pH Mastery: Navigating the Terrain of HEPES and Tris Buffers

In the realm of biological research and laboratory applications, the choice of a buffer is paramount for experiment precision and success. HEPES  and Tris stand out as two commonly employed buffers, each possessing unique characteristics.

pH and pKa Relationship

  • HEPES with a pKa around 7.5, HEPES excels in the neutral pH range. Its buffering capacity is most effective when the pH aligns with its pKa value.
  • Tris with a pKa around 8.1, performs exceptionally well in a higher pH range, particularly in studies involving proteins and enzymes.

Effective pH Range

  • The effective pH range of HEPES is typically defined as pH = pKa ± 1, ranging from 6.5 to 8.5. This stability makes HEPES a reliable choice for coping with diverse experimental conditions.
  • Tris's effective pH range is generally defined as pH = pKa ± 1, spanning from 7.1 to 9.1. This positions Tris as a robust buffer for alkaline experiments, such as DNA electrophoresis.

Temperature Impact on pKa

  • HEPES exhibits a relatively minor change in pKa with temperature (d(pKa)/dT), indicating its ability to maintain a stable pH during temperature variations. This is particularly crucial for studies requiring precise temperature control.
  • Tris on the other hand, undergoes a more substantial change in pKa with temperature, potentially affecting its buffering efficiency during temperature fluctuations. Careful consideration is advised for long-term experiments.

Enthalpy Change and Temperature Dependence

  • With an enthalpy change (ΔH) close to 0, the equilibrium constant of HEPES shows minimal variation with temperature changes. This contributes to the maintenance of stable reaction conditions during temperature fluctuations.
  • Similarly, Tris exhibits a small temperature-dependent change in equilibrium constant with ΔH close to 0, contributing to its widespread use in various experiments.


  HEPES buffer Tris buffer
pH and pKa Relationship
pKa ≈ 7.5 
pKa ≈ 8.1
Effective pH Range
pH = pKa ± 1 (6.5 to 8.5)
pH = pKa ± 1 (7.1 to 9.1)
Temperature Impact on pKa
Small change (d(pKa)/dT) Maintains stable pH with temp.
Significant change (d(pKa)/dT) May affect buffering efficiency
Enthalpy Change and Temp
ΔH close to 0  Minimal impact on equilibrium 
ΔH close to 0 Minimal impact on equilibrium


Unique Advantages of HEPES

  • Resistance to Metal Ion Interference: HEPES's inability to readily form coordination compounds with metal ions makes it particularly suitable for experiments where interference from metal ions needs to be avoided.
  • Applicability in Cation Exchange Chromatography: Due to its negatively charged nature, HEPES is especially suitable for cation exchange chromatography, a common application in many biological experiments.

Explanation of Advantages

Resistance to Metal Ion Interference: In certain biological experiments, especially those involving proteins and enzymes, the presence of metal ions can introduce interference with experimental results. HEPES's resistance to forming coordination compounds with metal ions makes it a more reliable buffer in such environments.

Applicability in Cation Exchange Chromatography: With its negative charge, HEPES is well-suited for cation exchange chromatography, a technique used for the analysis and separation of biomolecules with cationic properties. This characteristic makes HEPES an indispensable buffer in specific experimental conditions.

In summary, while both HEPES and Tris are reliable buffers, HEPES stands out with unique advantages in terms of stability, pH range, temperature stability, and resistance to metal ion interference. These qualities position HEPES as an ideal choice under specific experimental conditions. A thorough understanding of these properties in experimental design contributes to the successful execution of research.

Further Reading: Application of HEPES in biological science
Further Reading: Why use HEPES?
Further Reading:HEPES handling and storage tips that you must know
Further Reading: HEPES VS PBS 

Release date:2024.01.17