A method for detecting sialic acid by reverse phase chromatography
A technology of reversed-phase chromatography and sialic acid, applied in the field of chromatographic analysis, can solve the problems of low accuracy and low sensitivity, and achieve the effect of prolonging service life, high sensitivity and improving detection sensitivity.
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Embodiment 1
[0039] The optimization of embodiment 1 mobile phase and mobile phase gradient
[0040] According to the commercialized LudgerTag DMB sialic acid release and labeling kit, the present invention improves its mobile phase and mobile phase gradient, as follows.
[0041] The traditional mobile phase (Ludger kit) gradient elution program is shown in Table 1:
[0042] Table 1 Traditional mobile phase gradient elution program
[0043]
[0044] Mobile phase gradient elution program of the present invention is as shown in table 2:
[0045] Table 2 Mobile phase gradient elution program of the present invention
[0046]
[0047]
[0048] Use the traditional mobile phase and the patented mobile phase to detect the same DMB-labeled blank control and sialic acid standard on the same column in the same instrument:
[0049] 1. DMB marking conditions
[0050] (1) Blank: Add 5 μL ultrapure water and 20 μL DMB labeling reagent to a 0.5 mL centrifuge tube, mix well and centrifuge, heat...
Embodiment 2
[0057] A method utilizing reverse phase chromatography to detect sialic acid, comprising the following specific steps:
[0058] (1) The protein sample to be tested is desalted and dried in a vacuum concentrator for 1-2 hours.
[0059] The specific steps of desalination treatment are as follows: take a certain amount of protein sample to be tested and add it to a 10kD ultrafiltration concentration tube, add ultrapure water to 400μL, centrifuge at 13000rpm to change the liquid, and repeat the operation twice. After centrifugation, reverse the 10kD ultrafiltration tube containing the sample into a new centrifuge tube, centrifuge at 1000rpm for 1min, and transfer the collected sample to a 1.5mL centrifuge tube.
[0060] (2) Perform acid hydrolysis on the dried sample to obtain an acid hydrolyzate.
[0061] In this comparative example, 25 μL of 2mol / L glacial acetic acid solution was used for acid hydrolysis.
[0062] (3) DMB label the acid hydrolyzate obtained in step (2). Take ...
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