Every day scientists in laboratories across the world sit at their desks and painstakingly design experiments in the hope of making a discovery that will change how we think about a biological process. Because biological processes such as enzymatic activity are dependent on pH, one critical aspect of the experimental design is choosing a buffering system that will help maintain a stable pH without altering the results.
And, often, it is the choice of buffer that makes or breaks the experiment. It is possible that the buffer you are using in your lab might be the reason your experiment is failing. Here, you will find how a buffering system works, a description of the characteristics of a good buffer and a list of possible applications and characteristics of the most commonly used biological buffers.
What is a buffer?
A buffer consists of a weak acid (proton donor, HA) and its conjugate base (proton acceptor, A -). In water, HA can dissociate into A- and H+. H+ then reacts with water to form H3O+. In the aqueous buffer solution, H3O+, HA and H+ exist in equilibrium with each other. The buffering mechanism consists of two reversible reactions where the concentration of proton donor and proton acceptor are equal.
Then, when a strong acid or base is introduced into this system by the scientist or by enzymatic activity during the experiment, the new ions from the introduced acid or base (H + or OH-) are absorbed by the buffer and the pH remains stable preventing changes in protein structure and function.
Buffers cannot arbitrarily moderate any changes in ion concentration. Their optimal buffering capacity, or range, is defined by the dissociation constant, or ka, of the acid. We commonly discuss buffering capacity in terms of the pKa or the logarithmic constant of ka. We consider the buffering capacity of a specific buffer to be the pKa ± 1. For example, a buffer with a pH of 6.8 has a pH buffering range of 5.8-7.8.
What is a Good biological buffer?
Years ago, scientists performed biochemical experiments with inadequate buffers that greatly limited the impact of their research. These buffers exhibited high cell toxicity and could not support enzymatic activity throughout the procedures. Then, in 1966 Norman E. Good and his team designed a series of buffers specifically for biological research with the following characteristics:
- Buffers should have a pKa between 6.0 and 8.0 because the optimal pH for most biological reactions rests in this range.
- Buffers should have high water solubility and minimum solubility in organic solvents so it remains in the aqueous medium of the biological system.
- Buffers should not permeate cell membranes. The buffer should not accumulate in cellular organelles. This may not apply to your specific experiment. Zwitterionic buffers do not permeate cell membranes.
- Buffers should have minimal salt effects because ionic buffers can be problematic if the biological system being studied is negatively affected by salts.
- The buffer’s concentration, temperature and ionic composition of the medium should have a minimal effect on buffering capability (pKa).
- The formation of complexes between a metal ion and the buffer results in proton release, which affects the pH of the system and may have an adverse effect on experimental results. Thus, these ionic complexes should be soluble and their binding constant must be known. A buffer with a low metal-binding constant is suitable for the study of metal-dependent enzymatic reactions. If your experimental design requires the use of a metal, then you should choose a buffer that does not form a complex with that specific metal.
- Buffers should be stable and resist enzymatic and nonenzymatic degradation. And they should not interfere with enzyme substrates or resemble them.
- Buffers should not absorb light in the visible or ultraviolet regions of the spectrum to prevent interference in spectrophotometric assays.
- Their preparation and purification should be easy and inexpensive.
What are the most common buffers and how are they used?
The characteristics considered by Good and his team are a good starting point when choosing the buffer for your specific experiment. Here, we are including a Biological Buffer Selection Guide (click here or scroll down for the PDF) containing a list of the most common biological buffers and the specific techniques and experiments they are used for. We are also including a description of their properties including pH, buffering range, metal binding capabilities, advantages and disadvantages and links to the protocols for the stock solutions.
In addition, we are including a Guide (click here or scroll down for the PDF) of the most commonly used buffers in the lab describing their pH and composition.
10X Running buffer. (1970, January 01). Retrieved September 21, 2018, from http://cshprotocols.cshlp.org/content/2006/1/pdb.rec10475.full?text_only=true.
Ferreira, C. M., Pinto, I. S., Soares, E. V., & Soares, H. M. (2015). (Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ions – a review. RSC Advances, 5(39), 30989-31003. doi:10.1039/c4ra15453c.
Good, N., & Izawa, S. (1972).  Hydrogen ion buffers. Methods in Enzymology Photosynthesis and Nitrogen Fixation Part B, 53-68. doi:10.1016/0076-6879(72)24054-x.
Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S., & Singh, R. M. (1966). Hydrogen Ion Buffers for Biological Research*. Biochemistry, 5(2), 467-477. doi:10.1021/bi00866a011.
Laboratory Stock Solutions and Equipment. (1998). Current Protocols in Cell Biology, 00(1). doi:10.1002/0471143030.cba02as00.
Purich, D. L. (2010). Factors Influencing Enzyme Activity. Enzyme Kinetics: Catalysis & Control, 379-484. doi:10.1016/b978-0-12-380924-7.10007-9.
Tris-Glycine/MeOH Transfer Buffer. (1970, January 01). Retrieved September 21, 2018, from http://cshprotocols.cshlp.org/content/2015/5/pdb.rec087064.full?sid=3b0abcbe-5bcf-460f-87d8-48a5525a9d25.
SDS-PAGE Running Buffer. (1970, January 01). Retrieved September 21, 2018, from http://cshprotocols.cshlp.org/content/2014/7/pdb.rec081117.full?sid=f680f5fc-664e-4fe1-a840-07cec720d52e.
Zbacnik, T. J., Holcomb, R. E., Katayama, D. S., Murphy, B. M., Payne, R. W., Coccaro, R. C., . . . Manning, M. C. (2017). Role of Buffers in Protein Formulations. Journal of Pharmaceutical Sciences, 106(3), 713-733. doi:10.1016/j.xphs.2016.11.014.
Category Code: 79105, 79104, 88251
Biological Buffer Selection Guide
Guide: Commonly used Buffers
Fernanda Ruiz is a science content writer at Gold Biotechnology. She holds a bachelor's of science in biology from St. Mary's University and a PhD in molecular biology from Baylor College of Medicine.