Amide-sulfonyl composite hydrophilic medium and application thereof in separation of acidic human milk oligosaccharide and neutral human milk oligosaccharide

Abstract

The invention belongs to the field of food chemical analysis, and relates to an amide-sulfonyl composite hydrophilic medium and application thereof in separation of acidic human milk oligosaccharide and neutral human milk oligosaccharide. Compared with an existing commercial chromatographic column, the hydrophilic interaction chromatographic column of the amide-sulfonic compound type separation medium has the advantages of being low in operation pressure, excellent in separation efficiency, simple in elution condition and the like, base line separation of various acid human milk oligosaccharides and neutral human milk oligosaccharides can be achieved within short analysis time, and the method is suitable for large-scale industrial production. Wide application prospects are realized. The hydrophilic interaction chromatographic column of the amide-sulfonic compound type separation medium provided by the invention is operated at low pressure, is low in cost and has excellent separation performance.

Description

Technical Field

[0001] The invention belongs to the field of food chemical analysis and relates to an amide-sulfonic acid group composite hydrophilic medium and application thereof in separating acidic human milk oligosaccharides and neutral human milk oligosaccharides. Background Art

[0002] Human milk oligosaccharides (HMOs) are important components of breast milk and play a key role in infant growth, immune system development, and intestinal microbiota regulation. Due to their complex structure and diversity, the development of efficient, accurate, and economical separation and analysis methods has become a research hotspot. At present, liquid chromatography, especially hydrophilic interaction liquid chromatography, has become the mainstream method for separating and analyzing human milk oligosaccharides. Hydrophilic interaction chromatography columns often use amino-functionalized materials as fillers, which can bind to analytes through polar interactions to achieve separation. However, traditional hydrophilic interaction chromatography columns often have problems such as unsatisfactory separation effects and excessive operating pressure when separating acidic human milk oligosaccharides and neutral human milk oligosaccharides. For example, acidic human milk oligosaccharides such as 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) are easily electrostatically adsorbed or repelled by functional groups such as amino groups on the surface of the chromatographic column when they are negatively charged, making it difficult to separate them from neutral human milk oligosaccharides. In addition, existing hydrophilic interaction chromatography columns usually require higher operating pressures and have slow analysis speeds, limiting their application in high-throughput analysis.

[0003] In order to overcome these problems, researchers have proposed some new chromatographic column designs in recent years. However, existing technologies still face some challenges: although commercial chromatographic columns (such as AMINEX HPX-87H columns, ROA organic acid columns, and Glycan BEH amide columns) can achieve certain separation effects in some cases, they usually have high operating pressures and are expensive, and often need to be used in conjunction with ultra-high performance liquid chromatography systems, which increases experimental costs and operational complexity. Especially when dealing with complex samples containing acidic and neutral components, the separation efficiency and stability of existing technologies are still limited. Therefore, the development of a new mixed-mode chromatographic column that can effectively separate acidic human milk oligosaccharides and neutral human milk oligosaccharides and has the characteristics of low cost, low operating pressure, and high efficiency has important scientific research and application value.

[0004] The prior art (CN 116637600 A) discloses the separation of glucose, lactose, 2'-FL (2'-fucosyllactose) and LNnT (lactoyl-N-neotetraose) using an amide-sulfonic acid composite hydrophilic medium, wherein 2'-FL (2'-fucosyllactose) and LNnT (lactoyl-N-neotetraose) are both neutral human milk oligosaccharides, but acidic human milk oligosaccharides and neutral human milk oligosaccharides cannot be successfully separated. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an amide-sulfonic acid composite hydrophilic medium and its application in separating acidic human milk oligosaccharides and neutral human milk oligosaccharides in view of the deficiencies of the prior art.

[0006] Idea of ​​the invention: Generally speaking, in a HILIC column, the more surface modification groups there are, the stronger the mixed-mode hydrophilic effect is. In order to enhance the efficient separation, analysis and resolution of glucose, lactose, 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose and LNnT through a mixed-mode separation mechanism, the present invention introduces a large number of polar molecules (AMPS) to improve the hydrophilicity and electrostatic interaction of the stationary phase. Therefore, the present invention selects the Merrifield resin surface initiated atom transfer radical polymerization (SI-ATPR) method to modify more AMPS monomers. This method has the advantages of good controllability of the polymerization process and precise molecular structure design.

[0007] Among them, the mixed mode separation mechanism: the separation mechanism of the chromatographic column is mainly based on two interactions: electrostatic repulsion and hydrophilic interaction. Specifically, acidic human milk oligosaccharides such as 3'-sialyllactose and 6'-sialyllactose contain negative charges, and thus electrostatically repel the negatively charged groups on the surface of the chromatographic column filler, causing them to be eluted before neutral human milk oligosaccharides (such as glucose, lactose, 2'-fucosyllactose and LNnT). The neutral human milk oligosaccharides form a stable hydration layer with the surface of the chromatographic column filler through hydrophilic interaction, thereby achieving separation. The hydrophilic interaction chromatographic column of the amide-sulfonic acid composite separation medium provided by the present invention has high stability and durability, is suitable for long-term continuous analysis work, and the separation and analysis method has good repeatability and stability.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] The present invention discloses an amide-sulfonic acid composite hydrophilic medium, which is obtained by introducing amide-sulfonic acid groups into chloromethylated polystyrene microspheres by atom transfer radical polymerization reaction, and has the following structural formula:

[0010]

[0011] Wherein, PS represents polystyrene microspheres, and n is 1-8.

[0012] In some embodiments, the diameter of the chloromethylated polystyrene microspheres is 3 to 20 μm.

[0013] In some embodiments, the diameter of the amide-sulfonic acid composite hydrophilic medium is 3-20 μm.

[0014] In some embodiments, the loading amount of sulfonic acid groups in the amide-sulfonic acid composite hydrophilic medium is 1.6 mmol to 2.5 mmol sulfonic acid groups / g amide-sulfonic acid composite hydrophilic medium.

[0015] In some embodiments, preferably, the loading amount of sulfonic acid groups in the amide-sulfonic acid composite hydrophilic medium is 1.8 mmol to 2.2 mmol sulfonic acid groups / g amide-sulfonic acid composite hydrophilic medium.

[0016] In some embodiments, further preferably, the loading amount of sulfonic acid groups in the amide-sulfonic acid composite hydrophilic medium is 2.0 mmol to 2.1 mmol sulfonic acid groups / g amide-sulfonic acid composite hydrophilic medium.

[0017] Furthermore, the present invention discloses a method for preparing the above-mentioned amide-sulfonic acid composite hydrophilic medium, wherein chloromethylated polystyrene is dissolved in a first solvent for soaking and swelling, and then 2-acrylamide-2-methylpropanesulfonic acid, a catalyst, and a base are added to perform an atom transfer radical polymerization reaction to obtain the amide-sulfonic acid composite hydrophilic medium;

[0018] The structural formula of the amide-sulfonic acid composite hydrophilic medium is as follows:

[0019]

[0020] Wherein, PS represents polystyrene microspheres, and n is 1-8.

[0021] In some embodiments, the first solvent is any mixture of water and N,N-dimethylformamide in any proportion, a mixture of N,N-dimethylformamide and toluene in any proportion, a mixture of water and dimethyl sulfoxide in any proportion, a mixture of ethylene glycol and N,N-dimethylformamide in any proportion, and a mixture of water and methanol in any proportion, or a combination of several mixtures; and / or, the catalyst is any one of cuprous chloride, cuprous bromide, ferrous chloride and ferrous bromide, or a combination of several; and / or, the base is any one of 2,2'-bipyridine, tetramethylethylenediamine, pentamethyldiethylenetriamine and 1,1,4,7,10,10-hexamethyltriethylenetetramine, or a combination of several.

[0022] In some embodiments, preferably, the first solvent is a mixture of water and N,N-dimethylformamide in any proportion; and / or, the catalyst is cuprous chloride; and / or, the base is 2,2'-bipyridine.

[0023] There is no special requirement for the amount of the first solvent, as long as it can fully swell the chloromethylated polystyrene.

[0024] In some embodiments, the molar ratio of chlorine in the chloromethylated polystyrene to the 2-acrylamido-2-methylpropanesulfonic acid is 1:(30-110); and / or, the molar ratio of chlorine in the chloromethylated polystyrene to the catalyst and the base is 1:(1-2):(2-4).

[0025] In some embodiments, preferably, the molar ratio of chlorine in the chloromethylated polystyrene to the 2-acrylamido-2-methylpropanesulfonic acid is 1:110; and / or, the molar ratio of chlorine in the chloromethylated polystyrene to the catalyst and the base is 1:1:2.

[0026] In some embodiments, the atom transfer radical polymerization reaction is carried out under an inert gas; and / or the reaction temperature of the atom transfer radical polymerization reaction is 60° C. to 130° C.; and / or the reaction time of the atom transfer radical polymerization reaction is 5 to 15 hours.

[0027] In some embodiments, preferably, the atom transfer radical polymerization reaction is carried out under an inert gas; and / or the reaction temperature of the atom transfer radical polymerization reaction is 60° C. to 100° C.; and / or the reaction time of the atom transfer radical polymerization reaction is 8 to 12 hours.

[0028] In some embodiments, further preferably, the atom transfer radical polymerization reaction is carried out under an inert gas; and / or the reaction temperature of the atom transfer radical polymerization reaction is 80° C.; and / or the reaction time of the atom transfer radical polymerization reaction is 10 hours.

[0029] Wherein, the inert gas is preferably nitrogen.

[0030] During the soaking and swelling process, the time required is sufficient to allow the chloromethylated polystyrene microspheres to swell fully.

[0031] Furthermore, the present invention discloses a hydrophilic interaction chromatography column, comprising the above-mentioned amide-sulfonic acid composite hydrophilic medium.

[0032] Furthermore, the present invention discloses a method for preparing the above-mentioned hydrophilic interaction chromatography column, wherein the above-mentioned amide-sulfonic acid composite hydrophilic medium is dispersed in a second solvent to prepare a homogenate, and then acetonitrile is used as a displacement liquid, and filled into a stainless steel column under a pressure of 5MPa to 11MPa to obtain a hydrophilic interaction chromatography column of an amide-sulfonic acid composite separation medium.

[0033] In some embodiments, preferably, the above-mentioned amide-sulfonic acid composite hydrophilic medium is dispersed in a second solvent to prepare a homogenate, and then acetonitrile is used as a displacement fluid and filled into a stainless steel column at a pressure of 6 MPa to obtain a hydrophilic interaction chromatography column of an amide-sulfonic acid composite separation medium.

[0034] In some embodiments, the second solvent is any one of 10% v / v to 50% v / v ethylene glycol aqueous solution, 10% v / v to 50% v / v methanol aqueous solution, 10% v / v to 50% v / v carbon tetrachloride aqueous solution, 10% v / v to 50% v / v acetonitrile aqueous solution and 10% v / v to 50% v / v acetic acid aqueous solution, or a combination of several of them.

[0035] In some embodiments, preferably, the second solvent is a 10% v / v to 50% v / v ethylene glycol aqueous solution.

[0036] In some embodiments, further preferably, the second solvent is a 50% v / v ethylene glycol aqueous solution.

[0037] Wherein, after the second solvent is replaced by acetonitrile, the second solvent is discharged.

[0038] The use of the above-mentioned amide-sulfonic acid composite hydrophilic medium in the separation of acidic human milk oligosaccharides and neutral human milk oligosaccharides is also within the protection scope of the present invention.

[0039] The use of the above-mentioned hydrophilic interaction chromatography column or the hydrophilic interaction chromatography column prepared by the above-mentioned preparation method in the separation of acidic human milk oligosaccharides and neutral human milk oligosaccharides is also within the protection scope of the present invention.

[0040] Specifically, the test solution containing acidic human milk oligosaccharides and neutral human milk oligosaccharides is directly injected into a high performance liquid chromatograph for separation and purification. The specific chromatographic conditions are as follows:

[0041] Chromatographic column: the hydrophilic interaction chromatographic column mentioned above or the hydrophilic interaction chromatographic column prepared by the above preparation method, 4.6mm*250mm;

[0042] Mobile phase: Mobile phase A is 0.005mol / L-0.06mol / L ammonium acetate aqueous solution, mobile phase B is CH 3 CN, isocratic elution, the volume ratio of mobile phase A to mobile phase B was 20-30:70-80;

[0043] Flow rate: 0.5mL / min~1.0mL / min;

[0044] Column temperature: 30℃~55℃;

[0045] Injection volume: 5μL~10μL;

[0046] System operating pressure: 50bar~70bar;

[0047] Retention time: 25min~30min.

[0048] Specifically, preferably, the test solution containing acidic human milk oligosaccharides and neutral human milk oligosaccharides is directly injected into a high performance liquid chromatograph for separation and purification, and the specific chromatographic conditions are as follows:

[0049] Chromatographic column: the hydrophilic interaction chromatographic column mentioned above or the hydrophilic interaction chromatographic column prepared by the above preparation method, 4.6mm*250mm;

[0050] Mobile phase: Mobile phase A is 0.05 mol / L ammonium acetate aqueous solution, mobile phase B is CH 3 CN, isocratic elution, the volume ratio of mobile phase A to mobile phase B was 30:70;

[0051] Flow rate: 0.6 mL / min;

[0052] Column temperature: 30°C;

[0053] Injection volume: 5μL~10μL;

[0054] System operating pressure: 60bar;

[0055] Retention time: 25min~30min.

[0056] Specifically, the acidic human milk oligosaccharides are 3'-sialyllactose and 6'-sialyllactose; and the neutral human milk oligosaccharides are glucose, lactose, 2'-fucosyllactose and LNnT.

[0057] Specifically, the specific preparation method of the above-mentioned test solution containing acidic human milk oligosaccharides and neutral human milk oligosaccharides is as follows:

[0058] The strain DH1W6G6 (the strain DH1W6G6 has been disclosed in Chinese patent CN 119372233 A) was pre-cultured in LB medium at 25°C to 37°C overnight to prepare a seed solution. Subsequently, the seed solution was inoculated into the fermentation medium at an inoculum amount of 2.0% v / v-2.5% v / v, and cultured at 25°C to 37°C and 220 to 300 rpm. When the cell density (OD600) reached 0.6 to 0.8, isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.05 mmol / L to 0.15 mmol / L and lactose with a final concentration of 1 to 10 g / L were added respectively, and the fermentation temperature was reduced to 25°C to 28°C, and the culture was continued for 48 to 72 hours. After the culture is completed, the culture solution is centrifuged at 10,000 to 12,000 rpm for 10 to 15 minutes, and the supernatant is collected to obtain the 2'-fucosyllactose fermentation solution supernatant. The 2'-fucosyllactose fermentation solution supernatant is diluted with water, and then 3'-sialyllactose, 6'-sialyllactose, and LNnT are added to obtain a test solution containing acidic human milk oligosaccharides and neutral human milk oligosaccharides.

[0059] Specifically, preferably, the specific preparation method of the above-mentioned test solution containing acidic human milk oligosaccharides and neutral human milk oligosaccharides is as follows:

[0060] The strain DH1W6G6 (the strain DH1W6G6 has been disclosed in Chinese patent CN 119372233 A) was pre-cultured in LB medium at 37°C overnight to prepare seed liquid. Subsequently, the seed liquid was inoculated into the fermentation medium at an inoculum amount of 2.5% v / v and cultured at 37°C and 220rpm. When the cell density (OD600) reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1mmol / L and lactose with a final concentration of 5g / L were added respectively, and the fermentation temperature was reduced to 28°C, and the culture was continued for 72 hours. After the culture was completed, the culture liquid was centrifuged at 12,000rpm for 10 minutes, and the supernatant was collected to obtain the 2'-fucosyllactose fermentation liquid supernatant. The supernatant of the 2'-fucosyllactose fermentation broth was diluted with water, and then 3'-sialyllactose, 6'-sialyllactose and LNnT were added to obtain a test solution containing acidic human milk oligosaccharides and neutral human milk oligosaccharides.

[0061] Specifically, further preferably, in the test solution containing acidic human milk oligosaccharides and neutral human milk oligosaccharides, the concentration of 3'-sialyllactose is 0.2-10000 mg / L, the concentration of 6'-sialyllactose is 0.2-10000 mg / L, the concentration of glucose is 0.2-10000 mg / L, the concentration of lactose is 0.2-10000 mg / L, the concentration of 2'-fucosyllactose is 0.2-10000 mg / L, and the concentration of LNnT is 0.2-10000 mg / L.

[0062] Wherein, during the preparation process of the test solution containing acidic human milk oligosaccharides and neutral human milk oligosaccharides, deionized water can be appropriately used for dilution, or a centrifuge can be used for appropriate concentration according to the situation, so that the acidic human milk oligosaccharides and neutral human milk oligosaccharides reach the required concentration.

[0063] Beneficial effects:

[0064] (1) The present invention selects the Merrifield resin surface initiated atom transfer radical polymerization (SI-ATPR) method to modify more AMPS monomers (2-acrylamide-2-methylpropane sulfonic acid, having an amino group and a sulfonic acid group) to obtain an amide-sulfonic acid composite separation medium. This method has the advantages of good controllability of the polymerization process and precise molecular structure design.

[0065] (2) The amide-sulfonic acid composite separation medium provided by the present invention can be used to prepare a hydrophilic interaction chromatography column, which uses a mixed mode separation mechanism to separate 3'-sialyllactose, 6'-sialyllactose, glucose, lactose, 2'-fucosyllactose and LNnT. Among them, the mixed mode separation mechanism: the separation mechanism of the chromatographic column is mainly based on two interactions: electrostatic repulsion and hydrophilic interaction. Specifically, acidic human milk oligosaccharides such as 3'-sialyllactose and 6'-sialyllactose contain negative charges, and thus electrostatically repel the negatively charged groups on the surface of the chromatographic column filler, causing them to be eluted before neutral human milk oligosaccharides (such as glucose, lactose, 2'-fucosyllactose and LNnT). Neutral human milk oligosaccharides form a stable hydration layer with the surface of the chromatographic column filler through hydrophilic interaction, thereby achieving separation. The hydrophilic interaction chromatographic column of the amide-sulfonic acid composite separation medium provided by the invention has high stability and durability, is suitable for long-term continuous analysis work, and the separation and analysis method has good repeatability and stability.

[0066] (3) Compared with existing commercial chromatographic columns (such as AMINEX HPX-87H column, ROA organic acid column, Glycan BEH amide column), the hydrophilic interaction chromatographic column of amide-sulfonic acid composite separation medium provided by the present invention has the characteristics of low operating pressure, excellent separation efficiency, simple elution conditions, etc., and can achieve baseline separation of various acidic human milk oligosaccharides and neutral human milk oligosaccharides in a shorter analysis time, and has broad application prospects.

[0067] (4) Low-pressure operation of the hydrophilic interaction chromatography column of the amide-sulfonic acid composite separation medium provided by the present invention: the chromatography column can operate stably at an operating pressure of 60 bar, avoiding the equipment burden caused by high-pressure operation.

[0068] (5) The hydrophilic interaction chromatography column of the amide-sulfonic acid composite separation medium provided by the present invention is low-cost: the use of low-cost hydrophilic adsorbent significantly reduces the cost of chromatographic analysis.

[0069] (6) The hydrophilic interaction chromatography column of the amide-sulfonic acid composite separation medium provided by the present invention has excellent separation performance: the chromatographic column can efficiently separate common acidic human milk oligosaccharides and neutral human milk oligosaccharides, including 3'-sialyllactose, 6'-sialyllactose, glucose, lactose, 2'-fucosyllactose and LNnT, and can achieve baseline separation in a relatively short analysis time, and the purity of each separated substance is as high as 100%.

[0070] (7) The hydrophilic interaction chromatography column of the amide-sulfonic acid composite separation medium provided by the present invention is suitable for HPLC system, the analysis time is 25min to 30min, and separation is achieved by isocratic elution, avoiding the complicated operation required for gradient elution. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0072] Figure 1 The infrared spectra of chloromethylated polystyrene, 2-acrylamido-2-methylpropanesulfonic acid and PS-g-PAMPS resin.

[0073] Figure 2 These are SEM images of chloromethylated polystyrene and PS-g-PAMPS resin.

[0074] Figure 3 TEM images of chloromethylated polystyrene and PS-g-PAMPS resin.

[0075] Figure 4The chromatographic data diagrams are of 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), glucose, lactose, 2'-fucosyllactose (2'-FL) and LNnT separated by different chromatographic columns; wherein, 1 is 3'-SL, 2 is 6'-SL, 3 is glucose, 4 is lactose, 5 is 2'-FL, and 6 is LNnT.

[0076] Figure 5 Schematic diagram of the separation effect of 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), glucose, lactose, 2'-fucosyllactose (2'-FL) and LNnT under different mobile phase ratios; wherein, 1 is 3'-SL, 2 is 6'-SL, 3 is glucose, 4 is lactose, 5 is 2'-FL, and 6 is LNnT.

[0077] Figure 6 is the liquid phase peak time of each standard under different chromatographic columns and chromatographic conditions. DETAILED DESCRIPTION

[0078] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0079] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0080] Example 1: Preparation of amide-sulfonic acid composite hydrophilic medium

[0081] The preparation of the amide-sulfonic acid composite hydrophilic medium can be prepared by referring to the prior art (CN 116637600 A), or by the following method:

[0082] Weigh 1.0 g of chloromethylated polystyrene microspheres (PS-g-Cl, diameter about 5 μm, 1.0 g of chloromethylated polystyrene microspheres contains 2.0 mmol of chlorine) resin in a round-bottom flask, add 32 mL of a mixed solvent of water and N,N-dimethylformamide (the volume ratio of water to N,N-dimethylformamide is 1:3), and soak for 3 hours to ensure swelling; then, continue to add 2-acrylamido-2-methylpropanesulfonic acid (AMPS, 45.8 g, 0.22 mol), CuCl (0.198 g, 2.0 mmol), 2,2'-bipyridine (0.624 g, 4.0 mmol), stir and dissolve evenly, and then seal. Evacuate, and alternately introduce nitrogen 5 times under nitrogen protection, and perform atom transfer radical polymerization at 80 ° C for 10 hours. After the reaction is completed, wash with DMF three times to remove AMPS self-polymers. Then, soak the adsorbent in dilute hydrochloric acid for 0.5 hours to remove Cu + / Cu 2+ Then, it was washed three times with water and methanol at 30°C, each washing lasting 8 minutes, until no hydrochloric acid remained in the adsorbent. After vacuum drying to constant weight, PS-g-PAMPS resin, i.e., amide-sulfonic acid composite hydrophilic medium, was obtained.

[0083] The loading capacity of AMPS in PS-g-PAMPS resin was determined to be 2.08 mmol / g (i.e., the loading amount of sulfonic acid groups). The calculation formula is as follows:

[0084] Among them, W 1 and W 2 represent the mass of resin before and after the reaction, respectively, and 207.25 is the molecular weight of AMPS.

[0085] The infrared spectra of the raw material chloromethylated polystyrene, the raw material 2-acrylamido-2-methylpropanesulfonic acid and the product PS-g-PAMPS resin are shown in Figure 1 As shown in the figure, AMPS is at 1243cm -1 and 1081cm -1 There is an obvious absorption peak at 1201cm, which is the characteristic peak of O=S=O bond. After the introduction of AMPS to PS-g-Cl to form PS-g-PAMPS, -1 and 1039cm -1 The peaks at 1544cm -1 The peaks at belong to the NH bending vibration and CN stretching vibration on PS-g-PAMPS. This shows that PS-g-PAMPS has the characteristic peaks of PS-g-Cl and AMPS, and the modification of PS-g-Cl resin particles with AMPS was successful.

[0086] The scanning electron microscopy images of the raw material chloromethylated polystyrene (PS-g-Cl) and the product PS-g-PAMPS resin are shown in Figure 2 As shown in the figure, among which, Figure a and Figure c are scanning electron micrographs of PS-g-Cl, and Figure b and Figure d are scanning electron micrographs of PS-g-PAMPS. It can be seen from the figure that the diameter of PS-g-Cl and PS-g-PAMPS particles is uniform and consistent, about 5μm. Ten particles are randomly selected for measurement, and the variance of the results is very small, only 0.01. As shown in Figure c and Figure d, PS-g-Cl and PS-g-PAMPS particles are close to spherical. At the same time, the PS-g-PAMPS adsorbent combines the rigidity of polystyrene in the PS-g-Cl matrix and the hydrophilic and hydrophobic properties of AMPS, so this particle is suitable for filling into the analytical column. However, as shown in Figure a and Figure b, the surface morphology of PS-g-Cl and PS-g-PAMPS is quite different. The polymer layer of PS-g-Cl is like a corolla with an uneven surface; while the coating of PS-g-PAMPS presents a regular core-shell structure with a smooth and uniform surface.

[0087] TEM images of the raw material chloromethylated polystyrene (PS-g-Cl) and the product PS-g-PAMPS resin are shown in Figure 3 As shown, Figure a is the TEM image of PS-g-Cl, and Figure b is the TEM image of PS-g-PAMPS. As can be seen from the figure, PS-g-Cl is just an ordinary spherical particle with no obvious polymer coating on its surface. However, after modification with AMPS, it can be seen that the surface of PS-g-PAMPS is covered with a thin film (the red marked part in Figure b). This indicates that a hydrophilic shell has been formed, which can provide HILIC active sites for target molecules (sugars). Combining the results of IR spectroscopy and TEM, it can be concluded that the ideal hydrophilic composite material was successfully prepared through a carefully designed platform, and the ATRP method of AMPS has good feasibility for modifying the surface of PS-g-Cl balls.

[0088] By N 2 Adsorption-desorption experiments were performed to determine PS-g-Cl and PS-g-PAMPS. The experimental results showed that both PS-g-Cl and PS-g-PAMPS particles showed mesoporous structures. The specific surface area of ​​PS-g-Cl was 245.63 m 2 / g, the total pore volume is 0.5242cm 3 / g, and the average pore diameter is 6.0151nm. Correspondingly, the specific surface area of ​​PS-g-PAMPS is 226.55m 2 / g, the total pore volume is 0.2812cm 3 / g, and the average pore diameter is 3.1781nm. The results show that PS-g-PAMPS has a smaller specific surface area and total pore volume than PS-g-Cl, which may be because some of the modifier AMPS occupies the pores of PS-g-Cl to form a hydrophilic shell, which is consistent with the TEM results.

[0089] Example 2: Preparation of 2'-fucosyllactose fermentation broth supernatant

[0090] The strain DH1W6G6 used in this example has been disclosed in Chinese patent CN 119372233 A.

[0091] During conventional culture, strain DH1W6G6 was pre-cultured in 5 mL LB medium at 37°C overnight to prepare seed liquid. Subsequently, the seed liquid was inoculated into 25 mL fermentation medium at an inoculum size of 2.5% v / v and cultured at 37°C and 220 rpm. When the cell density (OD600) reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1 mmol / L and lactose with a final concentration of 5 g / L were added respectively, and the fermentation temperature was reduced to 28°C and cultured for 72 hours. After the culture was completed, 1 mL of the culture solution was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected to obtain the 2'-fucosyllactose fermentation liquid supernatant for standby use. The supernatant of the 2'-fucosyllactose fermentation broth contains 2'-fucosyllactose (concentration of about 6 g / L), glucose (concentration of about 1 g / L), and lactose (concentration of about 1 g / L).

[0092] Embodiment 3:

[0093] This example uses the PS-g-PAMPS resin prepared in Example 1.

[0094] The chromatographic column was filled with a homogenization method, and the obtained PS-g-PAMPS resin was filled into a 4.6mm*250mm stainless steel empty liquid chromatography column: the PS-g-PAMPS resin was dispersed in a 50% v / v ethylene glycol aqueous solution to prepare a homogenate, and acetonitrile was used as a displacement liquid, and filled into a stainless steel column at a pressure of 6MPa (the chromatographic column filling equipment was provided by MPAPOWER (Shanghai, China)). The obtained column was recorded as a mixed-mode chromatographic column (Mixed-HILIC column). The mixed-mode chromatographic column was used to investigate the hydrophilic chromatographic retention behavior of the chromatographic column in the hydrophilic chromatographic mode for separating 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), glucose, lactose, 2'-fucosyllactose (2'-FL) and LNnT.

[0095] In order to analyze the Mixed-HILIC column, an Agilent Technologies 1260 series HPLC system (USA) equipped with ELSD was used, and the isocratic elution method was used for analysis. The specific chromatographic operating conditions are as follows:

[0096] Chromatographic column: the filler is PS-g-PAMPS resin (prepared in Example 1), specification 4.6mm*250mm;

[0097] Flow rate: 0.6 mL / min;

[0098] Column temperature: 30°C;

[0099] Injection volume: 5μL or 10μL;

[0100] Mobile phase: Mobile phase A is 0.05 mol / L ammonium acetate aqueous solution, mobile phase B is CH 3 CN, isocratic elution, the volume ratio of mobile phase A to mobile phase B was 30:70;

[0101] Operating pressure: 60bar.

[0102] Among them, the operating pressure is 60bar, which greatly improves the stability and durability of the mixed-mode chromatographic column.

[0103] The 2'-fucosyllactose fermentation broth supernatant (containing 2'-fucosyllactose, glucose, and lactose) prepared in Example 2 was diluted twice with water (2'-fucosyllactose concentration was about 3 g / L, glucose concentration was about 0.5 g / L, and lactose concentration was about 0.5 g / L), and then 3'-sialyllactose (final concentration was 1 g / L), 6'-sialyllactose (final concentration was 1 g / L), and LNnT (final concentration was 1 g / L) were added to obtain the sample to be tested. The sample to be tested was directly injected into the HPLC system, and the mixed mode chromatographic column of the present embodiment was used to separate 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), glucose, lactose, 2'-fucosyllactose (2'-FL) and LNnT. Each substance was collected at its peak retention time, and the purity of each substance was tested after collection, and the purity was 100%.

[0104] Comparative Example 1:

[0105] Commercial chromatographic columns were used to separate and detect the samples to be tested in Example 3. The commercial chromatographic columns were AMINEX HPX-87H column, ROA-Organic Acid column, and Glycan BEH amide column.

[0106] AMINEX HPX-87H column: Bio-Rad brand, column specifications 300mm×7.8mm, hydrogen type, particle diameter 9μm, cross-linking degree 8%, pH applicable range 1~3.

[0107] ROA-Organic Acid column: phenomenex brand, cross-linking degree 8%, column size 100mm×4.6mm, particle diameter 8μm.

[0108] Glycan BEH amide column: Waters brand, the pore size of the filler is The particle diameter is 1.7 μm and the column size is 2.1 mm × 150 mm.

[0109] (1) The analytical equipment and parameters for the commercial AMINEX HPX-87H column and the commercial ROA-Organic Acid column were as follows: Agilent Technologies (CA, USA) 1260 series HPLC equipped with a differential refractive index detector (RID);

[0110] Chromatographic column: commercial AMINEX HPX-87H column or commercial ROA-Organic Acid column;

[0111] Mobile phase: Mobile phase A and mobile phase B are 0.005 mol / L H 2 SO 4 Aqueous solution;

[0112] Flow rate: 0.6 mL / min;

[0113] Column temperature: 55°C;

[0114] Injection volume: 10 μL;

[0115] Operating pressure: 80bar.

[0116] The sample obtained in Example 3 was injected into the HPLC system, and 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), glucose, lactose, 2'-fucosyllactose (2'-FL) and LNnT were separated using a commercial AMINEX HPX-87H column or a commercial ROA-Organic Acid column.

[0117] (2) The analytical equipment and parameters of the commercial Glycan BEH amide column were as follows: a ThermoFisher Scientific ultra-high performance liquid chromatography system equipped with an evaporative light scattering detector (ELSD);

[0118] Chromatographic column: commercial Glycan BEH amide column;

[0119] Mobile phase: Mobile phase A is 0.1 mol / L ammonium formate aqueous solution, and mobile phase B is 100% acetonitrile;

[0120] Gradient elution;

[0121] The Glycan BEH amide column was pre-equilibrated with 86.5% B. After injection, mobile phase B was maintained at 86.5% for 2 minutes, then gradually decreased to 78% in 34 minutes; mobile phase B was further decreased from 78.0% to 75.6% in 9 minutes, and then decreased from 75.6% to 50% in 2 minutes; finally, the mobile phase B concentration was increased to 86% in 0.5 minutes. The column was then re-equilibrated at 86.5% mobile phase B for 2.5 minutes before the next injection;

[0122] Flow rate: 0.6 mL / min;

[0123] Column temperature: 50°C;

[0124] Injection volume: 1 μL;

[0125] Collection time: 40min;

[0126] Operating pressure: 400bar.

[0127] The sample obtained in Example 3 was injected into the HPLC system, and a commercial Glycan BEH amide column was used to separate 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), glucose, lactose, 2'-fucosyllactose (2'-FL) and LNnT.

[0128] Embodiment 4:

[0129] 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), glucose, lactose, 2'-fucosyllactose (2'-FL) and LNnT were prepared into reference solution with concentration of 0.5g / L using deionized water. Each reference substance was injected using Mixed-HILIC column and corresponding liquid chromatography method of Example 3, AMINEX HPX-87H column, ROA-Organic Acid column, Glycan BEH amide column and corresponding liquid chromatography method in Comparative Example 1, and the liquid phase elution time of each sample under different chromatographic columns and chromatographic conditions was analyzed for comparison. The data graph of the specific reference substance liquid phase elution time is shown in Figure 6 Among them, the chromatographic column used in Figure a is a ROA-Organic Acid column; the chromatographic column used in Figure b is an AMINEX HPX-87H column, the chromatographic column used in Figure c is a Mixed-HILIC column, and the chromatographic column used in Figure d is a Glycan BEH amide column.

[0130] The experimental results of Example 3 and Comparative Example 1 are as follows Figure 4 As shown, Figure a is a liquid chromatogram separated by a commercial AMINEX HPX-87H column; Figure b is a liquid chromatogram separated by a commercial ROA-Organic Acid column; Figure c is a liquid chromatogram separated by a mixed mode chromatographic column prepared in Example 3; and Figure d is a liquid chromatogram separated by a commercial Glycan BEH amide column.

[0131] from Figure 4 As shown in Figure a, the commercial AMINEX HPX-87H column can separate some sugars under the pressure of 80 bar in the HPLC system, but its effect in separating 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), glucose, lactose, 2'-fucosyllactose (2'-FL) and LNnT is not as good as the mixed-mode chromatographic column prepared by the present invention, and the peaks of various substances in Figure a are seriously overlapped.

[0132] from Figure 4 As shown in Figure b, similar to the separation effect of the commercial AMINEX HPX-87H column, the separation effect of the commercial ROA-Organic Acid column mainly relies on size exclusion, the peaks between the analytes overlap severely, and the separation performance is limited.

[0133] from Figure 4 As shown in Figure d, although the Glycan BEH amide column can provide efficient separation effects and achieve baseline separation of six substances (3'-sialyllactose, 6'-sialyllactose, glucose, lactose, 2'-fucosyllactose and LNnT), it needs to be operated at an operating pressure of more than 400 bar in the ultra-high performance liquid chromatography system and requires gradient elution, which is relatively cumbersome and has high requirements on the equipment.

[0134] from Figure 4 As shown in Figure c, by using the mixed mode chromatographic column provided by the present invention for separation, 3'-sialyllactose, 6'-sialyllactose, glucose and lactose can be baseline separated. At the same time, a good separation between 2'-fucosyllactose and LNnT can also be achieved. As expected, due to the presence of sulfonic acid groups on the designed PS-g-PAMPS resin particles, the silicic acid residues of 3'-sialyllactose and 6'-sialyllactose are eluted first (electrostatic repulsion) and separated from the other four neutral sugars (i.e., glucose, lactose, 2'-fucosyllactose and LNnT). This result is consistent with the commercial AMINEX HPX-87H column ( Figure 4 Figure a) and commercial ROA-Organic Acid column ( Figure 4 Figure b) These two commercial H + The results of the chromatographic columns were consistent.

[0135] Example 5: Investigation of isocratic elution ratio

[0136] The experimental method is the same as that of Example 3, except that during isocratic elution, mobile phase A (0.05 mol / L ammonium acetate aqueous solution) and mobile phase B (CH 3 CN) were 20 / 80, 25 / 75, 30 / 70, 35 / 65, and 40 / 60, respectively. Figure 5 , the chromatographic separation is calculated by the experimental results, and the chromatographic separation R i1 / i2 Calculation disclosure:

[0137]

[0138] t Ri2 is the retention time of the latter of two adjacent peaks; t Ri1 W is the retention time of the previous peak between two adjacent peaks; i1 and W i2 Therefore, the base width of two adjacent peaks.

[0139] (1) When mobile phase A (0.05 mol / L ammonium acetate aqueous solution) and mobile phase B (CH 3 CN) is 20 / 80, the results in the figure show that the separation degree of all target substances is: R 3′-SL / 6′-SL =2.19, R 2′-FL / LNnT =2.78, R 葡萄糖 / 乳糖 =5.66, the separation degree of all substances is greater than 1.5, and baseline separation is achieved. The HPLC system pressure is 50 bar, but the analysis time is nearly 40 minutes, which affects the overall separation efficiency. Therefore, although this ratio can achieve separation, there may be some time efficiency issues in actual applications.

[0140] (2) When mobile phase A (0.05 mol / L ammonium acetate aqueous solution) and mobile phase B (CH 3 CN) is 25 / 75, the results in the figure show that the separation degree of all target substances is: R 3′-SL / 6′-SL =2.36, R 2′-FL / LNnT =2.78, R 葡萄糖 / 乳糖 =3.69, the separation degree of all substances is greater than 1.5, and baseline separation is achieved. The HPLC system pressure is 55 bar, but the analysis time is nearly 35 minutes, which affects the overall separation efficiency. Therefore, although this ratio can achieve separation, there may be some time efficiency issues in actual applications.

[0141] (3) When mobile phase A (0.05 mol / L ammonium acetate aqueous solution) and mobile phase B (CH3 CN) is 30 / 70, the results in the figure show that the separation degree of all target substances is: R 3′-SL / 6′-SL =1.61, R 2′-FL / LNnT =10.93, R 葡萄糖 / 乳糖 =1.55, the separation degree of all substances is greater than 1.5, and baseline separation is achieved. The HPLC system pressure is 60 bar, the analysis time is shortened to 27.5 min, and the separation efficiency is significantly improved. This mobile phase ratio is the best choice for achieving efficient separation.

[0142] (4) When mobile phase A (0.05 mol / L ammonium acetate aqueous solution) and mobile phase B (CH 3 CN) is 35 / 65, the results in the figure show that the separation degree of all target substances is: R 3′-SL / 6′-SL =0.81, R 2′-FL / LNnT =4.54, R 葡萄糖 / 乳糖 =0.82, HPLC system pressure is 63 bar, although the analysis time is shortened to 15 min, 2'-FL and LNnT can achieve baseline separation, but 3'-SL and 6'-SL cannot achieve baseline separation, and glucose and lactose cannot achieve baseline separation. Therefore, this ratio cannot be used in practice.

[0143] (5) When mobile phase A (0.05 mol / L ammonium acetate aqueous solution) and mobile phase B (CH 3 CN) is 40 / 60, the results in the figure show that the separation degree of all target substances is: R 3′-SL / 6′-SL =0.78, R 2′-FL / LNnT =4.51, R 葡萄糖 / 乳糖 =0.81, HPLC system pressure at 65 bar, although the analysis time is 15 min, 2'-FL and LNnT can achieve baseline separation, but due to the high proportion of ammonium acetate, 3'-SL and 6'-SL cannot be effectively separated, and glucose and lactose cannot be effectively separated, resulting in overlap between them and poor separation effect. Therefore, the mobile phase ratio of 40 / 60 is not suitable for the separation of acidic and neutral human milk oligosaccharides.

[0144] in conclusion:

[0145] Comparative experiments and examples have shown that the novel mixed-mode chromatographic column provided by the present invention exhibits excellent performance in separating acidic and neutral human milk oligosaccharides, has the advantages of low operating pressure, good separation effect, simple elution conditions, etc., and has broad application potential.

[0146] Example 6: Methodological Validation

[0147] 1. Main instruments

[0148] Agilent Technologies 1260 series HPLC system (USA) equipped with ELSD.

[0149] 2. The operating parameters of HPLC are as follows:

[0150] Chromatographic column: the filler is PS-g-PAMPS resin (prepared in Example 1), specification 4.6mm*250mm;

[0151] Flow rate: 0.6 mL / min;

[0152] Column temperature: 30°C;

[0153] Injection volume: 5μL or 10μL;

[0154] Mobile phase: Mobile phase A is 0.05 mol / L ammonium acetate aqueous solution, mobile phase B is CH 3 CN, isocratic elution, the volume ratio of mobile phase A to mobile phase B was 30:70;

[0155] Operating pressure: 60bar.

[0156] 3. Standard curve drawing

[0157] 3'-Sialyl lactose series standard solution: Mix 3'-Sialyl lactose with deionized water to prepare 0.2 mg / L to 3000 mg / L 3'-Sialyl lactose series standard solution.

[0158] 6'-Sialyl lactose series standard solution: Mix 6'-Sialyl lactose with deionized water to prepare 0.2 mg / L to 3000 mg / L 6'-Sialyl lactose series standard solution.

[0159] Glucose series standard solution: Mix glucose with deionized water to prepare 0.2 mg / L to 3000 mg / L glucose series standard solution.

[0160] Lactose series standard solution: Mix lactose with deionized water to prepare 0.2 mg / L to 3000 mg / L lactose series standard solution.

[0161] 2'-fucosyllactose series standard solution: Mix 2'-fucosyllactose with deionized water to prepare 0.2 mg / L to 3000 mg / L 2'-fucosyllactose series standard solution.

[0162] LNnT series standard solution: Mix LNnT with deionized water to prepare 0.2 mg / L to 3000 mg / L LNnT series standard solution.

[0163] 4. Methodological Validation

[0164] (1) Linear verification

[0165] The area integral value of the peak of the measured substance using the ELSD detector was used as a linear verification sample. The correlation coefficient and slope of the linear equation were recorded using 1 / X fitting. The results are shown in Table 1.

[0166] Table 1 Linearity results

[0167]

[0168]

[0169] The experimental results showed that the linear equation correlation coefficients of the six analytes were all greater than 0.990, indicating that the detection method had a good linear relationship in the concentration range of 0.2 mg / L to 3000 mg / L.

[0170] (2) Verification of quantification limit

[0171] To determine the limit of detection (LOD) and limit of quantification (LOQ), a signal-to-noise ratio (S / N) of 3 and 10 was selected, respectively, and three measurements were performed to ensure accuracy. The results are shown in Table 2.

[0172] Table 2 Results of LOD and LOQ

[0173]

[0174] The test results show that the detection limit and quantification limit on the Mixed-HILIC column provided by the present invention are in the range of 0.206 mg / L to 0.945 mg / L and 0.687 mg / L to 3.150 mg / L, which indicates that the accuracy of the method proposed by the present invention is acceptable.

[0175] (3) Accuracy verification

[0176] In order to study the accuracy, the recovery test was considered in the experiment. Six analytes (glucose, lactose, 2'-FL, 3'-SL, 6'-SL, LNnT) were added to the standard test solution of the corresponding components and analyzed 3 times. The spike concentration of each analyte standard was 0.2g / L. The related equation for the spike recovery is as follows: Recovery % = 100% × (spike sample measurement amount - sample measurement amount) / spike amount. The results are shown in Table 3.

[0177] Table 3 Results of spike recovery

[0178]

[0179]

[0180] The test results show that for the six analytes, the Mixed-HILIC column method provided by the present invention has high accuracy, and the spike recovery rate is 93.5% to 109.16%.

[0181] The present invention provides an amide-sulfonic acid composite hydrophilic medium and its application in separating acidic human milk oligosaccharides and neutral human milk oligosaccharides. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. An amide-sulfonic acid composite hydrophilic medium, characterized in that: The amide-sulfonic acid composite hydrophilic medium is obtained by introducing amide-sulfonic acid groups into chloromethylated polystyrene microspheres by atom transfer radical polymerization, and its structural formula is as follows: Wherein, PS represents polystyrene microspheres, and n is 1-8.

2. The amide-sulfonic acid composite hydrophilic medium according to claim 1, characterized in that: The diameter of the chloromethylated polystyrene microspheres is 3 to 20 μm.

3. The method for preparing the amide-sulfonic acid composite hydrophilic medium according to claim 1, characterized in that: The chloromethylated polystyrene is dissolved in the first solvent for soaking and swelling, and then 2-acrylamide-2-methylpropanesulfonic acid, a catalyst and a base are added to carry out an atom transfer radical polymerization reaction to obtain an amide-sulfonic acid composite hydrophilic medium; The structural formula of the amide-sulfonic acid composite hydrophilic medium is as follows: Wherein, PS represents polystyrene microspheres, and n is 1-8.

4. The preparation method according to claim 3, characterized in that: The first solvent is any mixture of water and N,N-dimethylformamide in any proportion, a mixture of N,N-dimethylformamide and toluene in any proportion, a mixture of water and dimethyl sulfoxide in any proportion, a mixture of ethylene glycol and N,N-dimethylformamide in any proportion, a mixture of water and methanol in any proportion, or a combination of several mixtures; and / or, the catalyst is any one of cuprous chloride, cuprous bromide, ferrous chloride and ferrous bromide, or a combination of several; and / or, the base is 2,2'-bipyridine, tetramethylethylenediamine, pentaethylenetriamine and 1,1,4,7,10,10 -hexamethyltriethylenetetramine or any combination thereof; and / or, the molar ratio of chlorine in the chloromethylated polystyrene to the 2-acrylamido-2-methylpropanesulfonic acid is 1:(30-110); and / or, the molar ratio of chlorine in the chloromethylated polystyrene to the catalyst and the base is 1:(1-2):(2-4); and / or, the atom transfer radical polymerization reaction is carried out under inert gas; and / or, the reaction temperature of the atom transfer radical polymerization reaction is 60°C-130°C; and / or, the reaction time of the atom transfer radical polymerization reaction is 5-15 hours.

5. A hydrophilic interaction chromatography column, characterized in that: The invention comprises the amide-sulfonic acid composite hydrophilic medium as claimed in claim 1.

6. The method for preparing a hydrophilic interaction chromatography column according to claim 5, characterized in that: The amide-sulfonic acid composite hydrophilic medium of claim 1 is dispersed in a second solvent to prepare a homogenate, and then acetonitrile is used as a displacement liquid, and filled into a stainless steel column at a pressure of 5MPa to 11MPa to obtain a hydrophilic interaction chromatography column of the amide-sulfonic acid composite separation medium; Wherein, the second solvent is any one or a combination of 10% v / v to 50% v / v ethylene glycol aqueous solution, 10% v / v to 50% v / v methanol aqueous solution, 10% v / v to 50% v / v carbon tetrachloride aqueous solution, 10% v / v to 50% v / v acetonitrile aqueous solution and 10% v / v to 50% v / v acetic acid aqueous solution.

7. Use of the amide-sulfonic acid composite hydrophilic medium according to claim 1 in the separation of acidic human milk oligosaccharides and neutral human milk oligosaccharides.

8. Use of the hydrophilic interaction chromatography column according to claim 5 or the hydrophilic interaction chromatography column prepared by the preparation method according to claim 6 in the separation of acidic human milk oligosaccharides and neutral human milk oligosaccharides.

9. The use according to claim 8, characterized in that: The test solution containing acidic human milk oligosaccharides and neutral human milk oligosaccharides was directly injected into the high performance liquid chromatography for separation and purification. The specific chromatographic conditions are as follows: Chromatographic column: the hydrophilic interaction chromatographic column according to claim 5 or the hydrophilic interaction chromatographic column prepared by the preparation method according to claim 6, 4.6 mm*250 mm; Mobile phase: Mobile phase A is 0.005mol / L-0.06mol / L ammonium acetate aqueous solution, mobile phase B is CH3CN, isocratic elution, the volume ratio of mobile phase A to mobile phase B is 20-30:70-80; Flow rate: 0.5mL / min~1.0mL / min; Column temperature: 30℃~55℃; Injection volume: 5μL~10μL; System operating pressure: 50bar~70bar; Retention time: 25min~30min; Preferably, The test solution containing acidic human milk oligosaccharides and neutral human milk oligosaccharides was directly injected into the high performance liquid chromatography for separation and purification. The specific chromatographic conditions are as follows: Chromatographic column: the hydrophilic interaction chromatographic column according to claim 5 or the hydrophilic interaction chromatographic column prepared by the preparation method according to claim 6, 4.6 mm*250 mm; Mobile phase: Mobile phase A is 0.05 mol / L ammonium acetate aqueous solution, mobile phase B is CH3CN, isocratic elution, the volume ratio of mobile phase A to mobile phase B is 30:70; Flow rate: 0.6 mL / min; Column temperature: 30°C; Injection volume: 5μL~10μL; System operating pressure: 60bar; Retention time: 25min~30min.

10. The use according to any one of claims 7 to 9, characterized in that: The acidic human milk oligosaccharides are 3'-sialyllactose and 6'-sialyllactose; the neutral human milk oligosaccharides are glucose, lactose, 2'-fucosyllactose and LNnT.

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