What is the isoelectric point?
Pick an amino acid, or type its pKa values, and the calculator gives its isoelectric point pI, the pH at which it carries no net charge. It also gives the average net charge at a pH you choose and which way the amino acid moves in electrophoresis.
- Answer
- Alanine: pI = 6.015
Alanine: pI = 6.015.
- pI
- 6.015
- Averaged
- (2.34 + 9.69) ÷ 2
- Side chain
- neutral: average of pKa₁ and pKa₂
- Net charge at the pH
- −0.00509
- In electrophoresis at the pH
- toward the positive electrode: pH above pI, net charge negative
Answer: Alanine: pI = 6.015. Alanine: pI = 6.015.
How to calculate
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.
Example with the default inputs (Amino acid Alanine, pH for the net charge (optional) 7.4): Alanine: pI = 6.015.
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)).
- 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
Each example is checked against the calculator on every build.
- Amino acid Alanine gives pI 6.015, Answer Alanine: pI = 6.015, Averaged (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
- Amino acid Aspartic acid gives pI 2.765, Averaged (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
- Amino acid Lysine gives pI 9.74, Averaged (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
- Amino acid Histidine 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
- Amino acid Tryptophan 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
- Amino acid Other: type the pKa values, pKa of the α-carboxyl group 2.34, pKa of the α-amino group 9.69, The side chain is Acidic, pH for the net charge (optional) 9 gives pI 6.015, Net charge at the pH −0.17, In electrophoresis at the pH 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⁺
- Amino acid Other: type the pKa values, pKa of the α-carboxyl group 2.18, pKa of the α-amino group 8.95, pKa of the side chain (optional) 10.53, The side chain is Basic gives pI 9.74, Side chain 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
How it works
Isoelectric point (OpenStax §26.2). pI is the average of the two pKa values on either side of the neutral form:
- neutral side chain: pI = (pKa₁ + pKa₂) ÷ 2
- acidic side chain: the average of the two lowest pKa values
- basic side chain: the average of the two highest pKa values
In general: sort the pKa values from low to high, count the basic groups b (the α-NH₃⁺ group, plus a basic side chain), and average the b-th and (b + 1)-th values. For typed values the page does the same: the α-CO₂H group is acidic, the α-NH₃⁺ group basic, and the side chain (if its pKa is given) acidic or basic as chosen. With no side chain pKa the side chain counts as neutral.
pKa values (OpenStax Table 26.1).
| Amino acid | pKa α-CO₂H | pKa α-NH₃⁺ | pKa side chain | Side chain |
|---|---|---|---|---|
| Alanine | 2.34 | 9.69 | — | neutral |
| Arginine | 2.17 | 9.04 | 12.48 | basic |
| Asparagine | 2.02 | 8.80 | — | neutral |
| Aspartic acid | 1.88 | 9.60 | 3.65 | acidic |
| Cysteine | 1.96 | 10.28 | 8.18 | acidic |
| Glutamine | 2.17 | 9.13 | — | neutral |
| Glutamic acid | 2.19 | 9.67 | 4.25 | acidic |
| Glycine | 2.34 | 9.60 | — | neutral |
| Histidine | 1.82 | 9.17 | 6.00 | basic |
| Isoleucine | 2.36 | 9.68 | — | neutral |
| Leucine | 2.36 | 9.60 | — | neutral |
| Lysine | 2.18 | 8.95 | 10.53 | basic |
| Methionine | 2.28 | 9.21 | — | neutral |
| Phenylalanine | 1.83 | 9.13 | — | neutral |
| Proline | 1.99 | 10.60 | — | neutral |
| Serine | 2.21 | 9.15 | — | neutral |
| Threonine | 2.09 | 9.10 | — | neutral |
| Tryptophan | 2.38 | 9.39 | — | neutral |
| Tyrosine | 2.20 | 9.11 | 10.07 | acidic |
| Valine | 2.32 | 9.62 | — | neutral |
Two values differ from OpenStax’s table, which prints 2.83 for tryptophan’s α-CO₂H and 9.60 for isoleucine’s α-NH₃⁺. Its own pI column (5.89 and 6.02) and PubChem (HSDB, from the Merck Index: 2.38 and 9.68) agree on 2.38 and 9.68, so the page uses those. OpenStax counts cysteine and tyrosine with the neutral amino acids, but their side chains lose H⁺ (thiol and phenol), so the rule averages their two lowest pKa values, which matches the table’s pI of 5.07 and 5.66.
Net charge at a pH (Henderson–Hasselbalch, OpenStax §26.2). Each acidic group adds −1 ÷ (1 + 10^(pKa − pH)); each basic group adds +1 ÷ (1 + 10^(pH − pKa)). The pH is optional (default 7.4); leave it empty to skip the charge. At the pI the charge is close to 0, but not exactly 0 when a third group is present.
Electrophoresis at the pH. Compared on the exact values: pH below pI, “toward the negative electrode: pH below pI, net charge positive”; pH above pI, “toward the positive electrode: pH above pI, net charge negative”; pH equal to pI, “no net movement: pH equals pI”.
Rules the page checks. Typed pKa values from −5 to 20; the pH from 0 to 14. A pI that is not 0 but smaller in size than 2⁻¹⁰²² (about 2.2 × 10⁻³⁰⁸) has no answer, because a number cannot keep its digits.
How results are written. The pI is the exact average, written with 6 significant figures, trailing zeros dropped (6.015, 9.74, 7.585), and m × 10ⁿ below 0.000001 in the sentence and the row (typed pKa values of 3 × 10⁻⁷ and −1 × 10⁻⁷ give pI = 1 × 10⁻⁷). The sentence reads “Alanine: pI = 6.015”, or “pI = 6.015” for typed values. The “Averaged” row reads “(2.34 + 9.69) ÷ 2”, the pKa values written the same way (9.60 as 9.6). The side chain row reads “neutral: average of pKa₁ and pKa₂”, “acidic: average of the two lowest pKa values” or “basic: average of the two highest pKa values”. The charge is a double-precision number shown to 3 significant figures.
Assumptions. Free amino acids in water near 25 °C, with pKa values far enough apart that each group ionizes on its own.
Worked examples by hand
Alanine. (2.34 + 9.69) ÷ 2 = 6.015 (OpenStax: 6.01).
Aspartic acid (acidic). The two lowest pKa values are 1.88 and 3.65: (1.88 + 3.65) ÷ 2 = 2.765 (table: 2.77).
Lysine (basic). The two highest are 8.95 and 10.53: (8.95 + 10.53) ÷ 2 = 9.74.
Histidine (basic). The two highest are 6.00 and 9.17: (6.00 + 9.17) ÷ 2 = 7.585 (table: 7.59).
Tryptophan. (2.38 + 9.39) ÷ 2 = 5.885 (table: 5.89).
Alanine’s charge at pH 9.00. α-CO₂H: −1 ÷ (1 + 10^(2.34 − 9.00)) = −0.9999998; α-NH₃⁺: 1 ÷ (1 + 10^(9.00 − 9.69)) = 0.830445. Net charge = −0.169555; pH 9.00 is above the pI, so it moves toward the positive electrode.
Other questions people ask
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.