Temperature dependence of HEPES buffer

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HEPES buffer is an important zwitterionic buffer used in biochemical experiments and molecular biology research to regulate the pH value of reaction systems. The pKa value of HEPES (N-2-hydroxyethylpiperazin-2-ethanesulfonic acid) varies with temperature and has a certain dependence. If the temperature is not well understood, this may have an impact on experimental design and results.

HEPES parameters:

English name


Molecular formula


Melting point

234-238 °C

PKa (25 ° C)


PH range


Maximum wavelength



1、 HEPES temperature change

HEPES is widely used in fields such as cell culture, enzyme reactions, and protein crystallization. Its pKa value is an indicator of acid-base properties, and the lower the pKa value, the stronger the acidity. At room temperature (25 º C), the pKa value of HEPES is approximately 7.48, but it drops to 7.31 at 37 º C, indicating that the acidity and alkalinity of HEPES may change at higher temperatures.

2、 Usage considerations

HEPES is widely used in biochemical experiments to regulate the acid-base balance of reaction systems, as it has relatively stable acid-base properties within the physiological pH range, and is often chosen as the reaction environment for biomolecules. However, due to the impact of temperature changes on the pKa value of HEPES, it is necessary to consider the effect of temperature on acid-base equilibrium in the experiment.

3、 Temperature in HEPES applications

1. In cell culture, the growth and metabolism of cells are influenced by the pH value in the buffer solution. Due to the fact that the pKa value of HEPES decreases with increasing temperature, its buffering capacity may slightly decrease at higher cultivation temperatures. In this case, researchers need to adjust the concentration of HEPES according to temperature to maintain a suitable acid-base environment.

2. In the study of enzyme reactions, the activity and stability of many enzymes are closely related to the acidity and alkalinity of the environment. As the temperature increases, the pKa value decreases, thereby increasing its acidity, which may affect the catalytic efficiency of the enzyme. In this case, researchers need to carefully consider the environmental temperature of using HEPES in the experiment to ensure the normal progress of enzyme reactions.

The temperature dependence of HEPES buffer is one of the important factors that need to be considered in experimental design. When selecting HEPES as a buffer, researchers should pay attention to adjusting the concentration of the buffer according to the temperature conditions of the experiment and the studied biological molecular characteristics to ensure the accuracy and reliability of the experimental results. As a professional manufacturer of biological buffer, Desheng can supply HEPES raw material powder with a purity of 99%, which has good water solubility and is suitable for various fields such as cell culture and cosmetics. If you have any interest, please feel free to contact us for purchase!