Chromatography and
Instrumentation
Chromatography
Components
Mixture
• Analyze
Separate
• Identify
• Purify
• Quantify
Chromatography is used by scientists to:
• Analyze – examine a mixture and its components
• Identify – determine the identity of a mixture or
components based on known
components example – to identify the mixture found in a fibre at a crime scene.
• Purify – separate components in order to isolate
study
one of interest for further
• Quantify – determine the amount of the a mixture
in the sample. Example – drugs in urine or blood samples
and/or the components present
Explanation of
Chromatography
Explanation:
•
•
•
•
Mixture is placed on stationary phase
Mobile phase passes through the stationary phase
Mobile phase dissolves the parts of the mixture
Mobile phase carries the individual parts a certain distance
through the stationary phase, depending on their solubility
in both of the phases
Definition of Chromatography
Definition:
Chromatography is a way to separate parts of a mixture
by using the different solubilites of the parts for a mobile
phase compared to the stationary phase
Terminology:
• Mobile Phase – gas or liquid that carries the parts
• Stationary Phase – the part of the apparatus that does
not move with the sample
Types of Chromatography
1.
2.
3.
4.
5.
Paper Chromatography
Thin Layer Chromatography
Column Chromatography
Gas Chromatography
High Performance Liquid Chromatpgraphy
Paper Chromatography
Paper =
Stationary
phase
Liquid in jar=
Mobile phase
Thin Layer Chromatography
Glass plate with solid
absorbant layer like
alumina/ silica on it
= Stationary phase
Solvent Liquid in jar =
Mobile phase
Used as way to identify
fibres in forensic work
Column Chromatography
Solid filling a glass
column= Stationary
phase
Liquid flowing
downwards under
gravity =
Mobile phase
Used as a purification
technique
Definition of Chromatography
Chromatography is a way to _______parts of a
________by using the different ___________of the parts
for a mobile phase compared to the stationary phase
Terminology:
• Mobile Phase –
• Stationary Phase –
Paper Chromatography
Stationary
phase = ?
Mobile phase
=?
Thin Layer Chromatography
Stationary phase=
Mobile phase =
Common application?
Column Chromatography
Stationary phase =
Mobile phase =
Application?
HPLC – High Performance
Liquid Chromatography
• A highly improved form of column chromatography.
Mobile phase =
• Solvent of high purity
• Instead of a solvent being allowed to drip through
a column under gravity, it is forced through
under high pressures - much faster.
HPLC – High Performance
Liquid Chromatography
Stationary phase
• Solid particles - but smaller
particle size which gives a
much greater surface area
for interactions between the
stationary phase and the
molecules flowing past it.
This allows a much better
separation of the
components of the mixture –
even for parts that are
similar to each other.
HPLC – High Performance
Liquid Chromatography
• Better detection methods can be
used - highly automated and
extremely sensitive.
Used for
measuring levels
of growth
promoters and
vitamins in meat
Gas Chromatography
• Mobile phase is a gas
• Stationary phase is a high boiling
point liquid absorbed onto a solid.
• How fast a particular compound
travels through the machine will
depend on how much of its time is
spent moving with the gas as opposed
to being attached to the liquid in
some way.
Gas Chromatogrpahy
• The injector for the sample is contained
in an oven
• It is hot enough so that all the sample
boils and is carried into the column as a
gas by the carrier gas
Gas Chromatography
• The coiled column is packed with finely
ground SOLID
• This is coated with a high boiling
LIQUID
• Column is cooler than the injector oven
How separation works on the
column
What happens to the parts of the mixture?
• May remain in the gas phase.
• May condense on the stationary phase.
• May dissolve in the liquid on the surface
of the stationary phase.
Hence different components will have
different retention times and separation
occurs
Gas chromatography
• The time taken for a particular compound
to travel through the column to the
detector is known as its retention time
HPLC – High Performance
Liquid Chromatography
• A highly improved form of column chromatography.
• How is the mobile phase different
than in Column chromatography?
HPLC – High Performance
Liquid Chromatography
Stationary phase
• What is different
about it compared to
normal column
Chromatography?
HPLC – High Performance
Liquid Chromatography
• What else is different about this
method?
Used for?
Gas Chromatography
• Mobile phase?
• Stationary phase?.
• How fast a particular
compound travels
through the machine will
depend on…
Gas chromatography
• Retention time means…
• An application is…
Illustration of Paper
Chromatography
Stationary Phase
Separation
Mobile Phase
Mixture
Components
Components
Affinity to Stationary
Phase
Affinity to Mobile
Phase
Blue
----------------
Insoluble in Mobile Phase
Black


Red


Yellow

        
Parts of mixtures identified
using Rf values:
Rf
=
Distance moved by spot (x)
Distance moved by solvent (y)
• A characteristic of a particular
solute in a particular solvent.
Seperating inks in a pen
using Paper chromatography
1. Saturate tank with vapour before
the experiment
2. Draw a line with pencil on the
bottom of chromatography paper
3. Solvent should be below line level
4. Add mixture to the line using a
capillary tube, and dry before use.
5. Calculate Rf value of any
components present
Thinking about this experiment..
1.
Why is a paper chromatography tank not used for a
considerable time after the chromatography solvent has
been added?
2. Why are two lines usually drawn on a paper chromatogram?
3. What type of mixture is suitable for seperation using paper
chromatography?
4. When two substances are found to have different Rf values,
what does this mean?
5. When two substances are found to have different Rf values,
what does this mean?
Instrumentation Part 2:
Spectroscopy
Spectroscopy
• Uses a range of techniques to
examine the composition, structure
and bonding of elements and
compounds.
1. Mass Spectrometer
2. Infra red Spectroscopy
3. Ultraviolet absorption Spectroscopy
Mass spectrometer in
determining relative atomic
masses
The mass spectrometer can be
used to measure relative
atomic masses.
It is also commonly used to
determine concentrations of
drugs in urine samples
Higher
Level
Fundamental processes that
occur in the mass spectrometer
1.
2.
3.
4.
5.
Vaporisation
Production of positive
ions
Acceleration
Separation
Detection
Higher
Level
Stage 1 - Vaporisation
The liquid sample is
injected into the
instrument.
There is a vacuum inside
the chamber so the liquid
turns into a gas –
becomes vaporised.
Higher
Level
Stage 2 – Ionisation
The vaporised sample passes into
the ionisation chamber.
The particles in the sample are
bombarded with a stream of
electrons.
The collisions will knock one or
more electrons out of the sample
particles to make positive ions.
Higher
Level
3 - Acceleration
Negatively charged plates in the
accelerator attract the positively
charged ions out of the ionisation
chamber and into the accelerator
As the ions pass through the
plates they accelerate and an ion
beam passes into the separator.
Negatively
charged
plates
Higher
Level
5. Detection
By changing the strength of the
magnetic field ions of different
masses are focused on the detector
The signal is amplified and can be
viewed on a computer screen
The signal is sent to a recorder
which traces out a mass spectrum.
The mass spectrum is interpreted
by the scientist.
Higher
Level
A mass spectrum
2. Infra-Red Spectroscopy
• Works by molecules absorbing Infrared energy
waves which cause particular bonds to vibrate
and stretch if present
• Allows the identification of functional groups in
a molecule
3. Ultraviolet Spectroscopy
• Works on the principle that molecules absorb
ultraviolet energy waves, and the amount of
energy absorbed will depend on the amount of
substance present – even for colourless molecules
Use – analysing the amount of drugs in a medicine