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Isoelectric Point Calculator

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pI = (pKa + (14−pKb))/2.
20 Amino Acid Reference.
Charge-vs-pH Map.
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How it Works

01Identify the Two Groups

An amphoteric molecule has at least one acidic and one basic group (e.g. amino acids: COOH + NH₂).

02Enter pKa (Acid) + pKb (Base)

pKa = -log Ka of the carboxylic / phenolic / acidic group; pKb = -log Kb of the amine / basic group.

03Apply pI = (pKa + (14 − pKb)) / 2

Convert pKb to conjugate-acid pKa (14 − pKb), average with the acid pKa.

04Get pI + Charge Map

pH at zero net charge. At pH < pI molecule is +; at pH > pI molecule is −.

What is an Isoelectric Point Calculator?

The isoelectric point (pI) is the pH at which an amphoteric molecule — one that contains both acidic and basic groups — has zero net electric charge. At this pH, the molecule exists predominantly as a zwitterion (a single molecule with equal positive and negative charges), the form with minimum solubility in water and the foundation of every protein-purification technique that exploits charge: ion-exchange chromatography, isoelectric focusing (IEF), 2D gel electrophoresis, isoelectric precipitation. Our Isoelectric Point Calculator implements the simple two-group formula pI = (pKa + (14 − pKb)) / 2 — average of the acid pKa and the conjugate-acid pKa of the base (recovered as 14 − pKb at 25 °C in water).

The formula is exact for any molecule with exactly one acidic group (carboxylic acid, phenol, sulfonic acid) and one basic group (amine, guanidine, imidazole). For amino acids without ionizable side chains (Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, Ser, Thr, Asn, Gln) the simple formula gives the textbook pI value (e.g. glycine pI = 5.97 from pKa1 = 2.34 and pKa2 = 9.60 → conjugate amine pKb = 4.40). For amino acids with ionizable side chains (acidic Asp 2.77, Glu 3.22; basic Lys 9.74, Arg 10.76, His 7.59; polar Tyr 5.66, Cys 5.07), pI is computed from the two pKa values flanking the zero-net-charge species — these values are tabulated in the result panel for reference. For peptides and proteins, use ExPASy's Compute pI/Mw tool with the Bjellqvist algorithm for sequence-based predictions.

Designed for biochemistry students learning amino-acid chemistry and amphoteric titration, protein chemists planning purification (ion-exchange selection, IEF gel pH gradients), pharmaceutical formulation scientists optimizing solubility, and food scientists working with milk caseins and soy proteins, the tool runs entirely in your browser — no account, no data stored.

Pro Tip: Pair this with our Buffer pH Calculator for buffer preparation at specific pH values, our Protein Solubility Calculator for Kjeldahl protein quantification, or our Molarity Calculator for stock preparation.

How to Use the Isoelectric Point Calculator?

Identify the Acidic and Basic Groups: An amphoteric molecule has at least one acidic (proton-donating) group AND at least one basic (proton-accepting) group on the same molecule. Examples: amino acids (COOH + NH₂), zwitterionic surfactants (sulfonate + quaternary amine), some pharmaceuticals (e.g. amphoteric antibiotics, alendronate).
Look Up or Measure pKa of the Acid: pKa = -log(Ka), the equilibrium constant for proton dissociation. Carboxylic acids: pKa 2-5. Phenols: pKa 8-11. Sulfonic acids: pKa 1-3. Look up on PubChem, the CRC Handbook, or measure by potentiometric titration.
Look Up or Measure pKb of the Base: pKb = -log(Kb). Aliphatic amines: pKb 3-5 (so conjugate-acid pKa = 9-11). Aromatic amines (anilines): pKb 9-10 (conjugate-acid pKa = 4-5). Imidazole (His side chain): pKb 8 (conjugate-acid pKa = 6). NOTE: many references quote pKa of the CONJUGATE ACID of the amine instead of pKb directly — convert with pKa_conj = 14 − pKb.
Apply pI = (pKa + (14 − pKb)) / 2: The calculator converts pKb to conjugate-acid pKa internally (subtract from 14), then averages with the acid pKa. Result is the pH at which the zwitterion form predominates — exactly zero net charge.
Read pI + Charge-vs-pH Map: Hero card shows pI to 3 significant figures. The 3-card charge map shows: at pH < pI molecule is positively charged; at pH = pI it's neutral (zwitterion); at pH > pI it's negatively charged. The 20-amino-acid reference table gives published pI values for sanity-checking.
Apply to Protein Purification: For ion-exchange chromatography, choose a buffer pH that gives the desired charge (1-2 pH units away from pI for tight binding). For isoelectric focusing, the protein migrates to its pI position in the pH gradient. For isoelectric precipitation (e.g. casein at pH 4.6), adjust pH to the pI to drop solubility and crash out the protein.

How is the isoelectric point calculated?

The isoelectric-point calculation comes from the requirement that net charge = 0. For a molecule with one acidic (HA) and one basic (B) group, the relevant equilibria are HA ⇌ H⁺ + A⁻ (with Ka) and BH⁺ ⇌ H⁺ + B (with Ka_conj). Setting the concentrations of A⁻ and BH⁺ equal (the only way net charge is zero) gives the pI formula.

Standard biochemistry textbook derivation; CRC Handbook of Chemistry and Physics; IUPAC Goldbook on isoelectric point.

Core Formula (Two-Group Amphoteric)

pI = (pKa + pKa_of_conjugate_base) / 2

where pKa_of_conjugate_base = 14 − pKb at 25 °C in water (using the relation Ka × Kb = K_w = 10⁻¹⁴).

Combined: pI = (pKa + (14 − pKb)) / 2

If both pKa values are given directly (more common in modern biochemistry), simply: pI = (pKa1 + pKa2) / 2 where pKa1 is the acid pKa and pKa2 is the conjugate-acid pKa of the base.

Worked Example — Glycine

Glycine has α-COOH (pKa1 = 2.34) and α-NH₃⁺ (pKa2 = 9.60). The base group is the α-amine (NH₂); its pKb = 14 − 9.60 = 4.40.

  • Method 1 (using pKa + pKb): pI = (pKa + (14 − pKb)) / 2 = (2.34 + (14 − 4.40)) / 2 = (2.34 + 9.60) / 2 = 5.97.
  • Method 2 (using both pKa values): pI = (pKa1 + pKa2) / 2 = (2.34 + 9.60) / 2 = 5.97.
  • Both methods give identical results — they are the same equation expressed differently.
  • At pH 5.97, glycine exists as the zwitterion ⁺H₃N−CH₂−COO⁻ with zero net charge. Below pH 5.97 it carries net positive charge (cationic form ⁺H₃N−CH₂−COOH); above pH 5.97 it carries net negative charge (anionic form H₂N−CH₂−COO⁻).

For Amino Acids with Ionizable Side Chains

When the side chain has its own ionizable group (Asp, Glu, Lys, Arg, His, Tyr, Cys), there are 3 pKa values total. The pI is the average of the TWO pKa values that flank the zero-net-charge species:

  • Acidic amino acids (Asp, Glu, Cys, Tyr): pI = (pKa1_COOH + pKa_side) / 2 — average of the two ACIDIC pKas.
  • Basic amino acids (Lys, Arg, His): pI = (pKa2_NH₃ + pKa_side) / 2 — average of the two BASIC pKas (high pH side).

Aspartate (Asp) example: pKa1 = 1.88 (α-COOH), pKa2 = 9.60 (α-NH₃⁺), pKa_side = 3.65 (β-COOH). Both COOH groups are acidic, so pI = (1.88 + 3.65) / 2 = 2.77.

Lysine (Lys) example: pKa1 = 2.18, pKa2 = 8.95, pKa_side = 10.53 (ε-NH₃⁺). Both NH₃⁺ groups are basic, so pI = (8.95 + 10.53) / 2 = 9.74.

Charge-vs-pH Behaviour

  • At pH below pI (pH < pI − 1 or so): the molecule has net positive charge — basic groups are protonated, acid groups are mostly protonated.
  • At pH = pI: net charge is exactly zero (zwitterion form). Solubility minimum; molecule is electrostatically neutral but still has + and − charges within itself.
  • At pH above pI (pH > pI + 1 or so): the molecule has net negative charge — acid groups are deprotonated, basic groups are mostly deprotonated.
  • At pH ≈ pI ± 0.5: mixture of charge states; molecule has small net charge that depends sensitively on pH; this is the "buffer region" near the pI.

Why pI Matters in Practice

  • Protein solubility: proteins are LEAST soluble at pH = pI (zero net charge → no electrostatic repulsion between molecules → aggregation and precipitation). The basis of "isoelectric precipitation" used to crash out proteins like casein (pI ≈ 4.6, the basis of cheese-making).
  • Ion-exchange chromatography: for cation exchangers (e.g. SP Sepharose), buffer pH below the protein's pI so the protein has + charge and binds. For anion exchangers (Q Sepharose), buffer pH above pI so the protein has − charge and binds. Elute by raising salt or adjusting pH toward pI.
  • Isoelectric focusing (IEF): separation of proteins in a pH gradient — each protein migrates to its specific pI and stops moving (zero net charge means zero migration in the electric field). Combined with SDS-PAGE this gives 2D gel electrophoresis, the standard proteomics method.
  • Pharmacokinetics: drug pI affects absorption, distribution (cross membranes more easily when neutral), and excretion. Charged forms are more water-soluble; neutral forms cross lipid bilayers.
  • Food chemistry: casein pI ≈ 4.6 (acidified milk → curd formation), gelatin pI ≈ 4.5-9.0 depending on processing (acid- vs alkali-extracted), egg-white ovalbumin pI ≈ 4.5.
Real-World Example

Isoelectric Point – Worked Examples

Example 1 — Glycine. α-COOH pKa = 2.34, α-amine pKb = 4.40 (from α-NH₃⁺ pKa = 9.60).
  • pI = (2.34 + (14 − 4.40)) / 2 = (2.34 + 9.60) / 2 = 5.97.
  • Matches CRC Handbook reference value of 5.97 for glycine.
  • At pH 5.97, glycine zwitterion ⁺H₃N−CH₂−COO⁻ has zero net charge.

Example 2 — General Amphoteric Compound. Carboxylic acid pKa = 4.50, primary amine pKb = 4.20 (so amine conjugate-acid pKa = 9.80).

  • pI = (4.50 + (14 − 4.20)) / 2 = (4.50 + 9.80) / 2 = 7.15.
  • Near-neutral pI suggests this could be a small-molecule amphoteric drug or a simple amino-acid analogue.
  • At physiological pH 7.4, the molecule has slight negative charge (just past pI by 0.25).

Example 3 — Aspartate (Asp). Three pKa values: α-COOH 1.88, α-NH₃⁺ 9.60, β-COOH (side chain) 3.65.

  • Asp has TWO acid groups (α-COOH and β-COOH) and one base (α-amine). It's an ACIDIC amino acid.
  • Use the simple two-pKa formula with the two ACIDIC pKa values: pI = (pKa1 + pKa_side) / 2 = (1.88 + 3.65) / 2 = 2.77.
  • If you tried to use only α-COOH and α-amine: pI = (1.88 + 9.60) / 2 = 5.74 — WRONG; ignores the second acidic group.
  • Reference value for aspartate: 2.77 ✓.

Example 4 — Lysine (Lys). α-COOH 2.18, α-NH₃⁺ 8.95, ε-NH₃⁺ (side chain) 10.53.

  • Lys has ONE acid (α-COOH) and TWO bases (α-amine and ε-amine). It's a BASIC amino acid.
  • Use the two BASIC pKa values: pI = (pKa2 + pKa_side) / 2 = (8.95 + 10.53) / 2 = 9.74.
  • At physiological pH 7.4, lysine carries net positive charge (~+0.9) — the basis of histone-DNA binding (DNA's negative phosphate backbone attracts the positive lysine residues).
  • Reference value: 9.74 ✓.

Example 5 — Casein at pH 4.6 (Industrial Application). Bovine α-casein has pI ≈ 4.6 (computed from the full sequence using ExPASy ProtParam).

  • At pH 6.7 (raw milk), casein carries net negative charge → highly soluble (clear milk solution).
  • Acidify milk (e.g. by lactic-acid bacteria fermentation) to pH 4.6 = casein's pI → zero net charge → minimum solubility → casein precipitates as curd.
  • This is the molecular basis of cheese-making — every cheese variety starts with isoelectric precipitation of milk casein.
  • Industrial casein production: acidify with HCl or H₂SO₄ to pH 4.6, centrifuge, wash, dry; yield ~95%.

Who Should Use the Isoelectric Point Calculator?

1
Biochemistry Students: Learning amphoteric ionization, amino-acid chemistry, zwitterion behaviour. Standard exam topic in physical biochemistry / general chemistry courses.
2
Protein Chemists: Choosing buffer pH for ion-exchange chromatography (cation-exchange below pI; anion-exchange above pI), planning isoelectric focusing (IEF) experiments, designing isoelectric precipitation protocols.
3
Pharmaceutical Formulation: Optimizing drug solubility (drugs are most soluble away from pI), targeting tissue distribution (charged form for water-soluble; neutral form for lipid-membrane crossing).
4
Food Scientists: Casein precipitation (cheese-making at pH 4.6), gelatin formulation (acid- vs alkali-processed have different pIs), soy protein isolate functionality.
5
Analytical Chemists: Capillary isoelectric focusing (cIEF), 2D gel electrophoresis, protein characterization by pI vs Mw mapping.
6
Bioprocess Engineers: Downstream protein purification — sizing ion-exchange columns based on pI vs operating pH, isoelectric precipitation as a coarse purification step.
7
Cosmetic Chemists: Zwitterionic surfactants and amphoteric ingredients (e.g. cocoamidopropyl betaine) — the pI determines mildness and skin-compatibility.

Technical Reference

Mathematical Derivation. For an amphoteric molecule with one acidic (HA) and one basic (B) group, the equilibria are: HA ⇌ A⁻ + H⁺ (Ka) and BH⁺ ⇌ B + H⁺ (Ka_conj). The species present are HA-BH⁺ (positively charged form), A⁻-BH⁺ (zwitterion, neutral), and A⁻-B (negatively charged form). Net charge is zero when [HA-BH⁺] = [A⁻-B] (cation = anion), which after applying both Henderson-Hasselbalch equations gives pH = pI = (pKa + pKa_conj) / 2. This is exact in the limit of dilute solutions where activity coefficients are unity.

The pKa + pKb = 14 Relation. For any acid HA and its conjugate base A⁻ in water at 25 °C: Ka × Kb = K_w = 10⁻¹⁴, so pKa + pKb = 14. This relation comes from the autoionization of water: H₂O ⇌ H⁺ + OH⁻ with K_w = [H⁺][OH⁻] = 10⁻¹⁴ at 25 °C. At other temperatures K_w changes (10⁻¹⁵ at 0 °C, 10⁻¹³ at 60 °C), so the pKa + pKb sum changes too — typically by < 0.5 pH units across the 0-100 °C range. For pI calculations within this range, the 14 approximation is acceptable; for high-precision work cite the actual K_w at the working temperature.

Reference Amino Acid pI Values (CRC Handbook).

  • Acidic amino acids (low pI): Aspartate (Asp, D) 2.77; Glutamate (Glu, E) 3.22.
  • Polar amino acids: Asparagine (Asn, N) 5.41; Glutamine (Gln, Q) 5.65; Serine (Ser, S) 5.68; Threonine (Thr, T) 5.60; Tyrosine (Tyr, Y) 5.66; Cysteine (Cys, C) 5.07.
  • Neutral / hydrophobic amino acids (pI near 6): Glycine (Gly, G) 5.97; Alanine (Ala, A) 6.00; Valine (Val, V) 5.96; Leucine (Leu, L) 5.98; Isoleucine (Ile, I) 6.02; Methionine (Met, M) 5.74; Phenylalanine (Phe, F) 5.48; Tryptophan (Trp, W) 5.89; Proline (Pro, P) 6.30.
  • Basic amino acids (high pI): Histidine (His, H) 7.59; Lysine (Lys, K) 9.74; Arginine (Arg, R) 10.76.

Why Side-Chain pI Calculation Differs. Amino acids without ionizable side chains have only TWO ionizable groups (α-COOH and α-NH₂); the simple two-pKa formula directly applies. Amino acids WITH ionizable side chains have THREE groups (α-COOH, α-NH₂, and the side-chain COOH or NH₂ or imidazole or thiol). The species transitions go through more than two charge states as pH rises:

  • Aspartate at increasing pH: +1 (all protonated) → 0 (α-COOH deprotonated) → -1 (β-COOH deprotonated) → -2 (α-NH₃⁺ deprotonated). pI is the pH between charges 0 and -1, where average charge crosses zero on the macroscopic scale.
  • The pI is the AVERAGE of the two pKa values that flank the zero-charge species: pKa1_COOH (1.88) and pKa_side (3.65). Result: pI = 2.77.
  • Same logic for basic amino acids — pI = average of the two pKa values flanking the zero-charge state.

Protein pI from Sequence — The Bjellqvist Algorithm. For peptides and proteins (more than ~30 residues), pI cannot be calculated from a few pKa values — it requires summing the contributions of every ionizable side chain. The standard algorithm is Bjellqvist (1993) which uses these table values:

  • α-COOH (C-terminal): 3.55 · α-NH₃⁺ (N-terminal): 7.50 · Asp side chain: 4.05 · Glu: 4.45 · Cys: 9.0 · Tyr: 10.0 · His: 5.98 · Lys: 10.0 · Arg: 12.0.
  • The algorithm iterates pH between 0 and 14 to find the pH at which sum-of-charges (positive groups − negative groups) equals zero. Implemented in ExPASy Compute pI/Mw (web.expasy.org/compute_pi/) and EMBOSS pepstats.
  • Accuracy: ±0.5 pH units for typical soluble proteins; less accurate for highly basic / acidic proteins, membrane proteins (buried residues have shifted pKas), and post-translationally modified proteins (phosphorylation, glycosylation shift pI).
  • For experimental pI: use 2D gel electrophoresis, capillary isoelectric focusing (cIEF), or chromatofocusing. Experimental pI typically differs from sequence-predicted by 0.3-1.0 pH units.

Practical Protein pI Examples (Sequence-Computed):

  • Pepsin: pI ≈ 1.0 (extremely acidic; functions in stomach acid).
  • Casein (α-casein): pI ≈ 4.6 (basis of cheese-making isoelectric precipitation).
  • Albumin (BSA): pI ≈ 4.7 (most-used carrier protein in immunoassays).
  • Ovalbumin (egg white): pI ≈ 4.5.
  • Hemoglobin (human): pI ≈ 6.8 (close to physiological pH 7.4).
  • Myoglobin: pI ≈ 7.4 (essentially neutral at physiological pH).
  • Histone H1: pI ≈ 11.0 (extremely basic; binds the negative DNA backbone in nucleosomes).
  • Lysozyme: pI ≈ 11.4 (basic; antimicrobial enzyme in tears, saliva).
  • Cytochrome c: pI ≈ 10.0 (basic; mitochondrial electron-transport protein).
  • Insulin: pI ≈ 5.4 (slightly acidic; basis of crystallization for storage).

Limitations of the Simple Formula.

  • Activity coefficients: the formula assumes ideal-solution thermodynamics; deviations of 0.1-0.3 pH units occur at high salt concentrations (> 0.5 M) or in mixed solvents.
  • Polyprotic species: for molecules with 3+ ionizable groups, the simple formula doesn't apply; use the appropriate two pKa values flanking the zero-charge state.
  • Intramolecular interactions: nearby groups affect each other's pKa (e.g. α-COOH and α-NH₃⁺ of glycine differ from free acetic acid + methylamine). Tabulated pKa values already account for this in single amino acids.
  • Temperature: pKa, pKb, and K_w all depend on T; tabulated values are at 25 °C. For physiological body T (37 °C) shifts are typically < 0.2 pH units.
  • Local environment in proteins: buried residues, salt bridges, hydrogen bonds shift pKa by 1-3 units from solution values. This is why ExPASy / Bjellqvist algorithms use empirically-derived "effective" pKa values different from free-amino-acid pKas.

Tools and References for Advanced Work. For sequence-based protein pI, use ExPASy Compute pI/Mw (web.expasy.org/compute_pi/) which is the gold-standard for proteomics. For experimental pI determination, capillary isoelectric focusing (cIEF) or 2D gel electrophoresis (IEF + SDS-PAGE). For drug pI prediction, ChemAxon Marvin (Chemicalize.com) or ACD/Labs Percepta provide pKa estimates from structure with reasonable accuracy. The CRC Handbook of Chemistry and Physics is the standard reference for amino acid pI and amphoteric molecule data.

Key Takeaways

Isoelectric point (pI) is the pH at which an amphoteric molecule has zero net charge — the pH at which it exists predominantly as a zwitterion. Math: pI = (pKa + (14 − pKb)) / 2, equivalent to averaging the acid pKa and the conjugate-acid pKa of the base (since pKa + pKb = 14 at 25 °C in water). Equivalently pI = (pKa1 + pKa2) / 2 when both pKa values are given directly. For amino acids with ionizable side chains: ACIDIC (Asp 2.77, Glu 3.22, Cys 5.07, Tyr 5.66): pI = (pKa1 + pKa_side) / 2. BASIC (Lys 9.74, Arg 10.76, His 7.59): pI = (pKa2 + pKa_side) / 2. Charge behaviour: at pH < pI molecule is +; at pH = pI it's neutral (zero net charge); at pH > pI it's −. Practical applications: protein solubility (minimum at pI), ion-exchange chromatography (cation-exchange below pI; anion-exchange above), isoelectric focusing (IEF), 2D gel electrophoresis, isoelectric precipitation (casein at pH 4.6 → cheese curd, the basis of cheese-making). For proteins, use ExPASy's Compute pI/Mw tool with the Bjellqvist algorithm — accurate to ±0.3-0.5 pH units from sequence alone.

Frequently Asked Questions

What is the Isoelectric Point Calculator?
It implements the standard isoelectric-point formula pI = (pKa + (14 − pKb)) / 2 for amphoteric molecules with one acidic and one basic ionizable group. The pI is the pH at which net charge is zero — the foundation of every protein-purification technique that exploits charge: ion-exchange chromatography, isoelectric focusing (IEF), 2D gel electrophoresis, isoelectric precipitation. The result panel includes a charge-vs-pH map (positive below pI, neutral at pI, negative above pI) and a 20-row reference table for all standard amino acids.

Pro Tip: Pair this with our Buffer pH Calculator for buffer preparation.

What's the formula for isoelectric point?
pI = (pKa + (14 − pKb)) / 2 for an amphoteric molecule with one acid (pKa) and one base (pKb). Equivalently, pI = (pKa1 + pKa2) / 2 when both pKa values are given (acid pKa and the conjugate-acid pKa of the base; pKa2 = 14 − pKb). The two forms are mathematically identical — the relation pKa + pKb = 14 at 25 °C in water lets you convert between them. The formula is exact for two-group amphoteric molecules; for amino acids with ionizable side chains use the average of the two pKa values flanking the zero-net-charge species.
What does the isoelectric point mean physically?
The pI is the pH at which an amphoteric molecule has zero net electric charge. The molecule still has internal + and − charges (it's a zwitterion), but they cancel out at this pH. Practical consequences: (1) Minimum solubility — no electrostatic repulsion between molecules → aggregation and precipitation (basis of isoelectric precipitation); (2) Zero electrophoretic mobility — no net force in an electric field, the basis of isoelectric focusing (IEF); (3) Maximum buffering capacity at pH = pI ± 0.5 — both pKa groups contribute; (4) Solubility / membrane permeability minimum — pharmaceutical molecules are least bioavailable at their pI.
How do I find pKa and pKb for a molecule?
Lookup sources: CRC Handbook of Chemistry and Physics, PubChem (pubchem.ncbi.nlm.nih.gov), the original synthesis paper, or commercial supplier datasheets. Software prediction: ChemAxon's Chemicalize.com, ACD/Labs Percepta, or pKaPlugin from Marvin (free for academic). Experimental measurement: potentiometric titration with a glass electrode is the gold-standard method (precision ±0.05 pH units); conductometric titration also works. For amino acids, the values are tabulated in every biochemistry textbook (CRC Handbook is most authoritative); the calculator's reference table includes all 20 standard amino acids.
What's the difference between pKa and pKb?
Both are equilibrium constants for proton transfer, but for opposite directions: pKa = -log Ka for acid dissociation (HA ⇌ A⁻ + H⁺); pKb = -log Kb for base protonation (B + H₂O ⇌ BH⁺ + OH⁻). They're related by Ka × Kb = K_w = 10⁻¹⁴, so pKa + pKb = 14 at 25 °C in water. The pKa of the conjugate acid of a base = 14 − pKb. Modern biochemistry typically quotes pKa for ALL ionizable groups (acidic and basic), referring to the proton-loss equilibrium of each species. Older references and some general-chemistry textbooks use pKb for basic groups; the calculator handles both conventions via the 14 − pKb conversion.
What's special about amino acids with ionizable side chains?
Amino acids without ionizable side chains (Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, Ser, Thr, Asn, Gln) have only TWO ionizable groups (α-COOH and α-NH₃⁺); the simple formula gives their pI directly (typically 5.5-6.3). Amino acids with ionizable side chains (Asp 2.77, Glu 3.22, Cys 5.07, Tyr 5.66, His 7.59, Lys 9.74, Arg 10.76) have THREE ionizable groups; pI is the average of the TWO pKa values that flank the zero-net-charge species. Acidic amino acids (Asp, Glu, Cys, Tyr — side chain acts as additional acid): pI = (pKa1 + pKa_side) / 2. Basic amino acids (Lys, Arg, His — side chain acts as additional base): pI = (pKa2 + pKa_side) / 2. This is why Asp has pI = 2.77 (very acidic) but Lys has pI = 9.74 (very basic).
How do I use pI for protein purification?
Ion-exchange chromatography: for cation exchangers (negatively-charged matrix like SP Sepharose, CM Cellulose, S Strong-cation), use buffer pH BELOW the protein's pI so the protein is positively charged and binds; elute by raising salt or pH. For anion exchangers (positively-charged matrix like Q Sepharose, DEAE, ANX Anion), use buffer pH ABOVE pI so the protein is negatively charged and binds. Isoelectric precipitation: adjust pH to pI to minimize protein solubility and crash it out (e.g. casein from milk at pH 4.6 → cheese curd). Isoelectric focusing (IEF): protein migrates in pH gradient until reaching its pI position (zero net charge → zero migration). 2D gel electrophoresis: IEF (1st dimension by pI) + SDS-PAGE (2nd dimension by Mw) — the proteomics standard.
What's the pI of a typical protein?
Most soluble proteins have pI in the 4-8 range, with strong clustering near pH 5-6 (the average of acidic Asp/Glu and basic Lys/Arg/His amino-acid frequencies in proteomes). Examples: Bovine serum albumin (BSA) pI = 4.7 (the most-used carrier protein); casein pI = 4.6 (cheese-making basis); insulin pI = 5.4; hemoglobin pI = 6.8; myoglobin pI = 7.4. Extreme pI examples: pepsin pI = 1.0 (functions in stomach acid pH 1-2); histone H1 pI = 11.0 (binds negative DNA backbone in chromatin); lysozyme pI = 11.4 (basic antimicrobial enzyme); cytochrome c pI = 10.0. The wide pI range is functional — extreme pIs often correlate with specific biological roles (DNA binding, acidic environments, etc.).
How accurate is the simple formula?
Exact for two-group amphoteric molecules (one acid + one base, no other ionizable groups). For amino acids without ionizable side chains, the simple formula matches CRC Handbook reference values to 0.01 pH units. Approximate (±0.3-0.5 pH units) for proteins due to local-environment effects on pKa values (buried residues, salt bridges, hydrogen bonds shift effective pKas by 1-3 units). For proteins use ExPASy Compute pI/Mw (web.expasy.org/compute_pi/) which applies the Bjellqvist (1993) algorithm with empirically-derived effective pKa tables. Experimental pI determination (2D gel, cIEF) typically differs from sequence-predicted by 0.3-1.0 pH units due to post-translational modifications, splice variants, and isoforms.
How does pI change with temperature?
Modestly — typically < 0.5 pH units across 0-100 °C. Three temperature-dependent factors: (1) K_w changes from 10⁻¹⁵ at 0 °C to 10⁻¹⁴ at 25 °C to 10⁻¹³ at 60 °C; the pKa + pKb sum changes from 15 to 14 to 13 across this range. (2) Individual pKa values shift — amine pKa typically decreases ~0.03 pH units per °C; carboxylic acid pKa is much less temperature-sensitive (changes ~0.005/°C). (3) Solvent dielectric changes affect ionic equilibria. For body temperature (37 °C) vs lab T (25 °C), expect pI shift of 0.1-0.3 pH units for typical molecules. For high-precision work cite pI at the specific temperature; for routine biochemistry the 25 °C value is acceptable for most purposes.
Can I use this for proteins?
Not directly — proteins have many ionizable residues (every Asp, Glu, Lys, Arg, His, Tyr, Cys side chain plus N-terminal NH₃⁺ and C-terminal COO⁻), and their pI requires summing all contributions. The simple two-pKa formula only works for two-group amphoteric molecules. For protein pI use ExPASy Compute pI/Mw (web.expasy.org/compute_pi/) which applies the Bjellqvist (1993) algorithm — paste in the protein sequence, get pI and Mw in seconds. Other tools: EMBOSS pepstats, IPC 2.0 (Isoelectric Point Calculator 2.0, isoelectric.org/calculator.html). For peptides < 30 residues, the same tools work. For experimental verification, use 2D gel electrophoresis, capillary isoelectric focusing (cIEF), or chromatofocusing.

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The ToolsACE Team

Our ToolsACE biochemistry team built this calculator on the standard isoelectric-point relation for amphoteric molecules — those that have both acidic (proton-donating) and basic (proton-accepting) groups in the same molecule. The simplest case (one acid + one base): <strong>pI = (pKa + (14 − pKb)) / 2</strong>, equivalent to the average of the two pKa values when you convert pKb of the base to pKa of its conjugate acid (pKa = 14 − pKb at 25 °C). The pI is the pH at which the molecule has zero net charge — at pH below pI, the molecule is positively charged; above pI, negatively charged. This pH-dependent charge behaviour drives every protein-purification technique that relies on isoelectric properties: ion-exchange chromatography, isoelectric focusing (IEF), 2D gel electrophoresis, and protein-precipitation methods. The calculator outputs pI to 3 significant figures, the conjugate-acid pKa equivalent (14 − pKb), and a 20-row reference table covering all standard amino acids with their published pI values from the CRC Handbook.

Standard biochemistry referencesCRC Handbook of Chemistry and PhysicsIUPAC amino-acid pKa data

Disclaimer

The simple two-pKa formula applies to amphoteric molecules with one acidic and one basic group. For amino acids with ionizable side chains, average the two pKa values flanking the zero-net-charge species (acidic AAs: pI = (pKa1 + pKa_side)/2; basic AAs: pI = (pKa2 + pKa_side)/2). For proteins, use ExPASy Compute pI/Mw which applies the Bjellqvist (1993) algorithm with accurate pKa tables; sequence-predicted pI is accurate to ±0.5 pH units. For experimental pI use 2D gel electrophoresis or capillary isoelectric focusing. References: CRC Handbook of Chemistry and Physics, IUPAC Goldbook, ExPASy ProtParam.