Isoelectric Point Calculator

Calculate the isoelectric point (pI) of amino acids and polyprotic species where the net electrical charge is exactly zero.

pKa 1 (Carboxyl Group)

pKa 2 (Amino Group)

pKa R (Side Chain - Optional)

Environmental pH

Isoelectric Point (pI)

5.97

Predicted Net ChargeNegative (-)

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The Point of Neutrality

The Isoelectric Point (pI) is the exact pH at which a specific molecule carries no net electrical charge.

This concept is absolutely vital in biochemistry, specifically regarding amino acids and proteins. Amino acids are "zwitterions"—they contain both a positively charged amino group and a negatively charged carboxyl group simultaneously.

Because the charge of these groups depends entirely on the pH of the surrounding blood or buffer, the overall net charge of the protein changes as the pH changes.

Why the Isoelectric Point Matters

Solubility: At the isoelectric point, molecules have no net charge. Because they are neutral, they stop repelling each other, clump together, and crash out of solution (precipitate). This is exactly how cheese is made—acid is added to milk until the casein proteins hit their pI and curdle.
Electrophoresis: When running proteins on an electrophoresis gel, they migrate toward the positive or negative anode based on their charge. If the gel is exactly at the protein's pI, the protein will not move at all.

Calculating the pI

To find the pI, you must average the two pKa values that bracket the neutral species.

The Formula

pI = (pKa₁ + pKa₂) / 2

Where:

pI=

Isoelectric Point

pKa₁=

pKa of the acidic bounding group

pKa₂=

pKa of the basic bounding group

The Complication of R-Groups

Simple amino acids like Glycine only have two pKa values (one for the carboxyl, one for the amino). You simply average them.

However, acidic amino acids (like Glutamic Acid) and basic amino acids (like Lysine) have a third pKa value on their side chain (the R-group).

For Acidic side chains: The neutral zwitterion exists between the two acidic pKa values. You average pKa1 (carboxyl) and pKaR (side chain).
For Basic side chains: The neutral zwitterion exists between the two basic pKa values. You average pKa2 (amino) and pKaR (side chain).

Frequently Asked Questions

If the surrounding pH is lower (more acidic) than the isoelectric point, the high concentration of protons in the environment will protonate the molecule. The molecule will take on an overall positive net charge.

If the surrounding pH is higher (more basic) than the isoelectric point, the environment will strip protons away from the molecule. The molecule will take on an overall negative net charge.

A whole protein has hundreds of amino acids, but the internal amino and carboxyl groups are locked up in peptide bonds. Only the side-chains (R-groups) and the very ends of the chain contribute to the charge. Computer algorithms iteratively test different pH values until the sum of all predicted side-chain charges exactly equals zero.

Water molecules normally form a hydration shell around charged proteins, keeping them dissolved. At the pI, the net charge is zero. The hydration shell breaks down, and the hydrophobic regions of the proteins stick to each other instead of the water, causing them to fall out of solution.

It is an advanced laboratory technique where a gel is created with a permanent pH gradient (e.g., pH 3 on the left, pH 10 on the right). When a voltage is applied, proteins move through the gel until they hit the exact spot where the pH matches their pI, at which point they lose their charge and stop moving permanently.

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