Biological Buffers

Biological reactions depend on the pH of the solution being kept within a specific range. This is accomplished with buffers which maintain the required pH parameters. Buffers are an everyday but crucial part of your lab supplies. You go through a lot of them and need to be sure that they are top-quality. United States Biological sells a wide variety of premium buffers and buffer components, and since we are using the same prices that have been in effect since 1995 for most of our biochemicals, you can afford to stock up.

 

Choosing the right buffer

Common Buffer Table (at 20°C)
Buffer	pKa	pH range	MW
HEPES	7.55	6.8-8.2	238.3
MOPS	7.28	6.5-7.9	209.3
MES	6.15	5.8-6.5	195.2
PIPES	6.8	6.1-7.5	302.4
TAPS	8.49	7.7-9.1	243.3
Tricine	8.15	7.4-8.8	179.2
Tris	8.2	7.5-9.0	121.1

 

H20100 HEPES	M3000 MES	P4200 PIPES

 

HEPES, MES, and PIPES are three of the biological buffers (zwitterionic) designed by Good, et al., and typically referred to as Good’s buffers. They are useful in cell culture media formulations. Selection of biological buffer systems should include the following criteria: exclusion by biological membranes, low absorption between 240 and 700nm, chemical stability, and stable to temperature and concentration changes.

 

Biological Buffers
Good's buffers are good buffers

Good buffers (Good’s buffers) are twelve buffering agents selected and described by Norman E. Good and colleagues in 1966.  Good developed buffers based on several criteria that made them useful candidates for biochemical research. He sought to identify buffering compounds which met several criteria likely to be of value in biological research.

1. pKa.  pKa values in the regions between 6-8 were selected because most biological reactions take place at near-neutral pH.
2. Solubility.  For ease in handling aqueous biological systems, good solubility in water is required.  Poor solubility in nonpolar solvents (fats, oils, organic solvents) was also required because this would tend to prevent the buffer from accumulating in cell membranes and other nonpolar compartments of the biological system.
3. Membrane impermeability.  Buffers were considered that did not readily pass through cell membranes, thus reducing the accumulation of the buffer compound within cells.
4. Minimal salt effects. Highly ionic buffers may cause problems or complications in some biological systems
5. Minimal temperature and ionic strength effects.  There should be a minimum influence of buffer concentration, temperature, and ionic strength of the medium on the pKa of the buffer.  For example, Tris (a frequently used, but not a Good buffer) has one of the largest pH temperature effects (ΔpKa/°C= -0.031 ) of all biological buffers.
6. Well-behaved cation interactions.  If the buffers form complexes with cationic ligands, the complexes formed should remain soluble.  Ideally, at least some of the buffering compounds will not form complexes.
7. Stability.  The buffers should be chemically stable, resisting enzymatic and non-enzymatic degradation.
8. Optical absorbance.  Buffers should not absorb in the visible or ultraviolet at wavelengths longer than 230nm so as not to interfere with commonly used spectrophotometric assays.
9. Ease of preparation.  Buffers should be easily prepared and purified from inexpensive materials.

 

Buffer	pK at 20°C	DpKa/°C
MES	6.15	-0.011
ADA	6.6	-0.011
PIPES	6.8	-0.0085
ACES	6.9	-0.02
Cholamine chloride	7.1	-0.027
BES	7.15	-0.016
TES	7.5	-0.02
HEPES	7.55	-0.014
Acetamidoglycine	7.7	--
Tricine	8.15	-0.021
Glycinamide	8.2	-0.029
Bicine	8.35	-0.018

 

We can get a flavor of Norm Good’s own words related to his biological buffers from the Citation Classic interview in Current Contents (1983) 40:23.

N. E. Good, G. D. Winget, W. Winter, T. N. Connolly, S. Izawa and R. M. M. Singh (1966). "Hydrogen Ion Buffers for Biological Research". Biochemistry 5:467-477.

Norman E. Good (1917-1992), a photosynthesis researcher and inventor of the "Good" buffers Norman E. Good Department of Botany and Plant Pathology Michigan State University East Lansing, MI 48824 July 5, 1983 “The idea of tailoring molecules for specific buffering purposes came from a game I used to play with one of my colleagues, Richard O’Brien, at the University of Western Ontario.  He was interested in the effects of ionization on the activity of nerve poisons and related drugs.  We developed between us a game of guessing pKa’s on the basis of oftentimes rather complex structures of the drugs he was studying.  Meanwhile I had been investigating electron transport and ATP synthesis in chloroplast lamellar preparations, and I had discovered that various anions commonly used in buffers could uncouple electron transport from phosphorylation. How then to avoid the anion uncoupling and still have essential control of hydrogen ion concentration?  Since the inner salt glycine had absolutely no uncoupling effect, it occurred to me that I might modify glycine in such a manner that it’s new pKa would bring it into an appropriate buffering range (glycine itself buffers at far too high a pH for chloroplast research). It seemed ovious that replacing the amino group of glycine with tris(hydroxymethyl)-aminomethane (tris) would produce an amino acid with a pKa rather similar to that of Tris itself.  Thus, Tricine was born. Subsequently, it also occurred to me that I could use sulfonic acids instead of carboxylic acids and thereby avoid much of the binding of polyvalent metal ions.  Consequently, when I moved to Michigan State University, I ordered all of the simple primary and secondary amines commercially available and started making the appropriate N-substituted taurines and glycines, drawing on the intuition developed in the above-mentioned pKa guessing game.  Hence the origin of the buffers listed in the paper. Almost all experimental biologists and not a few analytical chemists were interested in the new buffers and consequently the paper received instant attention.  The fact the buffers often proved superior to anything before available made their wide use inevitable.  It is a measure of their acceptance that the biological and chemical journals now allow the use of  the trivial names of several – e.g., Hepes, Tricine – without definition and without reference. In a desultory way, I have continued to make and introduce more buffers along the same lines and I have occasionally enlisted the assistance of real chemists.  A series of N-substituted 3-aminopropanesulfonic acids was prepared using propane sultone and, more recently, W.J. Ferguson and I (1) have introduced a series of N-substituted 3-amino-2hydroxypropanesulfonic acids, all excellent buffers.

A summary of available biological buffers was compiled in 1972 by me and my colleague, S. Izawa (2).”
1. Ferguson, W.J., Braumschweiger, K.L., Braumscheiger, W.R., Smith, J.R., McCormick, J.J., Wasman, C.C., Jarvis, N.P., Bell, D.H., and Good, N. E. Hydrogen Ion buffers for biological research.  Anal. Biochem. 104:300-310 (1980).
2. Good, N.E. and Izawa, S. Hydrogen Ion Buffers, Meth. Enzymology 24: 53-68 (1972).

US Biological Good Buffers

Catalog #	Product Name
B2102	Bis-Tris
C1076	CAPS, Free Acid
G8225	Glycylglycine (Gly-Gly, Diglycine)
H2030	HEPES Sodium Salt
H2010	HEPES Free Acid
M3000	MES Free Acid
M3005	MES, Sodium Salt
M4610	MOPS Free Acid
M4615	MOPS Sodium Salt
M4620	MOPSO Free Acid
P4200	PIPES Free Acid
P4205	PIPES, Sodium Salt
T1000	TAPS Free Acid
T1005	TAPS, Sodium Salt
T1010	TAPSO Free Acid
T1015	TAPSO, Sodium Salt
T2900	TES Free Acid
T2905	TES, Sodium Salt
T8400	Tricine
T8600	Tris Base Ultrapure
T8650	Tris Hydrochloride (Tris HCl)
T8601	Tris Base USP
T8600-20	Tris Hydrochloride Buffer (Tris HCl), 1M, pH 7.5
T8600-21	Tris Hydrochloride Buffer (Tris-HCl), 1M, pH 8.0
T8600-05	Tris Acetate