Isoelectric Point (pI) Calculator
Enter two pKa values for a simple molecule, or choose one of the 20 standard amino acids to see its isoelectric point (pI) instantly. The pI is the pH at which a molecule carries zero net charge. The calculator shows step-by-step working, the net charge at your chosen pH, and a charge vs pH profile so you can see exactly how ionisation changes across the pH scale.
What is the isoelectric point (pI)?
The isoelectric point, abbreviated pI or sometimes IEP, is the pH at which a molecule (most commonly an amino acid or protein) carries zero net electrical charge. At any pH below the pI, the molecule has gained protons and carries a net positive charge; at any pH above the pI, it has lost protons and carries a net negative charge. The pI is not the same as a pKa value: pKa describes the equilibrium for a single ionisable group, whereas pI describes the overall electrical balance across all ionisable groups on the molecule. For simple amino acids, the pI sits between the two pKa values that bracket the zwitterionic (electrically neutral) form. For amino acids without an ionisable side chain, that is always the average of pKa1 (alpha-carboxyl, typically 2 to 3) and pKa2 (alpha-amino, typically 9 to 10). For amino acids with acidic side chains (Asp, Glu), the pI falls between pKa1 and pKa3 because the molecule is anionic at all higher pHs. For amino acids with basic side chains (Arg, Lys, His), the pI falls between pKa2 and pKa3 because the side chain adds a second cationic group.
How to calculate the isoelectric point - the formula and worked example
For a neutral amino acid such as alanine (pKa1 = 2.34, pKa2 = 9.69): pI = (pKa1 + pKa2) / 2 = (2.34 + 9.69) / 2 = 6.02 For an acidic amino acid such as aspartic acid (pKa1 = 1.88, pKa2 = 9.60, pKa3 = 3.65): pI = (pKa1 + pKa3) / 2 = (1.88 + 3.65) / 2 = 2.77 For a basic amino acid such as lysine (pKa1 = 2.18, pKa2 = 8.95, pKa3 = 10.53): pI = (pKa2 + pKa3) / 2 = (8.95 + 10.53) / 2 = 9.74 The rule behind these formulas is that pI always lies halfway between the two pKa values that flank the neutral (uncharged) species. Sorting all pKa values and picking the pair whose average gives a pH at which no ionisation form dominates is the general method for more complex polyprotic molecules.
Why the isoelectric point matters in biochemistry and protein science
The pI has direct practical consequences in several areas of biochemistry: Solubility: Proteins are least soluble at their pI because the lack of net charge minimises electrostatic repulsion between molecules, allowing them to aggregate or precipitate. Casein in milk, for example, precipitates at pH 4.6, which is close to its pI. Electrophoresis: In gel electrophoresis, proteins migrate toward the electrode of opposite charge. A protein at a pH below its pI carries a positive charge and moves toward the cathode; above its pI it moves toward the anode. At its pI it stops. Isoelectric focusing (IEF) exploits this by running proteins through a pH gradient until each stops at its own pI. Ion-exchange chromatography: Knowing whether a protein is positively or negatively charged at a given buffer pH tells you which column resin (anion-exchange or cation-exchange) to use and what pH gradient to apply for elution. Crystallisation: Many protein crystallisation screens use pHs near the pI to reduce surface charge and promote ordered crystal packing.
Net charge as a function of pH - the Henderson-Hasselbalch approach
The net charge at any pH is calculated by summing the fractional charge contributions of every ionisable group, using the Henderson-Hasselbalch equation for each: For a carboxyl group (acidic, charge = -1 when deprotonated): z = -1 / (1 + 10^(pKa - pH)) For an amino group (basic, charge = +1 when protonated): z = +1 / (1 + 10^(pH - pKa)) The isoelectric point is the pH where the sum of all such terms equals zero. For complex proteins, this is found by iterative numerical search across pH 0 to 14. The charge vs pH chart in this calculator shows you the full titration curve so you can see not just the pI but also how quickly the charge changes with pH and what the charge is at any physiological or experimental pH you choose.
Standard amino acid pKa values and isoelectric points
| Amino acid | Code | pKa1 (COOH) | pKa2 (NH3) | pKa3 (SC) | pI | Class |
|---|---|---|---|---|---|---|
| Alanine | A | 2.34 | 9.69 | - | 6.01 | Nonpolar |
| Arginine | R | 2.17 | 9.04 | 12.48 | 10.76 | Basic |
| Asparagine | N | 2.02 | 8.8 | - | 5.41 | Polar |
| Aspartic acid | D | 1.88 | 9.6 | 3.65 | 2.77 | Acidic |
| Cysteine | C | 1.96 | 10.28 | 8.18 | 5.07 | Polar |
| Glutamic acid | E | 2.19 | 9.67 | 4.25 | 3.22 | Acidic |
| Glutamine | Q | 2.17 | 9.13 | - | 5.65 | Polar |
| Glycine | G | 2.34 | 9.6 | - | 5.97 | Nonpolar |
| Histidine | H | 1.82 | 9.17 | 6 | 7.59 | Basic |
| Isoleucine | I | 2.36 | 9.68 | - | 6.02 | Nonpolar |
| Leucine | L | 2.36 | 9.6 | - | 5.98 | Nonpolar |
| Lysine | K | 2.18 | 8.95 | 10.53 | 9.74 | Basic |
| Methionine | M | 2.28 | 9.21 | - | 5.74 | Nonpolar |
| Phenylalanine | F | 1.83 | 9.13 | - | 5.48 | Nonpolar |
| Proline | P | 1.99 | 10.6 | - | 6.3 | Nonpolar |
| Serine | S | 2.21 | 9.15 | - | 5.68 | Polar |
| Threonine | T | 2.09 | 9.1 | - | 5.6 | Polar |
| Tryptophan | W | 2.83 | 9.39 | - | 5.89 | Nonpolar |
| Tyrosine | Y | 2.2 | 9.11 | 10.07 | 5.66 | Polar |
| Valine | V | 2.32 | 9.62 | - | 5.97 | Nonpolar |
pKa values from Lehninger Principles of Biochemistry (7th ed.). pKa3 applies only to amino acids with ionisable side chains.
Frequently asked questions
What does isoelectric point mean?
The isoelectric point (pI) is the pH value at which a molecule carries zero net electrical charge. At this pH, the positive charges from protonated amino groups exactly cancel the negative charges from deprotonated carboxyl groups. The molecule is in its zwitterionic form and is least soluble in water.
How do I calculate the isoelectric point from pKa values?
For a molecule with two ionisable groups (like a simple amino acid without an ionisable side chain), pI = (pKa1 + pKa2) / 2. For amino acids with an acidic side chain (Asp, Glu), pI = (pKa_alpha_COOH + pKa_side_chain_COOH) / 2. For amino acids with a basic side chain (Arg, Lys, His), pI = (pKa_alpha_NH3 + pKa_side_chain_NH3) / 2. The general rule is: sort all pKa values and average the two that bracket the neutral (zero-charge) form.
Why are acidic amino acids positively charged at low pH?
At a pH well below all pKa values, all ionisable groups are in their protonated form: the carboxyl groups carry -COOH (neutral) and the amino group carries -NH3+ (positive). As pH rises and passes pKa1, the carboxyl loses a proton and becomes -COO- (negative), so the net charge drops. The net charge is only positive at very low pH before the first pKa is reached.
What is the difference between pI and pKa?
A pKa value describes the equilibrium for a single ionisable group: it is the pH at which that group is 50 percent protonated and 50 percent deprotonated. The pI is an overall property of the whole molecule: it is the pH at which the net sum of all charges across all ionisable groups equals zero. A simple diprotic amino acid has two pKa values and one pI; a polyprotic molecule with many ionisable groups has many pKa values but still just one pI.
Why is a protein least soluble at its isoelectric point?
Solubility in water depends partly on electrostatic repulsion between molecules: charged molecules repel each other and stay dispersed. At the pI, the net charge is zero, so this repulsion disappears and molecules can approach each other closely enough for hydrophobic interactions and hydrogen bonds to cause aggregation or precipitation. Adding salt (salting out) or adjusting pH away from the pI are the standard ways to redissolve a precipitated protein.
What are typical pI values for each amino acid class?
Neutral (nonpolar and polar) amino acids have pI values between about 5.4 and 6.3, close to pH 7. Acidic amino acids (Asp, Glu) have pI values around 2.8 to 3.2, well below neutrality. Basic amino acids have high pI values: histidine is about 7.6, lysine about 9.7, and arginine about 10.8. These differences reflect the extra ionisable group on the side chain shifting the electrical balance of the molecule.
How is isoelectric focusing (IEF) used in the lab?
Isoelectric focusing uses a pre-formed pH gradient (usually a polyacrylamide gel with ampholytes, or an immobilised pH gradient strip). When a voltage is applied, proteins migrate through the gradient until each reaches the pH equal to its pI. At that point the protein carries no net charge and stops moving. IEF gives very high resolution because proteins are separated purely by pI differences as small as 0.01 pH unit, and it is the first dimension of 2D gel electrophoresis.
Can I use this calculator for proteins, not just single amino acids?
This calculator uses the standard two-pKa or three-pKa formula for individual amino acids and simple molecules. For full protein pI calculation you need to know the pKa of every ionisable residue in the sequence (the N-terminus, C-terminus, and all Asp, Glu, His, Cys, Tyr, Lys, and Arg residues) and find the pH where their combined charge sums to zero. That requires a sequence-based tool such as ExPASy Compute pI/Mw or isoelectric.org. Use the custom pKa mode here to explore how individual pKa values influence the result.