# What is the isoelectric point?

Finds the isoelectric point (pI) of any of the 20 common amino acids, or of an amino acid with typed pKa values, as the average of the two pKa values around its neutral form, with its net charge at a chosen pH.

- Page: https://www.acalculator.org/chemistry/isoelectric-point-calculator
- JSON spec: https://www.acalculator.org/chemistry/isoelectric-point-calculator.json
- Version: 7c55ebc9851a

## Default answer

Example with the default inputs (Amino acid Alanine, pH for the net charge (optional) 7.4): Alanine: pI = 6.015.

## Inputs

| Key | Label | Description |
| --- | --- | --- |
| aa | Amino acid | One of the 20 common amino acids, or Other to type the pKa values. |
| p1 | pKa of the α-carboxyl group | The pKa of the α-CO₂H group, about 2. |
| p2 | pKa of the α-amino group | The pKa of the α-NH₃⁺ group, about 9 to 10. |
| pr | pKa of the side chain (optional) | The side chain’s pKa, if it has an acidic or basic group. |
| side | The side chain is | Acidic (loses H⁺ to go from 0 to −1, as in Asp) or basic (from +1 to 0, as in Lys); used with a side chain pKa. |
| ph | pH for the net charge (optional) | A pH at which to find the amino acid’s average net charge, such as 7.4. |

## Outputs

| Key | Label | Description |
| --- | --- | --- |
| result | Answer | The isoelectric point pI. |
| pi | pI | The pH at which the amino acid carries no net charge. |
| average | Averaged | The two pKa values whose average is the pI. |
| kind | Side chain | Neutral, acidic or basic, which sets the two pKa values used. |
| charge | Net charge at the pH | The average charge from the Henderson–Hasselbalch equation for each group. |
| moves | In electrophoresis at the pH | Which electrode the amino acid moves toward. |

## Method

pI = (pKa below the neutral form + pKa above it) ÷ 2: (pKa₁ + pKa₂) ÷ 2 for a neutral side chain, the two lowest pKa values for an acidic one, the two highest for a basic one; net charge = Σ basic groups 1 ÷ (1 + 10^(pH − pKa)) − Σ acidic groups 1 ÷ (1 + 10^(pKa − pH)).

## Assumptions

- pKa values for the free amino acids in water at 25 °C (OpenStax Table 26.1); in a peptide or protein the values shift, so a protein’s pI needs every ionizable group.
- The pKa values are far enough apart that each group ionizes on its own, as the averaging rule assumes.

## Worked examples

1. aa = ala gives pi = 6.015, result = Alanine: pI = 6.015, average = (2.34 + 9.69) ÷ 2. Source: OpenStax, Organic Chemistry, §26.2 Amino Acids, the Henderson–Hasselbalch Equation, and Isoelectric Points (pI rule; alanine pKa 2.34 and 9.69, pI 6.01), https://openstax.org/books/organic-chemistry/pages/26-2-amino-acids-and-the-henderson-hasselbalch-equation-isoelectric-points: (2.34 + 9.69) ÷ 2 = 6.015, which OpenStax writes 6.01.
2. aa = asp gives pi = 2.765, average = (1.88 + 3.65) ÷ 2. Source: OpenStax, Organic Chemistry, §26.1 Structures of Amino Acids, Table 26.1 The 20 Common Amino Acids in Proteins, https://openstax.org/books/organic-chemistry/pages/26-1-structures-of-amino-acids: pKa 1.88, 9.60, 3.65 and pI 2.77; OpenStax, Organic Chemistry, §26.2 Amino Acids, the Henderson–Hasselbalch Equation, and Isoelectric Points (pI rule; alanine pKa 2.34 and 9.69, pI 6.01), https://openstax.org/books/organic-chemistry/pages/26-2-amino-acids-and-the-henderson-hasselbalch-equation-isoelectric-points.
3. aa = lys gives pi = 9.74, average = (8.95 + 10.53) ÷ 2. Source: OpenStax, Organic Chemistry, §26.1 Structures of Amino Acids, Table 26.1 The 20 Common Amino Acids in Proteins, https://openstax.org/books/organic-chemistry/pages/26-1-structures-of-amino-acids: pKa 2.18, 8.95, 10.53 and pI 9.74.
4. aa = his gives pi = 7.585. Source: OpenStax, Organic Chemistry, §26.1 Structures of Amino Acids, Table 26.1 The 20 Common Amino Acids in Proteins, https://openstax.org/books/organic-chemistry/pages/26-1-structures-of-amino-acids: pKa 1.82, 9.17, 6.00 and pI 7.59.
5. aa = trp gives pi = 5.885. Source: OpenStax, Organic Chemistry, §26.1 Structures of Amino Acids, Table 26.1 The 20 Common Amino Acids in Proteins, https://openstax.org/books/organic-chemistry/pages/26-1-structures-of-amino-acids: pI 5.89; PubChem, L-Tryptophan (CID 6305), Dissociation Constants: pK1 = 2.38, pK2 = 9.39 (HSDB, Merck Index 2006), https://pubchem.ncbi.nlm.nih.gov/compound/6305.
6. aa = custom, p1 = 2.34, p2 = 9.69, side = acidic, ph = 9 gives pi = 6.015, charge = −0.17, moves = toward the positive electrode: pH above pI, net charge negative. Source: OpenStax, Organic Chemistry, §26.2 Amino Acids, the Henderson–Hasselbalch Equation, and Isoelectric Points (pI rule; alanine pKa 2.34 and 9.69, pI 6.01), https://openstax.org/books/organic-chemistry/pages/26-2-amino-acids-and-the-henderson-hasselbalch-equation-isoelectric-points: at pH 9.00, [A⁻]/[HA] = 10^(9.00 − 9.69) = 0.20, so 0.17 of the amino groups have lost H⁺.
7. aa = custom, p1 = 2.18, p2 = 8.95, pr = 10.53, side = basic gives pi = 9.74, kind = basic: average of the two highest pKa values. Source: OpenStax, Organic Chemistry, §26.1 Structures of Amino Acids, Table 26.1 The 20 Common Amino Acids in Proteins, https://openstax.org/books/organic-chemistry/pages/26-1-structures-of-amino-acids: pI 9.74; OpenStax, Organic Chemistry, §26.2 Amino Acids, the Henderson–Hasselbalch Equation, and Isoelectric Points (pI rule; alanine pKa 2.34 and 9.69, pI 6.01), https://openstax.org/books/organic-chemistry/pages/26-2-amino-acids-and-the-henderson-hasselbalch-equation-isoelectric-points.

## FAQ

### How do I calculate the isoelectric point of an amino acid?

Average the two pKa values on either side of the neutral (zwitterion) form. For an amino acid with a neutral side chain, that is pKa₁ and pKa₂: alanine’s are 2.34 and 9.69, so pI = (2.34 + 9.69) ÷ 2 = 6.015, which OpenStax writes as 6.01.

### Which pKa values do I average for acidic and basic amino acids?

For an acidic side chain (aspartic acid, glutamic acid, and the weakly acidic cysteine and tyrosine), the two lowest pKa values: aspartic acid gives (1.88 + 3.65) ÷ 2 = 2.765. For a basic side chain (lysine, arginine, histidine), the two highest: lysine gives (8.95 + 10.53) ÷ 2 = 9.74.

### What does the isoelectric point mean?

It is the pH at which the amino acid exists mostly as its neutral zwitterion, with no net charge. Below the pI it is mostly positive; above it, mostly negative.

### Which way does an amino acid move in electrophoresis?

At a pH below its pI it carries a positive charge and moves toward the negative electrode; above its pI it is negative and moves toward the positive electrode; at its pI it does not move. That is how electrophoresis separates amino acids and proteins.

### How is the net charge at a given pH worked out?

With the Henderson–Hasselbalch equation, pH = pKa + log([A⁻]/[HA]), for each group. An acidic group is ionized (−1) in the fraction 1 ÷ (1 + 10^(pKa − pH)); a basic group is protonated (+1) in the fraction 1 ÷ (1 + 10^(pH − pKa)). Alanine at pH 9.00 has a net charge of −0.17.

### Can I use this for a protein or peptide?

No. A protein’s pI depends on all its ionizable side chains and their shifted pKa values, so it needs every group in the sequence. Examples from OpenStax: lysozyme 11.0, pepsin about 1.0, hemoglobin 6.8.

## Sources

- OpenStax, Organic Chemistry, §26.2 Amino Acids, the Henderson–Hasselbalch Equation, and Isoelectric Points: the pI rule (average of the two pKa values around the neutral zwitterion; the two lowest for acidic side chains, the two highest for basic ones), alanine (2.34, 9.69, pI 6.01) and its charge at pH 9.00, and the Henderson–Hasselbalch equation. https://openstax.org/books/organic-chemistry/pages/26-2-amino-acids-and-the-henderson-hasselbalch-equation-isoelectric-points (retrieved 2026-10-19)
- OpenStax, Organic Chemistry, §26.1 Structures of Amino Acids, Table 26.1 The 20 Common Amino Acids in Proteins: pKa of α-CO₂H, α-NH₃⁺ and the side chain, and pI. https://openstax.org/books/organic-chemistry/pages/26-1-structures-of-amino-acids (retrieved 2026-10-19)
- PubChem, L-Tryptophan (CID 6305), Dissociation Constants: pK1 = 2.38, pK2 = 9.39 (HSDB, from the Merck Index, 2006). https://pubchem.ncbi.nlm.nih.gov/compound/6305 (retrieved 2026-10-19)
- PubChem, L-Isoleucine (CID 6306), Dissociation Constants: pK1 = 2.36, pK2 = 9.68 (HSDB, from the Merck Index). https://pubchem.ncbi.nlm.nih.gov/compound/6306 (retrieved 2026-10-19)