UNI T

52
Tests for functional groups,
separation and purification
of compounds

52.1 Specific chemical tests for functional

groups

In previous topics, you have met simple tests that provide information
about carbon compounds to be analysed.

Here are some examples:

• Is the compound acidic (suggests carboxylic acid)?

• Does the compound dissolve in water (suggests polar groups) or

not (suggests no polar groups)?

• Does the compound burn with a smoky flame (suggests high

carbon to hydrogen ratio, possibly aromatic) or non-smoky flame
(suggests low carbon to hydrogen ratio, probably non-aromatic)?

In this unit, you are going to look at specific chemical tests for
detecting the presence of the functional groups below:

• carbon-carbon double bond (C=C);

• alcohol functional group (–OH);

• aldehyde functional group (–CHO);

• ketone functional group ( C=O);

• carboxylic acid functional group (–COOH).

44 TOPIC 15 Analytical Chemistry

52.2 Test for carbon-carbon double bond

(C=C)

When bromine dissolved in 1,1,1-trichloroethane is added to a carbon

compound containing carbon-carbon double bond (C=C) at room
temperature, the orange bromine solution becomes colourless quickly.
This is because an addition reaction occurs.

C C + Br2 (in CH3CCl3) C C (in CH3CCl3)

Br Br

Aqueous bromine becomes colourless quickly when mixed with

a carbon compound containing carbon-carbon double bond. The test
for carbon-carbon double bond often uses aqueous bromine rather
than pure bromine.

C C + Br2 + H2O C C + HBr

Br OH

Method

• Add aqueous bromine dropwise to the carbon compound under

test slowly and shake the mixture.

• When added to a carbon compound containing carbon-carbon

double bond(s), the yellow-brown aqueous bromine becomes
colourless quickly (Fig. 52.1).

aqueous bromine

shake

alkene

Fig. 52.1 Add aqueous bromine dropwise to an alkene (left);

the yellow-brown aqueous bromine becomes colourless quickly (right)
UNIT 52 Tests for functional groups, separation and purification of compounds 45

52.3 Tests for the alcohol functional group

(–OH)

Oxidation by acidified aqueous solution of

potassium dichromate
Primary alcohols are oxidised by acidified aqueous solution of
potassium dichromate to aldehydes, which can be further oxidised
to carboxylic acids.
acidified K2Cr2O7(aq)
primary alcohol aldehyde
acidified K2Cr2O7(aq)
carboxylic acid

Secondary alcohols are oxidised by acidified aqueous solution of

potassium dichromate to ketones.
acidified K2Cr2O7(aq)
secondary alcohol ketone

Tertiary alcohols are not oxidised by acidified aqueous solution

of potassium dichromate.

Method

• Mix an acidified aqueous solution of potassium dichromate with

the carbon compound under test.

• Warm the mixture.

• A primary alcohol or a secondary alcohol turns the acidified

aqueous solution of potassium dichromate from orange to green
quickly (Fig. 52.2).

warm

primary alcohol or acidified

secondary alcohol K2Cr2O7(aq)

Fig. 52.2 When warmed with acidified aqueous solution of potassium dichromate,
primary or secondary alcohols turn the orange dichromate solution green
46 TOPIC 15 Analytical Chemistry

Oxidation by acidified aqueous solution of

potassium permanganate
Acidified aqueous solution of potassium permanganate oxidises primary
alcohols to carboxylic acids and oxidises secondary alcohols to ketones.

Method

• Mix an acidified aqueous solution of potassium permanganate

with the carbon compound under test.

• Warm the mixture.

• A primary alcohol or a secondary alcohol turns the acidified

aqueous solution of potassium permanganate from purple to
The permanganate ion is colourless◀ quickly (Fig. 52.3).
reduced to manganese(II)
ion. Concentrated solution of
Mn 2+ (aq) ion is pale pink in
colour. However, dilute solution
2+
of Mn (aq) ion seems to have
no colour at all. warm

primary alcohol or acidified

secondary alcohol KMnO4(aq)

Fig. 52.3 When warmed with acidified aqueous solution of potassium permanganate,
primary or secondary alcohols turn the purple permanganate solution
colourless

P
Practice 52.1

Phosphorus pentachloride (PCl5) can be used to test for the –OH group.

a) Describe what would be observed when phosphorus pentachloride is added to butan-1-ol.

b) A tertiary alcohol, X, is an isomer of butan-1-ol. Suggest a chemical test to distinguish X and butan-1-ol. State
the expected observation for each alcohol.
 UNIT 52 Tests for functional groups, separation and purification of compounds 47

Activity 52.1 52.4 Tests for the aldehyde and ketone

functional groups (–CHO and C=O)

Test with 2,4-dinitrophenylhydrazine

The reagent 2,4-dinitrophenylhydrazine reacts with carbonyl
compounds to form 2,4-dinitrophenylhydrazones, which are bright
red, orange or yellow precipitates.

For example, ethanal reacts with 2,4-dinitrophenylhydrazine according

to the equation below.

H3 C NO2 H3 C NO2

C O + H2N NH NO2 C N NH NO2 + H2O

H H
2,4-dinitrophenylhydrazine ethanal-2,4-dinitrophenylhydrazone

Method

• Add a solution of 2,4-dinitrophenylhydrazine to the carbon

compound under test.

• A carbonyl compound gives a bright red, orange or yellow

precipitate (Fig. 52.4).

mix with
carbonyl compound

2,4-dinitrophenylhydrazine 2,4-dinitrophenylhydrazone

Fig. 52.4 Carbonyl compound reacts with 2,4-dinitrophenylhydrazine to form an

orange 2,4-dinitrophenylhydrazone precipitate

2,4-dinitrophenylhydrazine 2,4-肼
48 TOPIC 15 Analytical Chemistry

Tests for aldehyde functional group (–CHO)

Aldehydes and ketones can be distinguished by a series of redox
reactions. Aldehydes are oxidised readily to carboxylic acids; ketones
are not oxidised easily.

Acidified aqueous solution of potassium

dichromate
Acidified aqueous solution of potassium dichromate turns from orange
to green when warmed with an aldehyde. The aldehyde is oxidised
to a carboxylic acid in the reaction.

Tollens’ reagent
Tollens’ reagent, also called ammoniacal silver nitrate solution,
contains the complex ion [Ag(NH3)2]+. It is a colourless chemical that
is made in a two-stage process:

1 Add sodium hydroxide solution to silver nitrate solution until a

brown precipitate is formed.

2 Add dilute aqueous ammonia dropwise until the brown precipitate

dissolves.

Tollens’ reagent is a weak oxidising agent and can react with an

aldehyde but not a ketone.

When an aldehyde reacts with Tollens’ reagent, the silver ion is

reduced and the aldehyde is oxidised.

RCHO + 2[Ag(NH3)2]+ + 3OH– RCOO– + 2Ag + 4NH3 + 2H2O

colourless carboxylate silver
solution ion mirror

The silver metal is observed as a silver mirror on the inside of

the reaction vessel.

Method

• Add a few drops of the carbon compound under test to 2 cm3 of

freshly prepared Tollens’ reagent in a test tube.

• Warm the mixture in a water bath at about 60 °C.

Tollens’ reagent
 UNIT 52 Tests for functional groups, separation and purification of compounds 49

• A silver mirror forms on the inside of the test tube if the carbon
compound is an aldehyde (Fig. 52.5).

warm with
an aldehyde

Tollens’ reagent silver mirror

Fig. 52.5 Warming Tollens’ reagent with an aldehyde gives a silver mirror

P
Practice 52.2

1 Two carbon compounds, X and Y, both with the molecular formula C5H10O, contain a carbonyl group.

a) Describe what you would observe when 2,4-dinitrophenylhydrazine is added to either of these compounds.

b) It is suspected that X is an aldehyde and Y is a ketone. Outline a chemical test you could carry out to
confirm this and describe the expected observation in each case.

c) Give the structural formulae of TWO possible isomers of Y which are ketones.

2 A carbon compound W reacts with 2,4-dinitrophenylhydrazine to form a yellow solid Z. The structure of Z is
shown below:
NO2
N
N H
NO2

Draw the structure of W.

50 TOPIC 15 Analytical Chemistry

52.5 Tests for the carboxylic acid

functional group (–COOH)

Test with aqueous solution of sodium

hydrogencarbonate
A carboxylic acid reacts with an aqueous solution of sodium
hydrogencarbonate, liberating carbon dioxide gas.

RCOOH + NaHCO3 RCOO– Na+ + CO2 + H2O

Method

• Mix the carbon compound under test with an aqueous solution

of sodium hydrogencarbonate.

• Effervescence occurs if the carbon compound is a carboxylic acid

(Fig. 52.6).

• The gas can be collected and bubbled into limewater which should
turn milky, proving that the gas produced is carbon dioxide.
Fig. 52.6 Effervescence occurs when
a carboxylic acid is mixed
with an aqueous solution of
sodium hydrogencarbonate Ester formation
A carboxylic acid can react with an alcohol to form an ester in the
presence of a concentrated acid catalyst.
H+
R1COOH + R2OH R1COOR2 + H2O

Method

• Mix the carbon compound under test with ethanol.

• Use concentrated sulphuric acid as a catalyst. Heat the mixture.

• A sweet, fruity smell from an ester can be detected if the carbon

compound is a carboxylic acid.
 UNIT 52 Tests for functional groups, separation and purification of compounds 51

P
Practice 52.3

Compound X (C4H8O2) reacts with hot dilute sulphuric acid. Compounds P (CH2O2) and Q (C3H8O) are produced.
Tests are performed on P and Q and the results are as follows:

Test 1 P gives effervescence with NaHCO3(aq).

Test 2 Q is oxidised by acidified K 2Cr 2O 7(aq). The oxidation product R gives an orange precipitate with
2,4-dinitrophenylhydrazine but gives a negative result when warmed with Tollens’ reagent.

a) With reference to Test 1, suggest ONE functional group that P may have.

b) i) What is the purpose of using Tollens’ reagent?

ii) State the expected observation if an oxidation product gives a positive result with Tollens’ reagent.

iii) Suggest one functional group that may be present in R.

c) Draw a possible structure of X. Explain your answer.

Activity 52.2 52.6 Specific chemical tests for detecting

the presence of functional groups

Table 52.1 summarises the specific chemical tests for detecting the
presence of functional groups discussed above.

Table 52.1 Specific chemical tests for detecting the presence of functional groups
52 TOPIC 15 Analytical Chemistry

E xamp e 52.1

O
O
X Y Z

Pr blem S lving
UNIT 52 Tests for functional groups, separation and purification of compounds 53

52.7 Common separation and purification

methods

The result of any organic reaction will be a mixture of substances. The

required product must be separated from the reaction mixture and
then purified. The separation and purification methods used depend
on whether the product is a solid or a liquid.

Table 52.2 lists the common separation and purification methods

for liquid products and solid products.

Table 52.2 Common separation and purification methods

for products
Type of product Separation and purification methods to employ
• distillation
Liquid product • fractional distillation
• liquid-liquid extraction
• recrystallisation
Solid product
• chromatography

52.8 Simple distillation

Simple distillation is a common method for separating a crude liquid

product from the reaction mixture if the liquid product has a much
lower boiling temperature than other substances in the mixture.
Fig. 52.7 shows the experimental set-up for simple distillation.

thermometer

water out
still head

condenser
pear-shaped
flask

water bath receiver adaptor

water in
reaction anti-bumping
mixture granules
heat conical flask

Fig. 52.7 Experimental set-up for simple distillation

54 TOPIC 15 Analytical Chemistry

Heat the mixture until it boils gently. When the vapour temperature
is approximately 2 °C below the boiling point of the liquid you are
about to collect, put the collecting flask in place. Collect the distillate
until the temperature of the vapour rises above the boiling point of
the liquid you are collecting. Stop heating.

Simple distillation can also be used to separate two volatile liquids

(with a large difference in boiling points) that form a solution.

52.9 Fractional distillation

Fractional distillation separates mixtures of liquids with close boiling

points. Fig. 52.8 shows the experimental set-up for fractional distillation.

thermometer

water out

still head
condenser
fractionating
column
receiver
adaptor
glass beads water in

distillate

water bath

a mixture of
miscible liquids anti-bumping
granules
heat

Fig. 52.8 Experimental set-up for fractional distillation

The column is filled with glass beads, acting as surfaces on which

the vapour from the mixture can condense and the condensed liquid
can vaporise when meeting hot vapour passing up the column.
 UNIT 52 Tests for functional groups, separation and purification of compounds 55

The vapour becomes richer and richer in the most volatile liquid
in the mixture as it rises up the column. The most volatile liquid
with the lowest boiling point distils over first, then the liquid with
the next lowest boiling point and so on.

P
Practice 52.4

In an experiment, ethanoic acid was prepared by heating ethanol with excess acidified K2Cr2O7(aq) under reflux.

a) What is meant by ‘heating under reflux’?

(Consider the physical changes that occur.)

b) Write the half equation for the reduction of dichromate ion.

c) What would be the colour of the reaction mixture after it was heated under reflux?

d) A solution containing both water and ethanoic acid was produced by distillation of the final reaction mixture.

i) Explain why the other products and any excess reactants were left behind in the distillation flask.

ii) Suggest a method to separate pure ethanoic acid (boiling point: 118 °C) from water.

52.10 Liquid-liquid extraction

Liquid-liquid extraction (also called solvent extraction) involves
using a solvent to extract the desired liquid product from the other
substances in the reaction mixture.

There are several solvents that can be used, but the choice depends
mainly on these features.

• The solvent added should be immiscible (i.e. not mix) with the
solvent containing the desired product.

• The desired product should have a higher solubility in the solvent

added than in the reaction mixture.

liquid-liquid extraction
56 TOPIC 15 Analytical Chemistry

Here is a summary of the process of liquid-liquid extraction.

1 Place the reaction mixture in a separating funnel, and then add

the chosen solvent — it should form a separate layer (Fig. 52.9).

separating funnel

solvent layer

reaction mixture

Fig. 52.9 During the extraction, add a solvent to the reaction Fig. 52.10 Hold the stopper and the tap firmly when shaking◀
mixture, giving two layers of liquids

During shaking, open the tap of 2 Place the stopper and gently shake the contents of the funnel for
the separating funnel regularly a while (Fig. 52.10).
to release any pressure that may
build up. 3 Allow the contents to settle into two layers.

4 Remove the stopper and open the tap to allow the lower layer
to drain into a flask. Then pour the upper layer into a separate
flask.

The desired product has been removed from the reaction mixture,
but is now mixed with the added solvent. You can use simple
distillation or fractional distillation to separate the desired product
from the solvent used.

If necessary, the product can be further purified by simple

distillation.
 UNIT 52 Tests for functional groups, separation and purification of compounds 57

E xamp e 52.2

X Y
58 TOPIC 15 Analytical Chemistry

P
Practice 52.5

The procedure below is used to extract caffeine from tea.

Step 1 Add 25 g of tea, 10 g of calcium carbonate and 250 cm3 of water to a large beaker.

Step 2 Boil the mixture gently for 15 minutes.

Step 3 Filter the mixture.

Step 4 Separate caffeine from the aqueous mixture using solvent extraction, with dichloromethane as the solvent.

Step 5 Dry the solution of caffeine in dichloromethane obtained in Step 4.

Step 6 Remove the dichloromethane.

(Density of dichloromethane = 1.32 g cm–3)

a) i) Draw a diagram of the apparatus used in Step 4, labelling the organic and aqueous layers that would be
observed at the end of Step 4.

ii) Outline how to carry out the solvent extraction in Step 4, including the necessary safety precautions.

b) Name a suitable drying agent for drying the solution of caffeine in Step 5.

c) Suggest a suitable way to remove the dichloromethane.

52.11 Recrystallisation
A solid product is removed from the reaction mixture by filtration.
It is then purified by recrystallisation. A solvent has to be found
in which the solid product is soluble when the solvent is hot, but
insoluble (or very much less soluble) when the solvent is cold.

Follow the steps below in the process (Fig. 52.11).

1 Dissolve the impure solid in the minimum volume of hot solvent.

2 Filter the hot mixture to remove any insoluble impurities. Retain

the filtrate.

3 Leave the filtrate to cool until crystals form, leaving the soluble
impurities in solution.

4 Collect the crystals by filtration.

 UNIT 52 Tests for functional groups, separation and purification of compounds 59

5 Wash the crystals with a small amount of cold solvent to remove

any of the filtrate which contains dissolved impurities.

6 Allow the solvent to evaporate from the crystals in the air or

place the crystals in a desiccator.

crystals
impure solid product insoluble impurities

filter paper
filter funnel
solvent

water

pure crystals forming

heat
1 2 3 4

crystals

crystals
or crystals
wash bottle
containing
filter paper drying agent
cold solvent

5 6

Fig. 52.11 Steps of recrystallisation

A minimum volume of hot solvent is used to dissolve the solid

product, making a saturated solution. The solution is cooled and the
solubility of the compound drops, causing it to recrystallise from the
solution. Impurities remain dissolved in the solution. A minimum
volume of hot solvent is used to ensure that as much of the solid
product is obtained as possible.
60 TOPIC 15 Analytical Chemistry

P
Practice 52.6

A crude sample of benzoic acid is purified by recrystallisation using water as a solvent. The steps are listed below.

Step 1 Dissolve the impure solid in the minimum volume of hot water.

Step 2 Filter the solution quickly.

Step 3 Allow the filtrate to cool.

Step 4 Filter the crystals formed from the remaining solution.

Step 5 Wash the crystals with a little cold water.

Step 6 Leave the crystals to dry.

Give reasons for each of the following steps.

a) The minimum volume of hot water is used in Step 1.

b) The filtrate is cooled in Step 3 before the crystals are filtered off.

c) The crystals are washed with cold water in Step 5.

52.12 Chromatography
In 1903, the Russian botanist Michel Tswett developed the technique
of chromatography to study plant pigments.

There are now a range of chromatography techniques that can

be used to

• separate and identify the components of a mixture of chemicals;

• check the purity of a chemical;

• identify the impurities in a chemical preparation;

• purify a chemical product.

 UNIT 52 Tests for functional groups, separation and purification of compounds 61

Principles of chromatography
All types of chromatography have a stationary phase and a mobile
phase.

• The stationary phase does not move and is normally a solid or

a liquid supported on a solid.

• The mobile phase does move, and is normally a liquid or a gas.

Chromatography depends on the movement of the mobile phase

through the stationary phase. A small amount of the mixture to be
analysed is added to the stationary phase. The mobile phase moves
through the stationary phase, carrying the components of the mixture
with it.

A mixture can be separated because the components distribute

themselves differently between the stationary and mobile phases,
according to their affinity for each phase.

In this unit, three types of chromatography will be studied:

• paper chromatography;

• thin layer chromatography; and

• column chromatography.

52.13 Paper chromatography (PC)

In paper chromatography, the stationary phase is the water trapped
in the fibres of the chromatography paper, and the mobile phase is
a developing solvent.

Producing a paper chromatogram

Dissolve the mixture to be analysed in a solvent. This solvent is
usually not the same as the mobile phase.

1 Spot the solution under test on a pencil line 1 cm from the base
of the chromatography paper. Pencil is used because it will not
run into the solvent.

stationary phase mobile phase paper chromatography

thin layer chromatography column chromatography
developing solvent
62 TOPIC 15 Analytical Chemistry

The lid prevents the evaporation 2 Add the developing solvent to a chromatography tank and covers
of the solvent. it with a lid◀. Let the tank stand for a while to allow the air inside
become saturated with the solvent vapour.

3 Place the prepared paper in the tank, checking that the spot is
above the level of the solvent (Fig. 52.12).

lid
glass rod to hang paper

strip of chromatography
paper

mixture

starting position
developing solvent

Fig. 52.12 A chromatography tank (the sample spot on the paper must be above the
level of the solvent)

4 The solvent rises up the paper, carrying the components of the

mixture with it.

5 Take the paper from the tank when the solvent gets near the
top. Mark the position of the solvent front. The resulting paper
is called a chromatogram (Fig. 52.13).

Fig. 52.13 A chromatogram

solvent front 沿 chromatogram

 UNIT 52 Tests for functional groups, separation and purification of compounds 63

solvent front
Interpreting a chromatogram
Suppose a brand of black ink is analysed using paper chromatography.
From the chromatogram shown in Fig. 52.14, you know that the black
ink contains three different dyes.

7.0 cm Separation of dyes occurs because of the different relative solubilities

of the dyes in the developing solvent (i.e. the mobile phase) and in
the water in the fibres of the paper (i.e. the stationary phase).

Dyes that are more soluble in the mobile phase than in the
stationary phase move rapidly up the paper, while those that are
more soluble in the stationary phase are not carried as far up the
2.4 cm paper (Fig. 52.15).

starting position
mixture of three
Fig. 52.14 A paper chromatogram dyes at start
obtained when a black ink
is analysed paper with water in fibres

developing solvent moves

developing solvent
paper with water in fibres

slow-moving dye more soluble fast-moving dye more soluble

in the stationary phase in the mobile phase

Fig. 52.15 The principles of paper chromatography

Identifying the component of a mixture by using

retention factor
The distance a component travels relative to the solvent is called the
retention factor (symbol: Rf).

distance travelled by the component

Rf =
distance travelled by the solvent

Fig. 52.14 shows that the red dye has travelled 2.4 cm from the
2.4 cm
starting position, so its Rf value = = 0.34.
7.0 cm

retention factor
64 TOPIC 15 Analytical Chemistry

Data tables list retention factors for a wide variety of chemicals,

using particular solvents under stated conditions. These allow the
identification of unknown components by comparison with these
retention factors.

Alternatively, spots of solutions of chemicals suspected of

being present in the unknown mixture can be run on the same
chromatography paper and a direct comparison can be made.

Making colourless components on a

Activity 52.3 chromatogram visible
Coloured components on a chromatogram are easy to see. Colourless
components can be made visible by using ultraviolet radiation or by
spraying with a chemical reagent that will react with the components
to form coloured products.

Ninhydrin forms a purple Fig. 52.16 shows a paper chromatogram of an amino acid mixture
compound with each amino after spraying with ninhydrin◀.
acid and makes each spot visible.

solvent front

amino acid mixture spot

starting position

Fig. 52.16 The components of an amino acid mixture are visible after spraying with
ninhydrin

A general visualising agent for most carbon compounds is iodine.

When the paper is placed in a tank containing iodine vapour, the
iodine is absorbed by the carbon compounds in the spots, turning
them brown.
 UNIT 52 Tests for functional groups, separation and purification of compounds 65

P
Practice 52.7

Some food colourings are a mixture of coloured substances. Paper chromatography can be used to separate the
coloured substances in food colourings.

A student carried out an experiment to test which food colouring was present in a drink. Samples of three food
colourings (X, Y and Z) were also used. The chromatogram obtained is shown below.

10 cm

solvent front

scale (cm)
5 cm

starting 0 cm
position X Y Z drink

a) Name the mobile phase and stationary phase in this paper chromatography.

b) Describe the principle underlying the separation of coloured substances in the food colourings by paper
chromatography.

c) State all the food colourings that contain more than one coloured substance.

d) Food colouring X is banned.

Explain how the student could tell that the drink tested did NOT contain the banned food colouring X.

e) Calculate the Rf value for the coloured substance in food colouring Y.

66 TOPIC 15 Analytical Chemistry

52.14 Thin layer chromatography (TLC)

Thin layer chromatography (TLC) is carried out in almost an identical
fashion to paper chromatography.

Thin layer chromatography uses a thin layer of either alumina

(Al2O3) or silica gel (SiO2), which is supported on a glass or plastic
plate. The thin layer is the stationary phase. The sample is spotted
onto the plate and placed in a developing solvent. The container is
covered with a lid (Fig. 52.17).

lid

TLC plate

mixture

developing solvent

Fig. 52.17 The apparatus used for thin layer chromatography (TLC)

Both alumina and silica gel can adsorb chemicals onto their
surfaces.

Components in the mixture separate because

• they differ in the extent to which they are soluble in the mobile
phase;

• they are adsorbed by the stationary phase to different extents.

The better adsorbed components travel more slowly up the TLC

plate.

Thin layer chromatograms are analysed by calculating the value

for the retention factor Rf for each component. Each component can
be identified by comparing its Rf value with known values recorded
using the same solvent system and stationary phase.

You can recover the separated components by scraping the areas

containing the spots into a suitable solvent.

Thin layer chromatography is quicker than paper chromatography

and can be used on smaller samples, making it useful in forensic
science, where it can be used to identify drugs and explosive residues.
 UNIT 52 Tests for functional groups, separation and purification of compounds 67

Activity 52.4

P
Practice 52.8

A student prepared aspirin from 2-hydroxybenzoic acid in the laboratory.

COOH COOH
OH OCOCH3

2-hydroxybenzoic acid aspirin

The crude aspirin was purified by recrystallisation. The student investigated the composition of both the crude and
recrystallised aspirin using thin layer chromatography (TLC).

Separate samples of 2-hydroxybenzoic acid, pure aspirin, the crude aspirin and the recrystallised aspirin were applied
to a chromatography plate. The chromatography was run.

The developed chromatogram is shown below.

solvent
front

key:
1 2-hydroxybenzoic acid
2 pure aspirin
starting CA crude aspirin
position 1 2 CA RP RP recrystallised aspirin

a) Describe how the student would carry out the experiment to produce the chromatogram.

b) Explain why the spots of 2-hydroxybenzoic acid and pure aspirin travel different distances up the chromatogram.

c) Analyse the chromatogram.

68 TOPIC 15 Analytical Chemistry

Pr blem S lving

52.15 Column chromatography (CC)

Column chromatography uses a powder, such as alumina or silica
gel as the stationary phase. This is packed into a narrow tube — the
column.

The mixture to be separated is dissolved in a suitable solvent

and placed on the top of the column. Fresh solvent is added to the
column. The solvent moves through the column and the components
in the mixture separate depending on their different solubilities in the
solvent (mobile phase) and their different adsorption by the stationary
phase. The process is called elution and the solvent added is called
the eluent.

elution eluent
 UNIT 52 Tests for functional groups, separation and purification of compounds 69

Fig. 52.18 shows the separation of two pigments in a green

pigment. Add eluent to the column until the bands have been eluted
and collect each component separately.

eluent
(mobile phase) eluent eluent eluent

sand
green pigment

later later later

alumina or silica gel

(stationary phase)
column
glass
wool

Fig. 52.18 Separation of two pigments in a green pigment by column chromatography

For colourless components, add fluorescent dyes to them and you

can see their movement under ultraviolet light.

Column chromatography has the advantage that fairly large

amounts can be separated and collected. It is not used to identify
the components of a mixture.
70 TOPIC 15 Analytical Chemistry

Table 52.3 summarises the stationary and mobile phases, and the
applications of the three types of chromatography discussed above.

Table 52.3 The stationary and mobile phases, and the applications
of three types of chromatography
Application
Type of chromatography Stationary phase Mobile phase Separation and Separation for
identification further use
Paper water in the paper fibres liquid solvent 4
thin layer of alumina
Thin layer or silica gel coated liquid solvent 4
onto a plate
alumina or silica gel in
Column liquid solvent 4
vertical glass column

E xamp e 52.3

Continued on next page

 UNIT 52 Tests for functional groups, separation and purification of compounds 71

P
Practice 52.9

1 The diagram shows the separation of pigments in a plant extract by column chromatography using hexane as
the eluent.

chlorophyll b

chlorophyll a

xanthophyll pigment
carotene pigment

glass wool

a) Briefly describe the principle underlying the separation of the pigments by using column chromatography.

b) Explain how changing the eluent would affect the separation process.

c) In another experiment, the pigments in plant extract are separated by using thin layer chromatography,
using the same stationary phase and the same mobile phase.

Which of the pigments would have the smallest Rf value? Explain your answer.

2 Rose petals contain a useful organic compound X. X can dissolve in warm hexane, and can be extracted from
the petals by using hexane.

X obtained from the extraction may contain other organic impurities. Suggest a method for separating X from
these impurities.
72 TOPIC 15 Analytical Chemistry

52.16 Choosing appropriate methods for

separating substances in a mixture

It is important that you choose the appropriate method for separating

the substances in a mixture.

For example, 1-chloropentane can be prepared by adding excess

phosphorus pentachloride to pentan-1-ol:

CH3(CH2)3CH2OH(l) + PCl5(s)
CH3(CH2)3CH2Cl(l) + HCl(g) + POCl3(l)

Phosphorus trichloride oxide, POCl3, has a boiling temperature

of 105 °C, which is very similar to that of 1-chloropentane (108 °C).
Therefore, it is not possible to separate the two by distillation of the
reaction mixture. Liquid-liquid extraction is needed for extracting
1-chloropentane from the reaction mixture.

E xamp e 52.4
 UNIT 52 Tests for functional groups, separation and purification of compounds 73

P
Practice 52.10

1 A liquid mixture consists of two organic compounds X and Y. The boiling point of X is 79.3 °C while that of
Y is 82.6 °C.

Explain why fractional distillation is NOT a suitable method to separate X from the mixture.

2 Outline how pentane can be obtained from a mixture of pentane, decane and water by physical methods.

(Boiling points: pentane = 36 °C, decane = 174 °C, water = 100 °C)

52.17 Checking the purity of a product

You can use melting points and boiling points to identify the products
obtained in organic preparations because substances have specific
temperatures at which they melt and boil. These data can be looked
up in databooks or databases stored on compounds.

A pure substance melts and boils over a very small temperature

range while a mixture does not. Impurities tend to lower the melting
point of a substance and raise its boiling point.

So after the separation and purification of the products from

organic preparations, you can check the purity of a solid product by
determining its melting point, and the purity of a liquid product by
determining its boiling point.

Melting point determination

The apparatus for measuring the melting pont of a solid product is
shown in Fig. 52.19.

thermometer

capillary tube stirrer

rubber band

solid product
hot liquid bath

Fig. 52.19 Apparatus used to measure the melting point of a solid product
74 TOPIC 15 Analytical Chemistry

The method is as follows:

• Insert some of the solid product into a capillary tube and then
attach the tube open end upwards to a thermometer with a rubber
band.

• Place the thermometer into a bath of liquid. The liquid must boil
at a higher temperature than the melting temperature of the solid
product being tested.

• Slowly heat the liquid bath with constant stirring. Notice the
temperature at which the solid product starts and finishes melting.

Compare the experimental value to the published value for the

melting point. The presence of impurities in a substance broadens its
melting temperature range. A pure compound will melt within 0.5 °C
of the true melting point.

In practice, the apparatus shown in Fig. 52.19 is often replaced

by various electrical devices (Fig. 52.20).
Fig. 52.20 An electrical device used to
determine melting point
Boiling point determination
The apparatus for measuring the boiling point of a liquid product is
shown in Fig. 52.21. If a large amount of the sample is available, the
boiling point can be measured using simple distillation apparatus.

thermometer
capillary tube
(sealed at 0.5 cm
from one end) stirrer
rubber band
ignition tube

liquid product

water

Fig. 52.21 Apparatus used to measure the boiling point of a liquid product
 UNIT 52 Tests for functional groups, separation and purification of compounds 75

A pure liquid product will boil over a very small range of

temperature, whereas an impure one will boil over a considerable
range.

The method is as follows:

• Place a small amount of the liquid product in the ignition tube

and, using a rubber band, attach it to the thermometer.

• Insert a capillary tube sealed at 0.5 cm from one end into the
liquid.

• Clamp the thermometer in the beaker of water.

• Slowly heat the water, stirring all the time. When the stream of
bubbles coming out of the capillary tube is rapid and continuous,
stop heating.

• Allow the beaker of water to cool, stirring continuously. The

bubbles slow down. The boiling point of the liquid product is
reached when the bubbles stop.

P
Practice 52.11

Acetanilide can be synthesised from aniline according to the following reaction scheme:

NH3 Cl–
+
NH2 N O
HCl 1 (CH3CO)2O C
– +
H 2O 2 CH3COO Na
CH3
aniline acetanilide

The crude solid product is separated from the reaction mixture.

a) Suggest how you can purify the crude solid product by using recrystallisation from an ethanol-water mixture.

b) Suggest how you can verify whether the recrystallised sample of acetanilide is pure.
 76 TOPIC 15 Analytical Chemistry

Have you mastered ?

Key terms

2,4-dinitrophenylhydrazine 2,4- 47 Tollens’ reagent 48

liquid-liquid extraction 55 stationary phase 61 mobile phase 61

paper chromatography 61 thin layer chromatography 61

Have you mastered?

column chromatography 61 developing solvent 61 solvent front 62

chromatogram 62 retention factor 63 elution 68 eluent 68

Checklist

After studying this unit, you should be able to

apply appropriate chemical tests to detect the presence of various functional groups in carbon
compounds: C=C, –OH, –CHO, C=O and –COOH;

separate and purify substances by the following methods:

• simple distillation;

• fractional distillation;

• liquid-liquid extraction;

• recrystallisation;

• chromatographic methods;

determine the Rf values of substances in a chromatogram;

justify the choice of an appropriate method used for separating substances in a mixture;

determine the melting point or boiling point of a substance;

check the purity of a substance by measuring its melting or boiling point.

(Put a ‘✔’ in the box if you have acquired the knowledge concerned.)
 UNIT 52 Tests for functional groups, separation and purification of compounds 77

Summary

1 The table below summarises results of chemical tests for some functional groups.

Functional group Test Observation(s)

add aqueous bromine to yellow-brown aqueous bromine becomes
–C=C–
carbon compound slowly colourless quickly

–OH warm with acidified K2Cr2O7(aq) colour change from orange to green
(primary and colour change from purple to colourless
secondary alcohols) warm with acidified KMnO4(aq)
(or very pale pink)
treat with 2,4-dinitrophenylhydrazine a bright red, orange or yellow precipitate forms

Have you mastered?

–CHO warm with Tollens’ reagent silver mirror forms
warm with acidified K2Cr2O7(aq) colour change from orange to green

C=O treat with 2,4-dinitrophenylhydrazine a bright red, orange or yellow precipitate forms

effervescence occurs; gas produced turns

mix with NaHCO3(aq)
limewater milky
–COOH
warm with ethanol in the presence of
a sweet, fruity smell is detected
concentrated sulphuric acid

2 The table below summarises common separation and purification methods for liquid and solid
products.

Type of product Method Remark(s)

• If a crude liquid product has a much lower boiling temperature
distillation than other substances in the reaction mixture, separate it by
distillation.
Liquid product • Separate mixtures of liquids with close boiling points by
fractional distillation
fractional distillation.
• Use a solvent to extract the desired liquid product from the
liquid-liquid extraction
reaction mixture.
• A solvent has to be found in which the solid product is soluble
recrystallisation
when the solvent is hot, but insoluble when the solvent is cold.
• This method involves a stationary phase and a mobile phase.
Solid product
• A mixture can be separated because the components distribute
chromotography
themselves differently between the stationary and mobile
phases, according to their affinity for each phase.
 78 TOPIC 15 Analytical Chemistry

3 The table below summarises the stationary and mobile phases, and the working principle of three
types of chromatography.

Type of
Stationary phase Mobile phase Working principle
chromatography
Separation occurs because each component has
Paper water in the paper fibres liquid solvent different solubilities in the mobile phase and in
the stationary phase.
thin layer of alumina
Thin layer or silica gel coated liquid solvent Separation occurs because each component has
onto a plate a different solubility in the mobile phase and a
alumina or silica gel in different adsorption by the stationary phase.
Have you mastered?

Column liquid solvent

vertical glass column

4 As long as the same solvent and type of chromotographic paper are used, a component in a mixture
can be identified from its retention factor Rf.

distance travelled by the component

Rf =
distance travelled by the solvent

5 The purity of a solid product may be checked by its melting point and that of a liquid product by
its boiling point.
 UNIT 52 Tests for functional groups, separation and purification of compounds 79

Unit Exercise

Note: Questions are rated according to ascending level of difficulty (from 1 to 5):
question targeted at level 3 and above; question targeted at level 4 and above;
question targeted at level 5.

Part I Knowledge and understanding

1 Complete the following concept map.

–OH
(1° and 2°)

turns from turns from

C=C (a) to (b) (c) to (d)

Br2(aq) warm with acidified –CHO

K2Cr2O7(aq)

warm with –OH

alcohol in the
(1° and 2°)
presence of

Unit Exercise
concentrated warm with acidified
acid catalyst identifying unknown KMnO4(aq) turns from
–COOH forms an (e) functional groups in (f) to (g)
carbon compounds

–CHO

NaHCO3(aq) 2,4-dinitrophenylhydrazine
–CHO

a (j) precipitate
–COOH liberates (h) gas
forms
Tollens’
reagent
C=O

(i) mirror forms

–CHO
 80 TOPIC 15 Analytical Chemistry

Part II Multiple choice questions

2 Which of the following compounds would form an 4 Safranal is one of the substances that contributes to
orange precipitate with 2,4-dinitrophenylhydrazine? the aroma of saffron.

A O

B
OH
Separate samples of safranal were tested with aqueous
bromine, 2,4-dinitrophenylhydrazine and Tollens’
reagent.

What are the final observations when safranal is tested

C O with each of those reagents?

OH Aqueous Tollens’
bromine 2,4-dinitrophenylhydrazine reagent

A yellow-brown orange yellow

D O
solution solution solution
B colourless orange silver
solution precipitate mirror
C yellow-brown orange silver
Unit Exercise

solution solution mirror

3 Compound X is treated with warm acidified D colourless orange yellow
aqueous solution of potassium dichromate. The precipitate solution
resulting product gives an orange precipitate with
2,4-dinitrophenylhydrazine but no observable change 5 Consider the two carbon compounds X and Y:
with Tollens’ reagent.
CH3CH(OH)CH2CHO CH3COCH2CH2OH
What could X be? X Y
A Pentan-1-ol Which of the following reagents can be used to
B Pentan-2-ol distinguish between them?
C Pentanal
D 2-methylbutan-2-ol A Acidified aqueous solution of potassium dichromate
B Dilute sulphuric acid
C Phosphorus pentachloride
D Tollens’ reagent

6 For which purpose is distillation used?

A To allow a liquid to boil without the loss of vapour

B To purify a liquid product
C To remove an involatile impurity
D To allow further reaction without the loss of product
(OCR Advanced Subsidiary, Chem. B (Salters), H033/01,
May 2017, 5)
 UNIT 52 Tests for functional groups, separation and purification of compounds 81

7 In the first stage of the synthesis of methyl 9 The structure of vitamin C is shown below.
3-nitrobenzoate, methyl benzoate, C6H5COOCH3, is
prepared by the reaction of benzoic acid with methanol HO OH
in the presence of concentrated sulphuric acid. When
C C
the reaction is complete, the sulphuric acid is neutralised
by the addition of aqueous sodium carbonate. The
O C CHCH(OH)CH2OH
simplest way of obtaining the impure methyl benzoate
O
from this mixture will be

A refluxing. Which of the following properties is vitamin C likely

B solvent extraction. to have?
C filtration. (1) It is soluble in water.
D recrystallisation. (2) It decolourises aqueous bromine rapidly.
(Edexcel IAL, Advanced, Unit 5, WCH05/01, Jun. 2014, 19) (3) It gives a silver mirror with Tollens’ reagent.

8 Ninhydrin is used in thin layer chromatography to help A (1) and (2) only
with the identification of amino acids. This is because B (1) and (3) only
the ninhydrin C (2) and (3) only
D (1), (2) and (3)
A reacts with amino acids to form a compound which
has an intense colour. 10 In which of the following reactions are the carbon
B reacts with amino acids to form compounds each compounds oxidised?
of which has a characteristic colour.
C increases the separation of the amino acids on the (1) CH3CH2=CH2 + acidified aqueous solution of

Unit Exercise
chromatogram. potassium permanganate
D ensures that the mobile phase maintains a nearly (2) CH3CH2CHO + Tollens’ reagent
constant pH for all the amino acids. (3) CH3COCH3 + 2,4-dinitrophenylhydrazine reagent
(Edexcel Advanced GCE, Unit 5, 6CH05/01R, A (1) and (2) only
Jun. 2014, 16) B (1) and (3) only
C (2) and (3) only
D (1), (2) and (3)
 82 TOPIC 15 Analytical Chemistry

Part III Structured questions

11 The structures of glucose and fructose are shown below.

OH
H O
C H C H

HO C H C O

H C OH H C OH

HO C H HO C H

H C OH H C OH

H C H H C H

OH OH

glucose fructose

a) Glucose and fructose are ‘structural isomers’. What is meant by this term?

b) State what will be observed when glucose is

Unit Exercise

i) warmed with Tollens’ reagent;

ii) treated with 2,4-dinitrophenylhydrazine.

12 Suggest a chemical test to distinguish one compound from the other in each of the following pairs. Your answer
should include the reagents used and the expected observations for both compounds.

O O

a) H C and CH3CH2 C

O CH2CH3 OH

b) CH3 C CH2 C CH3 and H C CH2 C H

O O O O

13 a) Suggest a chemical test to show the presence of each of the following:

i) HCl(g)

ii) C O functional group

b) Which of the following chemicals is most suitable for drying ethyl ethanoate?

anhydrous magnesium sulphate, concentrated sulphuric acid, solid sodium hydroxide

(HKDSE, Paper 2, 2014, 3(a)(i)–(ii))
 UNIT 52 Tests for functional groups, separation and purification of compounds 83

14 In an experiment, propanal is warmed with Tollens’ reagent.

a) State the expected observation.

b) Draw the structure of the organic product formed.

15 An organic compound X reacts with 2,4-dinitrophenylhydrazine to form a yellow solid Z. The structure of Z is shown
below:
H
H NO2
N
N

NO2

Given that the molecular formula of X is C9H8O, draw the structure of X.

(HKDSE, Paper 2, 2017, 1(a)(iii))

16 The molecular formula of X is C3H6O3. The carbon atoms in X are bonded directly to one another.

X gives effervescence when shaken with Na2CO3(aq).

When X is heated under reflux with acidified K2Cr2O7(aq), the product, Y, gives no observable change with
2,4-dinitrophenylhydrazine.

Unit Exercise
a) Give the structural formula of X.

b) Give the structural formula of Y.

c) When X is warmed with a little concentrated sulphuric acid, a small amount of a cyclic compound, Z, is formed.
Z has the molecular formula C6H8O4.

Draw the structure of Z.

17 Alcohol X is heated with acidified K2Cr2O7(aq) under reflux. X is oxidised.

a) State the colour change observed.

b) The oxidation results in the formation of either a ketone or a carboxylic acid.

Suggest a chemical test to show that the oxidation product is a

i) ketone;

ii) carboxylic acid.

Give your expected observation in each case.

c) Tests show that X is oxidised to a ketone. What type of alcohol is X?

 84 TOPIC 15 Analytical Chemistry

18 Four unlabelled reagent bottles each contains one of the colourless liquids listed below:

CH3CH2CH2CH2OH

CH3CH2COOCH3

CH3CH2CH2COOH

CH2=CHCH2COOH

Suggest chemical tests to distinguish the four liquids.

19 Outline how hex-1-ene can be obtained from a mixture of hex-1-ene, octane and water by physical methods.

(Boiling points: hex-1-ene = 63 °C, octane = 125 °C, water = 100 °C)
(HKDSE, Paper 2, 2013, 3(a))

20 Cloves are dried, unopened flower buds of an evergreen tree that grows in tropical climates.

Clove oil is extracted from dried cloves and a mixture of clove oil and water is obtained. This mixture is placed in
a separating funnel and 50 cm3 of liquid dichloromethane is added. The mixture is shaken and allowed to stand,
giving two layers as shown below.

stopper
Unit Exercise

aqueous layer
dichloromethane layer
tap

Most of the clove oil dissolves in the dichloromethane.

a) Describe how the dichloromethane layer is obtained free from the aqueous layer.

b) The dichloromethane, which boils at 40 °C and produces a toxic vapour, is then evaporated off to leave clove
oil.

Suggest a suitable method and the equipment used for removing the dichloromethane from the mixture.
 UNIT 52 Tests for functional groups, separation and purification of compounds 85

21 Aspirin and paracetamol are widely used painkiller.

O OH
H
O N

O O
HO
aspirin paracetamol

a) A student added small amounts of powdered aspirin and powdered paracetamol separately to aqueous solution
of sodium carbonate.

Describe what would be observed in each case and explain the difference in the observations.

b) An aspirin sample S was contaminated with an organic base Y.

i) Suggest how aspirin can be isolated from the aspirin sample S by using an organic solvent dichloromethane
and an aqueous solution of sodium hydroxide.

ii) Suggest how you can identify that the solid obtained is pure.

22 a) Scientists are investigating how best to recycle nylon-6,6. One approach is to break the polymer down into its
monomers which can be reused. The following flow chart shows how hexanedioic acid can be produced from
nylon-6,6.

Unit Exercise
heat under reflux recrystallise
nylon-6,6 with H2SO4 filter using water as hexanedioic acid
for 3 hours a solvent

filtrate

i) What is meant by ‘heating under reflux’?

ii) Describe the main steps involved in carrying out the recrystallisation of hexanedioic acid using water as a
solvent.

In your account, describe what property of hexanedioic acid this process depends upon.

b) The student adds excess sodium hydroxide solution to the filtrate. The student notices that the mixture develops
a ‘fishy’ smell characteristic of an amine.

Suggest the structural formula of the compound responsible for the ‘fishy’ smell.
(OCR Advanced Level Chem. B (Salters), Sample Question Paper, H433, 2016, 1(a)(i), (iii), (b))

23 What is meant by the ‘Rf value’ of a substance in a paper chromatogram?

(HKDSE, Paper 2, 2018, 3(a)(iii))
 86 TOPIC 15 Analytical Chemistry

24 Thin layer chromatography (TLC) is used to analyse a mixture of two amino acids. The result is shown below.

solvent front 8

Y 6

X
2

cm
starting position

a) Briefly describe the principle underlying the separation of amino acids X and Y by using thin layer chromatography.
Unit Exercise

b) Suggest ONE method that can be used to make the spots on the TLC plate visible.

c) The Rf values for some amino acids, recorded under identical conditions, are shown in the table below.

Amino acid Rf
Serine 0.21
Glycine 0.29
Alanine 0.55
Proline 0.64
Tyrosine 0.75
Valine 0.82

Determine the identity of the amino acids in the mixture.

 UNIT 52 Tests for functional groups, separation and purification of compounds 87

25 Inks are mixtures of different dyes.

A student used paper chromatography to separate the dyes in two samples of inks. The experimental set-up the
student used is shown below.

starting position
ink 1 ink 2 solvent

a) Explain why the dotted line of starting position

i) was drawn in pencil, NOT in ink;

ii) was drawn above the solvent level.

b) Name the mobile phase and stationary phase in paper chromatography.

Unit Exercise
c) Explain how the components in ink are separated into two or more spots.

d) The result of the experiment is shown below.

2.5 mm solvent front

50 mm

spot A

9.5 mm

4.0 mm

i) Calculate the Rf value for spot A.

ii) Suggest why the two samples of inks are NOT the same.

26 Describe how impurities affect the melting temperature of an impure solid.

 88 TOPIC 15 Analytical Chemistry

27 At a given pressure, a pure liquid boils at a constant temperature. This boiling point can be used to help identify
the liquid. One procedure for measuring the boiling point of an aldehyde is shown in the diagram.

Procedure

• Set up the apparatus as shown in the diagram.

Make sure that the bulb of the thermometer is about 3 cm above the level of the aldehyde.

thermometer
cork

heating aldehyde
bath
anti-bumping
granules

heat

• Add a few anti-bumping granules to the aldehyde.

Unit Exercise

• Heat the liquid in the beaker gently until the aldehyde begins to boil. The boiling point of the aldehyde is greater
than 150 °C.

• Adjust the heat source so that the aldehyde boils gently.

• Record the temperature when it becomes steady.

• Record the atmospheric pressure.

a) State the purpose of the anti-bumping granules.

b) Give ONE reason why it is essential for the cork to have the hole, labelled X, in it.

c) State TWO properties that a liquid must have to make it suitable for use in the heating bath in this boiling point
determination.

d) State why the atmospheric pressure was recorded.

e) State why the boiling point of a compound may be insufficient on its own to identify the compound.
(AQA Advanced GCE, Unit 6T, P13, May 2013, 8)

28 A pale yellow waxy solid was obtained in an experiment. The melting point of the solid was believed to be about
38 °C.

Devise a simple experiment that can be used to find the melting point of the solid.