Isoelectric point

Isoelectric point

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The isoelectric point sometimes abbreviated to IEP, is the pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...

 at which a particular molecule
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...

 or surface carries no net electrical charge
Electric charge
Electric charge is a physical property of matter that causes it to experience a force when near other electrically charged matter. Electric charge comes in two types, called positive and negative. Two positively charged substances, or objects, experience a mutual repulsive force, as do two...

.

Amphoteric molecules called zwitterion
Zwitterion
In chemistry, a zwitterion is a neutral molecule with a positive and a negative electrical charge at different locations within that molecule. Zwitterions are sometimes also called inner salts.-Examples:...

s contain both positive and negative charges depending on the functional groups present in the molecule. The net charge on the molecule is affected by pH of their surrounding environment and can become more positively or negatively charged due to the loss or gain of protons (H+). The pI is the pH value at which the molecule carries no electrical charge or the negative and positive charges are equal.

Surfaces naturally charge to form a double layer
Double layer (interfacial)
A double layer is a structure that appears on the surface of an object when it is placed into a liquid. The object might be a solid particle, a gas bubble, a liquid droplet, or a porous body. The DL refers to two parallel layers of charge surrounding the object...

. 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. Again, the pI is the pH value of the solution at which the surfaces carries no net charge.

The pI value can affect the solubility of a molecule at a given pH. Such molecules have minimum solubility
Solubility
Solubility is the property of a solid, liquid, or gaseous chemical substance called solute to dissolve in a solid, liquid, or gaseous solvent to form a homogeneous solution of the solute in the solvent. The solubility of a substance fundamentally depends on the used solvent as well as on...

 in water or salt solutions at the pH that corresponds to their pI and often precipitate out of solution
Solution
In chemistry, a solution is a homogeneous mixture composed of only one phase. In such a mixture, a solute is dissolved in another substance, known as a solvent. The solvent does the dissolving.- Types of solutions :...

. Biological amphoteric molecules such as protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s 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
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...

 below their pI, proteins carry a net positive charge; above their pI they carry a net negative charge. Proteins can, thus, be separated according to their isoelectric point (overall charge) on a polyacrylamide gel using a technique called isoelectric focusing
Isoelectric focusing
Isoelectric focusing , also known as electrofocusing, is a technique for separating different molecules by their electric charge differences...

, which uses a pH gradient to separate proteins. Isoelectric focusing is also the first step in 2-D gel polyacrylamide gel electrophoresis
Two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins...

.

Calculating pI values


For an amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...

 with only one amine
Amine
Amines are organic compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are derivatives of ammonia, wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group. Important amines include amino acids, biogenic amines,...

 and one carboxyl group, the pI can be calculated from the mean
Mean
In statistics, mean has two related meanings:* the arithmetic mean .* the expected value of a random variable, which is also called the population mean....

 of the pKa
PKA
PKA, pKa, or other similar variations may stand for:* pKa, the symbol for the acid dissociation constant at logarithmic scale* Protein kinase A, a class of cAMP-dependent enzymes* Pi Kappa Alpha, the North-American social fraternity...

s of this molecule.

For amino acids with more than two ionizable groups, such as lysine
Lysine
Lysine is an α-amino acid with the chemical formula HO2CCH4NH2. It is an essential amino acid, which means that the human body cannot synthesize it. Its codons are AAA and AAG....

, the same formula is used, but this time the two pKa's used are those of the two groups that lose and gain a charge from the neutral form of the amino acid. Lysine
Lysine
Lysine is an α-amino acid with the chemical formula HO2CCH4NH2. It is an essential amino acid, which means that the human body cannot synthesize it. Its codons are AAA and AAG....

 has a single carboxylic pKa and two amine pKa values (one of which is on the R-group
Side chain
In organic chemistry and biochemistry, a side chain is a chemical group that is attached to a core part of the molecule called "main chain" or backbone. The placeholder R is often used as a generic placeholder for alkyl group side chains in chemical structure diagrams. To indicate other non-carbon...

), so fully protonated lysine has a +2 net charge. To get a neutral charge, we must deprotonate the lysine twice, and therefore use the R-group
Side chain
In organic chemistry and biochemistry, a side chain is a chemical group that is attached to a core part of the molecule called "main chain" or backbone. The placeholder R is often used as a generic placeholder for alkyl group side chains in chemical structure diagrams. To indicate other non-carbon...

 and amine pKa values (found at List of standard amino acids).


The pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...

 of an electrophoretic gel is determined by the buffer
Buffer solution
A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a...

 used for that gel. If the pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...

 of the buffer is above the pI of the protein being run, the protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

 will migrate to the positive pole (negative charge is attracted to a positive pole). If the pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...

 of the buffer is below the pI of the protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

 being run, the protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

 will migrate to the negative pole of the gel (positive charge is attracted to the negative pole). If the protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

 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.

Examples

glycine pK = 2.72, 9.60 lysine pK = 2.15, 9.16, 10.67 Adenosine monophosphate pK = 0.9, 3.8, 6.1

In these three examples the isoelectric point is shown by the green vertical line. In glycine
Glycine
Glycine is an organic compound with the formula NH2CH2COOH. Having a hydrogen substituent as its 'side chain', glycine is the smallest of the 20 amino acids commonly found in proteins. Its codons are GGU, GGC, GGA, GGG cf. the genetic code.Glycine is a colourless, sweet-tasting crystalline solid...

 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
Zwitterion
In chemistry, a zwitterion is a neutral molecule with a positive and a negative electrical charge at different locations within that molecule. Zwitterions are sometimes also called inner salts.-Examples:...

 at the isoelectric point, but the equilibrium constant for the isomerization reaction in solution
H2NCH2CO2H H3N+CH2CO2-

is not known.

Lysine
Lysine
Lysine is an α-amino acid with the chemical formula HO2CCH4NH2. It is an essential amino acid, which means that the human body cannot synthesize it. Its codons are AAA and AAG....

 has an amino group on the side-chain, so the isoelectric point is the average of the amino group pK values. The concentration of the neutral species, lysine (LH), is a maximum at the isoelectric point, but the concentration is less than 100% because the difference in pK values is only about 1.5. Lysine may exist as a zwitterion in solution.

The third example, adenosine monophosphate
Adenosine monophosphate
Adenosine monophosphate , also known as 5'-adenylic acid, is a nucleotide that is used as a monomer in RNA. It is an ester of phosphoric acid and the nucleoside adenosine. AMP consists of a phosphate group, the sugar ribose, and the nucleobase adenine...

 is shown to illustrate the fact that a third species may, in principle, be involved. In actual fact the concentration of (AMP)H32+ is negligible at the isoelectric point in this case.

Ceramic materials


The isoelectric points (IEP) of metal oxide ceramics are used extensively in material science in various aqueous processing steps (synthesis, modification, etc.). For these surfaces, present as colloids or larger particles in aqueous solution, the surface is 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
    Vanadium(V) oxide
    Vanadium oxide is the chemical compound with the formula V2O5. Commonly known as vanadium pentoxide, this brown/yellow solid is the most stable and common compound of vanadium. Upon heating it reversibly loses oxygen...

     (vanadia) V2O5: 1-2 (3)
  • silicon dioxide
    Silicon dioxide
    The chemical compound silicon dioxide, also known as silica , is an oxide of silicon with the chemical formula '. It has been known for its hardness since antiquity...

     (silica) SiO2: 1.7-3.5
  • silicon carbide
    Silicon carbide
    Silicon carbide , also known as carborundum, is a compound of silicon and carbon with chemical formula SiC. It occurs in nature as the extremely rare mineral moissanite. Silicon carbide powder has been mass-produced since 1893 for use as an abrasive...

     (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
    Manganese(IV) oxide
    Manganese oxide is the inorganic compound with the formula . This blackish or brown solid occurs naturally as the mineral pyrolusite, which is the main ore of manganese and a component of manganese nodules. The principal use for MnO2 is for dry-cell batteries, such as the alkaline battery and the...

     MnO2: 4-5
  • delta-MnO2 1.5, beta-MnO2 7.3
  • titanium(IV) oxide (titania) (rutile
    Rutile
    Rutile is a mineral composed primarily of titanium dioxide, TiO2.Rutile is the most common natural form of TiO2. Two rarer polymorphs of TiO2 are known:...

     or anatase
    Anatase
    Anatase is one of the three mineral forms of titanium dioxide, the other two being brookite and rutile. It is always found as small, isolated and sharply developed crystals, and like rutile, a more commonly occurring modification of titanium dioxide, it crystallizes in the tetragonal system; but,...

    ) TiO2: 3.9-8.2
  • silicon nitride
    Silicon nitride
    Silicon nitride is a chemical compound of silicon and nitrogen. If powdered silicon is heated between 1300° and 1400°C in an atmosphere of nitrogen, trisilicon tetranitride, Si3N4, is formed. The silicon sample weight increases progressively due to the chemical combination of silicon and nitrogen...

     Si3N4: 6-7
  • iron (II, III) oxide
    Magnetite
    Magnetite is a ferrimagnetic mineral with chemical formula Fe3O4, one of several iron oxides and a member of the spinel group. The chemical IUPAC name is iron oxide and the common chemical name is ferrous-ferric oxide. The formula for magnetite may also be written as FeO·Fe2O3, which is one part...

     (magnetite) Fe3O4: 6.5-6.8
  • gamma iron (III) oxide
    Maghemite
    Maghemite is a member of the family of iron oxides. It has the same structure as magnetite, that is, it is spinel ferrite and is also ferrimagnetic.Maghemite can be considered as an Fe-deficient magnetite with formula...

     (maghemite) Fe2O3: 3.3-6.7
  • cerium(IV) oxide
    Cerium(IV) oxide
    Cerium oxide, also known as ceric oxide, ceria, cerium oxide or cerium dioxide, is an oxide of the rare earth metal cerium...

     (ceria) CeO2: 6.7-8.6
  • chromium(III) oxide
    Chromium(III) oxide
    Chromium oxide is the inorganic compound of the formula Cr2O3. It is one of principal oxides of chromium and is used as a pigment. In nature, it occurs as the rare mineral eskolaite.-Structure and properties:...

     (chromia) Cr2O3: 7 (6.2-8.1)
  • gamma aluminium oxide
    Aluminium oxide
    Aluminium oxide is an amphoteric oxide with the chemical formula 23. It is commonly referred to as alumina, or corundum in its crystalline form, as well as many other names, reflecting its widespread occurrence in nature and industry...

     (gamma alumina) Al2O3: 7-8
  • thallium(I) oxide Tl2O: 8
  • alpha iron (III) oxide
    Hematite
    Hematite, also spelled as haematite, is the mineral form of iron oxide , one of several iron oxides. Hematite crystallizes in the rhombohedral system, and it has the same crystal structure as ilmenite and corundum...

     (hematite) Fe2O3: 8.4-8.5
  • alpha aluminium oxide
    Aluminium oxide
    Aluminium oxide is an amphoteric oxide with the chemical formula 23. It is commonly referred to as alumina, or corundum in its crystalline form, as well as many other names, reflecting its widespread occurrence in nature and industry...

     (alpha alumina, corundum) Al2O3: 8-9
  • silicon nitride
    Silicon nitride
    Silicon nitride is a chemical compound of silicon and nitrogen. If powdered silicon is heated between 1300° and 1400°C in an atmosphere of nitrogen, trisilicon tetranitride, Si3N4, is formed. The silicon sample weight increases progressively due to the chemical combination of silicon and nitrogen...

     Si3N4: 9
  • yttrium(III) oxide
    Yttrium(III) oxide
    Yttrium oxide is Y2O3. It is an air-stable, white solid substance. Yttrium oxide is used as a common starting material for both materials science as well as inorganic compounds.-Materials science:...

     (yttria) Y2O3: 7.15-8.95
  • copper(II) oxide
    Copper(II) oxide
    Copper oxide or cupric oxide is the higher oxide of copper. As a mineral, it is known as tenorite.-Chemistry:It is a black solid with an ionic structure which melts above 1200 °C with some loss of oxygen...

     CuO: 9.5
  • zinc oxide
    Zinc oxide
    Zinc oxide is an inorganic compound with the formula ZnO. It is a white powder that is insoluble in water. The powder is widely used as an additive into numerous materials and products including plastics, ceramics, glass, cement, rubber , lubricants, paints, ointments, adhesives, sealants,...

     ZnO: 8.7-10.3
  • lanthanum(III) oxide
    Lanthanum(III) oxide
    Lanthanum oxide is La2O3, an inorganic compound containing the rare earth element lanthanum and oxygen. It is used to develop ferroelectric materials, and in optical materials.-Properties:...

     La2O3: 10
  • nickel(II) oxide
    Nickel(II) oxide
    Nickel oxide is the chemical compound with the formula NiO. It is notable as being the only well characterized oxide of nickel . The mineralogical form of NiO, bunsenite, is very rare. It is classified as a basic metal oxide...

     NiO: 10-11 (9.9-11.3)
  • lead(II) oxide
    Lead(II) oxide
    Lead oxide is the inorganic compound with the formula PbO. Lead oxide occurs in two polymorphs, red, having a tetragonal crystal structure and yellow, having an orthorhombic crystal structure...

     PbO: 10.7-11.6
  • magnesium oxide
    Magnesium oxide
    Magnesium oxide , or magnesia, is a white hygroscopic solid mineral that occurs naturally as periclase and is a source of magnesium . It has an empirical formula of and consists of a lattice of Mg2+ ions and O2– ions held together by ionic bonds...

     (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
Aluminosilicate
Aluminosilicate minerals are minerals composed of aluminium, silicon, and oxygen, plus countercations. They are a major component of kaolin and other clay minerals....

 (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
Barium titanate
Barium titanate is the inorganic compound with the chemical formula BaTiO3. Barium titanate is a white powder and transparent as larger crystals...

, 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
Barium carbonate
Barium carbonate , also known as witherite, is a chemical compound used in rat poison, bricks, ceramic glazes and cement.Witherite crystallizes in the orthorhombic system...

 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
Electrokinetic phenomena
Electrokinetic phenomena are a family of several different effects that occur in heterogeneous fluids or in porous bodies filled with fluid. The term heterogeneous here means a fluid containing particles...

 generally measure zeta potential
Zeta potential
Zeta potential is a scientific term for electrokinetic potential in colloidal systems. In the colloidal chemistry literature, it is usually denoted using the Greek letter zeta, hence ζ-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
Adsorption
Adsorption is the adhesion of atoms, ions, biomolecules or molecules of gas, liquid, or dissolved solids to a surface. This process creates a film of the adsorbate on the surface of the adsorbent. It differs from absorption, in which a fluid permeates or is dissolved by a liquid or solid...

 positive or negative charges. In this context, specific adsorption is understood as adsorption occurring in a Stern layer
Double layer (interfacial)
A double layer is a structure that appears on the surface of an object when it is placed into a liquid. The object might be a solid particle, a gas bubble, a liquid droplet, or a porous body. The DL refers to two parallel layers of charge surrounding the object...

 or chemisorption
Chemisorption
Chemisorption is a sub-class of adsorption, driven by a chemical reaction occurring at the exposed surface. A new chemical species is generated at the adsorbant surface...

. 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 predominate 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.

Further reading

  • Nelson DL, Cox MM (2004). Lehninger Principles of Biochemistry. W. H. Freeman; 4th edition (Hardcover). ISBN 0-7167-4339-
  • Kosmulski M. (2009). Surface Charging and Points of Zero Charge. CRC Press; 1st edition (Hardcover). ISBN 978-1-4200-5188-9

External links