company news about Don't buy the wrong one, PIPES and HEPES buffers are very different

  In biochemical experiments and cell culture, choosing the appropriate buffer is one of the key steps to ensure the accuracy and stability of the experiment. Among them, PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)) and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) are two commonly used buffers. Although they all play an important role in adjusting the pH of solutions, their properties, application scenarios and precautions are quite different. The following is a detailed analysis of the significant differences between PIPES and HEPES to help scientific researchers and laboratory technicians correctly select and use these two buffers.

一. Chemical properties and solubility of PIPES and HEPES
 

1. HEPES
 

(1) Chemical properties: HEPES is a zwitterionic buffer containing amino and sulfonic acid groups in its molecular structure, which enables it to buffer changes in hydrogen ion concentration over a wide pH range (6.8-8.2) and maintain the stability of the solution pH.
 

(2) Solubility: HEPES is highly soluble in water, which allows it to be easily used in a variety of biochemical experimental systems without the need for additional dissolution steps.

2. PIPES
 

(1) Chemical properties: PIPES is also an ideal buffer with a slightly narrower pH buffer range than HEPES, at 6.1-7.5. The piperazine ring and sulfonic acid groups in its molecular structure give it the ability to buffer pH under specific conditions.
 

(2) Solubility: Unlike HEPES, PIPES itself is insoluble in water, but soluble in NaOH aqueous solution. This property requires specific dissolution treatment before use, and its solubility properties need to be considered in actual experiments.

2. Application scenarios and functional differences between PIPES and HEPES
 

1. Application of HEPES
 

(1) Cell culture: HEPES is widely used in cell culture media of various types of organisms because it is non-toxic to cells and does not interfere with normal biochemical reactions, especially in experimental systems that require a long period of time to maintain a stable pH value.
 

(2) Protein and enzyme research: In protein stability research, HEPES is often used as a buffer because it can keep the solution pH constant for a long time, which is beneficial to the maintenance of protein structure and function. At the same time, it is also used in the research of highly volatile and pH-sensitive proteins and enzymes.
 

(3) Molecular biology: In DNA/RNA extraction, PCR diagnostic kits and biochemical diagnostic kits, HEPES is an important buffer component to ensure the stability of pH during the experiment.
 

2. Application of PIPES
 

(1) Protein purification: PIPES has special applications in the field of protein purification, such as the purification of tubulin using phosphocellulose chromatography and the purification of recombinant GTP-binding proteins ARF1 and ARF2 by gel filtration. Its characteristic of not forming stable complexes with most metal ions makes it perform well in solution systems containing metal ions.
 

(2) Chromatographic analysis: In cation exchange chromatography, PIPES is often used as a component of binding buffer or eluent, but its concentration needs to be strictly controlled to avoid experimental errors caused by excessive ionic strength or changes in pKa value.
 

(3) Specific biochemical experiments: Although the application range of PIPES is relatively narrow, it plays an irreplaceable role as a buffer in certain specific experiments, such as calcium phosphate and DNA precipitation system, AFM and electroporation experiments.

III. Precautions for the use of PIPES and HEPES buffers
 

1. Precautions for HEPES
 

(1) Interference: Although HEPES does not interfere with biochemical processes in most cases, it interferes with the reaction between DNA and restriction enzymes, so it is not suitable for experiments that require accurate control of DNA enzyme cutting. At the same time, it is not suitable for Lowry's method to determine protein content, which may affect the accuracy of the determination results.
 

(2) Concentration selection: When using HEPES, the appropriate concentration should be selected according to the experimental requirements. Generally, the culture medium often contains 20mmol/L HEPES to achieve sufficient buffering capacity.
 

2. Precautions for PIPES
 

(1) Solubility: Before using PIPES, make sure that it is completely dissolved in the NaOH aqueous solution to avoid interference of undissolved particles in the experiment.
 

(2) Free radical formation: PIPES can form free radicals, so it is not suitable for use in experiments that need to avoid redox reactions.
 

(3) Concentration dependence: The pKa value of PIPES is concentration-dependent, and its ionic strength is relatively large. In experiments such as cation exchange chromatography, special attention should be paid to the selection and control of concentration.