The isoelectric point (pI, pH(I), IEP), is the pH at which a particular molecule carries no net electrical charge in the statistical mean. The standard nomenclature to represent the isoelectric point is pH(I), although pI is also commonly seen, and is used in this article for brevity. The net charge on the molecule is affected by pH of its surrounding environment and can become more positively or negatively charged due to the gain or loss, respectively, of protons (H+).
Surfaces naturally charge to form a double layer. In the common case when the surface charge-determining ions are H+/OH−, the net surface charge is affected by the pH of the liquid in which the solid is submerged.
The pI value can affect the solubility of a molecule at a given pH. Such molecules have minimum solubility in water or salt solutions at the pH that corresponds to their pI and often precipitate out of solution. Biological amphoteric molecules such as proteins contain both acidic and basic functional groups. Amino acids that make up proteins may be positive, negative, neutral, or polar in nature, and together give a protein its overall charge. At a pH below their pI, proteins carry a net positive charge; above their pI they carry a net negative charge. Proteins can, thus, be separated by net charge in a polyacrylamide gel using either preparative gel electrophoresis or isoelectric focusing, which uses a pH gradient to separate proteins. Isoelectric focusing is also the first step in 2-D gel polyacrylamide gel electrophoresis.
Calculating pI values
The pH of an electrophoretic gel is determined by the buffer used for that gel. If the pH of the buffer is above the pI of the protein being run, the protein will migrate to the positive pole (negative charge is attracted to a positive pole). If the pH of the buffer is below the pI of the protein being run, the protein will migrate to the negative pole of the gel (positive charge is attracted to the negative pole). If the protein is run with a buffer pH that is equal to the pI, it will not migrate at all. This is also true for individual amino acids.
|glycine pK = 2.72, 9.60||adenosine monophosphate pK = 0.9, 3.8, 6.1|
In these two examples the isoelectric point is shown by the green vertical line. In glycine the pK values are separated by nearly 7 units so the concentration of the neutral species, glycine (GlyH), is effectively 100% of the analytical glycine concentration. Glycine may exist as a zwitterion at the isoelectric point, but the equilibrium constant for the isomerization reaction in solution
- H2NCH2CO2H ⇌ H3N+CH2CO2−
is not known.
The other example, adenosine monophosphate is shown to illustrate the fact that a third species may, in principle, be involved. In fact the concentration of (AMP)H32+ is negligible at the isoelectric point in this case. If the pI is greater than the pH, the molecule will have a positive charge.
Estimation of isoelectric point of peptides and proteins
Current algorithms for estimating isoelectric points of peptides and proteins depends primarily on the model proposed by Bjellqvist and co-workers. This model is based on the determination of the pK differences between closely related immobilines, by focusing the same sample in overlapping pH gradients. Some improvements in the methodology (especially in the determination of the pK values) have been published since the Bjellqvist publication. As an alternative, Cargile and co-workers has followed another approach: their algorithm accounts for the effect of adjacent amino acids ±3 residues away from a charged aspartic or glutamic acid, the effects on free C terminus, as well as applies a correction term to the corresponding pK values. They also applied genetic algorithm to a 5000-peptide training set to derive the results, which have shown isoelectric point not only depends on individual amino acid, but also on the interactions between different amino acids present in the peptide sequence. In 2012, Perez-Riverol and co-workers published method based on a support vector machine algorithm.
The isoelectric points (IEP) of metal oxide ceramics are used extensively in material science in various aqueous processing steps (synthesis, modification, etc.). In the absence of chemisorbed or physisorbed species particle surfaces in aqueous suspension are generally assumed to be covered with surface hydroxyl species, M-OH (where M is a metal such as Al, Si, etc.). At pH values above the IEP, the predominate surface species is M-O−, while at pH values below the IEP, M-OH2+ species predominate. Some approximate values of common ceramics are listed below (Haruta and Brunelle, except where noted). The exact value can vary widely, depending on material factors such as purity and phase as well as physical parameters such as temperature. In addition, precise measurement of isoelectric points is difficult and requires careful techniques, even with modern methods. Thus, many sources often cite differing values for isoelectric points of these materials.
Examples of isoelectric points
The following list gives the pH25 °C of isoelectric point at 25 °C for selected materials in water:
Note: The list is ordered by increasing pH values.
- tungsten(VI) oxide WO3: 0.2-0.5
- antimony(V) oxide Sb2O5: <0.4 to 1.9
- vanadium(V) oxide (vanadia) V2O5: 1-2 (3)
- silicon dioxide (silica) SiO2: 1.7-3.5
- silicon carbide (alpha) SiC: 2-3.5
- tantalum(V) oxide, Ta2O5: 2.7-3.0
- tin(IV) oxide SnO2: 4-5.5 (7.3)
- zirconium(IV) oxide (zirconia) ZrO2: 4-11
- manganese(IV) oxide MnO2: 4-5
- indium tin oxide ITO: 6
- delta-MnO2 1.5, beta-MnO2 7.3
- titanium(IV) oxide (titania) (rutile or anatase) TiO2: 3.9-8.2
- silicon nitride Si3N4: 6-7
- iron (II, III) oxide (magnetite) Fe3O4: 6.5-6.8
- gamma iron (III) oxide (maghemite) Fe2O3: 3.3-6.7
- cerium(IV) oxide (ceria) CeO2: 6.7-8.6
- chromium(III) oxide (chromia) Cr2O3: 7 (6.2-8.1)
- gamma aluminium oxide (gamma alumina) Al2O3: 7-8
- thallium(I) oxide Tl2O: 8
- alpha iron (III) oxide (hematite) Fe2O3: 8.4-8.5
- alpha aluminium oxide (alpha alumina, corundum) Al2O3: 8-9
- silicon nitride Si3N4: 9
- yttrium(III) oxide (yttria) Y2O3: 7.15-8.95
- copper(II) oxide CuO: 9.5
- zinc oxide ZnO: 8.7-10.3
- lanthanum(III) oxide La2O3: 10
- nickel(II) oxide NiO: 10-11 (9.9-11.3)
- lead(II) oxide PbO: 10.7-11.6
- magnesium oxide (magnesia) MgO: 12-13 (9.8-12.7)
Mixed oxides may exhibit isoelectric point values that are intermediate to those of the corresponding pure oxides. For example, Jara et al. measured an IEP of 4.5 for a synthetically prepared amorphous aluminosilicate (Al2O3-SiO2). The researchers noted that the electrokinetic behavior of the surface was dominated by surface Si-OH species, thus explaining the relatively low IEP value. Significantly higher IEP values (pH 6 to 8) have been reported for 3Al2O3-2SiO2 by others (see Lewis). Lewis also lists the IEP of barium titanate, BaTiO3 as being between pH 5 and 6, while Vamvakaki et al. reported a value of 3, although these authors note that a wide range of values have been reported, a result of either residual barium carbonate on the surface or TiO2-rich surfaces.
The farther the pH of an Amino Acid solution is from its pl the greater the electric charge on that population of molecules.
Isoelectric point versus point of zero charge
The terms isoelectric point (IEP) and point of zero charge (PZC) are often used interchangeably, although under certain circumstances, it may be productive to make the distinction.
In systems in which H+/OH− are the interface potential-determining ions, the point of zero charge is given in terms of pH. The pH at which the surface exhibits a neutral net electrical charge is the point of zero charge at the surface. Electrokinetic phenomena generally measure zeta potential, and a zero zeta potential is interpreted as the point of zero net charge at the shear plane. This is termed the isoelectric point. Thus, the isoelectric point is the value of pH at which the colloidal particle remains stationary in an electrical field. The isoelectric point is expected to be somewhat different than the point of zero charge at the particle surface, but this difference is often ignored in practice for so-called pristine surfaces, i.e., surfaces with no specifically adsorbed positive or negative charges. In this context, specific adsorption is understood as adsorption occurring in a Stern layer or chemisorption. Thus, point of zero charge at the surface is taken as equal to isoelectric point in the absence of specific adsorption on that surface.
According to Jolivet, in the absence of positive or negative charges, the surface is best described by the point of zero charge. If positive and negative charges are both present in equal amounts, then this is the isoelectric point. Thus, the PZC refers to the absence of any type of surface charge, while the IEP refers to a state of neutral net surface charge. The difference between the two, therefore, is the quantity of charged sites at the point of net zero charge. Jolivet uses the intrinsic surface equilibrium constants, pK− and pK+ to define the two conditions in terms of the relative number of charged sites:
For large ΔpK (>4 according to Jolivet), the predominant species is MOH while there are relatively few charged species - so the PZC is relevant. For small values of ΔpK, there are many charged species in approximately equal numbers, so one speaks of the IEP.
- Acceptable variants on pH(I) would include pHI, pHIEP, etc; the main point is that one cannot take the 'power' of I, rather one measures the pH subject to a nominated condition.
- IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). XML on-line corrected version: http://goldbook.iupac.org (2006-) created by M. Nic, J. Jirat, B. Kosata; updates compiled by A. Jenkins. ISBN 0-9678550-9-8. doi:10.1351/goldbook. Last update: 2014-02-24; version: 2.3.3. DOI of this term: doi:10.1351/goldbook.I03275.
- For derivation of this expression see acid dissociation constant
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- Calculation of protein isoelectric point — free online and offline program to calculation pI and more theoretical information about this subject.
- prot pi - protein isoelectric point — free online program for calculating pI of proteins, also allowing for multiple subunits and posttranslational modifications. Prot pi also shows a plot of protein charge versus pH.
- Isoelectric point determination and Charge versus pH plot of amphoteric molecules (e.g., amino acids) by a free suite of spreadsheets for computing acid-base equilibria.