To know how to prepare 50mM Phosphate buffer of pH 6.8, first, you need to know three factors that are needed to prepare a phosphate buffer (in fact any buffer system). These are the concentration of the buffer, pH value, and final volume of the buffer you want to prepare. For example, in the title, two factors are known that are concentration and pH value of the buffer. The third one (final volume) can be 50 ml, 100 ml, or more than that as you need.
So, come to the topic, How to prepare 50mM Phosphate buffer of pH 6.8? To make a 50mM 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. In the same way, potassium dihydrogen phosphate is acid and dipotassium hydrogen phosphate is salt for potassium phosphate buffer. Read more: What is a buffer system and what is its importance?
As I said before, There are three factors that we need to consider to make a buffer. In this regard, the pH, and concentration of the buffer are mentioned which are 6.8 pH and 50 mM. Now let’s assume we are going to make 100 ml. So, the next step is to determine what amount of which components are needed to achieve the goal. Keep reading down.
I’m sure you heard of the Henderson-Hasselbalch equation (). It is the formula fundamentally used in the lab to prepare different any kind of buffers (e.g. phosphate buffer, acetate buffer, tris-HCl buffer, etc.). You will need to use this formula to calculate the concentration of the individual components of the phosphate buffer or any buffer. However, you need to know the pKa of the buffer that you are going to prepare.
The molar concentration of individual components
pKa value of a buffer system is the negative logarithm of the acid dissociation constant the key compound of the buffer. For the phosphate buffer, the pKa value that we use is the negative logarithm of the acid dissociation constant of the second ionization of the phosphoric acid.
The pKa value (specifically pKa2) of the phosphate buffer is 7.21. Now, using pH and pKa values you can calculate the molar concentration of individual components of the phosphate buffer. Read more: How to calculate pKa of phosphate buffer?
Now, solving equation-1 and equation-2, you will get molar concentration of individual components; [Salt]= 14.003 mM and [Acid]=35.997 mM. After that, it’s easy for you to calculate the required amount of both components using the molarity formula.
Weights of the individual components
In this calculation, I have used sodium dihydrogen phosphate and disodium hydrogen phosphate (basically sodium phosphate buffer), however, you can opt to use either combination of sodium and potassium or only sodium or only potassium to prepare phosphate buffer. It’s entirely up to you and your need.
In the picture given below, I have illustrated the calculation of the required weight for the NaH2PO4. Now, we have the following information regarding the NaH2PO4; molarity: 35.997 mM (0.035997 M), molecular weight: 119.98 g/mol, total volume: 100 ml (0.1 L). Using the molarity formula, the required weight of the NaH2PO4 can be calculated which is 0.4319 g.
In the same way, you can also calculate the required amount of Na2HPO4 which is 0.1988 g. Now, put these components in a 100 ml volumetric flask and add 50 ml of distilled water. Dissolve the components completely and make the final volume 100 ml. In this way, you can prepare 100 ml of 50 mM phosphate buffer of pH 6.8. This is a manual calculation for the preparation of phosphate buffer.
However, if you want to save time in calculating these things, you can use our online phosphate buffer calculator. I have recently developed an online calculator, especially for preparing phosphate buffer. You will find it more useful than any other online calculator available for the preparation of phosphate buffer. After reading this post, I’m sure you are clear on how to prepare 50mM Phosphate buffer of pH 6.8