Method for detecting enterotoxin C in milk by combining multidimensional liquid chromatography with mass spectrometry
A multi-dimensional liquid chromatography, two-dimensional liquid chromatography technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of food matrix interference, false positives, false negatives, etc., to reduce test costs, accurate identification, automation high degree of effect
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Embodiment 1
[0051] This embodiment provides a two-dimensional liquid chromatography platform, and the two-dimensional liquid chromatography platform includes a first-dimensional liquid chromatography column, a second-dimensional liquid chromatography column, a trapping column and a six-way valve switching device. The first-dimensional liquid chromatography column, the trapping column and the second-dimensional liquid chromatography column are connected through a six-way valve switching device.
[0052] Two six-way valves are used to build an online two-dimensional liquid chromatography analysis platform, and the connection method is as follows: figure 1 As shown, the first six-port valve port 1 is connected to the syringe, the first six-port valve ports 2 and 5 are connected to the sample loop, the first six-port valve port 3 is connected to the first-dimensional ion-exchange column, and the first six-port The first six-way valve port 4 is connected to the first-dimensional chromatographi...
Embodiment 2
[0055] In this example, the conditions of the first-dimensional ion-exchange liquid chromatography were optimized according to the relevant properties such as the molecular weight of the enterotoxin C protein is 27.6 KDa, and the isoelectric point PI=8.6. Specifically include the following:
[0056] (1) Selection of chromatographic column
[0057] The isoelectric point of enterotoxin C is 8.6, which belongs to basic protein and is suitable for cation exchange chromatography column. Preferably, chromatographic column: strong cation exchange chromatographic column (Shimadzu Shim-pack PA-SP, 5 μm, 8 × 100mm, ), trapping column (Yuexu WelchXtimate C8, 5μm, 2.1×10mm, )
[0058] (2) Optimization of mobile phase pH
[0059] 20mM dipotassium hydrogen phosphate was selected as the buffer salt system, and two pH conditions of pH=6 and pH=5.3 were investigated. When the pH=6, enterotoxin C was not retained in the chromatographic separation process. When the pH was lowered to 5.3, e...
Embodiment 3
[0069] In this example, the conditions of the second-dimensional reversed-phase liquid chromatography were optimized. Specifically include the following:
[0070] (1) Selection of chromatographic column
[0071] The molecular weight of enterotoxin C is 27.6kDa. It is a medium polar compound. It can be separated by C8 column or C4 column. After investigation, the separation effect of C8 column is better than that of C4 column, and it has a good peak shape, such as Figure 6 shown. Preferably, chromatographic column: C8 chromatographic column (Yuexu Welch Xtimate C8, 5 μm, 2.1 × 250mm, ).
[0072] (2) Verification of elution position (combined with SDS-PAGE gel electrophoresis to verify the elution position of enterotoxin in the second dimension)
[0073] SDS-PAGE gel electrophoresis was used to verify the fractions of enterotoxin C second dimension chromatogram with different separation time, Figure 7 It is the gel image after silver staining, the band position shown on ...
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