How to calculate pKa of phosphate buffer?

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(Last Updated On: June 24, 2017)
Henderson-Hasselbalch equation

Henderson-Hasselbalch equation for the calculation of the concentration of the individual components of a buffer system.

The pKa of phosphate buffer can be calculated by using a mathematical expression. Phosphate buffer is an important buffer system used in biochemistry laboratories. Phosphate buffer is essential for the experiments involving proteins and enzymes. It maintains the pH of the solution and also prevents the drastic change in the pH. Therefore, we have to be familiar with the preparation of the phosphate buffer and how does it work. Read more: What is a buffer system and its importance?


Calculation of pKa of phosphate buffer

Since phosphate buffer consists of two components; dihydrogen phosphate (H2PO4) and hydrogen phosphate (HPO4-2), there is a dissociation constant that we call acidity constant of phosphate buffer. The phosphoric acid is a tri protic acid and, therefore, there are three acidity constants for this acid. However, our concern for the pKa of phosphate buffer is with the acidity constant of dihydrogen phosphate (H2PO4). Acidity constants of phosphoric acid are, H3PO4 (), H2PO4 (),  and HPO4-2 ()

Phosphate buffer involves in the ionization of H2PO4 to HPO4-2 and vice versa. Therefore, we will use acidity constant K2 to determine the pKa value.  Since pK1 is a negative logarithm of the acidity constant, pKa will be log(K2) or log(6.2*10-8) or 7.21. Therefore, the pKa value of phosphate buffer is 7.21. We can now use it to prepare the phosphate buffer of pH 7.21±1 with a definite ionic strength. Knowing the pKa of a buffer is very important during the buffer solution preparation.

Preparation of the phosphate buffer

Using the Henderson-Hasselbalch equation, you can calculate the required concentration of each component of the phosphate buffer of the desired pH. You just have to put pKa that you determined above (7.21) and the desired pH of the solution (that can be 7.21±1) and you will be able to determine the concentration of H2PO4 and HPO4-2. It’s just easy to calculate the concentration of individual components H2PO4 and HPO4-2 using the mathematical expression Henderson-Hasselbalch equation as given below. For phosphate buffer, H2PO4 is the acid and HPO4-2. is the salt. Read more: How to prepare 100 ml of 50 mM Phosphate buffer of pH 6.8?

Using the Henderson-Hasselbalch equation; you can calculate the required components of the phosphate buffer or if you want you can also use an online phosphate buffer calculator. I have recently developed an online calculator only for you so that you can easily determine necessary amounts of individual components of the phosphate buffer without any calculations.

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