Why do biological buffering HEPES 7365-45-9 solutions need to follow the principle of ready to use and mix

In the field of biological experiments, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES buffer) solution is widely used in cell culture, molecular biology, and other scenarios as a "key role" in maintaining system pH stability. However, experimenters must strictly follow the principle of "ready to use", which has profound scientific basis behind it.

1, Chemical stability: the 'invisible disruptor' of light and temperature

The unique amphiphilic structure of HEPES molecules endows them with excellent buffering ability within the pH range of 6.8-8.2. But this stability is not absolute, as light and temperature are two potential threats. Experiments have shown that HEPES is prone to decomposition under strong light or ultraviolet radiation, producing oxidizing substances such as hydrogen peroxide. In fluorescence microscopy observation experiments, if HEPES solution exposed to light for a long time is used, the fluorescence signal will be distorted due to interference from oxidation products, seriously affecting the accuracy of experimental results.

HEPES powder

Meanwhile, although high temperatures can be temporarily tolerated, prolonged processing can accelerate molecular dissociation, leading to a decrease in buffering capacity. In PCR experiments, short-term high-temperature cycling has limited impact on HEPES, but once long-term high-temperature incubation is involved, its buffering efficiency will be greatly reduced, thereby interfering with the experimental process.

2, Microbial contamination: the 'fatal hazard' of solution quality

Long term storage of HEPES solution can easily become a breeding ground for microorganisms. Some bacteria metabolize to produce acidic substances such as lactic acid and acetic acid, or release alkaline ammonia substances, directly disrupting the acid-base balance of the solution. In open cell culture experiments, unsterilized HEPES solution may deviate from the target pH range within a few hours due to microbial contamination, resulting in a significant decrease in cell survival rate.

More seriously, some microorganisms can directly utilize the sulfonic acid groups or nitrogen atoms in HEPES as a nutrient source, further weakening its buffering efficiency. Therefore, aseptic operation and ready to use are key to avoiding microbial contamination risks and ensuring solution quality.

3, Metal ion interference: a potential disruptor in experimental systems

The sulfonic acid groups and nitrogen atoms in HEPES molecules have coordination ability and can form stable complexes with divalent or trivalent metal ions. In routine biological experiments, this characteristic may cause problems. For example, when copper ions bind to HEPES, they inhibit enzyme activity that depends on copper ions; Although calcium and magnesium ions have a relatively small impact, caution is still necessary in high-sensitivity experiments.

Long term storage of HEPES solution may cause metal ions to precipitate from the container or contaminate the system with reagents, resulting in fluctuations in buffering capacity. In PCR reactions, the coordination between trace metal ions and HEPES may alter the ion strength of the reaction system, affect amplification efficiency, and interfere with experimental results.

4, Experimental accuracy requirement: the "butterfly effect" of small changes

In high-precision experiments such as protein crystallization and enzyme kinetics research, even small changes in HEPES concentration can trigger chain reactions. Diluting HEPES solution will reduce its buffer capacity, making the system unable to effectively resist the acid-base fluctuations generated by cellular metabolism; Concentrated solutions may affect protein solubility due to changes in ion strength.

Product packaging

Hubei Xindesheng Material Technology Co., Ltd. specializes in producing high-quality biological buffering agents such as Hepes. Since its establishment, Desheng has been dedicated to the research and production of in vitro diagnostic reagents and has rich production experience. Our professional technical team not only ensures product quality, but also provides customized solutions and timely resolution of after-sales issues. If you are interested in our products, please click on the official website to learn more details or contact me!