How to prepare 100 ml of 50 mM Phosphate buffer of pH 6.8?

Preparation of 50 mM phosphate buffer

Preparation of 50 mM phosphate buffer

To prepare 50 mM phosphate buffer of pH 6.8, you may use either sodium salt or potassium salt of the orthophosphoric acid. There are two components of the phosphate buffer; acid and salt (conjugate base). For sodium phosphate buffer, acid is sodium dihydrogen phosphate and salt is disodium hydrogen phosphate. While for potassium phosphate buffer, acid is potassium dihydrogen phosphate and salt is dipotassium hydrogen phosphate.


To prepare buffer, you need to consider three factors; concentration, pH, and volume of the buffer that you need to prepare. In this case, the pH, concentration, and the volume are 6.8, 50 mM and 100 ml.

Henderson Hassel Balch equation (pH = pKa + log[Salt]/[Acid]) is most often used in the lab to prepare buffers such as phosphate buffer. You may use this formula to calculate the concentration of the individual components of the phosphate buffer. However, you will also need to know the pKa of the buffer that you are going to prepare. As follow;

pKa value of the phosphate buffer (especially pKa2) is the negative logarithm of acid dissociation constant of the second ionization of the phosphoric acid and the value is 7.19. Now, using pH and pKa values you can calculate the amount of individual components of the phosphate buffer.

pH = pKa + log[Salt]/[Acid]
6.8 = 7.19 + log[Salt]/[Acid]

log[Salt]/[Acid] = -0.39

[Salt]/[Acid] = 0.407

[Salt] = 0.407[Acid] —–equation-1

Since, [Salt]+[Acid]=50 mM ——-equation-2

Now, solving equation-1 and equation-2, you will get [Salt]= 14.463 mM and [Acid]=35.536 mM. Once you get the concentration of individual components, you can easily prepare the solution of the 50 mM phosphate buffer by taking required amount of the individual components. For acid (NaH2PO4), the required amount (in gram) can be calculated by using the molarity formula as follows, (Since 100 ml=0.1 L, 35.536 mM=0.0355 M and molecular weight of the Na2HPO4 is 137.993 g/mol)

M=[(given weight)/(molecular weight)]/(volume in litre)

0.0355=[(given weight)/(137.992)]/0.1

[(given weight)/(137.992)]=0.0036

Given weight =0.0036 x 137.992

Given weight = 0.489g

In the same way, you can calculate the amount of salt (Na2HPO4) that is 0.205g. Now, weigh the respective amount of the individual components and keep them in a beaker and then add about 50 ml of distilled water and dissolve the components. After dissolution, add more distilled water to the solution to make total volume 100ml with the help of volumetric flask. In this way, you can prepare 100ml of 50 mM phosphate buffer of pH6.8.

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