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To prepare a 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.
In the same way, potassium dihydrogen phosphate is acid and dipotassium hydrogen phosphate is salt for potassium phosphate buffer. Read more: What are a buffer system and its importance?
For the preparation of phosphate buffer, you need to consider three factors. These factors are; concentration, pH, and final volume of the buffer. In this case, the pH, concentration, and volume are 6.8, 50 mM, and 100 ml.
Henderson-Hasselbalch equation; is most often used in the lab to prepare different types of buffers such as phosphate buffer. You may 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?
Since,
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
Here, 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 to 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 your time in calculating these things, you can use our online phosphate buffer calculator. I have recently developed an online calculator especially for the preparation of phosphate buffer. You will find it more useful than any other online calculators available for the preparation of phosphate buffer.
bonhi
wrong calculation
Biochempages
Thank you so much for your time in pointing out mistakes. I really appreciate you and I have already corrected the wrong value of pKa. Thanks again.
اغانى شعبى 2017
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Biochempages
Thank you for your time in reading the article. I really appreciate it.
Nur Syafiqah Muhammed
Hai, great explanation.
But how about if the pH that I want to make is higher than the pKa value, does it mean that I have to switch the position of [A]/[HA]??
Biochempages
Thank you for your question. Buffer works best within a certain range of pH and that is pKa±1. Therefore, in the case of phosphate buffer you can have pH higher than pKa (7.21). The maximum pH that you can have is 8.21. One more thing, you don’t need to invert the ratio but just follow the calculations, you will end up at what amount you will need to prepare the buffer of desired pH. If you want to use our online calculator for the phosphate buffer preparation, please visit the link given below. https://www.biochempages.com/2017/06/online-phosphate-buffer-calculator.html
kodel
How molecular weight can be 137.993 g/mol,
Biochempages
Sorry, I have already updated the right molecular weight of the NaH2PO4 that is 119.98, not 137.993. Thank you for your comment.
Zia khan
How to prepare 100 ml of 50 mM phosphate buffer of pH 7.0?
Biochempages
Hi, Thank you for your question. To prepare 100 ml of 50 mM phosphate buffer of pH 7.0, please follow one of our best online calculator Online phosphate buffer calculator. You will find this calculator the most useful. Best regards!!