Download chromatography

Chromatography
Learning objective:
To be able to describe the method
of chromatography and its
applications
Chromatography
• Chromatography is used to separate pure
substances from a mixture of substances,
such as a cell extract.
• It is based on different substances having
different solubilities in different solvents.
• A simple and common form of
chromatography uses filter paper
Clip
Direction of
solvent
movement
Filter paper
cylinder
Solvent
Concentrated
spot of
chemicals to
be separated
and identified
Paper chromatography is
a technique used for the
separation & identification
of relatively small chemical
substances by a moving
solvent on sheets or strips
of filter paper
Substances to be identified
are ‘spotted’ near one end
of the filter paper
As the solvent moves up the paper, different molecules move at
different rates with the smallest molecules moving the fastest
The technique is used for small molecules such
as amino acids, small peptides and sugars
Mark the solvent
front & allow
paper to dry
Solvent
front
Purple spots
develop located at
different distances
from the origin line
Amino acid spots
origin
solvent
Chromatography separates small molecules
in a mixture on the basis of size
As the solvent moves up the paper,
molecules move at different rates
When the spots are colourless (most
amino acids), a locating agent is needed to
visualise their positions on the
chromatography paper
amino acid spots
on the origin line
•
•
The chromatogram can be analysed by measuring
the distance travelled by the solvent front, and
the distance from the origin to the centre of each
spot.
This is used to calculate the Rf (relative front)
value for each spot:
distance moved by spot
Rf 
distance moved by solvent
•
An Rf value is characteristic of a particular solute
in a particular solvent. It can be used to identify
components of a mixture by comparing to tables
of known Rf values.
lid
solvent
front
chromatography
tank
chromatography
paper
spots
y
x
origin
solvent
Rf = x/y
X
Rf 
Y
The Rf value is
always a value less
than one as the
solvent front always
moves further than
the solute molecules
X3
Y
X1
X4
X2
X5
The mixture of
unknown amino
acids is seen to
contain four different
amino acids
Of these four
amino acids,
two can be positively
identified
The mixture contains
four amino acids; two
unknown together
with arginine & leucine
• Sometimes chromatography with a single
solvent is not enough to separate all the
constituents of a mixture.
• In this case the separation can be improved
by two-dimensional chromatography, where
the chromatography paper is turned through
90° and run a second time in a second
solvent.
• Solutes that didn't separate in one solvent
will separate in another because they have
different solubilities.
rotate
through
90°
original
spot
first run
second run
original
spot
Solvent
front
Paper dried and
rotated clockwise
through 90o
Solvent
front
Mixture of
amino acids
on origin line
First solvent
Second solvent
Two-way chromatography provides better separation of substances that behave
in a similar fashion in the first solvent.
A second run in a different solvent resolves two very close spots more clearly
Different types of chromatography
• Paper chromatography is the simplest, but does not
always give very clean separation.
Different types of chromatography
• Thin layer chromatography (tlc) uses
a thin layer of cellulose or silica
coated onto a plastic or glass sheet.
This is more expensive, but gives
much better and more reliable
separation.
Different types of chromatography
• Column chromatography uses a glass
column filled with a cellulose slurry. Large
samples can be pumped through the
column and the separated fractions can be
collected for further experiments, so this is
preparative chromatography as opposed to
analytical chromatography.
Different types of chromatography
• High performance liquid
chromatography (HPLC) is an
improved form of column
chromatography that delivers
excellent separation very quickly.
Different types of chromatography
• Electrophoresis uses an electric
current to separate molecules on the
basis of charge. It can also be used to
separate on the basis of molecular
size, and as such is used in DNA
sequencing