The little corner of ϕ - χ DOI : 10.5281/zenodo.

6346860

Difference between Isoelectric point (IEP), Point of

zero charge (PZC), and Isoionic point (IIP)
Issue:
- What is the definition of Isoelectric point (IEP), Point of zero charge (PZC), and Isoionic point (IIP)?
- What is the difference between IEP, PZC, and IIP?
- Why IEP, PZC, and IIP are important?

Contents:
- Definitions and differences
- Examples : proteins, carbons, and metal oxides

Introduction
Colloids in an aqueous medium become naturally charged to
form an electrical double layer. The protonation/deprotonation
capacity of the colloid surface is a key parameter for the
charge transfer between solvent and particle. In this case, the
net surface charge is affected by the pH of the medium. Of
particular importance is the pH value at which the surface is
electrically neutral as the electric repulsion between colloids
is minimal. Depending on how and what is measured, this pH
is either called Isoelectric Point (IEP), Point of Zero Charge
(PZC), or Isoionic Point (IIP).

However, these 3 terms are often confused and misused in

many articles while they are very different. This paper aims
to define these 3 terms and explain when and how they should
be used but also compared to each other.
Figure 1. Ionic concentration and potential difference as a
1. Principle function of distance from the charged surface of a particle
suspended in a dispersion medium.
1.1 Context
The surface charge developed by particles in water attracts charge, it is then of importance to know the pH value, at which
counterions forming the electrical double layer (Figure 1). the net surface charge equals zero.
Two characteristic potentials are defined:

• the surface potential, i.e. the potential at the surface of
the particle (particle/solution interface);
• the zeta potential, i.e. the potential at the shear plane.
The protonation/deprotonation capacity of the surface is a
key parameter for the charge transfer between solvent and
particle. Thus, H + and OH − are potential-determining ions
as they can modulate the surface charge and potential decay.
Particle interfaces tend to be positively charged at low pH,
and negatively charged at high pH. Likewise, the charge can
also be affected by the presence of electrolytes (acids, bases,
and salts) in solution.
As the ion distributions and the physical properties of dis-
persions depends on the sign and magnitude of the surface
1.2 Point of zero charge (PZC)
There are several different definitions of the point of zero
charge depending on which one of the categories of surface
charge is equal to zero [1]:
• point of zero charge (PZC) is the pH at which the
net surface charge (surface potential) of total particle
surface is equal to zero;
• point of zero net charge (PZNC) is the pH value at
which the intrinsic surface charge is equal to zero;
• point of zero net proton charge (PZNPC) is the pH
value at which the net proton surface charge is equal to
zero;
• point of zero salt effect (PZSE) is the pH value at

1 A. Hutin – Zenodo – Researchgate

The little corner of ϕ - χ
which the effect of electrolyte concentration on a pH-
dependent charge is zero.
But, mostly, authors used PZC as a general term for all types
without making any difference.
At the PZC, there is no surface charge to be neutralized by
ions in the diffuse layer, and any adsorbed ions that exist must
be bound in surface complexes. Therefore, the PZC can be
measured by ascertaining the pH value at which a perfect
charge balance exists among the ions in an aqueous solution
with which particles have been equilibrated.
This does not mean that the surface has no charge at PZC, but
rather that there are equal amounts of positive and negative
charges.
PZC can be determined using potentiometric titration of col-
loidal dispersions [2]. Once satisfactory graphs are obtained
(acid/base amount—pH, and pH—zeta potential), the PZC is
established as the common intersection point (cip) of the lines.
Therefore, PZC is also sometimes referred to as cip.
1.3 Isoelectric point (IEP)
IUPAC gives the following definition : ”The pH value at
which the net electric charge of an elementary entity is zero.
pI is a commonly used symbol for this kind-of-quantity. It
should be replaced by pH(I) because it is a pH determined
under that particular condition” [3].
However, this definition is not accurate, it is better to define
IEP for materials showing a pH dependent surface charging
as the ”pH value at which the electrokinetic potential equals
zero”, i.e. zero velocity for particles subjected to an external
electric field. This doesn’t mean there is zero charge: for
proteins at IEP, there will be positive and negative charges
present at the same extent. Furthermore, it does not matter
how the charges are distributed.
It is of importance to notice that IEP is strictly determined
by electrokinetics (e.g. electrophoresis). This term cannot be
used with other measurement techniques.
1.4 Isoionic point (IIP)
According to IUPAC, ”IIP is the pH value at which the net
electric charge of an elementary entity in pure water equals
zero” [3]. Again, this definition can be confusing and it is
better to define IIP as the ”pH at which the net protonic charge
of an amino acid or a protein is zero” [4]. It is important to
highlight that IIP only relates to protons and only applies to a
zwitterionic substance in pure water.
Bryan [5] used an operational definition of IIP as the pH
of a solution of the substance in water which contains, in
addition to the substance itself, no ions other than those
2 A. Hutin – Zenodo – Researchgate
DOI : 10.5281/zenodo.6346860
arising from the dissociation of the aqueous solvent (H + or
OH − ). This particular pH serves as an important reference
point in acid-base titration studies of amino acids and pro-
teins. As an example, he considered an isoionic solution
of glycine. The following species can be present in aque-
ous solution: H + , OH − , + H3 NCH2CO2 H, + H3 NCH2CO− 2,
H2 NCH2CO2 H and H2 NCH2CO− 2 . The concentration of H +
is equal to :
√
[H + ] = K1 K2
with K1 and K2 the equilibrium constants of the glycine.
The operationally defined IIP is dependent on amino acid
concentration. At high concentration the operationally IIP
approaches its theoretical value, but as concentration goes to
zero it approaches a value of 7. The fact that the operationally
defined IIP of glycine is always less than 7 is an indication
that when glycine is dissolved in water, the protonated amino
group of the molecule has a greater tendency to loose a proton
to the solvent than the carboxylate group has to gain a proton
from the solvent. The result is a net production of hydrogen
ions.
IIP can be measured through potentiometric titrations car-
ried out at different ionic strengths. It is obtained as the
intersection point among the different titration curves [6, 7].
2. Differences
2.1 IEP vs PZC
The equivalence IEP = PZC occurs if there is no adsorption
of other ions than the potential determining ions like H + and
OH − at the surface. This is often the case for pure (pristine
surface) oxides in water. Other potential-determining ions
include:
• ions that are constituents of the surface and can cross
to the aqueous phase,
• specifically-adsorbing ions that form chemical bonds
(complex) with surface ions,
• ions forming precipitates that accumulate at the parti-
cle/solution interface.
In these cases, the x-axis would not be the pH but the concen-
tration (or activity) of these ions.
In the presence of specific adsorption ions, i.e. counterions
held only by electrostatic forces, PZC and IEP generally have
different values. For example, in Figure 2 each Ca2+ ions
carries two charges, they cause the potential to become less
negative more rapidly with distance than equal concentrations
of monovalent ions but leave the surface potential unaffected.
Strictly, these ions cannot reverse the zeta potential, but it can
approach zero.
The little corner of ϕ - χ
Figure 2. Effect of Ca2+on electric potential as a function of
distance from the particle surface.
The PZC is obtained by acid-base titrations of colloidal dis-
persions while monitoring the electrophoretic mobility of the
particles and the pH of the suspension. Several titrations are
required to distinguish PZC from IEP, using different elec-
trolytes (including varying the electrolyte ionic strength).
Note that both surface and zeta potentials reverse sign (i.e.,
cross the zero-potential line) at the same pH. The pH where
the zeta potential reverses is the IEP and where the surface
potential reverses is the PZC.
For simple materials, e.g. inorganic oxides, zero velocity usu-
ally means zero absolute charge (rather than zero net charge).
If there is only one charged species so it cannot be ”iso”,
hence IEP doesn’t apply but PZC does.
2.2 IEP vs IIP
IIP is for protons only while IEP is for all types of charges
such as bound counterions. Therefore, unless the entire charge
is proton, IIP and IEP are different [8]. So, IIP and IEP should
coincide when the electrolyte concentration is zero.
3. Examples
3.1 Proteins
The net electrical charge of proteins is a parameter that strongly
affects their physicochemical behavior in living organisms.
The most common surface charge determining ions are H +
and OH − . The pH value at which the surface of the protein is
electrically neutral is particularly important because at this pH
the electrical repulsion between proteins is minimal. There-
fore, they can easily coagulate and precipitate.
This pH value is called either IIP or IEP. As discussed before,
there is a conceptual difference between these two terms. The
3 A. Hutin – Zenodo – Researchgate
DOI : 10.5281/zenodo.6346860
IIP is defined as the pH value at which a zwitterionic molecule
has an equal number of positive and negative charges and no
adsorbed ionic species. IEP is the pH value at which the zeta
potential (or surface potential), equivalent to the net charge of
the molecule including bound ions, is zero.
The differences between IEP and IIP for proteins, and their
shifts with salt type and concentration, have been widely stud-
ied [7, 9]. These differences depend also on the experimental
conditions and the experimental method used [6, 10, 11].
The concept of PZC should not be used for proteins due to
their zwitterionic nature. It is more appropriate to use IEP and
IIP which only includes protonic charge.
3.2 Carbons
The chemical and electrochemical surface properties of car-
bons are of critical importance when used as catalyst supports
and absorbents [12]. It has been suggested that the IEP values
are only representative of the external surface charges of car-
bon particles in solution, whereas the PZC varies in response
to the net total (external and internal) surface charge of the
particles [13, 14].
The difference PZC − IEP can be interpreted as a measure
of surface charge distribution of porous carbons [15]. Values
greater than zero indicate more negatively charged external
than internal particle surfaces, and values close to zero cor-
respond to a more homogeneous distribution of the surface
charges.
3.3 Metal oxides
Oxide particles in aqueous solution form a monolayer of sur-
face surface hydroxyl groups that may become protonated
or deprotonated depending on the pH. The particles develop
electrical charges that are either positive or negative:
+
MOH + H(aq) ⇌ MOH2+
−
MOH + OH(aq) ⇌ MO− + H2 O
If the concentrations of the two sites MOH2+ and MO− of the
particle are equals, there will be no net charge on the surface.
This condition is the PZC (or more accurately the point of
zero proton charge). At pH values lower than the PZC, the
sites become protonated and MOH2+ predominates; the oxide
behaves as a Brönsted acid and as an anion exchanger. At pH
values higher than the PZC, MO− predominates and the oxide
behaves as a Brönsted base and a cation exchanger. Mixed ox-
ides can have both exchange types, depending on the relative
pK values of the different surface sites.
As seen before, PZC can be measured by potentiometric titra-
tion if there are no others ions that H + and OH − in solution.
However, with electrolytes, other ions A− and B+ may absorb
The little corner of ϕ - χ
onto the surface sites as follow:
MOH2+ + A− + −
(aq) ⇌ MOH2 A
− +
MOH2+ + B+ +
(aq) ⇌ MO B + 2H(aq)
Thus, the net surface charge not only depends on the H + and
OH − ions, but also on the concentration of the electrolytes.
A simple and fast method that gives a reasonable approxi-
mation to the PZC consists in locating the intersection of
one or more titration curves performed with constant ionic
strength and different amounts of solid, and that of a blank
solution (i.e., without the solid). This is called the potentio-
metric mass titration method (PMT). This method is explained
by Ibanez et al. [16] by analyzing the plot in Figure 3.
Figure 3. Determination of PZC of a simple metal oxide by
PMT. (Modified from [16])
The points a, b, and c correspond to the blank solution, and
d, e, f , and g correspond to the sample. The blank solution
consists of a solution of an inert, supporting electrolyte (e.g.,
KNO3 ); thus, the point at a corresponds to the natural pH of
this solution. Upon addition of NaOH, the pH of the blank
increases to b. By doing a potentiometric titration with a
strong acid (e.g., HNO3), the blank curve is generated (c is
an arbitrary final point).
The initial pH of the sample solution (inert electrolyte so-
lution and the hydrated oxide MOH) is at d (i.e., below the
PZC due to the removal of H + from the solution by the neutral
surface sites in MOH to form MOH2+ . Then, addition of the
same volume of NaOH to this suspension of the sample brings
it to the point e, which is below b. This is because the OH −
added play three roles: they serve to neutralize the H + and
the MOH2+ created during the a → d step, and to remove H +
from the resulting MOH sites so as to create basic sites MO−
at the oxide surface. By doing a potentiometric titration, the
H + added from e to f (Region I) play two roles: some of
4 A. Hutin – Zenodo – Researchgate
DOI : 10.5281/zenodo.6346860
them neutralize the OH − , and the others neutralize the MO− .
This is the reason for the smaller pH values in this curve as
compared to those in the blank curve. On the other hand, the
H + added from f to g (Region II) also play two roles: to
protonate the neutral MOH sites (major role) and to neutralize
the small amount of OH − remaining in solution (minor role).
The difference in pH between the sample and blank curves
in this region will therefore be essentially proportional to the
amount of solid sample in the suspension.
Between the end of Region I and the beginning of Region
II, there is a small region (or even a single point f ) where all
the H + added serve to neutralize the OH − , just as they do in
the blank curve. For this reason both curves intersect at this
point, which is then identified as the PZC. This intersection
should appear at the same pH value for any amount of oxide
placed in the solution.
Ibanez et al. [16] present a simple experimental method to
aproximate the PZC for several metal oxides TiO2 , SiO2 ,
Al2 O3 , ZnO, and MgO. Vakros et al. [17] also proposed a
simplified PMT with a single determination curve intersecting
the blank curve which gives a very good approximation to the
true value. In addition, the PZC determined using this method
is independent of the charging mechanism assumed for the
oxide surface, of the structure of the double layer, and of the
surface-site density.
Conclusion
Although there are many confusions regarding the use of these
3 terms, it is easy to differentiate them both by their theoreti-
cal definition and by their method of measurement.
The point of zero charge (PZC) is the pH at which the net
surface charge (surface potential) of total particle surface is
equal to zero. This does not mean that the surface has no
charge at PZC, but rather that there are equal amounts of
positive and negative charges. PZC can be determined using
potentiometric titration of colloidal dispersions.
The isoelectric point (IEP) is the pH value at which the elec-
trokinetic potential equals zero. This does not mean there is
zero charge. IEP is strictly determined by electrokinetics (e.g.
electrophoresis).
The isoionic point (IIP) is the pH at which the net protonic
charge of a zwitterionic substance as an amino acid or a pro-
tein is zero. IIP can be measured through potentiometric
titrations carried out at different ionic strengths.
It is important to conclude concerning the measurement of
the pH which is a key element for these 3 parameters. This
is because the determination and accuracy of pH is highly de-
pendent on electrolyte concentrations. At high ionic strength
(> 10 mM), pH depends on the activity of hydrogen ions and
The little corner of ϕ - χ
no longer to the hydrogen concentration, which can lead to a
significant deviation during its determination.
Reading tips
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✓ Comparison of Different Methods for the Point of
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al., Ind. Eng. Chem. Res., 2011, 50, 17, 10017–10023.
✓ A comparison of point of zero charge measurement
methodology. E. Cristiano et al. Clays Clay Miner. 59,
107–115 (2011).
✓ The pH dependent surface charging and points of
zero charge. VIII. Update, M. Kosmulski, Advances
in Colloid and Interface Science, 2020, 275.
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A. Hutin, Difference between Isoelectric point (IEP), Point of
zero charge (PZC), and Isoionic point (IIP). The little corner
of ϕ - χ, 2022, DOI: https://doi.org/10.5281/zenodo.6346860.