Buffers and Guidelines on the use of buffers in HPLC Methods

A buffer solution is a solution that resists changes to a solution’s pH, when small quantities of acid or alkali are added to the solution.

Use of buffer plays an important role in HPLC separation, change can separate two closely eluting peaks and may merge two separate peaks. The most commonly used buffers in LC are citrate, acetate and phosphate buffer.

Choosing the right buffer

Many factors can influence the choice of the buffer, points to be taken into consideration for selecting the right buffer:

• Required pH of mobile phase
• The UV cut-off value
• A volatile buffer should be used for MS-based chromatography.

Buffer pH

pH plays an important role in determining retention time and shape of the peak. A silica-based column should not be used for buffer solutions with Very low pH and High values; as at low pH stationary phase bond may get removed and there is chance of silica getting dissolved in high pH.

Buffer Solubility and Concentration

Buffer solubility and concentration play a vital role in method development. The best buffer is the one that is fully soluble and gives reproducible result at the lowest concentration. Higher buffer concentration may increase the viscosity and precipitation of the buffer which may lead to backpressure.

Preparation of buffer solutions: Weigh the required quantity of buffer in the volumetric flask and then add solvent as required. Adjust the pH of the solution and then fill the flask to the mark. Sonicate the buffer solution for complete degassing of the solution. Add the organic phase and filter the mixture with 0.45µm filter or 0.22 µm based upon the requirement of its application.