By James Ashenhurst

Isoelectric Points of Amino Acids (and How To Calculate Them)

Last updated: March 26th, 2024 |

Isoelectric Points Of Amino Acids (And How To Calculate Them)

The isoelectric point of an amino acid is the pH at which it bears no net charge.

  • Amino acids have an amino group and a carboxylic acid 
  • Although they are often drawn in their neutral form, in aqueous solution at pH 7 (physiological pH) their structure is more accurately described as a “zwitterion” (an internal salt) – the product of an acid-base reaction between the carboxylic acid and the amine.
  • The zwitterionic form has two point charges but has a net charge of 0.
  • In practice, the charges on an amino acid only balance out to zero at one specific pH value, called the isoelectric point pI. At this pH, the amino acid will not migrate in an applied electric field.
  • For amino acids with neutral sidechains, the pI can be calculated by averaging the pKa values of the carboxylic acid and ammonium groups
  • For amino acids with acidic sidechains, the pI can be calculated by averaging the pKa values of the two most acidic groups
  • For amino acids with basic sidechains, the pI can be calculated by averaging the pKa values of the least acidic groups

summary of the isoelectric point and how to calculate it

Table of Contents

  1. Amino Acids as Zwitterions
  2. The Isoelectric Point, pI, Is the pH At Which Negative and Positive Charges are Balanced
  3. Calculating Isoelectric Point pI from pKa Values
  4. pI Calculations for Amino Acids With Acidic Side Chains
  5. pI Calculations for Amino Acids With Basic Side Chains
  6. Summary
  7. Notes
  8. Quiz Yourself!
  9. (Advanced) References and Further Reading

1. Amino Acids As Zwitterions

Amino acids contain a carboxylic acid and an amino group. We often like to draw them like this:

Drawing of amino acids in their neutral form with carboxylic acid and amines

Although simple, these drawings don’t accurately convey the physical properties of these molecules, especially under physiological conditions.

When a carboxylic acid (pH 4) is added to a solution containing an amine (R-NH2), an acid-base reaction quickly occurs, resulting in formation of the ammonium salt R-NH3(+) with a pKa of about 9.

This is a favorable equilibrium because the reaction proceeds from a stronger acid and a stronger base to a weaker acid and a weaker base. [See article: How To Use a pKa Table]

Favorable acid base reactions proceed between carboxylic acid and amine to give carboxylate and ammonium

Should amino acids behave any differently than the carboxylic acids and amines we’re familiar with?

No! Amino acids are acids. They are also bases containing an amino group. The term amphoteric is often used to describe amino acids, meaning that they are capable of acting as both acids and bases.

When dissolved in water (especially when dissolved in the polar solvent water!) we should likewise expect an acid-base reaction between the carboxylic acid and the amine.

Here’s what it would look like for the simplest amino acid, glycine. The pKa of the carboxylic acid is 2.24  and the pKa of the amino group is 9.60.

zwitterionic form of amino acids is more accurate than their neutral form after acid base

Glycine has become a salt, with a negatively charged carboxylate group and a positively charged ammonium group.

These “internal salts” are known as zwitterions. Note that, as drawn, the molecule bears no net charge since the positive and negative charges balance out.

The high melting point of glycine [233°C] and other amino acids is more consistent with this zwitterionic structure than the neutral form that is often drawn.

2. The Isoelectric Point, pI, Is The pH At Which Negative and Positive Charges Are Balanced

In practice, the zwitterionic amino acid will only have a net charge of zero at a very narrow range of pH values.

The pH at which the negative and positive charges are in balance is known as the isoelectric point.

At pH values below and above the isoelectric point, the molecule will bear a net positive or net negative charge, respectively.

It’s possible to test this by applying a sample of the amino acid to specially treated paper or gel and applying an electric field at different pH values – a technique known as electrophoresis. A molecule with a net charge of zero will not migrate in an electric field, whereas one bearing a positive or negative charge will migrate towards the cathode or anode, respectively. 

Why might this be? Let’s have a look at a typical amino acid in its zwitterionic form.

The carboxylate group is a weak base, and the ammonium salt is a weak acid.

If the pH is decreased to a low enough value (e.g. pH 1) then the carboxylate salt will be protonated to give the neutral carboxylic acid, and the molecule will have a net charge of +1.

at strongly acidic pH carboxylate of amino acid will be protonated and net charge is plus 1

If the pH is increased to a high enough value (e.g. pH 14) then the ammonium salt will be deprotonated to give the neutral amine. The molecule will have a net charge of -1.

under strongly basic conditions ammonium of amino acid will be deptoronated

Putting all of these acid-base reactions together, from low pH to high pH we get:

three ph dependent structures of a typical amino acid - conjugate base

For a typical amino acid, there will be a range of pH values where the positively charged form dominates, another where the neutral form dominates, and finally one where the negatively charged one dominates.

We could even make a sketch of mol percentage versus pH, and imagine it would look something like the following.

sketch of mole percentage versus pH for 3 forms of amino acid

The isoelectric pointpI should be right in the middle of the two points A and B where there each acid is equal in concentration to its conjugate base.

[Note 1 – for more detailed calculations, refer to this site]

3. The Formula for Calculating Isoelectric Point, pI

If only there were some formula we could use for figuring out the pH of points A and B on the graph above, where the acid and its conjugate base are present in equal concentration.

Thankfully, our old friend the Henderson-Hasselbalch equation gives us the answer!

When an acid is present in equal concentration with its conjugate base, the pH of the solution will simply be equal to the pKa of the acid.

[For a more detailed treatment, see Note 2]

This means that the two points A and B on the sketch above correspond to the pKa values of the amino acid.

So, in order to obtain the isoelectric point, all we have to do is average these two values.

Thankfully, the pKa values for all proteinogenic amino acids have been determined experimentally.

The pKa values for glycine, for example,  are 2.34 (for the carboxylic acid) and 9.60 (for the ammonium).

Adding the two pKa values together and dividing by 2 gives us a value of 5.97 for the isoelectric point.

calculation of isoelectric points simple formula is to average two pka values of amino acid

Here is a table with some structures and pKa values for amino acids.

pka values and structures of selected amino acids glycine alanine tryptophan

This table omits the amino acids with acidic and basic side chains, in addition to cysteine and tyrosine (all of which have 3 pKa values!).

See if you can calculate the isoelectric points.

Click to Flip

4.  Acidic Side Chains

Obtaining pI through averaging the two pKa values of the amino acid is simple enough.

But what happens when the amino acid contains an acidic side chain, such as in Glutamic acid (Glu) and aspartic acid (Asp)? How will that affect the isoelectric point?

First, let’s just take a guess. Do you think the pI will become lower or higher as a result of the acidic side chain?

Click to Flip

It may be helpful to walk through the changes in the molecule as we proceed from extremely low pH to high pH.

Let’s use aspartic acid as the example.

the four possible structures for an amino acid with acidic sidechain - pI will be average of two most acidic pKa values

  • At extremely low pH (e.g. pH 1), both carboxylic acids should be protonated along with the amino group, for a net charge of +1.
  • As acidity decreases, the most acidic carboxylic acid will be deprotonated to give us the zwitterionic form (net charge = 0)
  • With a further increase in basicity, the second carboxylic acid is deprotonated to give a net negatively charged form (net charge = -1)
  • at very high basicity (e.g. pH 14) the ammonium will be deprotonated, giving a doubly negatively charged species (net charge = -2)

Since the net neutral species is formed through loss of a proton on the most acidic site (pKa1 = 1.88 for aspartic acid ), and destroyed through loss of a proton on the second-most acidic site (pKa2 = 3.65 for aspartic acid) the concentration of the neutral species should be at its maximum at the point midway between these two values.

See if you can calculate the pI of aspartic and glutamic acid.

Click to Flip

5.  Basic Side Chains

There are also amino acids with basic side chains, such as lysine, arginine and histidine.

These amino acids have functional groups that will affect the isoelectric point in the opposite direction.

For lysine, the different forms of the amino acid would look like this:

ph dependent structures of amino acid with basic sidechain - to obtain pI average the two highest pKa values

  • at extremely low pH, the carboxylic acid and both amine groups are protonated to give a species with a net charge of +2
  • At intermediate pH, the carboxylic acid is deprotonated to give a species with a net charge of +1
  • At basic pH, the most acidic ammonium will be deprotonated to give the neutral species with a (net) charge of 0
  • At strongly basic pH, both ammoniums will be deprotonated to give a species with a net charge of -1.

The isoelectric point will be the pH at which the molecule bears a net charge of zero.

The neutral species is formed through deprotonation of the most acidic ammonium (pKa2 = 8.95) and destroyed through deprotonation of the least acidic ammonium (pKa3 = 10.53).

So averaging these two pKa values should give us the isoelectric point, pI.

See if you can calculate the pI of these basic amino acids.

Click to Flip

6. Summary

There is more to isoelectric point than just the calculation of pI values for individual amino acids!  The same concepts also apply to peptides and proteins, each of which will have a pI value that is influenced by the characteristics of its side chains.

These differences form the basis of electrophoresis, an effective technique for the separation of these molecules through adjustment of pH and application of an electric field.


Note 1.  More detailed calculations of titration curves can be obtained with the help of software such as Hyperquad – see here.

Note 2. The calculation ends up being so simple because the second term of the Henderson Hasselbalch equation ends up cancelling out:

Quiz Yourself!

Click to Flip

Click to Flip

Click to Flip

(Advanced) References and Further Reading

  • This site contains an isoelectric point calculator that will calculate the isoelectric point of amino acid sequences.
  • Definition of isoelectric point from IUPAC:
  • pKa values of amino acids are from the CRC Handbook of Chemistry and Physics.


Comment section

4 thoughts on “Isoelectric Points of Amino Acids (and How To Calculate Them)

  1. Great explanations! Shouldn’t the pI equation that is in the dotted border in section 3. be pI= (pKa1+pKa2)/2? The formula in the dashed border is missing a denominator of 2.

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