Method for separating fructo-oligosaccharide isomer by liquid chromatography

Abstract

The invention relates to the technical field of separation of fructo-oligosaccharide isomers, and provides a method for separating fructo-oligosaccharide isomers by liquid chromatography, which comprises the following steps: detecting and separating fructo-oligosaccharide isomers in a sample solution by adopting an HPLC-CAD (High Performance Liquid Chromatography-Computer Aided Design) method; chromatographic conditions in the HPLC-CAD method are as follows: water is taken as a mobile phase A, pure acetonitrile is taken as a mobile phase B, an elution mode is gradient elution, and a chromatographic column is a porous graphitized carbon chromatographic column. According to the technical scheme, the problem that the fruit-type fructo-oligosaccharide in the high-purity raw material of the fruit-type fructo-oligosaccharide is difficult to effectively separate in the prior art is solved.

Description

Technical Field

[0001] This invention relates to the field of fructooligosaccharide isomer separation technology, specifically, to a method for separating fructooligosaccharide isomers by liquid chromatography. Background Technology

[0002] Fructose oligosaccharides (FOS), as an important functional oligosaccharide, are widely used in food, health products, and pharmaceuticals due to their excellent properties such as regulating intestinal flora balance, promoting mineral absorption, and having a low glycemic index. FOS raw materials typically contain structural analogs with different degrees of polymerization and glycosidic bonds. Depending on the linkage mode and degree of polymerization of the fructose units, various isomers such as sucrose-type and fructose-type are formed. Accurate separation of these FOS isomers remains a significant challenge. Currently, hydrophilic interaction chromatography (HILIC) is commonly used for the liquid chromatography separation of FOS. HILIC chromatography is a chromatographic technique used to improve the retention behavior of highly polar substances that are poorly retained in reversed-phase chromatography. It achieves this by using a highly polar stationary phase combined with a mobile phase consisting of a high proportion of organic phase and a low proportion of aqueous phase. However, HILIC chromatography can currently only separate FOS with different degrees of polymerization; it cannot effectively separate FOS isomers. Chromatographic separation of fructooligosaccharide isomers is crucial for the purification and accurate quantification of fructooligosaccharides.

[0003] In recent years, the application of porous graphitized carbon chromatography columns in the field of sugar analysis has received increasing attention. Currently, porous graphitized carbon chromatography columns have been applied to the separation and analysis of human milk oligosaccharides, but there are no reports on their application in the separation and analysis of fructooligosaccharides. Furthermore, for fructooligosaccharides with the same degree of polymerization, problems such as excessively long retention times and peak tailing easily occur under conventional chromatographic conditions, making it difficult to achieve effective separation of fructooligosaccharides from sucrose-type fructooligosaccharide monomers.

[0004] Therefore, this invention proposes a method for separating fructooligosaccharide isomers by liquid chromatography, which can effectively separate fructooligosaccharides from high-purity sucrose-type fructooligosaccharide raw materials. This is of great significance for the purification and accurate quantification of fructooligosaccharides and the efficient application of fructooligosaccharides. Summary of the Invention

[0005] This invention proposes a method for separating fructooligosaccharide isomers by liquid chromatography, which solves the problem of the difficulty in effectively separating fructooligosaccharides from high-purity sucrose-type fructooligosaccharide raw materials in related technologies.

[0006] The technical solution of the present invention is as follows: This invention proposes a method for separating fructooligosaccharide isomers by liquid chromatography, comprising the following steps: using HPLC-CAD to detect and separate fructooligosaccharide isomers in a sample solution; The chromatographic conditions in the HPLC-CAD method include: water as mobile phase A, pure acetonitrile as mobile phase B, gradient elution, and the content of mobile phase B is expressed as a volume percentage. When the sample solution is a fructooligosaccharide solution, the gradient elution procedure is as follows: 0~20.0min, 5%~15%B; 20.1~25.0min, 100%B; 25.1~30.0min, 5%B; When the sample solution is a fructotetrasaccharide solution, the gradient elution procedure is as follows: 0~15.0min, 5%~15%B; 15.1~20.0min, 100%B; 20.1~27.0min, 5%B; When the sample solution is a fructooligosaccharide solution, the gradient elution procedure is as follows: 0~15.0min, 8%~10%B; 15.0~25.0min, 10%~20%B; 25.1~35.0min, 8%B; When the sample solution is a fructooligosaccharide solution, the gradient elution procedure is as follows: 0~10.0min, 10%B; 10.0~20.0min, 10%~15%B; 20.1~30.0min, 10%B; When the sample solution is a fructooligosaccharide solution, the gradient elution procedure is as follows: 0~15.0min, 8%~9%B; 15.0~25.0min, 9%~15%B; 25.1~35.0min, 8%B; When the sample solution is a fructooligosaccharide solution, the gradient elution procedure is as follows: 0~15.0min, 7%~9%B; 15.0~25.0min, 9%~20%B; 25.1~35.0min, 7%B; When the sample solution is a fructooligosaccharide solution, the gradient elution procedure is as follows: 0~15.0min, 8%~10%B; 15.0~25.0min, 10%~15%B; 25.1~35.0min, 8%B; When the sample solution is a fructooligosaccharide solution, the gradient elution procedure is as follows: 0~20.0min, 8%~10%B; 20.0~30.0min, 10%~17%B; 30.1~35.0min, 8%B; The chromatographic conditions in the HPLC-CAD method also include: the chromatographic column is a porous graphitized carbon column.

[0007] As a further technical solution, the porous graphitized carbon chromatographic column is specifically a Hypercarb chromatographic column.

[0008] As a further technical solution, the chromatographic conditions in the HPLC-CAD method also include: column temperature of 30~35℃, for example, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, preferably 30℃, 35℃.

[0009] As a further technical solution, the chromatographic conditions in the HPLC-CAD method also include: an injection volume of 2~5μL, for example, 2μL, 3μL, 4μL, or 5μL, preferably 2μL.

[0010] As a further technical solution, the chromatographic conditions in the HPLC-CAD method also include: a flow rate of 0.8~1.0μL, for example, 0.8μL, 0.9μL, or 1μL, preferably 0.8μL.

[0011] As a further technical solution, the CAD detector parameters in the HPLC-CAD method are specifically as follows: nebulization temperature 33~35℃, for example, 33℃, 34℃, 35℃, preferably 35℃; nitrogen pressure 30.0~35.0psi, for example, 30.0psi, 31.0psi, 32.0psi, 33.0psi, 34.0psi, 35.0psi, preferably 35.0psi.

[0012] As a further technical solution, the method for preparing the sample solution includes the following steps: mixing the sample to be tested and water evenly to obtain the sample solution; The concentration of the sample solution is 8~10 mg / mL, for example, it can be 8 mg / mL, 9 mg / mL, or 10 mg / mL, preferably 10 mg / mL.

[0013] In this invention, the sample to be tested includes one of the following: sucrose trisaccharide sample, sucrose tetrasaccharide sample, sucrose pentasaccharide sample, sucrose hexasaccharide sample, sucrose heptasaccharide sample, sucrose octasaccharide sample, sucrose nonasaccharide sample, and sucrose decasaccharide sample.

[0014] As a further technical solution, the detection and separation using HPLC-CAD method also includes the detection of standard solutions.

[0015] As a further technical solution, the method for preparing the standard solution includes the following steps: mixing the standard sample and water evenly to obtain the standard solution.

[0016] As a further technical solution, the concentration of the standard solution is 1~2 mg / mL.

[0017] In this invention, the standard sample includes one of the following: sucrose trisaccharide standard sample, sucrose tetrasaccharide standard sample, sucrose pentasaccharide standard sample, sucrose hexasaccharide standard sample, sucrose heptasaccharide standard sample, sucrose octasaccharide standard sample, sucrose nonasaccharide standard sample, sucrose decasaccharide standard sample, fructose standard sample, glucose standard sample, sucrose standard sample, frucrose disaccharide standard sample, frucrose trisaccharide standard sample, frucrose tetrasaccharide standard sample, frucrose pentasaccharide standard sample, frucrose hexasaccharide standard sample, and frucrose heptasaccharide standard sample.

[0018] The working principle and beneficial effects of this invention are as follows: 1. This invention proposes a method for separating structure-related impurities in high-purity fructooligosaccharide raw materials. HPLC-CAD is used to detect and separate fructooligosaccharide isomers in the sample solution. During detection, a porous graphitized carbon column is employed, and the chromatographic conditions affecting separation by the porous graphitized carbon column are optimized. Under optimized conditions, when detecting different fructooligosaccharide monomer raw materials, the porous graphitized carbon column, combined with a specific chromatographic elution program, can effectively separate fructooligosaccharide isomers from fructooligosaccharide types, with good repeatability and high stability. Furthermore, the use of a low organic phase ratio of pure acetonitrile and water for gradient elution with the porous graphitized carbon column not only saves costs but also effectively avoids the precipitation of high-polymerization-degree fructooligosaccharides in a high-organic phase, solving the solubility problem caused by the use of a high proportion of organic phase in existing HILIC chromatographic analysis of fructooligosaccharides.

[0019] 2. Using HPLC-CAD with a specific gradient elution program, sample solutions of fructooligosaccharide (FOS), fructooligosaccharide (FOS), fructooligosaccharide (FOS), fructooligosaccharide (FOS), fructooligosaccharide (FOS), fructooligosaccharide (FOS), fructooligosaccharide (FOS), fructooligosaccharide (FOS), and fructooligosaccharide (FOS) were separated and determined. The results were compared with standard solutions. The components and impurities in the test samples were qualitatively identified based on the retention time of each compound. Specifically, the FOS trisaccharide sample contained sucrose and fructooligosaccharide impurities; the FOS tetrasaccharide sample contained FOS trisaccharide and fructooligosaccharide impurities; the FOS pentasaccharide sample contained fructose pentasaccharide impurities; and the FOS hexasaccharide sample contained fructose bisaccharide, fructose pentasaccharide, and fructose hexasaccharide impurities. The hepta-saccharide sample contained fructo-hepta-saccharide and fructo-hexa-saccharide impurities; the fructo-octa-saccharide sample contained fructo-octa-saccharide and fructo-nona-saccharide impurities; the fructo-nona-saccharide sample contained fructo-octa-saccharide, fructo-nona-saccharide, and fructo-deca-saccharide impurities; and the fructo-deca-saccharide sample contained fructo-nona-saccharide and fructo-deca-saccharide impurities. Furthermore, due to the lack of standards, some trace impurities in the raw materials could not be qualitatively confirmed. Based on the separation principle of porous graphitized carbon chromatographic columns, involving the dispersion forces between the analyte and the mobile phase and graphitized carbon, as well as the charge interactions between polar compounds and the graphitized carbon surface, it was concluded that the higher the degree of polymerization of the analyte, the stronger its retention capacity. Therefore, it was speculated that the unidentified chromatographic peaks mainly consisted of compounds with a degree of polymerization greater than 10. Attached Figure Description

[0020] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0021] Figure 1 This is a chromatogram of the fructooligosaccharide solution in Example 1 of the present invention; Figure 2 This is the chromatogram of the fructotetrasaccharide solution in Example 2 of the present invention; Figure 3 This is the chromatogram of the fructooligosaccharide solution in Example 3 of the present invention; Figure 4 This is the chromatogram of the fructooligosaccharide solution in Example 4 of the present invention; Figure 5 This is the chromatogram of the fructooligosaccharide solution in Example 5 of the present invention; Figure 6 This is the chromatogram of the sucrose octaose solution in Example 6 of the present invention; Figure 7 This is the chromatogram of the fructooligosaccharide solution in Example 7 of the present invention; Figure 8 This is the chromatogram of the sucrose decaose solution in Example 8 of the present invention; Figure 9 The chromatogram of the fructooligosaccharide solution in Comparative Example 1 of this invention is shown below. Figure 10 The chromatogram of the fructooligosaccharide solution in Comparative Example 2 of this invention is shown below. Figure 11 The chromatogram of the fructotetrasaccharide solution in Comparative Example 3 of this invention is shown below. Figure 12 This is the chromatogram of the fructooligosaccharide solution in Comparative Example 4 of the present invention; Figure 13 This is the chromatogram of the fructosaccharide solution in Comparative Example 5 of the present invention; Figure 14 The chromatogram of the fructooligosaccharide solution in Comparative Example 6 of this invention is shown below. Figure 15 The chromatogram of the fructooligosaccharide solution in Comparative Example 7 of this invention is shown below. Figure 16 The chromatogram of the fructooligosaccharide solution in Comparative Example 8 of this invention is shown below. Figure 17 The chromatogram of the sucrose decaose solution in Comparative Example 9 of this invention is shown below. Figure 18 This is a chromatogram of six sucrose pentasaccharide solutions used in the repeatability test of this invention. in, Figures 1-18 In the diagram, the horizontal axis represents time (min) and the vertical axis represents signal strength (pA). Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] In the following examples and comparative examples, the preparation method of the standard solution includes the following steps: 1 mg of each of the following standard samples (trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, octasaccharide, nonasaccharide, and decasaccharide) were placed in brown screw-top bottles. 0.5 mL of ultrapure water was added to each bottle, and the mixtures were stirred thoroughly to obtain standard solutions of 2 mg / mL for 2 mg / mL: 2 mg / mL ... 5 mg of fructose, glucose, and sucrose standards were placed in brown screw-top bottles, and 5 mL of ultrapure water was added to each bottle. The mixtures were then thoroughly mixed to obtain fructose, glucose, and sucrose standard solutions with a concentration of 1 mg / mL, respectively. 1 mg of each of the following standard samples (fructo-biose, fructo-triose, fructo-tetraose, fructo-pentose, fructo-hexaose, and fructo-heptaose) were placed in brown screw-top bottles, and 0.5 mL of ultrapure water was added to each. The mixtures were stirred thoroughly to obtain fructo-biose, fructo-triose, fructo-tetraose, fructo-pentose, fructo-hexaose, and fructo-heptaose standard solutions with a concentration of 2 mg / mL. All the above standard solutions should be sealed and refrigerated, and allowed to stand until they return to room temperature before use. Fructose, GBW 10063, purity 99.7%±0.5%, k=2; Glucose, GBW 10062, purity 99.6%±0.5%, k=2; Sucrose, GBW 10067, purity 99.7%±0.8%, k=2; The standard sample of sucrose trisaccharide, with an HPLC purity of ≥98%, was purchased from Shanghai Tixiai Chemical Trading Co., Ltd. The standard sample of fructotetrasaccharide, specifically fructotetrasaccharide trihydrate, with an HPLC purity of ≥98%, was purchased from Shanghai Tixi Chemical Trading Co., Ltd. Standard samples of sucrose penta-sugar, sucrose hexa-sugar, sucrose hepta-sugar, sucrose octa-sugar, sucrose nona-sugar, and sucrose deca-sugar, all with HPLC purity ≥98%, were purchased from Chengdu Ruifensidedan Biotechnology Co., Ltd. The standard samples of fructo-biose, fructo-triose, fructo-tetraose, fructo-pentose, fructo-hexaose, and fructo-heptaose all had an HPLC purity of ≥98% and were purchased from Xinyang Zhongjian Metrology Biotechnology Co., Ltd. Acetonitrile, chromatographic grade, purchased from Merck, Germany; Ultrapure water, chromatographic grade, purchased from Milli-Q, USA.

[0024] Example 1 A method for separating fructooligosaccharide isomers by liquid chromatography includes the following steps: detecting and separating fructooligosaccharide isomers in a sample solution using HPLC-CAD. The sample solution is a fructooligosaccharide solution, and the preparation method of the fructooligosaccharide solution includes the following steps: Weigh 10 mg of the fructosaccharide sample into a brown screw-top bottle, add 1 mL of ultrapure water, mix well, and obtain a fructosaccharide solution with a concentration of 10 mg / mL. The CAD detector parameters in the HPLC-CAD method are as follows: Atomization temperature: 35℃; PFV value: 1.00; Nitrogen pressure: 35.0 psi; The chromatographic conditions for the HPLC-CAD method are as follows: Chromatographic column: Hypercarb porous graphitized carbon column, 100 mm × 4.6 mm, 3 μm; Thermo Fisher Scientific, USA; Column temperature: 35℃; Flow rate: 0.8 mL / min; Injection volume: 2 μL; Water was used as mobile phase A, and pure acetonitrile was used as mobile phase B. The content of mobile phase B was expressed as a volume percentage. The elution method was gradient elution, and the gradient elution program was as follows: 0~20.0min, 5%~15%B; 20.1~25.0min, 100%B; 25.1~30.0min, 5%B; The chromatographic separation of the substances in the fructooligosaccharide solution was examined, and the obtained chromatogram is shown below. Figure 1 As shown; from Figure 1 As can be seen, under this gradient elution procedure, the main component and various impurity peaks in the fructotriose solution are well separated. Compared with the peak area and retention time of the standard solution, in addition to the fructotriose main component, the fructotriose solution also contains sucrose and fructotetraose impurities.

[0025] Example 2 A method for separating fructooligosaccharide isomers by liquid chromatography includes the following steps: detecting and separating fructooligosaccharide isomers in a sample solution using HPLC-CAD. The sample solution is a fructotetrasaccharide solution, and the preparation method of the fructotetrasaccharide solution includes the following steps: Weigh 10 mg of the fructotetrasaccharide sample into a brown screw-top bottle, add 1 mL of ultrapure water, mix well, and obtain a fructotetrasaccharide solution with a concentration of 10 mg / mL. The CAD detector parameters in the HPLC-CAD method are as follows: Atomization temperature: 35℃; PFV value: 1.00; Nitrogen pressure: 35.0 psi; The chromatographic conditions for the HPLC-CAD method are as follows: Chromatographic column: Hypercarb porous graphitized carbon column, 100 mm × 4.6 mm, 3 μm; Thermo Fisher Scientific, USA; Column temperature: 30℃; Flow rate: 0.8 mL / min; Injection volume: 2 μL; Water was used as mobile phase A, and pure acetonitrile was used as mobile phase B. The content of mobile phase B was expressed as a volume percentage. The elution method was gradient elution, and the gradient elution program was as follows: 0~15.0min, 5%~15%B; 15.1~20.0min, 100%B; 20.1~27.0min, 5%B; The chromatographic separation of each substance in the fructotetrasaccharide solution was examined, and the obtained chromatogram is shown below. Figure 2 As shown; from Figure 2 As can be seen, under this gradient elution procedure, the main component and various impurity peaks in the fructotetrasaccharide solution are well separated. Compared with the peak area and retention time of the standard solution, in addition to the fructotetrasaccharide main component, the fructotetrasaccharide solution also contains fructotriose and fructopentose impurities.

[0026] Example 3 The only difference between this embodiment and Embodiment 2 is that, in this embodiment, the sample solution is a fructooligosaccharide solution, and the preparation method of the fructooligosaccharide solution includes the following steps: Weigh 10 mg of the fructosine sample to be tested into a brown screw-top bottle, add 1 mL of ultrapure water, mix well, and obtain a fructosine solution with a concentration of 10 mg / mL. In the HPLC-CAD method, water is used as mobile phase A, and pure acetonitrile is used as mobile phase B. The content of mobile phase B is expressed as a volume percentage. The elution method is gradient elution, and the gradient elution program is as follows: 0~15.0min, 8%~10%B; 15.0~25.0min, 10%~20%B; 25.1~35.0min, 8%B; The chromatographic separation of each substance in the fructooligosaccharide solution was examined, and the obtained chromatogram is shown below. Figure 3 As shown; from Figure 3 As can be seen, under this gradient elution procedure, the main component and impurity peaks in the fructosine solution are well separated. Compared with the peak area and retention time of the standard solution, the fructosine solution contains fructosine impurities in addition to the fructosine main component.

[0027] Example 4 The only difference between this embodiment and Embodiment 2 is that, in this embodiment, the sample solution is a fructooligosaccharide solution, and the preparation method of the fructooligosaccharide solution includes the following steps: Weigh 10 mg of the fructosaccharide sample to be tested into a brown screw-top bottle, add 1 mL of ultrapure water, mix well, and obtain a fructosaccharide solution with a concentration of 10 mg / mL. In the HPLC-CAD method, water is used as mobile phase A, and pure acetonitrile is used as mobile phase B. The content of mobile phase B is expressed as a volume percentage. The elution method is gradient elution, and the gradient elution program is as follows: 0~10.0min, 10%B; 10.0~20.0min, 10%~15%B; 20.1~30.0min, 10%B; The chromatographic separation of the substances in the fructooligosaccharide solution was examined, and the obtained chromatogram is shown below. Figure 4 As shown; from Figure 4 As can be seen, under this gradient elution program, the main component and impurity peaks in the fructosaccharide solution are well separated, and the analysis can be completed within 30 minutes. Compared with the peak area and retention time of the standard solution, in addition to the fructosaccharide main component, the fructosaccharide solution also contains fructosaccharide, fructosaccharide, and fructosaccharide impurities.

[0028] Example 5 The only difference between this embodiment and Embodiment 2 is that, in this embodiment, the sample solution is a fructooligosaccharide solution, and the preparation method of the fructooligosaccharide solution includes the following steps: Weigh 10 mg of the fructosuccinate sample into a brown screw-top bottle, add 1 mL of ultrapure water, mix well, and obtain a fructosuccinate solution with a concentration of 10 mg / mL. In the HPLC-CAD method, water is used as mobile phase A, and pure acetonitrile is used as mobile phase B. The content of mobile phase B is expressed as a volume percentage. The elution method is gradient elution, and the gradient elution program is as follows: 0~15.0min, 8%~9%B; 15.0~25.0min, 9%~15%B; 25.1~35.0min, 8%B; The chromatographic separation of the substances in the fructooligosaccharide solution was examined, and the obtained chromatogram is shown below. Figure 5 As shown; from Figure 5 As can be seen, under this gradient elution procedure, the main component and impurity peaks in the fructosine solution are well separated. Compared with the peak area and retention time of the standard solution, in addition to the fructosine main component, the fructosine solution also contains fructosine and fructohexasine impurities.

[0029] Example 6 The only difference between this embodiment and Embodiment 2 is that, in this embodiment, the sample solution is a fructooligosaccharide solution, and the preparation method of the fructooligosaccharide solution includes the following steps: Weigh 10 mg of the fructosaccharide sample to be tested into a brown screw-top bottle, add 1 mL of ultrapure water, mix well, and obtain a fructosaccharide solution with a concentration of 10 mg / mL. In the HPLC-CAD method, water is used as mobile phase A, and pure acetonitrile is used as mobile phase B. The content of mobile phase B is expressed as a volume percentage. The elution method is gradient elution, and the gradient elution program is as follows: 0~15.0min, 7%~9%B; 15.0~25.0min, 9%~20%B; 25.1~35.0min, 7%B; The chromatographic separation of each substance in the sucrose octaose solution was examined, and the obtained chromatogram is shown below. Figure 6 As shown; from Figure 6 As can be seen from this gradient elution procedure, the main component and impurity peaks in the fructosaccharide solution are well separated. Comparing the peak area and retention time of the standard solution, in addition to the main component fructosaccharide, the fructosaccharide solution also contains fructose octaose and fructosaccharide nonaose impurities. Among them, the fructose octaose standard solution can only be inferred from the retention time pattern due to the lack of standard products.

[0030] Example 7 The only difference between this embodiment and Embodiment 2 is that, in this embodiment, the sample solution is a fructooligosaccharide solution, and the preparation method of the fructooligosaccharide solution includes the following steps: Weigh 10 mg of the fructosine sample to be tested into a brown screw-top bottle, add 1 mL of ultrapure water, mix well, and obtain a fructosine solution with a concentration of 10 mg / mL. In the HPLC-CAD method, water is used as mobile phase A, and pure acetonitrile is used as mobile phase B. The content of mobile phase B is expressed as a volume percentage. The elution method is gradient elution, and the gradient elution program is as follows: 0~15.0min, 8%~10%B; 15.0~25.0min, 10%~15%B; 25.1~35.0min, 8%B; The chromatographic separation of each substance in the fructooligosaccharide solution was examined, and the obtained chromatogram is shown below. Figure 7 As shown; from Figure 7 As can be seen from this gradient elution procedure, the main component and impurity peaks in the fructosine solution are well separated. Comparing the peak area and retention time of the standard solution, in addition to the main component fructosine, the fructosine solution also contains fructosine octasate, fructosine nonaose, and fructosine decaose impurities. Among them, the fructosine standard solution can only be inferred from the retention time pattern due to the lack of standard products.

[0031] Example 8 The only difference between this embodiment and Embodiment 2 is that, in this embodiment, the sample solution is a fructooligosaccharide solution, and the preparation method of the fructooligosaccharide solution includes the following steps: Weigh 10 mg of the fructodextrose sample into a brown screw-top bottle, add 1 mL of ultrapure water, mix well, and obtain a fructodextrose solution with a concentration of 10 mg / mL. In the HPLC-CAD method, water is used as mobile phase A, and pure acetonitrile is used as mobile phase B. The content of mobile phase B is expressed as a volume percentage. The elution method is gradient elution, and the gradient elution program is as follows: 0~20.0min, 8%~10%B; 20.0~30.0min, 10%~17%B; 30.1~35.0min, 8%B; The chromatographic separation of each substance in the sucrose decaose solution was examined, and the obtained chromatogram is shown below. Figure 8 As shown; from Figure 8 As can be seen from this gradient elution procedure, the main component and impurity peaks in the fructodecose solution are well separated. Comparing the peak area and retention time of the standard solution, in addition to the fructodecose main component, the fructodecose solution also contains fructononose and fructose impurities. Among them, the fructose standard solution can only be inferred from the retention time pattern due to the lack of standard products.

[0032] Comparative Example 1 The only difference between this comparative example and Example 1 is that in this comparative example, the sample solution is a fructooligosaccharide solution, and the gradient elution program is as follows: 0~20.0min, 10%B; 20.1~25.0min, 100%B; 25.1~30.0min, 10%B; The chromatographic separation of the substances in the fructooligosaccharide solution was examined, and the obtained chromatogram is shown below. Figure 9 As shown; from Figure 9 As can be seen from this gradient elution program, the main component of sucrose and impurities in the sucrose solution are eluted within 20 minutes, indicating a relatively strong elution capacity. However, the separation between the sucrose main component peak and the impurity peak is relatively poor.

[0033] Comparative Example 2 The only difference between this comparative example and Example 1 is that in this comparative example, the sample solution is a fructooligosaccharide solution, and the gradient elution program is as follows: 0~20.0min, 5%~10%B; 20.1~25.0min, 100%B; 25.1~30.0min, 5%B; The chromatographic separation of the substances in the fructooligosaccharide solution was examined, and the obtained chromatogram is shown below. Figure 10 As shown; from Figure 10As can be seen from this gradient elution procedure, the separation of the main component of sucrose trisaccharide and impurities in the sucrose trisaccharide solution is relatively good, but the detection sensitivity is relatively low. Compared with the impurity peaks obtained in Example 1, the number of impurity peaks is reduced, and the impurity peaks cannot be fully separated within 20 minutes.

[0034] Comparative Example 3 The only difference between this comparative example and Example 2 is that in this comparative example, the sample solution is a fructotetrasaccharide solution, and the gradient elution procedure is as follows: 0~15.0min, 5%~20%B; 15.1~17.0min, 100%B; 17.1~27min, 5%B; The chromatographic separation of each substance in the fructotetrasaccharide solution was examined, and the obtained chromatogram is shown below. Figure 11 As shown; from Figure 11 As can be seen, under this gradient elution program, the separation of the fructotetrasaccharide main component and impurities in the fructotetrasaccharide solution is relatively poor.

[0035] Comparative Example 4 The only difference between this comparative example and Example 3 is that the chromatographic conditions in the HPLC-CAD method in this comparative example are as follows: Column temperature: 30℃; Flow rate: 1 mL / min; Injection volume: 5 μL; Chromatographic column: Shodex Asahipak NH2P-50 4E amino column, 250mm×4.6mm, 5μm, Showa Denko Corporation, Japan; The chromatographic separation of each substance in the fructooligosaccharide solution was examined, and the obtained chromatogram is shown below. Figure 12 As shown; from Figure 12 As can be seen from the data, when using an amino chromatography column instead of a porous graphitized carbon chromatography column, the separation degree between the main component of sucrose pentose and fructose in the sucrose pentose solution is poor. This shows that the amino chromatography column cannot achieve the separation of sucrose-type oligofructose and fructose-type oligofructose isomers.

[0036] Comparative Example 5 The only difference between this comparative example and Example 4 is that in this comparative example, the sample solution is a fructooligosaccharide solution, and the gradient elution procedure is as follows: 0~15.0min, 10%B; 15.0~20.0min, 10%~15%B; 20.1~25.0min, 15%~50%B; 25.1~30.0min, 10%B; 30.1~40.0min, 10%B; The chromatographic separation of the substances in the fructooligosaccharide solution was examined, and the obtained chromatogram is shown below. Figure 13As shown; Under this gradient elution procedure, the main impurities in the fructosaccharide solution can be identified as fructose pentose and fructose hexasaccharide, and the separation degree between fructose hexasaccharide and fructosaccharide is relatively good, but the elution time is relatively long compared with Example 4.

[0037] Comparative Example 6 The only difference between this comparative example and Example 5 is that in this comparative example, the sample solution is a fructooligosaccharide solution, and the gradient elution procedure is as follows: 0~15.0min, 10%B; 15.0~20.0min, 10%~15%B; 20.1~25.0min, 10%~50%B; 25.1~30.0min, 100%B; 30.1~40.0min, 10%B; The chromatographic separation of the various substances in fructooligosaccharides was examined, and the obtained chromatograms are shown below. Figure 14 As shown; Under this gradient elution procedure, the separation of the main component of fructosuccinate from fructosuccinate and fructosuccinate in the fructosuccinate solution is relatively poor, and the elution time is relatively long.

[0038] Comparative Example 7 The only difference between this comparative example and Example 6 is that in this comparative example, the sample solution is a sucrose octaose solution, and the gradient elution procedure is as follows: 0~15.0min, 10%B; 15.1~20.0min, 10%~100%B; 20.1~25.0min, 100%B; 25.1~30.0min, 10%B; The chromatographic separation of the various substances in fructooligosaccharide was examined, and the obtained chromatogram is shown below. Figure 15 As shown; Under this gradient elution program, the separation degree between the main component of fructosaccharide and impurities in the fructosaccharide solution is relatively poor, and the elution time is relatively long.

[0039] Comparative Example 8 The only difference between this comparative example and Example 7 is that in this comparative example, the sample solution is a fructooligosaccharide solution, and the gradient elution procedure is as follows: 0~15.0min, 10%B; 15.1~20.0min, 10%~15%B; 20.1.0~25.0min, 15%B; 25.1~30.0min, 100%B; 30.1~40.0min, 10%; The chromatographic separation of the various substances in fructooligosaccharide was examined, and the obtained chromatogram is shown below. Figure 16 As shown; Under this gradient elution program, the separation degree between the main component of fructosine and impurities in the fructosine solution is relatively poor, and the elution time is relatively long.

[0040] Comparative Example 9 The only difference between this comparative example and Example 7 is that in this comparative example, the sample solution is a sucrose decaose solution, and the gradient elution procedure is as follows: 0~20.0min, 8%~10%B; 20.0~30.0min, 10%~17%B; 30.1~35.0min, 8%B; The chromatographic separation of the various substances in fructooligosaccharides was examined, and the obtained chromatogram is shown below. Figure 17 As shown; Under this gradient elution procedure, the separation degree between the main component of fructodesugar and impurities in the fructodesugar solution is relatively poor, and the elution time is relatively long.

[0041] Repeatability test The sample solution is a fructooligosaccharide solution, and the preparation method of the fructooligosaccharide solution includes the following steps: Weigh 5 mg of the fructosine sample to be tested into a brown screw-top bottle, add 1 mL of ultrapure water, mix well, and obtain a fructosine solution with a concentration of 5 mg / mL; prepare 6 parallel samples. The chromatographic conditions for the HPLC-CAD method are as follows: Chromatographic column: Hypercarb porous graphitized carbon column, 100 mm × 4.6 mm, 3 μm; Thermo Fisher Scientific, USA; Column temperature: 35℃; Flow rate: 0.8 mL / min; Injection volume: 2 μL; Water was used as mobile phase A, and pure acetonitrile was used as mobile phase B. The content of mobile phase B was expressed as a volume percentage. The elution method was gradient elution, and the gradient elution program was as follows: 0~15.0min, 8%~10%B; 15.0~25.0min, 10%~20%B; 25.1~35.0min, 8%B; Record the chromatograms of six sucrose pentose solutions, and the overlay chromatograms of the six injections are shown below. Figure 18 As shown; Figure 18 The eight peaks in the image are labeled 1, 2, 3, 4, 5, 6, 7, and 8 from left to right, representing peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, and peak 8 respectively. The retention time and relative area data of the eight peaks are recorded in Tables 1-8.

[0042] Table 1. Record of retention time and relative area of ​​peak 1

[0043] Table 2. Record of retention time and relative area of ​​peak 2

[0044] Table 3. Record of retention time and relative area of ​​peak 3

[0045] Table 4. Record of retention time and relative area of ​​peak 4

[0046] Table 5. Record of retention time and relative area of ​​peak 5

[0047] Table 6 records the retention time and relative area data of peak 6.

[0048] Table 7. Record of retention time and relative area of ​​peak 7

[0049] Table 8 records the retention time and relative area data of peak 8.

[0050] from Figure 18 As can be seen from the data in Tables 1-8, the method for separating fructooligosaccharides from high-purity sucrose-type fructooligosaccharide raw materials used in this invention has good repeatability and high stability.

[0051] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for separating isomers of oligofructose by liquid chromatography, characterized in that, The method comprises the following steps: The HPLC-CAD method is used to detect and separate the fructooligosaccharide isomers in the sample solution; The chromatographic conditions in the HPLC-CAD method comprise: water as mobile phase A, pure acetonitrile as mobile phase B, and gradient elution, and the content of the mobile phase B is in percentage by volume; When the sample solution is a kestose solution, the gradient elution program is as follows: 0~20.0min, 5%~15%B; 20.1~25.0min, 100%B; 25.1~30.0min, 5%B; When the sample solution is a nystose solution, the gradient elution program is as follows: 0~15.0min, 5%~15%B; 15.1~20.0min, 100%B; 20.1~27.0min, 5%B; When the sample solution is a kestose solution, the gradient elution program is as follows: 0~15.0min, 8%~10%B; 15.0~25.0min, 10%~20%B; 25.1~35.0min, 8%B; When the sample solution is a kestose solution, the gradient elution program is as follows: 0~10.0min, 10%B; 10.0~20.0min, 10%~15%B; 20.1~30.0min, 10%B; When the sample solution is a kestose solution, the gradient elution program is as follows: 0~15.0min, 8%~9%B; 15.0~25.0min, 9%~15%B; 25.1~35.0min, 8%B; When the sample solution is a kestose solution, the gradient elution program is as follows: 0~15.0min, 7%~9%B; 15.0~25.0min, 9%~20%B; 25.1~35.0min, 7%B; When the sample solution is a kestose solution, the gradient elution program is as follows: 0~15.0min, 8%~10%B; 15.0~25.0min, 10%~15%B; 25.1~35.0min, 8%B; When the sample solution is a kestose solution, the gradient elution program is as follows: 0~20.0min, 8%~10%B; 20.0~30.0min, 10%~17%B; 30.1~35.0min, 8%B; The chromatographic conditions in the HPLC-CAD method further comprise: the chromatographic column is a porous graphitized carbon chromatographic column.

2. The method of claim 1, wherein the oligofructose isomers are separated by liquid chromatography. The porous graphitized carbon chromatographic column is specifically a Hypercarb chromatographic column.

3. The method of claim 1, wherein the oligofructose isomers are separated by liquid chromatography. The chromatographic conditions in the HPLC-CAD method further comprise: the column temperature is 30~35 DEG C.

4. The method of claim 1, wherein the oligofructose isomers are separated by liquid chromatography. The chromatographic conditions in the HPLC-CAD method further comprise: the injection amount is 2~5 muL.

5. The method of claim 1, wherein the oligofructose isomers are separated by liquid chromatography. The chromatographic conditions in the HPLC-CAD method further comprise: the flow rate is 0.8~1.0 muL.

6. The method of claim 1, wherein the oligofructose isomers are separated by liquid chromatography. The CAD detector parameters in the HPLC-CAD method are specifically: the atomization temperature is 33~35 DEG C, and the nitrogen pressure is 30.0~35.0 psi.

7. The method of claim 1, wherein the oligofructose isomers are separated by liquid chromatography. The preparation method of the sample solution comprises the following steps: mixing the sample to be tested and water uniformly to obtain a sample solution. The concentration of the sample solution is 8-10 mg / mL.

8. The method of claim 1, wherein the oligofructose isomers are separated by liquid chromatography. The detection and separation by the HPLC-CAD method further comprises detecting a standard solution.

9. The method of claim 8, wherein the oligofructose isomers are separated by liquid chromatography. The preparation method of the standard solution comprises the following steps: uniformly mixing a standard sample and water to obtain a standard solution.

10. The method of claim 9, wherein the oligofructose isomers are separated by liquid chromatography. The concentration of the standard solution is 1-2 mg / mL.

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