Application and synthesis of 4-hydroxyethyl piperazine ethanesulfonic acid 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) is an important hydrogen ion buffer, which has good buffering capacity in the range of pH 6.8 to 8.2 and can control a constant pH for a long time. The use concentration is 10~50 mmol/L, and the general culture medium contains 20 mmol/L 4-hydroxyethyl piperazine ethanesulfonic acid, which can have good buffering capacity and has no toxic effect on cells. Method of use (1) Hepes buffer solution can be directly added to the prepared culture solution according to the required concentration, and then filtered for sterilization. 2.38 grams of HEPES are added to every 1000 ml of culture solution, and the pH is adjusted to 7.2 with lNNaOH after being dissolved, and then used after filtering for sterilization. At this time, the use concentration of HEPES is 10 mmol/L (2) It can also be prepared into 100x storage solution (1mol/L). Before use, take 99ml of culture solution and add 1ml of storage solution. The final application concentration is still 10mmol/L. Preparation method of lmol/L (100x) hepes buffer solution: dissolve 23.8g of HEPES in 90ml of double-distilled water, adjust the pH to 7.5-8.0 with lNNaOH, and then use water to fix the volume to 100ml, filter and sterilize, sub-pack small bottles (2ml/bottle), and store at 4 ℃ or - 20 ℃. Applied HEPES (4-hydroxyethyl piperazine chemicalbook ethanesulfonic acid) is a non-ionic amphoteric buffer solution with high polarity, inert to a variety of chemical reagents and enzymes, and does not participate in and interfere with the process of biochemical reaction, It has no inhibitory effect on enzyme chemical reaction, etc., so it can be specially used for the research of organelles and highly variable, pH sensitive proteins and enzymes. The preparation method directly uses 1,2-dichloroethane as the solvent, and adds hydroxyethyl piperazine (5.00g, 0.02mol), potassium carbonate K2CO3 (6.00g, 0.04mol), 50mL1,2-dichloroethane into a 100mL three-mouth bottle equipped with mechanical stirring and thermometer, heating in the oil bath at 90 ℃ (boiling point of 1,2-dichloroethane at 85 ℃), stirring reaction for 20h. Stop the reaction, filter and wash the filtered salt with 200mL ethyl acetate (EA). The filtrate was dried to obtain 2.6g HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) solid. Use as biochemical reagent use as biological buffer; Reaction buffer, pre-hybridization buffer and hybridization buffer for separating and analyzing RNA nuclear components; For RNA and T4RNA use Molecular biological grade for RNA3 '- terminal labeling with T4RNA ligase, separation and analysis of reaction buffer components of nuclear RNA, pre-hybridization buffer to alleviate hybridization buffer components, cell-cell adhesion, short-term cell aggregation culture, and buffer for tissue and cell cleaning