Download Paper chromatography and electrophoresis

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General principles
• Substances are separated according to
their differential solubility between the
stationary phase, represented by the
water bonded to the cellulose molecules
of paper, and the moving phase, the
solvent.
• As the solvent front advances it carried
the components at different rates.
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
The tank should be
saturated with solvent
vapour
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
Investigating the composition of a
protein.
• The peptide bonds can be readily
hydrolysed by heating with 6M HCl at
100oC for 10-24 hours in an evacuated
tube.
• The amino acids released can then be
analysed by chromatography or
electrophoresis.
Choice of solvent
• The general principle is “like dissolves
like”.
• If a non-polar solvent is used the
hydrophobic amino acids will move much
further up the paper, as they are more
soluble in non-polar solvents.
• In contrast if a polar solvent is used the
hydrophilic amino acids will advance
further up the paper as they are now
more soluble.
Mark the solvent
front & allow
paper to dry
Spray the dry
paper with locating
agent (ninhydrin)
to make the
colourless spots
visible
Solvent
front
Dry the paper
with gentle heat
to develop
the amino acid
spots
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
Rf values can be
compared with
published values to
identify the amino
acids.
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
X
5
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
Solvent
front
Paper dried and
rotated clockwise
through 90o
Solvent
front
Mixture of
amino acids
on origin line
Second solvent
First 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
• Electrophoresis, like chromatography, is a technique
for the separation of molecules
• Both chromatography and electrophoresis may be used
for the separation of small molecules
• Electrophoresis, unlike chromatography, can be used
for the separation of relatively large molecules
• Electrophoresis is used mainly for separating mixtures
of proteins, peptides and DNA fragments
• Electrophoresis separates molecules from a mixture
according to their charge and size
• The technique involves passing a direct current
through either filter paper or a gel
Contact wick
making electrical
connection
between buffer
and gel
Electrophoresis gel
saturated with buffer
solution (to keep the pH
constant)
Negative
electrode
Positive
electrode
Buffer
Sample of
solution mixture placed
Buffer in a slot cut in
the gel
tank
CATHODE
Different molecules
show as different
bands after staining
ANODE
-
+
Sample slots
in the gel
GEL
Preparation of samples
• Filter paper wicks are soaked in the test
solution.
• They are then inserted in fine cuts made
in the thin layer of silica gel.
• NB Further separation can be achieved
using filter paper in combination with
chromatography in another direction.
Separation of Serum Proteins
albumin
Direction
of
migration
gamma
globulin
(mainly
antibodies)
alpha 1
globulin
alpha 2
globulin
beta
globulin
Blood serum contains
many different blood
proteins
A serum sample is
placed in a slot in the gel
The pH of the buffer is
adjusted to make all the
proteins negatively charged
When the direct current
is passed through the gel,
all the proteins will move
to the anode
After staining, the proteins
will show up as different
bands in different positions
from the origin slot
Amino acids are AMPHOTERIC
Amphoteric
molecules behave
as both acids and
bases
H+
H+
The amino group of the amino acid is a basic group
The carboxylic group is an acid group
Acids behave as proton donors
Bases behave as proton acceptors
Protons are hydrogen ions (H+)
A proton is the nucleus of a hydrogen atom
Amino acids possess a carboxylic
acid end and an amino end: The
amino group behaves as a base
The R group may also
possess a carboxylic acid
OR an amino group
In solution the acid group behaves
as a proton donor and the amino
group (basic) as a proton acceptor
In solution, the acid group
becomes negatively charged and
the amino group becomes
positively charged
H+
Neutral amino acids with no acid
or amino groups in the R group
have one negative and one positive
charge. Overall they are electrically
neutral molecules
The hydrogen atom contains a single
positively charged particle in its nucleus
This positively charged particle is a proton
Spinning around the nucleus in orbit is
a single negatively charged electron
++
In solution, the hydrogen atom of the
carboxylic acid group of the amino acid
releases a proton (H+)
The electron remains with the
carboxylic acid group which becomes
negatively charged
H+
H+
This is an ACIDIC amino acid as it
possesses TWO carboxylic acid groups
In solution the acid groups donate a
proton and the amino group accepts
a proton
The negative electrons remain
with the carboxylic acid groups
H+
The amino acid now has TWO
negatively charged carboxylic acid
groups and ONE positively charged
amino group
Overall, this acidic amino acid is
negatively charged
H+
H+
This is a BASIC amino acid as it
possesses TWO amino groups
In solution the acid group
donates a proton and the
amino groups accept a proton
The negative electron remains
with the carboxylic acid group
H+
The amino acid now has TWO
positively charged amino acid
groups and ONE negatively charged
carboxylic acid group
Overall, this basic amino acid is
positively charged
acidic amino acid
moves
towards
neutral amino acid
basic amino acid
stays at
the origin
ANODE
(+)
direct current
passed through
the gel and later
sprayed with
ninhydrin
moves
towards
CATHODE
(-)
mixture of the
three amino
acids placed
in well
GEL SOAKED IN BUFFER SOLUTION
The gel is examined under uv