Chromatography trace for protein purification BIOLOGICAL BUFFERS

Biological Buffers

The pKa of a buffer is commonly perceived as the pH of the said buffer when the concentrations of the two buffering species are equal, and where the maximum buffering capacity is achieved. However, it is often forgotten, that when defined as above, pKa depends on buffer concentration and temperature. To avoid this problem the concept of "thermodynamic" pKa⁰ was introduced. pKa⁰ is pKa of the buffer at infinite dilution (buffer concentration=0) and 25^oC. Thus, pKa⁰ is a true constant specific for a given buffer.

Note that, depending on the nature of the buffer, the pH (and pKa) of the buffer solution may increase or decrease upon dilution, and this effect may be significant. Additionally, small changes in temperature can also cause noticeable changes in the pH of the buffer solution. For the buffers shown on the brown background, the buffers' concentrations have especially strong influfluences on the buffers' pKa values.

For your convenience, the biological buffers table contain values of pKa⁰, d(pKa⁰)/dt at 298.25 K, and a calculator that allows you to estimate pKa values of each buffer at temperatures form 3^oC to 37^oC, and concentrations from 1 to 500mM for "white background" buffers and 1 to 130mM for "brown background" buffers.

To use the calculator, enter the buffer's concentration and temperature, then click on the corresponding = button.^**

pKa⁰ (25 ^oC) d(pKa)/dt
ΔH⁰, kJ/mol Calculate pKa at
given concentration
and temperature Buffer name/dissociation type Dissociation step UV
limit Notes

1.92 -0.0006
1.1 mM ^oC
pKa = Maleic acid
H₂L = H⁺ + HL^-1 Maleic acid, biological buffers structure 270nm Other pKa: 6.23. Can react with strong nucleophiles (e.g. thiols) by Michael addition.

2.148 +0.0047
-8.0 mM ^oC
pKa = Phosphoric acid
H₃L = H⁺ + H₂L^-1 H₃PO₄ = H⁺ + H₂PO₄^-1 Clear Other pKa: 7.198, 12.35. Stabilizes many enzymes. Precipitates bivalent cations.

3.128 -0.0024
4.07 mM ^oC
pKa = Citric acid
H₃L = H⁺ + H₂L^-1 Citric acid, biological buffers formula Clear Other pKa: 4.761, 6.396. Binds to some proteins. Forms complexes with many metals.

3.40
mM 25^oC
pKa = Malic acid
H₂L = H⁺ + HL^-1 Malic acid, biological buffers structure Clear Other pKa: 5.11. Is chiral. Forms complexes with some metals.

3.75
mM 25^oC
pKa = Formic acid
HL = H⁺ + L^-1 Formic acid, biological buffers formula Clear Slightly volatile, usually is sold containing 5-10% of water - difficult to dose in exact amounts.

3.86
mM 25^oC
pKa = Lactic acid
HL = H⁺ + L^-1 Lactic acid, biological buffers structure Clear Is chiral. Usually is sold as ~85% solution in water - difficult to dose in exact amounts.

4.207 -0.0018
3.0 mM ^oC
pKa = Succinic acid
H₂L = H⁺ + HL^-1 Succinic acid, biological buffers formula Clear Other pKa: 5.636.

4.756 +0.0002
-0.41 mM ^oC
pKa = Acetic acid
HL = H⁺ + L^-1 Acetic acid, biological buffers structure Clear Volatile.

5.03 (at 20^oC)
mM 20^oC
pKa = Pivalic (trimethylacetic) acid
HL = H⁺ + L^-1 Pivalic (trimethylacetic) acid, biological buffers formula Clear Volatile, has bad odor and relatively low solubility in water.

5.11
mM 25^oC
pKa = Malic acid
HL^-1 = H⁺ + L^-2 Malic acid, biological buffers structure Clear Other pKa: 3.40. Is chiral. Forms complexes with some metals.

5.23 -0.014 mM ^oC
pKa = Pyridine
HL⁺ = H⁺ + L pyridine, biological buffers stucture 275nm Volatile and toxic.

5.333 -0.018
31.11 mM ^oC
pKa = Piperazine
H₂L⁺² = H⁺ + HL⁺ piperazine, biological buffers formula Clear Other pKa: 9.731.

5.39 (at 20^oC)
mM 20^oC
pKa = Picolinic acid
HL^± = H⁺ + L^-1 Picolinic acid, biological buffers formula Other pKa: 0.99 at 20^oC.

5.636 +0.0002
-0.5 mM ^oC
pKa = Succinic acid
HL^-1 = H⁺ + L^-2 Succinic acid, biological buffers structure Clear Other pKa: 4.207.

6.05 -0.017
29.5 mM ^oC
pKa = L-Histidine
H₂L⁺ = H⁺ + HL^± Histidine, biological buffers structure 235nm Other pKa: 1.80, 9.34. Chiral. Forms complexes with Me²⁺, forms complexes with itself. It is a primary amine, and therefore can form Schiff’s bases with aldehydes/ ketones.

6.20 -0.0087
14.8 mM ^oC
pKa = MES
HL^± = H⁺ + L^-1 MES, biological buffers formula Clear Other pKa: <3. Weakly binds Ca, Mg, Mn. Negligible binding with Cu(II).^1,2

6.484 -0.017
28.4 mM ^oC
pKa = Bis-tris
HL⁺ = H⁺ + L Bis-tris, biological buffers formula Clear Binds Ca(II), Sr(II), Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Pb(II); weakly binds Mg(II), Ba(II), Mn(II).¹¹

6.65 -0.03
mM ^oC
pKa = Bis-tris propane
H₂L⁺² = H⁺ + HL⁺ Bis-tris propane, biological buffers structure Clear Other pKa: 9.11.

6.844 -0.0072
12.23 mM ^oC
pKa = ADA
HL^-1 = H⁺ + L^-2 ADA, biological buffers structure 260nm Other pKa: 1.59, 2.48. Binds Cu(II), Co(II), Zn(II), Mn(II), Ni(II), Ca(II), weakly binds Mg(II).^3,4 Free acid is poorly soluble in water.

6.847 -0.018
30.43 mM ^oC
pKa = ACES
HL^± = H⁺ + L^-1 ACES, biological buffers formula 230nm Other pKa: <3. Binds Cu(II), Co(II), Zn(II), Nil(II), weak binding to Ca(II), Mg(II), negligible binding to Mn(II).^1,5

6.90 -0.015
25.0 mM ^oC
pKa = MOPSO
HL^± = H⁺ + L^-1 MOPSO, biological buffers structure Clear Other pKa: <2. Chiral, some metal binding.

6.960 -0.0072
12.3 mM ^oC
pKa = PIPES
(HL^±)^-1 = H⁺ + L^-2 PIPES, biological buffers formula Clear Other pKa: <3. Negligible binding to metals.^1,2 Can form radicals, should be avoided in studies of oxidative compounds and redox processes in biochemistry.^6,7 Free acid is poorly soluble in water.

6.993 -0.021
36.64 mM ^oC
pKa = Imidazole
HL⁺ = H⁺ + L Imidazole, biological buffers formula 235nm Forms complexes with Me²⁺, and forms complexes with histidine. Strongly nucleophilic, catalyzes wide range of chemical transformations.

7.184 -0.012
21.1 mM ^oC
pKa = MOPS
HL^± = H⁺ + L^-1 MOPS, biological buffers structure Clear Other pKa: <2. Negligible binding to metals.² Partially degrades on autoclaving in the presence of glucose.

7.187 -0.014
24.25 mM ^oC
pKa = BES
HL^± = H⁺ + L^-1 BES, biological buffers formula Clear Other pKa: <3. Binds Cu(II), negligible binding to Ca(II), Mg(II) and Mn(II).¹

7.198 -0.0022
3.6 mM ^oC
pKa = Phosphoric acid
H₂L^-1 = H⁺ + HL^-2 H₂PO₄^-1 = H⁺ + HPO₄^-2 Clear Other pKa: 2.148, 12.35. Stabilizes many enzymes. Precipitates bivalent cations.

7.50 -0.019
32.13 mM ^oC
pKa = TES
HL^± = H⁺ + L^-1 TES, biological buffers formula Clear Other pKa: <3. Binds Cu(II), Co(II), Zn(II), Mn(II). Negligible binding to Ca(II), Mg(II) and Mn(II).^1,8

7.564 -0.012
20.4 mM ^oC
pKa = HEPES
HL^± = H⁺ + L^-1 HEPES, biological buffers structure Clear Other pKa: <3. Negligible binding to Ca(II), Mg(II) and Mn(II).¹ Is oxidized by Cu(II).⁹ Can form radicals, should be avoided in studies of redox processes in biochemistry.^6,7

7.576 -0.018
30.18 mM ^oC
pKa = DIPSO
HL^± = H⁺ + L^-1 DIPSO, biological buffers formula Clear Other pKa: <2. Chiral. Binds Co(II), Ni(II).¹⁰

7.635 -0.023
39.09 mM ^oC
pKa = TAPSO
HL^± = H⁺ + L^-1 TAPSO, biological buffers structure Clear Other pKa: <2. Chiral. Binds Co(II), Ni(II).¹⁰

7.762 -0.020
33.6 mM ^oC
pKa = TEA (Triethanolamine)
HL⁺ = H⁺ + L TEA (Triethanolamine), biological buffers formula Clear Binds Co(II), Ni(II), Cu(II), Zn(II), Cd(II).¹¹ Can form radicals in the presence of strong oxidants, exercise caution during studies of redox processes.

7.77 -0.016
27.4 mM ^oC
pKa = N-Ethylmorpholine
HL⁺ = H⁺ + L N-Ethylmorpholine, biological buffers structure Clear

7.85 -0.013
mM ^oC
pKa = POPSO
(HL^±)^-1 = H⁺ + L^-2 POPSO, biological buffers formula Clear Other pKa: <2. Chiral, is a mixture of two diastereomers. Can form radicals, should be avoided in studies of redox processes in biochemistry.^6,7 Free acid is poorly soluble in water.

7.957 -0.013
21.3 mM ^oC
pKa = EPPS, HEPPS
HL^± = H⁺ + L^-1 EPPS, HEPPS, biological buffers structure Clear Other pKa: <2. Binds metals. Can form radicals, should be avoided in studies of redox processes in biochemistry.^6,7

7.94 -0.014
23.70 mM ^oC
pKa = HEPPSO
HL^± = H⁺ + L^-1 HEPPSO, biological buffers formula Clear Other pKa: <2. Chiral.

8.072 -0.028
47.45 mM ^oC
pKa = Tris
HL⁺ = H⁺ + L Tris, biological buffers structure Clear Binds Cu(II), Ni(II).^11,12 Binds Co(II), Zn(II), Cd(II), Pb(II); weakly binds Ca(II), Mg(II), Ba(II), Mn(II).¹¹ It is a primary amine, and therefore can form Schiff’s bases with aldehydes/ ketones. Inactivates DEPC. Is involved in some enzymatic reactions (e.g. alkaline phosphatase).

8.135 -0.018
31.37 mM ^oC
pKa = Tricine
HL^± = H⁺ + L^-1 Tricine, biological buffers structure Clear Other pKa: 2.023. Binds Cu(II), Co(II), Zn(II), Ni(II), Cd(II), Pb(II) Ca(II), Mg(II) and Mn(II).^1,4 Is photooxidized by flavines.

8.265 -0.026
43.40 mM ^oC
pKa = Glycylglycine
HL^± = H⁺ + L^-1 Glycylglycine, biological buffers formula Clear Other pKa: 3.14. Binds Cu(II) Mn(II), weakly binds Ca(II) and Mg(II).¹ Is a primary amine, therefore it can form Schiff’s bases with aldehydes/ ketones.

8.334 -0.016
26.34 mM ^oC
pKa = Bicine
HL^± = H⁺ + L^-1 Bicine, biological buffers structure Clear Other pKa: 2.0. Binds Cu(II), Co(II), Zn(II), Mn(II), Ca(II), Mg(II).^1,8 Is slowly oxidized by ferricyanide.

8.44 -0.024
40.4 mM ^oC
pKa = TAPS
HL^± = H⁺ + L^-1 TAPS, biological buffers formula Clear Other pKa: <2. Binds Co(II), Ni(II).¹⁰

8.50 -0.022
mM ^oC
pKa = Morpholine
HL⁺ = H⁺ + L Morpholine, biological buffers formula Clear

8.54 -0.028
mM ^oC
pKa = N-Methyldiethanolamine
HL⁺ = H⁺ + L N-Methyldiethanolamine, biological buffers structure Clear

8.801 -0.029
49.85 mM ^oC
pKa = AMPD (2-amino-2-methyl-1,3-propanediol)
HL⁺ = H⁺ + L AMPD (2-amino-2-methyl-1,3-propanediol), biological buffers formula Clear It is a primary amine, and therefore can form Schiff’s bases with aldehydes/ ketones.

8.883 -0.026
42.08 mM ^oC
pKa = Diethanolamine
HL⁺ = H⁺ + L Diethanolamine, biological buffers structure Clear Binds Ni(II), Cu(II), Zn(II), Cd(II).¹¹

9.138 -0.025
43.19 mM ^oC
pKa = AMPSO
HL^± = H⁺ + L^-1 AMPSO, biological buffers structure Clear Other pKa: <2. Binds Co(II), Ni(II).¹⁰ Is chiral.

9.237 -0.008
13.8 mM ^oC
pKa = Boric acid
HL = H⁺ + L^-1 H₃BO₃ = H⁺ + H₂BO₃^-1 Clear Forms covalent complexes with mono- and oligosaccharides, ribose subunits of nucleic acids, pyridine nucleotides.

9.43 -0.023
39.55 mM ^oC
pKa = CHES
HL^± = H⁺ + L^-1 CHES, biological buffers formula Clear Other pKa: <3.

9.780 -0.026
44.2 mM ^oC
pKa = Glycine
HL^± = H⁺ + L^-1 Glycine, biological buffers structure Clear Other pKa: 2.351. Interferes with Bradford protein assay. Is a primary amine, therefore it can form Schiff’s bases with aldehydes/ ketones.

9.825 -0.027
46.67 mM ^oC
pKa = CAPSO
HL^± = H⁺ + L^-1 CAPSO, biological buffers formula Clear Other pKa: <2. Is chiral.

9.498 -0.030
50.52 mM ^oC
pKa = Ethanolamine
HL⁺ = H⁺ + L Ethanolamine, biological buffers formula Clear Binds Co(II), Ni(II), Cu(II), Zn(II), Cd(II); weakly binds Mn(II).¹¹ It is a primary amine, and therefore can form Schiff’s bases with aldehydes/ ketones.

9.694 -0.032
54.05 mM ^oC
pKa = AMP (2-amino-2-methyl-1-propanol)
HL⁺ = H⁺ + L AMP (2-amino-2-methyl-1-propanol), biological buffers structure Clear It is a primary amine, and therefore can form Schiff’s bases with aldehydes/ ketones.

9.731 -0.025
42.89 mM ^oC
pKa = Piperazine
HL⁺ = H⁺ + L Piperazine, biological buffers formula Clear Other pKa: 5.333.

10.499 -0.028
48.1 mM ^oC
pKa = CAPS
HL^± = H⁺ + L^-1 CAPS, biological buffers structure Clear Other pKa: <2.

10.55 -0.026
mM ^oC
pKa = 1,3-Diaminopropane
HL⁺ = H⁺ + L 1,3-Diaminopropane, biological buffers structure Clear Other pKa: 8.88. Forms strong complexes with many metals. It is a primary amine, and therefore can form Schiff’s bases with aldehydes/ ketones.

10.7
mM 25^oC
pKa = CABS
HL^± = H⁺ + L^-1 CABS, biological buffers formula Clear Other pKa: <2.

11.123 -0.031
mM ^oC
pKa = Piperidine
HL⁺ = H⁺ + L Piperidine, biological buffers structure Clear

^* Significant deviations exist in the reported values of pKa and other thermodynamic constants of most common buffers due to them being determined by methods of different accuracy. Additionally, many online resources provide pKa values of biological buffers at unspecified or wrongly specified ionic strengths. We attempted to provide the most consistent data available. pKa⁰, d(pKa)/dt and ΔH⁰ are compiled mostly from
- CRC Handbook of Chemistry & Physics, 93th edition: Dissociation Constants of Organic Acids and Bases.
- Goldberg, R. N., Kishore, N., Lennen, R. M. J. Phys. Chem. Ref. Data, 31, 2002, 231-370, as well as some other original publications.
If you have have experimentally observed significantly different values, please report them to support@reachdevices.com.

^**Temperature dependence of pKa is presumed to be linear.
White background buffers:
- for concentrations 1 to 200mM, the Debye-Hückel model is used, and the resulting pKa is presented in brown font.
- for concentrations 201 to 500mM, the Davies model is used, and the resulting pKa is presented in blue font.
Light brown background buffers:
- for concentrations 1 to 50mM, the Debye-Hückel model is used, and the resulting pKa is presented in brown font.
- for concentrations 51 to 130mM, the Davies model is used, and the resulting pKa is presented in blue font.
For a detailed explanation of pKa vs pKa⁰, and formulas used in the calculator, click on this link.

¹ - Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S., Singh. R. M. M. Biochemistry, 1966, 5, 467-477.

² - Kandegedara, A., Rorabacher, D. B. Anal. Chem., 1999, 71, 3140-3144.

³ - Lance, E. A., Rhodes, C.W. , Nakon, R. Anal. Biochem., 1983, 133, 492-501.

⁴ - Nakon, R. Anal. Biochem., 1979, 95, 527-532.

⁵ - Pope, J. M., Stevens, P. R., Angotti, M. T., Nakon. R. Anal. Biochem., 1980, 103, 214-221.

⁶ - Grady, J. K., Chasteen, N. D., Harris. D. C., Anal. Biochem., 1988, 173, 111-115.

⁷ - Kirsch, M., Lomonosova, E. E., Korth, H.-G., Sustmann, R. de Groot, H. The Journal of Biological Chemistry, 1998, 273, 12716-12724.

⁸ - Nakon, R., Krishnamoorthy. C. R. Science, 1983, 221, 749-750.

⁹ - Hegetschweiler, K., Saltman, P. Inorg. Chem., 1986, 25, 107-109.

¹⁰ - Machado, C. M. M., Gameiro, P., Soares, H. M. V. M. J. Solution Chem., 2008, 37, 603-617.

¹¹ - Scheller, K. H., Abel, T. H., Polanyi, P. E., Wenk, P. K., Fischer, B. E., Sigel. H. Eur. J. Biochem., 1980, 107, 455-466.

¹² - Bai, K. -S., Martell. A. E., J. Inorg. Nucl. Chem., 1969, 31, 1697–1707.

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