SO4 to the mixture.
compounds in phthalein
dye test is listed below. 4. Heat the solution in a water bath for 2-3 minutes.
5. Cool the mixture and pour it into another test tube containing 1drop 1N NaOH.
5. Dilute the whole mixture with 1 ml of water.
6. Observe the change in the colour in a white background.
7. If Fluorescence colour exists the view it in a black background.
Observations: Phenol, o-Cresol m-Cresol 1-Naphthol,2-Naphthol Resorcinol Hydroquinone

RED-PINK BLUE - VIOLET GREEN YELLOW-GREEN deep PURPLE

Questions

7 . AMINES

Amines are classified as primary, secondary and tertiary Amines. Primary Amines
are the one in which the nitrogen atom is bonded to one hydrocarbon atom and two
THEORY: hydrogen atoms. Secondary Amines are bonded with two hydrocarbon atom and
one hydrogen atom. Tertiary Amines are Amines in which all the hydrogen atoms
are replaced by hydrocarbon atoms.

The following are the tests carried to identify the presence of amino group
present in the organic compound.

8.1. Carbylamine test

8.2. Libermann’s test (Nitrous acid test / Azo-dye test
TESTS 8.3. Hinsberg test

NOTE: Amines are organic compounds which is basic in nature, so they dissolve in
mineral acids like Hydrochloric acid. But this test is not a confirmation test for Amines.

26
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Amines are basic in nature. So, it turns Universal litmus paper BLUE. This test is
also not a confirmation test for Amines.

7.1 . Carbylamine test:

Note: Both aliphatic and This test is also called Isocyanide test, because when Amines are treated
aromatic Amines give
positive for this test. with Chloroform in the presence of alkali, Isocyanide is formed.
Secondary and tertiary
Amines do not give this
test.

Materials Required: ✓ 1N NaOH:

✓ Chloroform
Precautions: ❖ Isocyanides are highly poisonous, do not smell the gas directly from the mouth of the test tube.
❖ The reaction is extremely sensitive! Caution! Isonitriles are very poisonous! The reaction
conducted under the hood! At the end of the work, the remains must decompose with
concentrated hydrochloric acid.

Procedure: 1. Add 2 drops (0.01 g) of Unknown sample to a clean micro test tube.
2. Add 5 drops of Chloroform and shake well.
3. Add 2 drops of 1N NaOH
4. Heat the solution in a water bath gently for about 2 minutes.
5. Observe the odor of the resulting mixture.
Observations: An unpleasant odor confirms the presence of primary Amine.

Questions

7.2 . Libermann’s test (Nitrous acid)/ Azo-dye test:

Complications: This test can be used to identify primary, secondary, and tertiary Amines.
Nitrous acid will react with
amides and phenols.
Most of the Phenols that do
not have substituents in the Primary aliphatic Amines react with Nitrous acid to give Nitrogen gas, which is seen as
para position, reacted with
Nitrous acid to form the bubbles.
p-nitrozophenols that in the
presence of concentrated
Sulfuric acid is condensed
with excess Phenol to form
intensely colored reaction
products.

Compounds having a
Secondary Amines react with Nitrous acid to form a YELLOW oily Nitrosamine.
methylene group adjacent
to a carbonyl group give a
positive test.

27
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Alkyl mercaptans yield red
thionitroso compounds.

Tertiary Amines react with Nitrous acid to form soluble Nitrite salts.

Aromatic Amines form a diazonium salts when treated with Nitrous acid HNO2. These
diazo compounds couple with Phenols like β-naphthol's forms an ORANGE azo dye
which is insoluble in water, and at higher temperatures the formed diazonium compounds
undergoes decomposition.

Materials Required, ✓ 1N HCl

Preparation: ✓ 1N NaNO2 (7%)
✓ 10% 2-Naphthol: Dissolve 10 g of 2-Naphthol in 100 ml of 1N NaOH.

Precautions: ❖ Nitrosamines are highly toxic; they do not have to get on your skin!
❖ Temperature for azo dye test should be maintained between 0-5oC.

Procedure: I. 1 o,2 o and 3o aliphatic Amines

1. Add 2 drops (0.01 g) of Unknown sample to a clean micro test tube.
2. Add 5 drops of 1N HCl solution.
3. Cool the solution in ice bath for 0-5oC.
4. To the cooled solution add 3 drops of 1N NaNO2 and mix well.
5. Test the solution for the presence of free nitrous acid.

II. Aromatic Amines:

1. Add 2 drops (0.01 g) of Unknown sample to a clean micro test tube.
2. Add 5 drops of 1N HCl solution.
3. Cool the solution in ice bath for 0-5oC.
4. To the cooled solution add 3 drops of 1N NaNO2 and mix well.
5. Keep the solution in the Ice bath and add 2 drops of 10% β-Naphthol alkaline solution
6. Stir the mixture with a glass rod and observe the changes in the test tube.

Observations: I. Appearance of bubbles shows the presence of primary Amines, YELLOW oily liquid shows
the presence of secondary Amines and formation of Nitrite salts conforms the presence of tertiary
Amines.

28
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 II. The presence of RED-ORANGE dye conforms the presence of aromatic Amines.

Questions

7.3 . Hinsberg test:

This test is useful if you have already established that you have an Amine. The reagent
used in this test is Benzenesulfonyl chloride. Amines reacts with Benzenesulfonyl chloride
in the alkaline medium.
Primary Amines react with Benzene sulfonyl chloride to produce substituted Sulfonamide
which contains an acidic hydrogen and dissolve in basic medium.

A secondary Amine forms a substituted sulfonamide which is insoluble in alkali because

it does not have acidic hydrogen.

A tertiary Amine

Materials Required: ✓ 5N NaOH

✓ Benzenesulfonyl chloride
✓ Conc. HCl

Precautions: ❖ Benzenesulfonyl chloride is very irritating to skin, eyes and mucous membranes. May emit
toxic fumes when heated to high temperatures. Use gloves and work in a fume hood or under
a snorkel.

Procedure: 1. Add 2 drops (0.01 g) of Unknown sample to a clean micro test tube.
2. Add 2 drops of 5N NaOH solution
3. Add 2 drops of Benzenesulfonyl chloride to it.

29
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 4. Shake the mixture well and add 0.5 ml of water to dilute the solution.
5. Cool the mixture under tap water and observe the formation of precipitate.
6. If precipitate formed then treat it with conc. HCl.

Observations: Primary Amines: Secondary Amines: Tertiary Amines:

No reaction or precipitate, on adding conc. Precipitate in test tube Precipitate in the test tube but dissolves in
HCl insoluble material is seen. but does not dissolve in conc. HCl.
conc. HCl.

Questions

8 . ETHERS

Ethers (R-O-R') as a class of compounds are relatively unreactive and only slightly
THEORY: polar. The Ether oxygen is slightly nucleophilic and will form a complex on
treatment with Iodine, and can be protonated by concentrated Sulfuric acid.

The following test can be carried out to detect the Ether's functional group.

TESTS
8.1. Zeisel test
8.2. Acidic Hydrolysis

8.1 . Zeisel test:

Zeisel test is a chemical test for the presence of Esters or Ethers in a chemical substance.
The ensuing reaction results in the cleavage of the Ether or the Ester into an alkyl iodide
and respectively an Alcohol or a Carboxylic acid. by distilling the Iodomethane into a
solution of Silver nitrate, which precipitates Silver iodide.

Materials Required: ✓ conc. Acetic acid

✓ conc. Hydrogen iodide
✓ 5% AgNO3

Precautions: ❖

30
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Procedure: 1. Add 0.5 ml of Unknown sample to a clean micro test tube.
2. Add 2 drops of conc. Acetic acid
3. Add 2 drops of conc. Hydrogen iodide to it.
4. Distillate the Iodomethane into a solution of Silver nitrate.

Observations: YELLOW Precipitation of Silver iodide.

Questions

8.2 . Acidic Hydrolysis:

Ether undergoes acidic hydrolysis to form Alcohol, and this can be tested easily in laboratories.
Firstly, on hydrolyzing Ethoxy ethane under acidic conditions, we get Ethanol, and by putting a
few drops of Acetic acid to it, we can get a fruity smelling compound characteristic of Ester ethyl
acetate. Thus, this two steps test can be used to identify that the starting compound was an Ether,
i.e., ethoxy ethane.

Materials Required: ✓ conc. Acetic acid

✓ conc. H2SO4

Precautions: ❖

Procedure: 1. Add 2 drops of Unknown solution to a well plate.

2. Add 2 drops of conc. H2SO4 to it.
3. Add 2 drops of conc. Acetic acid
Observations: Fruity smelling

Questions Does it possible to apply alcohol's tests, instead of addition of Acetic acid?

9 . CARBOHYDRATES

Carbohydrates are combinations of the chemical elements carbon and hydrogen

plus oxygen.
THEORY: Carbohydrates are usually neutral, mono and di-Saccharides are solid and dissolve
in Water, but polysaccharides usually do not dissolve in water The -OH groups of
carbohydrates are primarily responsible for their solubility.

TESTS The following are the tests to identify the presence of Carbohydrates.

31
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 9.1. Molisch’s test
9.2. Benedict’s test
9.3. Iodine test

9.1 . Molisch’s Test:

Molisch’s test is a general test for Carbohydrates. This test is given by almost all the

Note: To give a positive

Carbohydrates. In this test, concentrated Sulfuric acid converts the given Carbohydrate
test, the carbohydrate must into Furfural or its derivatives, which react with α-naphthol to form a PURPLE-colored
have at least five carbons.
(It is so because it involves product. The chemical reaction is given below.
the formation of furfural
derivatives that contain five
carbon atoms).
Proteins and lipids having
an attached carbohydrate
can also give this test
positive.

Materials Required, ✓ Molisch’s reagent: Dissolve 5 gm of 1-Naphthol in 100 ml Ethanol.

Preparation: ✓ Conc. H2SO4

Precautions: ❖ Molisch's Reagent is highly Flammable due to alcohol content.

❖ Store the prepared reagent in a bottle with tight fitting cap and away from open flames
❖ Don’t add too much Molisch’s reagent.
❖ Don’t pour sulfuric acid directly into the solution. Otherwise, charring of carbohydrates will
occur and a black ring will be formed, giving a false negative test.

Procedure: 1. Dissolve 0.01 g of the unknown in 0.5 mL of Water in a clean micro test tube.
2. Add 2 drops of Molisch reagent slowly.
3. Add 1 drop of conc. H2SO4 along the sides of the test tube.
4. The acid layer forms a layer at the bottom. Note the junction of the two layers.

Observations: Glucose Starch Sucrose Lactose

PURPLE ring PURPLE ring PURPLE ring PURPLE ring

Questions How Oligosaccharides and polysaccharides give positive result for this test?
Does Glycerin give positive Molisch test?

32
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 9.2 . Benedict’s test:

This test is based on the ability of reducing sugars to undergo oxidation in alkaline
solutions. In the presence of an alkali, Sodium carbonate converts Glucose to enediol
(reducing sugars undergo tautomerization to form enediols). These enediols reduce the

Note: Aldehydes will react
with the Cu2+ from the
Copper(II) sulfate to form
copper(I) oxide
Most Ketones and aromatic
Aldehydes will not react.
Cupric ions (Cu+2) to form Cuprous ions (Cu+). The Cuprous ions from cuprous hydroxide.
Upon heating, it is converted to Cuprous oxide that forms RED precipitates.
The Citrate ions present in the reagent release the Cuprous ions slowly for reduction and
prevent the formation of Cu(OH)2 until the oxidation-reduction process is completed.
It is a test for reducing sugars. Carbohydrates having free functional group, that is not
involved in a glycosidic bond, give this test positive. All monosaccharides and
disaccharides except sucrose give positive Benedict’s test. This test is negative for
polysaccharides.

Materials Required, ✓ Benedict’s reagent: Mix 2 g of Copper sulfate pentahydrate (CuSO4.5H2O), 10 g of Sodium
Preparation: carbonate (Na2CO3), and 15 g of Sodium citrate in 100 ml distilled water.

Precautions: ❖

Procedure: 1. Dissolve 0.01 g of the unknown in 0.5 mL of Water in a clean micro test tube.
2. Add 5 drops of Benedict’s reagent to it.
3. Heat the solution in a water bath gently for about 2 minutes.
4. Cool the solution and observe the solution.

Observations: Glucose Starch Sucrose Lactose

RED precipitate No precipitate No precipitate RED precipitate

Questions How Benedict’s test gives positive for aldehydes?

Predict the product formed when cyclohexane carbaldehyde reacts with Benedict’s reagent.
Give some examples of reducing sugars.

9.3 . Iodine test:

33
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 This test is specific for polysaccharides. Starch reacts with Iodine solution forms
complex BLUE colour solution. On heating the BLUE colour disappears and on cooling
the BLUE colour reappears. The colour varies with the concentration of the starch.

Materials Required, ✓ Iodine solution: Dissolve 3 g of Potassium iodide (KI) in 40ml of Water. To that solution add
Preparation: 1g of solid Iodine (I2) and dilute the solution to 100 ml.

Precautions: ❖ The iodine solution color is brown and should be stored in a dark place.

Procedure: 1. Dissolve 0.01 g of the unknown in 0.5 mL of Ethanol in a clean micro test tube.
2. Add 2 drops of Iodine solution.
Observations: Amylase or Starch Dextrin Glycogen

BLUE REDDISH-PURPLE REDDISH-BROWN

Questions Does Cellulose give positive Iodine test result?

10 . AMINO ACIDS

Protein has high molecular mass long chain polymer composed of α-amino acids. The
amino acids are crystalline solids with surprisingly high melting points. It is difficult to
THEORY:
pin the melting points down exactly because the amino acids tend to decompose before
they melt. Decomposition and melting tend to be in the 200 - 300°C range.

The following are the tests carried to find the presence of Amino acids in a
given sample.

TESTS
10.1. Ninhydrin test
10.2. Copper Complex (Biuret Test)
10.3. Xanthoproteic test

10.1 . Ninhydrin Test:

34
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
 Amino acids reacts with Ninhydrin give coloured solution from deep blue to violet.

Note: For ammonia, primary/secondary amines, and amino acids, deep purple colour is obtained.
For hydroxyproline and proline, a yellow colour is obtained.
For asparagine, brown colour is obtained

Materials Required, ✓ 1% Ninhydrin reagent: Dissolve 1 g of Ninhydrin in 100 ml Ethanol. But this solution of
Preparation: ninhydrin is unstable and can be kept for two days.

Procedure: 1. Dissolve 0.01 g of the unknown in 0.5 mL of Water in a clean micro test tube.
2. Add 2 drops of 1% Ninhydrin reagent to it.
3. Heat the solution in a water bath gently for about 5 minutes.

Observations: VIOLET-BLUE colouration confirms the presence of Proteins.

Questions Which Amino acid does not react with ninhydrin? Draw it is structure.
Why is Ninhydrin used for fingerprints?

10.2 . Copper Complex (Biuret Test):

In the presence of peptides, a Copper (II) ion forms VIOLET-BLUE colored

coordination complexes in an alkaline solution.

Materials Required: ✓ 10% CuSO4.5H2O

✓ 1N NaOH

Procedure: 1. Dissolve 0.01 g of the unknown in 0.5 mL of Water in a clean micro test tube.
2. Add 3 drops of 1N NaOH solution to it.
3. Add 3 drops of 10% CuSO4.5H2O solution to it.
Observations: VIOLET-BLUE colouration confirms the presence of Proteins.

Questions Could Fehling's test be used instead of Copper Complex test?

10.3 . Xanthoproteic Test:

35
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq

Note: It is a specific test
for detecting aromatic
amino acids that contain
active benzene ring or
aromatic nucleus.
Materials Required:
Precautions:
Procedure:
Observations:
Questions:
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq
Treating Aromatic amino acids with the concentrated Nitric acid leads to the Nitration of
the aromatic ring and formation of YELLOW nitro-products (nitro derivatives). When
the strong basic solution is added the colour of obtained products turns darker (from
YELLOW to ORANGE). The positive result of Xanthoproteic reaction gives: Tyrosine,
Tryptophan and Phenylalanine (only after extended heating time). It is difficult to Nitrate
Phenylalanine under normal conditions so it does not respond to this test without extended
heating.
✓ Conc. HNO3
✓ 5N NaOH
❖ Concentrated acids should be handled with care.
1. Dissolve 0.01 g of the unknown in 0.5 mL of Water in a clean micro test tube.
2. Add 2 drops of Conc. HNO3.
3. Mix and heat (In case of protein solution, initially white precipitate appears due to
denaturation of protein, which turns yellow on heating).
4. Cool under tap water
5. Add 5 drops of 5N NaOH to make the solution alkaline
YELLOW-ORANGE colouration confirms the presence of Proteins.
36
 37
Department of Chemistry, Faculty of science and health,
Koya University, Koya KOY45, Kurdistan Region - F.R. Iraq