A kind of high performance liquid chromatography detection method of Pseudomonas aeruginosa exotoxin A
A technology of Pseudomonas aeruginosa and exotoxin, which is applied in the field of HPLC analysis and detection of Pseudomonas aeruginosa exotoxin A, can solve the problems of low resolution, influence analysis accuracy, poor repeatability, etc., achieve good separation effect and shorten detection time. Effect
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Embodiment 1
[0041] Take 8g of Pseudomonas aeruginosa fermentation broth, transfer it to a 250mL Erlenmeyer flask, then measure 75mL of methanol with a graduated cylinder for soaking, then add 1mL of 2N NaOH solution, seal it, place the Erlenmeyer flask on a shaker, and shake it at room temperature at a speed of 250rpm After extraction for 1 hour, 40 mL of the extract was taken in a 50 mL centrifuge tube and centrifuged at 4000 rpm for 15 minutes to obtain a pretreated Pseudomonas aeruginosa exotoxin A fermentation liquid, and the supernatant was filtered to obtain a sample solution.
[0042] The Pseudomonas aeruginosa exotoxin A sample solution obtained through the above treatment was analyzed by HPLC method. The sample solution can be directly injected for analysis. The chromatographic column is a TC-C18 chromatographic column (5 μm, 250 mm×4.6 mm), an ultraviolet detector, a detection wavelength of 210 nm, a flow rate of 1.0 mL / min, and a column temperature of 35° C.
Embodiment 2
[0044] The mobile phase is methanol, acetonitrile and water; the volume ratio of methanol, acetonitrile and water in the initial mobile phase is 10:15:75, and the elution is 3 minutes; the volume ratio of methanol, acetonitrile and water is continuously adjusted to 30:35 : 35, eluted for 10 minutes; then the volume ratio of methanol, acetonitrile and water was continuously adjusted to 30:40:30, and eluted for 5 minutes; then the volume ratio of methanol, acetonitrile and water was continuously adjusted to 35:45:20 , eluted for 3 minutes; then the volume ratio of methanol, acetonitrile and water was adjusted to 10:15:75, eluted for 3 minutes, and equilibrated.
[0045] Under this method, the retention time of the reference substance is 12.15min, the resolution is 1.05, and the number of theoretical plates reaches 15800.
Embodiment 3
[0047] The mobile phase is methanol, acetonitrile and water; the volume ratio of methanol, acetonitrile and water in the initial mobile phase is 10:15:75, and the elution takes 5 minutes; the volume ratio of methanol, acetonitrile and water is continuously adjusted to 30:35 : 35, eluted for 15 minutes; then the volume ratio of methanol, acetonitrile and water was continuously adjusted to 30:40:30, and the elution was performed for 10 minutes; then the volume ratio of methanol, acetonitrile and water was continuously adjusted to 35:45:20 , eluted for 5 minutes; then the volume ratio of methanol, acetonitrile and water was adjusted to 10:15:75, eluted for 5 minutes, and equilibrated.
[0048] Under this method, the peak eluting time is about 20 minutes, the resolution is 1.09, and the number of theoretical plates is 12300.
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