Unit 2

Learning aim C: Undertake

chromatographic techniques to identify
components in mixtures

LEARNING OBJECTIVES:
You
LO1: MUST demonstrate how to read a
chromatogram.
LO2: SHOULD accurately calculate Rf values
on a chromatogram.
LO3: COULD describe the solubility of a
substance using the rF values and the
correct scientific terminology.
You are a newly appointed
technical assistant at a large
chemical plant,
Chemcalequip.
As part of your induction
period and to progress in
your role, you have to
demonstrate skills in a
range of practical
You need to demonstrate
that you can carry out
different chromatographic
techniques to separate
and identify components in
mixtures. In a report of the
techniques you have used,
you will need to explain the
techniques, analyse and
You will need to present
evidence of your practical
skills analysis and
evaluation in a report.
Criteria covered by this task:
To achieve the criteria you must show that you are
able to:

C.D3 Evaluate the chromatographic techniques

used in relation to outcomes and suggest
improvements.

C.M3 Analyse own chromatograms and relate the

factors that affect the separation of mixtures to the
quality of results obtained.

C.P5 Correctly use chromatographic techniques to

produce chromatograms.

C.P6 Explain the use of chromatographic techniques

to separate mixtures.
How chromatography
works
In all types of chromatography, two phases are
present: a stationary phase, which does not
move, and a mobile phase, which flows over
the stationary phase.

The mobile phase is a liquid or gas that

acts as a solvent. The substance to be
tested is dissolved in the mobile phase.

If a liquid solvent is used, it can be

either aqueous (water) or non-
aqueous (not water). Examples of
non-aqueous solvents include
alcohols and ethanoic acid.
Dynamic equilibrium
The mobile phase contains the dissolved substances of
interest. It is passed over the stationary phase.
As the mobile phase interacts with
the stationary phase, the phases
exchange particles at the same
rate. A dynamic equilibrium is
established.

The position of the equilibrium

determines how far the substance
travels through the stationary phase.
The more particles that stay in the
mobile phase, the further the
dissolved substance will travel.
 Paper chromatography
Paper chromatography is
used to separate and identify
components of mixtures.
Dots of single dyes are
placed alongside a dot
of the unknown mixture.
The solvent is drawn up the paper
by capillary action.
As the solvent moves up
the paper, the pattern of the
single dyes can be compared
to that of the mixture.
This technique can be used to identify additives in food.
Which ink do you think?
Rf values
Calculating Rf values
Reference material
Scientists can compare the
Rf value of an unknown amino acid Rf value
substance to Rf values of alanine 0.38
known compounds of that
serine 0.27
type.
leucine 0.75
The Rf values of compounds
such as amino acids are lysine 0.14
published in data books and valine 0.61
can be looked up. The amino
phenylalanine 0.68
acids present can be
identified by comparison of glysine 0.26
the Rf values obtained and cysteine 0.40
those in the data book.
 Thin layer chromatography
Remember that all chromatography involves a stationary
phase and a mobile phase.
glass plate
In thin layer chromatography
(TLC) the stationary phase is a
layer of silica gel fixed onto a
plate. The plate may be glass,
foil or plastic, for example.

The mobile phase is a solvent

that travels up the plate, silica gel
carrying the substances.

In paper chromatography, what are

the stationary and mobile phases?
 How does TLC work?
TLC uses the same basic principle as paper chromatography:
capillary action draws the solvent up the plate.

A dry sample of the substance of interest is placed in the

silica gel. Then, as the solvent moves up the gel, it dissolves
the sample and carries it up the plate.

The distance each substance travels depends on its affinity

(attraction) with the gel. The weaker the attraction between the
substance and the gel, the further the substance will travel.

Affinity determines the position of the dynamic equilibrium

established between the mobile and stationary phases. Affinity
depends on the bonding of the solvent, substance and gel.
 TLC or paper
chromatography
Thin layer chromatography has a number of advantages over
paper chromatography.

1. The glass plate is rigid, not flexible like paper, so it is easy

to control.

2. After separation, the substances in the mixture can be

recovered. The silica gel holding the separated substance
is scraped off the glass plate and added to a solvent.
The substance will dissolve and the silica gel can easily
be removed by filtration.

The glass plates can be re-coated with

silica gel and used over and over again.
 UV and locating agents
Many substances are white or colourless,
and so are not visible on a TLC plate.

One way of making

colourless substances
show up is to use UV light.
This usually works well for
organic compounds.

An alternative method is to use a chemical locating agent

a chemical that reacts with the substance to form
a coloured compound.

For example, when ninhydrin is exposed to an organic

compound, it stains it purple-brown.
Homework
Complete the questions on
chromatography and calculating Rf
values.

Bring to the next lesson.