Buffers are often overlooked and taken for granted by laboratory scientists, until the day comes when a bizarre artifact is observed and its origin is traced to a bad buffer.
The simplest definition of a buffer is a solution that resists changes in hydrogen ion concentration as a result of internal and environmental factors. Buffers essentially maintain pH for a system. The effective buffering range of a buffer is a factor of its pKa, the dissociation constant of the weak acid in the buffering system. Many things, such as changes in temperature or concentration, can affect the pKa of a buffer.
In 1966, Norman Good and colleagues set out to define the best buffers for biochemical systems (1). By 1980, Good and his colleagues identified twenty buffers that set the standard for biological and biochemical research use (2,3). Good set forth several criteria for the selection of these buffers:
1. A pKa between 6 and 8. Most biochemical experiments have an optimal pH in the range of 6–8. The optimal buffering range for a buffer is the dissociation constant for the weak acid component of the buffer (pKa) plus or minus pH unit.
2. Solubility in water. Biological reactions, for the most part, occur in aqueous environments, and the buffer should be water-soluble for this reason.
3. Exclusion by biological membranes. This isn’t important for all biochemical reactions. However, if this is an important criterion for your particular experiment, it is helpful to remember that zwitterionic buffers (positive and negative charges on different atoms within the molecule) do not pass through biological membranes. Examples of zwitterionic buffers include MOPS and HEPES; Tris and phosphate buffers do not isomerize into zwitterions.
4. Minimal salt effects. In other words, the buffer components should not interact or affect ions involved in the biochemical reactions being explored.
5. Minimal effects on dissociation from changes in temperature and concentration. Usually there is some change in dissociation with a change in concentration. If this change is small, stock solutions can usually be diluted without changing the pH of the buffer. However, with some buffers changes, in concentration produce more dramatic changes in pKa, and stock solutions cannot be diluted without significantly affecting pH.
For instance, the pH of Tris decreases approximately 0.1 pH unit per tenfold dilution, and the pH could change dramatically if you dilute a working solution and are at the limits of the optimal buffering range of the Tris (optimal buffering range pH 7.3–9.3 at 20°C). Note that Tris is not one of Good’s buffers.
Temperature changes can be a problem too, and again, Tris provides a cautionary example of a commonly used buffer because it exhibits a large shift in dissociation with a change in temperature. For example, if you prepare a Tris buffer at pH 7.0 in the cold room at 4.0°C, and perform a reaction in that same buffer at 37°C, the pH will drop to 5.95.
If you have a Tris buffer prepared at 20°C with a pKa of 8.3, it would be an effective buffer for many biochemical reactions (pH 7.3–9.3), but the same Tris buffer used at 4°C becomes a poor buffer at pH 7.3 because its pKa shifts to 8.8.
So the take home message: Make the buffer at the temperature you plan to use it. If your experiment will involve a temperature shift, select a buffer with a range that can accommodate any shift in dissociation as a result of the change in temperature.
6. Well defined or nonexistent interactions with mineral cations. If the buffer and cations in your system react, your buffer becomes less effective because it cannot handle additional hydrogen ions. If a complex forms between the buffer and a required cofactor, say a metal cation like zinc or magnesium, your reaction also might be compromised.
For instance, having excessive amounts of a chelating agent in an enzymatically driven reaction could cause problems (like too high a concentration of EDTA in a PCR amplification, for instance).
Tris buffers again give us problems, because Tris contains a reactive amine group. If you are trying to make Tris buffer that is RNase free, the amine group on the Tris molecule will react with diethylpyrocarbonate, the chemical typically used to pretreat aqueous solutions that will be use for RNA work. So, do not DEPC-treat Tris-containing solutions. HEPES also reacts DEPC. (Remember too, that MOPS is a much better buffer for most RNA work!)
Take home message: Buffers are not inert. Be careful which ones you chose.
7. Chemical stability. The buffer should be stable and not break down under working conditions. It should not oxidize or be affected by the system in which it is being used. Try to avoid buffers that contain participants in reactions (e.g., metabolites).
8. Light absorption. The buffer should not absorb UV light at wavelengths that may be used for readouts in photometric experiments.
9. Ease of Use. The buffer components should be easy to obtain and prepare.
Good’s selection criteria defined several characteristics of buffers for biochemical reactions that has stood the test of time. His work set the standard for the many benefits of buffers in biological research. No matter what buffer you choose, you need to consider effects of temperature and environment on the buffer and ensure that the buffer you choose will be compatible with your system.
For additional help, access the Student Resource Center. Whether it’s pouring gels, making buffers or optimizing protocols, we’re here to help with anything your lab throws at you.
- Good, N.E., et al. (1966) Hydrogen Ion Buffers for Biological Research. Biochemistry. 5(2): 467–77.
- Good, N.E. and Izawa, S. (1972) Hydrogen Ion Buffers. Methods Enzymol. 24: 53–68.
- Ferguson, W.J., et al. (1980) Hydrogen Ion Buffers for Biological Research. Anal. Biochem. 104(2): 300–10.