Molecules are separated using high performance liquid chromatography; this depends on their affinity for the stationary phase. Symmetrical peak shape is the ultimate goal of chromatography, so it requires appropriate mobile phases and sometimes buffer solutions to separate analytes. A buffer is just something that resists changes, a buffer solution is a solution that resists changes in the pH of a solution when a tiny acid or base is added.
For example, in HPLC, if the pH can be changed by adding the sample to the mobile phase, then the pH can be controlled by adding a buffer to the mobile phase to resist the pH change. Since the retention time (RT) of ionizable molecules is extremely sensitive to the pH of the mobile phase, it must be controlled by adding buffers.
Different types of buffers are used in HPLC, but phosphate and acetate are the most commonly used buffers for HPLC with UV detection. Commonly, a buffer pH range from 2.00 to 8.00 and a concentration 10 to 50 mM is adequate for the separation of analytes in the silica-based HPLC column.
Here are mentioned some buffers that commonly used in HPLC method development.
| pKa | Common Buffers of HPLC | The Range of pH |
| 4.8, and 9.2 | Ammonium acetate | 3.8 to 5.8, and 8.2 to 10.2 |
| 2.1 | KH2PO4 or Phosphoric acid | 1.1 to 3.1 |
| 3.8, and 9.2 | Ammonium format | 2.8 to 4.8, and 8.2 to 10.2 |
| 7.2 | KH2PO4 or K2PO4 | 6.2 to 8.2 |
| 9.2 | Ammonia or Ammonium hydroxide | 8.2 to 10.2 |
| 4.8 | Acetic Acid or Potassium acetate | 3.8 to 5.8 |
| <2 | Trifluoroacetic acid | 1.5 to 2.5 |
| 9.2 | Borate | 8.2 to 10.2 |
| 3.8 | Formic acid | 2.8 to 4.8 |