Isoelectric Point Calculator

Introduction

The isoelectric point (pI) is the pH at which an amino acid or simple peptide has zero net electrical charge. Below the pI the molecule is more protonated and tends to be net positive; above the pI it loses protons and tends to be net negative. This calculator applies the standard textbook rule for amino acids: average the two pKa values that flank the neutral zwitterion. Use the pKa calculator for pKa conversions and the pH calculator when you need hydrogen-ion concentration from pH.

Isoelectric point formula

For a simple amino acid with only an alpha-carboxyl group and an alpha-amino group, the zwitterion lies between the two dissociation steps, so pI = (pKa1 + pKa2) / 2. If a side chain also ionizes, include pKaR and choose the two pKa values on either side of the net-zero species. This flanking rule is a practical shortcut for amino acids whose pKa values are well separated.

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  No ionizable side chain: average pKa1 and pKa2.

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  Acidic side chain such as Asp or Glu: average the two lowest pKa values.

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  Basic side chain such as Lys, Arg, or His: average the two highest pKa values.

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  The result is a pH value, not a concentration or equilibrium constant.

Why charge changes with pH

Amino acids are ampholytes: they can donate and accept protons. At low pH, carboxyl groups are mostly COOH and amino groups are mostly NH3+, giving positive net charge. As pH rises past pKa1, the carboxyl group becomes COO− and the zwitterion often dominates. Above later pKa values, amino or side-chain groups lose protons, driving net charge negative. The buffer pH calculator uses the same pKa logic through the Henderson-Hasselbalch equation.

Acidic versus basic side-chain rules

Aspartic acid and glutamic acid contain side-chain carboxyl groups. Their neutral form is bracketed by the alpha-carboxyl and side-chain carboxyl pKa values, so pI is low. Lysine and arginine contain extra basic groups; their neutral form is bracketed by the two higher deprotonations, so pI is high. Histidine is a borderline basic amino acid with an imidazole pKa near physiological pH, making its pI especially relevant in protein active sites.

Standard pI values for the 20 amino acids

The following values are commonly cited textbook values at about 25 °C in dilute aqueous solution; exact numbers vary by source and ionic strength. They are useful for checking electrophoresis behavior, protein purification, and peptide solubility.

Worked examples from common amino acids

Glycine uses pKa1 = 2.34 and pKa2 = 9.60, giving pI = 5.97. Alanine uses 2.34 and 9.69, giving 6.02. Aspartic acid is acidic, so the two lowest values 1.88 and 3.65 give 2.77. Lysine is basic, so the two highest values 8.95 and 10.53 give 9.74. Glutamic acid uses 2.19 and 4.25, giving 3.22. These benchmarks are embedded in the calculator tests.

• Neutral side-chain examples cluster near pI 5.5–6.3.

• Acidic amino acids have low pI values and migrate toward the anode above their pI.

• Basic amino acids have high pI values and remain cationic until higher pH.

• For a full titration curve, combine pKa values with stoichiometry using the titration calculator.

Laboratory applications

pI guides isoelectric focusing, ion-exchange chromatography, peptide solubility, and formulation. Proteins are often least soluble near their pI because electrostatic repulsion is minimal, whereas peptides far from their pI carry charge and interact strongly with ion exchangers. For concentration preparation before electrophoresis or chromatography, pair this tool with the molarity calculator. Formal terminology for isoelectric point is defined by the IUPAC Gold Book, and practical amino-acid pKa data are tabulated by CRC Press.

Limitations for peptides and proteins

This calculator is intentionally scoped to single amino acids and simple peptides where the pI is well approximated by flanking pKa values. Large proteins require summing many ionizable groups and solving for the pH where the total charge equals zero. Neighboring residues, post-translational modifications, terminal blocking groups, ionic strength, solvent composition, and temperature can all shift apparent pKa and pI. See the ExPASy pI/Mw tool and LibreTexts biochemistry coverage for broader protein calculations.