Isoelectric Point Calculator

Created by Komal Rafay
Reviewed by Steven Wooding
Based on research by
P. Novák; V. Havlíček. Protein Extraction and Precipitation ScienceDirect (April 2016)
Last updated: Apr 25, 2022

Our isoelectric point calculator is here to help you determine the pH value of a molecule, which is free of any net charge.

In chemistry, isoelectric point and "the point of zero charge" are usually used interchangeably. However, there are some exceptions, and we will discuss them along with how to calculate the isoelectric point and what the isoelectric point equation looks like.

So, let's get started.

What is isoelectric point?

The pH value determines whether a substance is acidic or basic by nature. And we know that a pH value lesser than seven indicates acidic and above 7 indicates basic substance.

The isoelectric point is the pH value at which a molecule has zero net electrical charges and is considered neutral.

One important thing to remember is that pH values below zero and above 14 are possible. For instance, concentrated HCl (hydrochloric acid) has a pH of -1, and concentrated NaOH (sodium hydroxide) has 15.

Isoelectric point calculator usage

Our isoelectric point calculator is a tool you would love to use on the go. It determines the isoelectric point of molecules based on their pKa and pKb values. What are pKa and pKb values, you might wonder? These are the dissociation constants of acids and bases. The dissociation constant represents the capability of a substance to dissociate into ions in a solution.

Now, coming back to the point at hand, how to use the isoelectric point calculator? Follow the given steps, and that's it:

1. Input the pKa value of the molecule.
2. Input the pKb value of the molecule.
3. The result is that molecule's isoelectric point (pI value).

This value indicates the pH at which your molecule carries no net electric charge.

Let's consider an example. Say you have molecule X, its $pKa = 3.7$ and $pKb = 9.4$. Input these values in the calculator and the result is $6.55$

Remember, our tool works in reverse and any other order you want to. As long as you have any two of the three values to input, the calculator will work fine for you.

Isoelectric point formula

The isoelectric point formula is fairly simple.

$pI = \frac {(pKa + pKb)}{2}$

where:

• $pKa$ – Dissociation constant of acid;
• $pKb$ – Dissociation constant of base; and
• $pI$ - Isoelectric point.

Adding the pKa and pKb values and dividing them by two is all you have to do to determine the $pI$ value of your molecule. It is just like estimating the mean of any two numbers.

How to calculate isoelectric point

The isoelectric point impacts the solubility of a molecule at a given pH. So, knowing how to calculate the isoelectric point is essential.

You already know how this tool works. Now let's take a look at using the isoelectric point equation: $pI = (pKa + pKb) / 2$

According to this equation, you need:

1. The $pKa$ value of the molecule; and
2. The $pKb$ value of the molecule.

And the result is the $pI$ value.

Suppose you have $pKa = 2.2$ and $pKb = 7.5$.

1. First, add 2.2 to 7.9, which is 9.7.
2. Now, divide 9.7 by 2, which results in 4.85.
3. 4.85 is the isoelectric point.

Isoelectric point in chemistry and biochemistry

The isoelectric point is an essential topic in chemistry and biochemistry. It tells us when a particular molecule has attained the electrically neutral state, and the pI can affect the solubility of said molecule.

Let's look at the isoelectric points of specific biomolecules.

• Isoelectric point of amino acids
Amino acids are zwitterions when they attain their isoelectric point. The acidic amino acids have pI values between a pH of 5.0 to 7.0, while the basic amino acids have a higher pH to attain their isoelectric point.

• Isoelectric point of proteins
The knowledge of isoelectric points is significant in protein studies. It helps in methods used for protein separation like:

• Ion-exchange chromatography – Chromatographic separation method that separates proteins in a solution/environment based on their net charge; and
• Protein electrophoresis – A test that measures the amount of a specific protein, in the blood, based on its net electrical charge.

The algorithms that calculate the isoelectric point of proteins and peptides use the Henderson Hasselbalch equation.

Isoelectric point and point of zero charge

As we mentioned before, the isoelectric point and point of zero charge are interchangeable, but there are situations when they are distinct. They both represent the pH at which there is no electric charge.

The isoelectric point represents only the external surface charges, while the point of zero charge represents the particle's external and internal surface charge in a solution.

FAQ

How can I calculate the isoelectric point?

The formula to calculate the isoelectric point of a substance is:

pI = (pKa + pKb) / 2

where:

• pKa – Dissociation constant of acid;
• pKb – Dissociation constant of base; and
• pI – Isoelectric point.

So, to compute the isoelectric point:

1. First, you add the pKa and pKb values.
2. Then, divide the sum by 2.
3. The result is the isoelectric point.

What is the isoelectric point of pka = 3.2 and pKb = 8.1?

The isoelectric point is 5.65 for pKa = 3.2 and pKb = 8.1.

This isoelectric point value shows that your acidic molecule is electrically neutral at a pH value of 5.65. Remember, the pH of a molecule is affected by the pH of its surroundings

Can a molecule have more than one pka value?

Yes, a molecule can have more than one pKa value. A molecule with multiple pKa values is an indication of more than one ionizable functional group. It means it has more than one hydrogen atom, which can be transferred or deprotonated in an acid-base reaction.

How does the isoelectric point effect solubility of a molecule?

The isoelectric point is a pH where a molecule is free of net charge and is electrically neutral. If there is no charge on the molecule, it will not interact with its surroundings. In the case of a solute, it will not dissolve in the solvent and instead form precipitates.

Komal Rafay
pKa
pKb
Isoelectric point (pI)
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