Phosphate buffer is very commonly used in the laboratory. Its applications range widely, from enzymatic assays to histology. Wherever it is necessary to maintain a neutral pH under variable conditions, phosphate buffer is ideal. Another major advantage is the ability to control the buffer’s pH and adjust it to specific requirements (e.g. the optimal pH for the activity of particular enzymes).
What does the phosphate buffer consist of?
There are two types of phosphate buffer. The first is based on sodium salts, the second on potassium salts. Both buffers contain salts of dihydrogen phosphate and hydrogen phosphate.
| Sodium-phosphate buffer | Potassium-phosphate buffer |
| Disodium phosphate Sodium hydrophosphate pH 5,7 – 8,0 | Potassium dihydrophosphate Potassium hydrophosphate pH 5,8 – 8,0 |
Oba bufory posiadają podobną objętość buforową, jednak ich unikalne właściwości mogą wpływać na ich zastosowanie.
Which phosphate buffer should I choose?
Both the sodium phosphate buffer and the potassium phosphate buffer have similar pH-maintaining capabilities. Under certain specific conditions, their differing properties may significantly affect the test result or the stability of the system.
| Property | Sodium-phosphate buffer | Potassium-phosphate buffer |
| Solubility | At low temperatures and during freezing, it may precipitate, causing cloudiness and the formation of aggregates. | Better solubility at low temperatures. Does not cause cloudiness or precipitation during freezing. |
| Interaction with SDS | Does not react with SDS | Potassium ions react with SDS, leading to precipitation. Many people recommend avoiding this buffer in SDS-PAGE. |
| pH range | Effective buffering within the pH range of 5.7–8.0. Maximum buffering capacity at pH 6.8. | Effective buffering within the pH range of 5.8–8.0. Maximum buffering capacity at pH 6.8. |
| Mobility (CZE) | It may cause molecules to migrate more quickly | Slower migration |
How to prepare a sodium-phosphate buffer?
To prepare a 0.1 M sodium phosphate buffer, you must first prepare 0.2 M solutions of sodium dihydrogen phosphate (V) and sodium hydrogen phosphate (V).
Solution A (0.2 M sodium dihydrogen phosphate, NaH₂PO₄)
Dissolve 27.8 g of NaH₂PO₄ in 800 ml of water, transfer the solution volumetrically to a 1000 ml measuring flask and make up to the mark.
Solution B (0.2 M sodium hydrogen phosphate, Na₂HPO₄)
Dissolve 53.65 g of Na₂HPO₄·7H₂O or 71.7 g of Na₂HPO₄·12H₂O in 800 ml of water. Transfer to a 1000 ml volumetric flask and make up to the mark.
To obtain a buffer with the correct pH, mix solutions A and B as shown in the table below and make up to a final volume of 200 ml with water.
| pH | ml solution A (0,2 M NaH2PO4) | ml solution B (0,2 M Na2HPO4) |
| 5,7 | 93,5 | 6,5 |
| 5,8 | 92,0 | 8,0 |
| 5,9 | 90,0 | 10,0 |
| 6,0 | 87,7 | 12,3 |
| 6,1 | 85,0 | 15,0 |
| 6,2 | 81,5 | 18,5 |
| 6,3 | 77,5 | 22,5 |
| 6,4 | 73,5 | 26,5 |
| 6,5 | 68,5 | 31,5 |
| 6,6 | 62,5 | 37,5 |
| 6,7 | 56,5 | 43,5 |
| 6,8 | 51,0 | 49,0 |
| 6,9 | 45,0 | 55,0 |
| 7,0 | 39,0 | 61,0 |
| 7,1 | 33,0 | 67,0 |
| 7,2 | 28,0 | 72,0 |
| 7,3 | 23,0 | 77,0 |
| 7,4 | 19,0 | 81,0 |
| 7,5 | 16,0 | 84,0 |
| 7,6 | 13,0 | 87,0 |
| 7,7 | 10,5 | 90,5 |
| 7,8 | 8,5 | 91,5 |
| 7,9 | 7,0 | 93,0 |
| 8,0 | 5,3 | 94,7 |
I recommend preparing the buffer fresh and using it within two weeks. Solutions A and B can be stored for longer and used as starting solutions for preparing the working buffer.
How to prepare a potassium-phosphate buffer?
To prepare a 0.1 M potassium phosphate buffer, you must first prepare 0.2 M solutions of potassium dihydrogen phosphate and potassium hydrogen phosphate.
Solution A (0.2 M potassium dihydrogen phosphate, KH₂PO₄)
Dissolve 34.84 g of KH₂PO₄ in 800 ml of water, transfer the solution by volume to a 1000 ml volumetric flask and make up to the mark.
Solution B (0.2 M potassium hydrogen phosphate, K₂HPO₄)
Dissolve 34.84 g of K₂HPO₄·3H₂O in 800 ml of water. Transfer to a 1000 ml volumetric flask and make up to the mark.
To obtain a buffer with the correct pH, mix solutions A and B as shown in the table below and make up to a final volume of 200 ml with water.
| pH | ml solution A (0,2 M KH2PO4) | ml solution B (0,2 M K2HPO4) |
| 5.8 | 8.5 | 91.5 |
| 6.0 | 13.2 | 86.8 |
| 6.2 | 19.2 | 80.8 |
| 6.4 | 27.8 | 72.2 |
| 6.6 | 38.1 | 61.9 |
| 6.8 | 49.7 | 50.3 |
| 7.0 | 61.5 | 38.5 |
| 7.2 | 71.7 | 28.3 |
| 7.4 | 80.2 | 19.8 |
| 7.6 | 86.6 | 13.4 |
| 7.8 | 90.8 | 9.2 |
| 8.0 | 94.0 | 6.0 |
I recommend preparing the buffer fresh and using it within two weeks. Solutions A and B can be stored for longer and used as starting solutions for preparing the working buffer.
Literature:
Solution calculator: https://www.sigmaaldrich.com/PL/pl/support/calculators-and-apps/buffer-calculator
Kłyszejko-Stefanowicz, L. (red.). (2003). Ćwiczenia z biochemii. Wydawnictwo Naukowe PWN.