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
Do you know how to write the structure of an amino acid?
Can you draw and explain the structure of a zwitter ion and
how is it affected by acidic and basic pH
Can you explain two techniques which separate amino acids on
basis of their ionisation at different pH?
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)