Question 1

How do I choose a buffer for a given pH?

Accepted Answer

Pick a buffer whose pKa is as close as possible to your target pH, and ideally within one unit of it. A buffer works because both the acid and conjugate base forms are present in meaningful amounts, and that is only true near the pKa. At exactly one pH unit away from the pKa the mixture is already about 91 percent one form and 9 percent the other, so its capacity to soak up added acid or base is nearly exhausted. For a target of pH 7.4, that points to buffers like TES, HEPES, or MOPS.

Question 2

Why is the buffering range pKa plus or minus 1?

Accepted Answer

It comes straight out of the Henderson-Hasselbalch equation. At a pH one unit above the pKa the ratio of conjugate base to acid is 10 to 1, meaning about 91 percent of the buffer is in the base form and only 9 percent remains as acid to neutralize any base you add. Push further and one form essentially runs out, so the pH starts moving freely. Within pKa plus or minus 1 both forms are still abundant enough to resist change in either direction, which is why that is the accepted working window.

Question 3

Why does Tris pH change so much with temperature?

Accepted Answer

Tris has an unusually large temperature coefficient of about minus 0.028 pH units per degree Celsius, far bigger than most buffers. A Tris buffer adjusted to pH 8.00 at room temperature will actually sit near pH 8.59 in a 4 degree cold room and near pH 7.66 at 37 degrees. That is a swing of nearly a full pH unit across normal working temperatures, and it is a classic source of failed experiments. The fix is to adjust the pH at the temperature where the buffer will actually be used, and to prefer a buffer like phosphate or HEPES when temperature will vary.

Question 4

What are Good's buffers?

Accepted Answer

Good's buffers are a family of zwitterionic buffers, including MES, PIPES, MOPS, HEPES, TES, TAPS, CHES, and CAPS, selected by Norman Good and colleagues for biological work. They were chosen to have pKa values spanning the physiological range, high water solubility, minimal binding of metal ions, low membrane permeability, and little absorbance in the visible or near-UV range. Those properties make them far less likely to interfere with enzymes, cells, and spectrophotometric assays than older buffers like phosphate or citrate.

Question 5

When should I avoid phosphate buffer?

Accepted Answer

Phosphate is cheap, biologically relevant, and barely shifts with temperature, but it has real drawbacks. It precipitates with calcium and many divalent metal cations, it inhibits a number of enzymes including several kinases and phosphatases, and it participates directly in reactions where phosphate is a substrate or product. It also buffers poorly between about pH 8.5 and 10 because that region sits between its second and third pKa values. In those situations a Good's buffer such as HEPES or Tris is usually the better choice.

Buffer	Watch out for
Tris	Large temperature shift (−0.028/°C); also reacts with aldehydes and interferes with some protein assays
Phosphate	Precipitates with calcium and many divalent metals; inhibits several kinases and phosphatases; poor buffering pH 8.5–10
Citrate	Strong metal chelator, so it strips required cofactors from many enzymes
Borate	Complexes with cis-diols, so it binds sugars, glycerol, and some nucleotides
Carbonate	Exchanges CO₂ with the air, so the pH drifts in an open vessel
Glycine	An amino acid, so it can take part in reactions and interfere with amine assays

Buffer pKa Table

How to pick a buffer

Buffers to be careful with

FAQ