Amino acids consist of carboxylic acids (acidic) and amines
(basic). Acids contain a hydrogen atom which is a proton with one electron,
technically an H ion is just a proton, and hence an acid is a proton donor. As
well as, bases are proton acceptors. Moreover, there are two options for
amines, they can either be neutral (COOH) or lose a proton (COO-). The proton
count on each will be determined by the acidity of the solution and that amino
acids can only be neutral or zwitterion (overall neutral but have formal
charges across the molecule).
A zwitterion is sometimes called dipolar ions. This is due
to having a negative end (anion) and a positive end (cation). At a neutral PH,
amino acids exist in their zwitterion form (dipolar form). This means that the
amino acids contain a positive charge and a negative charge.
a) At the low PH of
0/1 the carboxylic group is protonated. At this acidic PH, amino acids are
positively charged species.
b) As the PH
increased, the carboxylic acid group loses its hydrogen to form a zwitterion.
Ion exchange chromatography:
c) The zwitterion form
usually persists until around a PH of 9. At this basic PH, the low H+ causes
the amino acids to lose its hydrogen therefore forming a negatively charged
Ion exchange chromatography: This
method can be used to purify mixture of proteins based on their net charge. So
ion exchange chromatography separates charged molecules and polar molecules.
Hydrophobic molecules cannot be separated by this method. There are two
different phases known as the stationary phase and the mobile phase. The interaction
between these two phases varies for different molecules. One again ion exchange
chromatography separates ions or polar or charged molecules. In this case of
ion exchange chromatography, we run a column, meaning it’s a structure made
with polymers which creates chamber. Inside that chamber we add the stationary
An amino acid has different charged
properties based on the PH of the solution it is in. PH dictates whether the
amine and carboxyl group in an amino acid will be protonated or deprotonated. Overall,
charge differences are affected by PH. This is because increasing PH in general
tends to deprotonate functional groups and makes the net charge on an amino
acid more negative. However, if PH is decreased then it protonates functional groups
and make the net charge more positive.
Ion exchange chromatography can
separate amino acids on the basis of their net charge. You have column filled
with resin beads that are either negatively charged or positively charged. As a
result, amino acids will stick to the column with certain timings based on
On the diagram above, charged
molecules attached to the beads hold onto the amino acids to be separated. The
ion exchange chromatography can be classified into two types which are the
anion exchange chromatography and the cation exchange chromatography. This
method is based on the reversible electrostatic interactions of proteins with
the separation matrix. Mechanism of protein separation has two types which are
PH based binding and salt based binding. The differences between the net
surface charges on the solute molecules, is what the PH based binding depends
As you can see above, the value which
is the PH underneath pi, the protein will gain itself a net positive charge. It
will also bind reversibly to the width of the surface of a cation exchange
resin which is negatively charged group based on the PH. Note that
At PH value below the pi, the protein
will have a net positive charge and will tend to bind reversibly to the width
of the surface of a cation exchange resin that is one that has negatively
charged groups at the PH. (1)
Another method by which proteins can
be cleansed based on size is gel electrophoresis. It uses an electric field to
move proteins down a gel at different rates. The buffers maintain the PH level.
The proteins net charge depends on the PH it’s in. As the protein is placed
into the buffer with specific PH, the difference in the buffer PH relative to
the isoelectric point of the protein determines the magnitude and type of
charge on the protein.
If the buffer solution has been used
which has a lower PH then, all the -COOH groups present will exist as -COOH
groups, and not as ions. All the -NH2 groups will have picked up hydrogen ions
to form -NH3+. This means that all amino acids will carry a positive charge and
all will move towards the negative electrode. But if the buffer has a lower PH
then all the -NH2 groups will be the same and won’t change, also the -COOH
groups present will exist as -COO- ions. Overall, all the amino acids will
carry a negative charge and as a result of this it will move towards the opposite
direction which is the positive electrode.