Chromatography is a laboratory technique that is often used for the separation and purification of proteins.

In this process, the separation capability of the system is directly connected to pH variations. If the mobile phase (solvent) pH is near the pKa, for example, small changes in pH can strongly affect retention, and consequently the separation results. Since the retention of ionizable compounds is especially sensitive to this mobile phase pH, a buffer should be added to the system in order to control this variable.

We have reviewed the most relevant literature in chromatography to make a list of the most common biological buffers used for this technique. We found that although there is a consensus that Tris and MES are often the best choices, other buffers have also been used in cation exchange chromatography, anion exchange chromatography, high-performance liquid chromatography (HPLC) and other similar techniques. Check out our list!

 

1) BIS-TRIS buffer

Useful pH range: 5.8 - 7.2
pKa (25°C): 6.46
Molecular weight: 209.2g/mol

Used as: a buffer during anion exchange chromatography¹

Concerns: several components in a chromatographic system may be competing for metal binding with this buffer²

Read more about Hopax BIS-TRIS

Further reading:The uses of Bis-Tris Buffer - A starting guide

2) BES buffer

Useful pH range: 6.4 - 7.8
pKa (25°C): 7.09
Molecular weight: 213.2g/mol

Used as: a binding buffer and eluent in cation exchange chromatography / as a buffer in gel filtration chromatography³

Read more about Hopax BES

3) Bicine buffer

Useful pH range: 7.6 - 9.0
pKa (25°C): 8.26
Molecular weight: 163.2 g/mol

Used as: a mobile phase buffer and eluent in cation exchange chromatography

Read more about Hopax Bicine

4) CAPS buffer

Useful pH range: 9.7 - 11.1
pKa (25°C): 10.40
Molecular weight: 221.32g/mol          

Used as: a binding buffer and eluent in cation exchange chromatography⁴

Read more about Hopax CAPS

5) HEPES buffer

Useful pH range: 6.8 - 8.2
pKa (25°C): 7.48
Molecular weight: 238.3g/mol

Uses:

• as a binding buffer and eluent in cation exchange chromatography^5,6
• in a two-stage reverse dialysis method for in vitro release testing (no interference from the buffer with a HPLC was found)⁷

Read more about Hopax HEPES

Further reading:Why use HEPES?

6) MES buffer

Useful pH range: 5.5 - 6.7
pKa (25°C): 6.10
Molecular weight: 195.2g/mol

Used as:

• a buffer in capillary electrochromatography⁸
• a buffer in gel-filtration chromatography⁹
• a buffer in phosphocellulose column chromatography¹⁰
• a buffer in hydrophobic interaction chromatography¹⁰
• a buffer in cation exchange chromatography¹¹

Read more about Hopax MES

Further reading:15 uses of MES Buffer you didn't know

7) MOPS buffer

Useful pH range: 6.5 - 7.9
pKa (25°C): 7.14
Molecular weight: 209.3g/mol

Used as: a running buffer for protein purification in chromatography

Read more about Hopax MOPS

Further reading:Why use MOPS?

8) PIPES buffer

Useful pH range: 6.1 - 7.5
pKa (25°C): 6.76
Molecular weight: 302.37g/mol

Used as:

• a buffer in cation exchange chromatography
• a buffer in phosphocellulose chromatography to purify microtubule proteins¹²

Concerns: when used in cation exchange chromatography, it should be used in lower concentrations due to its large ionic strength and dependence of concentration on pKa.

Read more about Hopax PIPES

Further reading:Why use PIPES Buffer

9) TAPS buffer

Useful pH range: 7.7 - 9.1
pKa (25°C): 8.40
Molecular weight: 243.28g/mol

Used as:

• a buffer in planar chromatography to separate dyes¹³
• a buffer in ion exchange chromatography for enzyme purification¹⁴

Read more about Hopax TAPS

10) Tris buffer

Useful pH range: 7.5 - 9.0
pKa (25°C): 8.06
Molecular weight: 121.14g/mol

Used as:

• as a buffer and eluent in anion exchange chromatography¹⁵
• as a buffer in capillary electrochromatography due to its low ionic mobility¹⁶

Read more about Hopax Tris

Further reading:The uses of Tris buffer - A starting guide

Hopax Biological Buffers

Hopax Fine Chemicals is among the largest manufacturers of biological buffers in the world. Our products are shipped daily to top research centers and biotech companies in Europe, America and Asia.

What we offer:

• 30 buffers straight from our manufacturing sites
• Small and bulk packages
• International quality standards
• Worldwide shipping to your door
• Assistance with shipping
• After-sales service with English speaking staff

Check the full list of Hopax biological buffers

Please contact us for the high-quality Bio-buffer!!

References:

¹ Donoghue, M., Hsieh, F., Baronas, E., Godbout, K., Gosselin, M., Stagliano, N., Donovan, M., Woolf, B., Robison, K., Jeyaseelan, R., Breitbart, R.E., & Acton, S. (2000). A novel angiotensin-converting enzyme–related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circulation Research, 87(5)

² Wang, F., Chmil, C., Pierce, F., Ganapathy, K., Gump, B. B., MacKenzie, J. A., Metchref, Y. & Bendinskas, K. (2013). J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 934, 26–33.

³ Schirch, V., Hopkins, S., Villar, E., & Angelaccio, S. (1985). Serine hydroxymethyltransferase from Escherichia coli: purification and properties. Journal of Bacteriology, 163(1), 1-7.

⁴ Thomas, B. R., Vekilov, P. G., & Rosenberger, F. (1996). Heterogeneity determination and purification of commercial hen egg-white lysozyme. Acta Crystallographica Section D: Biological Crystallography, 52(4), 776-784.

⁵ Yan, L. J., & Sohal, R. S. (1998). Mitochondrial adenine nucleotide translocase is modified oxidatively during aging. Proceedings of the National Academy of Sciences, 95(22), 12896-12901.

⁶ Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I., Brenner, C., Larochette, N., Prévost, M., Alzari, P. & Kroemer, G. (1999). Mitochondrial release of caspase-2 and-9 during the apoptotic process. The Journal of Experimental Medicine, 189(2), 381-394.

⁷ Xu, X., Khan, M. K., & Burgess, D. J. (2012). A Two-Stage Reverse Dialysis In Vitro Dissolution Testing Method for Passive Targeted Liposomes. Int. J. Pharm., 426, 211–218.

⁸ Boughtflower, R. J., Underwood, T., & Paterson, C. J. (1995). Capillary electrochromatography—Some important considerations in the preparation of packed capillaries and the choice of mobile phase buffers. Chromatographia, 40(5-6), 329-335.

⁹ Weisenberg, R. C. (1972). Microtubule formation in vitro in solutions containing low calcium concentrations. Science, 177(4054), 1104-1105.

¹⁰ Alonso, A. D. C., Zaidi, T., Novak, M., Grundke-Iqbal, I., & Iqbal, K. (2001). Hyperphosphorylation induces self-assembly of τ into tangles of paired helical filaments/straight filaments. Proceedings of the National Academy of Sciences, 98(12), 6923-6928.

¹¹ Cook, T. A., Urrutia, R., & McNiven, M. A. (1994). Identification of dynamin 2, an isoform ubiquitously expressed in rat tissues. Proceedings of the National Academy of Sciences, 91(2), 644-648.

¹² Clayton, L., Quinlan, R. A., Roobol, A., Pogson, C. I., & Gull, K. (1980). A comparison of tubulins from mammalian brain and Physarumpolycephalum using SDS—polyacrylamide gel electrophoresis andpeptide mapping. FEBS Letters, 115(2), 301-305.

¹³ Nurok, D., Frost, M. C., & Chenoweth, D. M. (2000). Separation using planar chromatography with electroosmotic flow. Journal of Chromatography A, 903(1), 211-217.

¹⁴ Anderson, M. P., & Gronwald, J. W. (1991). Atrazine resistance in a velvetleaf (Abutilon theophrasti) biotype due to enhanced glutathione S-transferase activity. Plant Physiology, 96(1), 104-109.

¹⁵ Huisman, T. H., & Dozy, A. M. (1965). Studies on the heterogeneity of hemoglobin: IX. The use of tris (hydroxymethyl) aminomethane—HCl buffers in the anion-exchange chromatography of hemoglobins. Journal of Chromatography A, 19, 160-169.

¹⁶ Altria, K. D., Smith, N. W. & Turnbull, C. H. (1998). J. Chromatogr. B: Biomed. Sci. Appl., 717, 341–353.