Luminol, the luminescent substrate of horseradish peroxidase HRP, is chemically named 3-aminophthalic acid hydrazide, and sometimes also contains isoluminol and its derivatives such as ABEI, ITCI, etc., which are the reagents of this type of reagent Collectively. The synthesis process of this kind of reagent directly affects its luminescence performance, which in turn affects the detection result.
The synthesis process of luminol, isoluminol and its derivatives are all based on 3-aminophthalic hydrazide. The synthesis steps are divided into three steps. Here is a brief introduction to their synthesis trilogy:
1. Synthesis of 3-nitrophthalic acid
Luminol and isoluminol are both prepared by the reaction of nitrophthalic acid and hydrazine, and it is an important raw material for the synthesis process. Raw materials can be purchased directly, the cost will be higher, or they can be produced on their own. Mix 12 mL of concentrated sulfuric acid and 12 g of phthalic anhydride and heat, slowly add 10 mL of fuming nitric acid dropwise, and control the temperature at 100-110°C.
After the reaction is completed, it is cooled to obtain the product 3-nitrophthalic acid and the by-product 4-nitrophthalic acid. Using different water solubility, add water and filter to obtain the crude 3-nitrophthalic acid solid, and then use water for the solid Recrystallize to obtain the product.
Three steps of luminol synthesis
2. Synthesis of 3-nitrophthalic hydrazide
Put 1.3 g of 3-nitrophthalic acid and 2 mL of 10% hydrazine hydrate in a 100 mL two-necked flask, heat to dissolve, add 4 mL of triethylene glycol, fix the two-necked flask, add zeolite, The catheter connects the flask to the water pump through a safety bottle. Turn on the water pump and heat the flask to maintain the temperature at 210-220°C. After the reaction is complete, stop heating and pumping. Cool to 100°C, heat water, cool to room temperature and filter, collect light yellow crystals to obtain 3-nitrophthalic acid hydrazide.
3. Synthesis of luminol 3-aminophthalic hydrazide
The product from the previous step was transferred to a beaker, and sodium hydroxide solution was added to dissolve it. Add 4 g of sodium dithionite dihydrate, heat and stir, and keep boiling for 5 minutes. After a little cooling, 2.6 mL of glacial acetic acid was added, and the mixture was cooled to room temperature in a cold water bath to precipitate light yellow crystals, which were washed with suction and water for three times to obtain the final product luminol.
The above is the synthesis steps of luminol. Similarly, the by-product 4-nitrophthalic acid can be made into isoluminol, or the synthesis of isoluminol derivatives can be continued. The luminescent substrates currently synthesized by Desheng include luminol, isoluminol, and acridinium ester, a direct chemiluminescence reagent.
