high performance liquid chromatography

High-performance liquid chromatography is used to separate molecules; it is depending on their affinity towards the stationary phase. Symmetric peak shape is an ultimate goal of chromatography so it requires the suitable mobile phase, sometimes requiring a buffer solution to separate the analytes. A buffer is just something that resists change, and a buffer solution is a solution that resists the pH change of a solution upon adding a minute acid or base.

For example, in HPLC, if pH can be shifted by adding a sample to a mobile phase, then a buffer can be added to the mobile phase to resist pH changes, this gives the control on the pH. As the retention time (RT) of ionizable molecules is extremely sensitive to the pH of the mobile phase, it is required to control it by the addition of buffer.

There are different types of buffers are used in high-performance liquid chromatography, but the phosphate and acetate are the most popular buffers for HPLC with UV detection. Usually, 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.

pKaCommon Buffers of HPLCThe Range of pH
4.8, and 9.2Ammonium acetate3.8 to 5.8, and 8.2 to 10.2
2.1KH2PO4 or Phosphoric acid1.1 to 3.1
3.8, and 9.2Ammonium format2.8 to 4.8, and 8.2 to 10.2
7.2KH2PO4 or K2PO46.2 to 8.2
9.2Ammonia or Ammonium hydroxide8.2 to 10.2
4.8Acetic Acid or Potassium acetate3.8 to 5.8
<2Trifluoroacetic acid1.5 to 2.5
9.2Borate8.2 to 10.2
3.8Formic acid2.8 to 4.8

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