Isoelectric Point Calculator - Amino Acid pI from pKa Values

An isoelectric point calculator is the fastest way to find the pH at which an amino acid carries no net electrical charge. You enter pKa1, pKa2, and the side-chain pKaR, and the calculator averages the two values that bracket the zwitterion to return the pI in one step.

• • Biochemistry homework: Check pI values for glycine, alanine, aspartic acid, lysine, and the other standard amino acids against the textbook.
• • Protein purification planning: Pick the buffer pH for ion-exchange chromatography so the target binds the resin and elutes predictably.
• • 2D gel electrophoresis setup: Set the IEF pH gradient endpoints so the protein migrates to its known pI in the first dimension.
• • Peptide pI estimation: Approximate the pI of a synthetic peptide from the side-chain pKa values of its residues before synthesis.

The isoelectric point is the pH at which the positive and negative charges on an amino acid exactly balance, so the molecule behaves as a neutral zwitterion in an electric field. Below the pI the molecule carries a net positive charge and migrates toward the cathode; above the pI it migrates toward the anode. That pH controls behaviour in ion-exchange columns, IEF gels, and any solution where pH-driven precipitation matters.

Once you know the pI, the Buffer pH Calculator shows how to choose a buffer pH at least one unit from the pI so the amino acid stays charged during ion exchange.

The calculator reads the alpha-carboxyl pKa1, the alpha-amino pKa2, and the side-chain pKaR, then picks the averaging rule based on whether the side chain is neutral, acidic, or basic. It returns the pI plus the dominant charge the molecule carries below, at, and above pI.

• pKa1: Negative base-10 log of the alpha-carboxyl dissociation constant. Typical range 1.8 to 2.5 for the 20 standard amino acids.
• pKa2: Negative base-10 log of the alpha-amino dissociation constant. Typical range 8.8 to 10.6 for the 20 standard amino acids.
• pKaR: Negative base-10 log of the side-chain dissociation constant. Enter 0 for neutral side chains, the side-chain COOH pKa for acidic residues, or the side-chain NH/imidazole/guanidino pKa for basic residues.
• sideChainType: Selects the averaging branch. Neutral averages pKa1 and pKa2, acidic averages the two lowest, basic averages the two highest.

Each pKa you enter is the pH at which half of the molecules of that group are protonated. The alpha-carboxyl pKa1 is the lowest pKa in almost every amino acid because the carboxyl is the most acidic site, and the alpha-amino pKa2 is the highest because the protonated amine is the most basic. The side-chain pKaR sits between those two values for acidic residues (Asp, Glu, Cys, Tyr) and above pKa2 for basic residues (Lys, Arg, His).

According to the ExPASy Compute pI/Mw tool (SIB Swiss Institute of Bioinformatics), the theoretical isoelectric point of a protein is computed from the pK values of its residues using the Bjellqvist algorithm, the same averaging rule applied here to free amino acids.

If you need to convert the resulting pI to [H+] or [OH-] in mol/L, the pH & pOH Calculator handles the reverse conversion.

Four ideas explain every pI the calculator returns, from how a pKa describes an ionizable group to why neutral amino acids cluster near pH 6 while acidic residues drop below pH 3.

When the molecule is a peptide or protein rather than a free amino acid, Protein Molecular Weight Calculator gives the residue-by-residue molecular weight that feeds into a pI prediction.

Enter the three pKa values for your amino acid, pick the side-chain type, and read the pI, dominant charge forms, and zwitterion pH window from the results.

1. 1 Look up pKa1, pKa2, and pKaR: Pull the alpha-carboxyl pKa1, alpha-amino pKa2, and side-chain pKaR from a reference table such as the CRC Handbook or the Lehninger pKa table. Use 0 for pKaR if the side chain is not ionizable.
2. 2 Pick the side-chain charge type: 'Neutral' for Gly, Ala, Val, Leu, Ser, Thr, Asn, Gln, Phe, Trp, Met, Pro, Ile; 'Acidic' for Asp, Glu, Cys, Tyr; 'Basic' for Lys, Arg, His.
3. 3 Enter the three pKa values: Type pKa1 in the first box, pKa2 in the second, and pKaR (or 0) in the third. Each box accepts 1 to 14 with two-decimal precision.
4. 4 Confirm the side-chain type: Make sure the dropdown matches the chemistry: 'Neutral' for the standard cluster near pH 6, 'Acidic' for residues below pH 5, 'Basic' for residues above pH 7.
5. 5 Pick a working buffer at least one pH unit from pI: Choose a buffer pH at least 1 pH unit above or below the pI so the amino acid carries a stable net charge during ion exchange or electrophoresis.

For glycine with pKa1 = 2.34, pKa2 = 9.60, pKaR = 0, and a neutral side chain, the calculator returns pI = 5.97. A 50 mM phosphate buffer at pH 7.4 keeps glycine anionic during anion-exchange chromatography.

If the amino acid is part of a larger peptide and you need the protein-level solubility profile around the pI, the Protein Solubility Calculator carries the same logic forward to the full protein.

An isoelectric point calculator turns three pKa values from a reference table into the pH that controls how an amino acid behaves in solution.

• • Skip the averaging by hand: Replace the manual two-pKa or three-pKa average with one click, especially when comparing many amino acids.
• Pick the right ion-exchange buffer: Choose a buffer pH at least one unit from the pI so the target binds the column and elutes predictably.
• • Set up 2D gel electrophoresis: Plan the pH gradient endpoints for the first-dimension IEF strip so the protein focuses at its known pI before SDS-PAGE.
• • Confirm the dominant charge form: See the net charge below, at, and above pI so you can decide which electrode the molecule migrates toward.

When you have a stock solution and need the working concentration in mg/mL, the Protein Concentration Calculator converts absorbance and dilution into concentration.

Five variables drive the pI the calculator returns and explain why similar amino acids give different isoelectric points.

• • The averaging rule assumes pKa values are well separated (at least 2 pH units apart) and ignores overlap between adjacent dissociations. Side chains with pKa values close to pKa1 or pKa2 deviate from the ideal picture.
• • The calculator treats the molecule as a single amino acid with three independent pKa values. Real peptides and proteins carry many overlapping ionizable groups, so the predicted pI ignores local electrostatic effects.
• • pKa values are tabulated for dilute aqueous solution at 25 C. Cosolvents, high salt, or non-aqueous mobile phases can shift pKa by up to 1 pH unit.

According to the PDB-101 educational portal (RCSB Protein Data Bank, NSF/DOE/NIH funded), protein structure and biological function are governed by the pK values and charges of each residue, so the same averaging rule that gives a single-amino-acid pI here is the starting point for full-protein pI prediction in 2D gel and ion-exchange chromatography.

To estimate how much strong acid or base a buffer can absorb at the pH you picked, the Buffer Capacity Calculator returns the beta value at any pKa, total concentration, and acid fraction.