What this calculator does
The isoelectric point is the pH at which a molecule carries no net charge. For a simple amino acid it is the average of the two pKa values either side of the neutral form: glycine, at 2.34 and 9.60, has a pI of 5.97.
At that pH the molecule exists as a zwitterion, with a positive and a negative charge cancelling rather than with no charges at all. This matters practically because solubility is lowest at the pI, where there is no net charge to keep molecules apart, which is exactly what isoelectric precipitation exploits.
The formula
The two pKa values flanking the neutral species are averaged. For an amino acid with no ionisable side chain, those are the carboxyl and amino groups. For one with a charged side chain, the relevant pair is different and depends on whether the side chain is acidic or basic, so the two values entered must be chosen accordingly.
| Term | Meaning |
|---|---|
| Isoelectric point (pI) | The pH of zero net charge. |
| Zwitterion | A molecule carrying both a positive and a negative charge that cancel. |
| Amphoteric | Able to act as either acid or base, which amino acids are. |
| Isoelectric precipitation | Separating a protein by adjusting the pH to its pI, where solubility is lowest. |
The inputs explained
| Field | What to enter |
|---|---|
| Lower pKa (e.g. α-carboxyl group) | The lower pKa flanking the neutral form. For a simple amino acid this is the carboxyl group, around 2. |
| Upper pKa (e.g. α-amino group) | The upper pKa. For a simple amino acid this is the amino group, around 9.5. |
When to use it
Separating proteins
Isoelectric focusing separates proteins by driving them through a pH gradient until each reaches its pI and stops.
Precipitating a protein
Adjusting pH to the pI minimises solubility, which is a standard purification step.
Predicting electrophoretic direction
Above its pI a molecule is negatively charged and migrates to the anode; below, the reverse.
Worked examples
Every figure in the tables below is produced by this page’s own calculator at build time, so the numbers and the tool always agree. Select any row to load that scenario.
What is the pI for each amino acid?
Several pKa pairs with the resulting isoelectric point.
| Lower pKa (upper fixed at 9.60) | Isoelectric point (pI) | Lower pKa used | pKa span |
|---|---|---|---|
| pKa₁ = 1.82 | 5.71 | 1.82 | 7.78 |
| pKa₁ = 2.09 | 5.85 | 2.09 | 7.51 |
| pKa₁ = 2.19 | 5.90 | 2.19 | 7.41 |
| pKa₁ = 2.34 | 5.97 | 2.34 | 7.26 |
Questions
What happens at the isoelectric point?
The molecule carries no net charge, existing as a zwitterion with equal positive and negative charges. Solubility is at its minimum because there is no net charge to produce electrostatic repulsion between molecules, and it will not migrate in an electric field.
How do I handle an amino acid with a charged side chain?
Use the two pKa values that flank the neutral form, which are not necessarily the carboxyl and amino ones. For acidic side chains average the two lowest pKa values; for basic ones average the two highest. Using the wrong pair gives a badly wrong answer.
Why is solubility lowest at the pI?
Because charged molecules repel each other and stay dispersed. At the pI the net charge is zero, so that repulsion disappears and molecules aggregate and precipitate. This is the basis of isoelectric precipitation, a standard first step in protein purification.
Does this work for proteins?
Only roughly. A protein has many ionisable groups and its pI emerges from all of them together, so the two-pKa average does not apply. Protein pI values are calculated computationally from the full sequence or measured directly by isoelectric focusing.
For pKa from Ka, see the pKa calculator. For buffer pH near a pKa, see the Henderson-Hasselbalch calculator.