Biological Buffers Selection Guide

Biological buffers are organic substances that maintain a constant pH over a given range by neutralizing the effects of hydrogen ions. Buffers are commonly used in research labs, especially in applications involving protein electrophoresis, polymerase chain reaction and Western blotting. Buffers are vital components for modeling biological systems and have many uses in cell culture, molecular biology, nucleic acid and protein purification, and transformation and transfection.

Buffers are aqueous solutions that resist changes in pH, or the acidity or basicity of a solution relative to other substances.  Buffers in a biological system keep the pH (potential hydrogen) constant when there are changes in the hydrogen [H⁺] ion. Acids give up protons, bases accept them. Buffer solutions are composed of a mixture of weak acid, which acts as a proton donor, and its conjugate base, which acts as an electron acceptor, or a mixture of weak base with a conjugate acid. The buffering of a solution results from two reversible reaction equilibria in a solution wherein the concentration of a proton donor and its conjugate proton acceptor are equal.



Most simple buffers effectively work in the pH scale of pKa ±1.  

Two common buffer examples include tris(hydroxymethyl)aminomethane (tris)-buffered saline (TBS) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). TBS has a neutral pH and is often used as a wash or dilution buffer in biological assays. HEPES is a zwitterionic buffer that is commonly used in cell culture.  

Buffers are typically classified into seven main groups based on the R-group in their chemical makeup and the pH range of their effectiveness. They can be further delineated by grade. At the acidic end of the pH scale, at a range from 5.5 to 8.3 are the morpholinic buffers such as MES, MOPS, and MOBS. Tris is a mid-range buffer stable at pH of 7 to 9.6. Cyclohexylamino buffers such as CHES and CAPS are effective at higher pH values from 8.6 to 11.4.

The strength of an acid is measured using a pKa value, or the negative log of the acid dissociation constant. A low pKa value indicates a strong acid and the greater its ability to donate a proton or fully dissociate in water. The pKa predicts what a molecule will do at a specific pH and is constant for each type of molecule.

pKa values can be determined by titration. A calibrated and automated titrator is used. The free acid of the material to be measured is titrated with a suitable base, and the titration curve is recorded. The pH of the solution is monitored as increasing quantities of a base is added to the solution. pKa is a thermodynamic value and pH is dependent on temperature. The most common pKa constants are available in the publication from R. Goldberg, et al., 2002.¹

Buffer capacity is the ability of the buffer to maintain the pH of a solution and generally depends on the concentration of hydrogen ions in a buffer solution. Buffers that contain high concentrations offer high buffering capacity and have a low pH. However, pKa remains unaffected by concentration. A buffer capacity of 1 means 1 mole of acid or base is added to 1 liter of buffer and the pH changes by 1 unit. The buffer capacity of a mixed weak acid-base buffer is much greater when the individual pKa values are in proximity with each other.

A buffering range or buffer range indicates the pH level that a buffer maintains over the course of an experiment.

Buffer grade indicates the quality and impurity levels appropriate for different applications and corresponding regulatory standards. We provide six grades of buffers for general lab use, diagnostic manufacturing, and applications that may need trace metal testing or materials of a specified purity.

There are several criteria to consider when selecting a buffer. It is best to choose a buffer with a pKa value near the desired target pH of the chemical solution that is most appropriate for the specific application. Other factors to consider include buffer concentration, solubility, biological membrane permeability, minimal salt effects, temperature, cationic interactions, metal interactions, ionic strength, and optical absorbance and stability from any enzymatic and non-enzymatic reactions.² Also, regulatory or purity needs for each specific application should be considered.