A method for accurately detecting the content of hexamethylenediamine in fermentation broth
A hexamethylenediamine, precise technology, applied in the field of accurate detection of hexamethylenediamine content in fermentation broth, can solve the problems of low detection value, interference of quantitative detection of hexamethylenediamine, etc.
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Embodiment 1
[0037] Example 1: 1,7-Heptanediamine is the establishment of a calibration curve detection method for hexamethylenediamine cell culture as an internal standard
[0038] 1. Construction of simulated cell culture system containing hexamethylenediamine
[0039] Yeast powder with a final concentration of 5g / L, 10g / L, 15g / L, 20g / L, 25g / L, 30g / L, 35g / L and 40g / L was added to the SOB medium. Add 25mg / L, 50mg / L, 100mg / L, 150mg / L, and 200mg / L standard hexamethylenediamine in the SOB medium that has been added with 5g / L yeast powder, and simultaneously add 0.1g / L of 1 , 7-Heptanediamine standard was used as internal standard. Then add 1 mL of derivatization reagent dansyl chloride with a concentration of 5 g / L for derivatization reaction, extract with ether, blow dry with nitrogen blower, and then redissolve with acetonitrile solution. Filter through a 0.22 μm filter membrane, and detect the concentration of dansyl chloride derivatives of hexamethylenediamine and 1,7-heptanediamine by...
Embodiment 2
[0058] Example 2: Establishment of a calibration curve detection method for hexamethylenediamine fermentation broth with 1,4-butanediamine dihydrochloride as an internal standard
[0059] 1. Simulated construction of hexamethylenediamine fermentation broth system
[0060] Yeast powder with a final concentration of 5g / L, 10g / L, 15g / L, 20g / L, 25g / L, 30g / L, 35g / L and 40g / L was added to the SOB medium. Add 25mg / L, 50mg / L, 100mg / L, 150mg / L, 200mg / L hexamethylenediamine standard substance respectively in the SOB culture medium that has added 5g / L yeast powder, simultaneously all add 100mg / L 1, 4-Butanediamine dihydrochloride standard was used as internal standard. Then add 0.5 mL of derivatization reagent dansyl chloride with a concentration of 10 mg / mL for derivatization reaction, extract with ether, blow dry with nitrogen blower, and then redissolve with acetonitrile solution. Filter through a 0.22 μm filter membrane, and use HPLC to detect the concentration of dansyl chloride d...
Embodiment 3
[0076] Embodiment 3: Double internal standard curve method detects the content of hexamethylenediamine in the fermented liquid
[0077] E.coli BL21(DE3) was used for batch fermentation in 250mL Erlenmeyer flasks. The fermentation medium was SOB medium supplemented with ampicillin (50mg / L). After 6 hours of fermentation, 0.1mM IPTG was added to induce adipodex Catalytic synthesis of amines. After 35 hours of induced expression, samples were taken to detect the content of hexamethylenediamine in the fermentation broth.
[0078] Take 500 μL hexamethylenediamine fermentation broth and 500 μL saturated NaHCO 3 Mix and adjust pH to 9.4 with NaOH. In order to improve the accuracy of calibration, two substances with a final concentration of 100mg / L 1,7-heptanediamine and 100mg / L 1,4-butanediamine dihydrochloride were added to the fermentation supernatant sample as internal standards. Calibrate the detection of hexamethylenediamine. Add 1 mL of the freshly prepared derivatization r...
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