Detection method of phenyl content in polysaccharide microspheres

Abstract

The present application relates to the technical field of polysaccharide analysis, and in particular to a method for detecting the phenyl content of polysaccharide microspheres coupled. The method comprises the following steps: mixing polysaccharide microspheres coupled with functional groups and a first acid agent to break the ether bond in the functional group to obtain a dissociation solution; mixing the dissociation solution and a first solvent to obtain a sample to be tested; mixing a standard substance and a second acid agent, and diluting the obtained mixed solution with a second solvent to prepare standard solutions of different concentrations; testing the absorbance values ​​of the standard solutions of different concentrations and the sample to be tested at a wavelength λ, and drawing a standard curve according to the concentration and absorbance values ​​of the standard solutions of different concentrations, and calculating the concentration of phenyl in the sample to be tested according to the standard curve. The above method can overcome the problem of the difficulty in quantitative detection of phenyl functional groups on polysaccharide microspheres, the structural characterization of polysaccharide microspheres, and the front-end structural design of polysaccharide microspheres, and the testing method is simple.

Description

Technical Field

[0001] The present application relates to the technical field of polysaccharide analysis, and in particular to a method for detecting the phenyl content of polysaccharide microspheres coupled thereto. Background Art

[0002] Polysaccharides are a class of macromolecules widely found in animals, plants and microorganisms. Researchers are constantly looking for new sources of polysaccharides from seaweed, microorganisms and plants. These polysaccharides can be prepared into polysaccharide microspheres through advanced technologies such as emulsification and suspension polymerization. The polysaccharide microspheres have uniform particle size and regular shape and can be used as chromatography fillers. By precisely controlling the cross-linking degree and polymerization degree of polysaccharides, the pore size and specific surface area of ​​the filler can be adjusted to meet the separation requirements of different molecular sizes. Polysaccharide microsphere chromatography fillers have excellent biocompatibility and biodegradability, and their application in the biomedical field is receiving increasing attention. For example, polysaccharide microsphere chromatography fillers prepared using intelligent response materials such as pH-sensitive and temperature-sensitive materials can achieve intelligent separation and purification of biomolecules. In high-throughput screening platforms, polysaccharide microsphere chromatography fillers can be used to quickly identify and purify active ingredients. With the advancement of technology and the continuous expansion of application fields, polysaccharide chromatography fillers not only play a role in the traditional field of biomacromolecule separation, but are also used in small molecule drugs, food industry, environmental monitoring and other fields. In addition, polysaccharide microsphere chromatography fillers can be combined with other chromatography techniques (such as ion exchange chromatography and affinity chromatography) to form a multidimensional separation system, significantly improving the separation efficiency.

[0003] Physical or chemical modification of polysaccharide microspheres can enhance their specific properties. For example, physical modification of polysaccharide microspheres can enable them to withstand the pressure and flow rate in industrial production and be used as fillers in gel filtration chromatography, affinity chromatography, hydrophobic chromatography, ion exchange chromatography and mixed mode chromatography. Chemical modification of polysaccharide microspheres by introducing specific functional groups such as carboxyl and amino groups can enhance their specific adsorption capacity and selectivity, and the introduction of functional groups containing ether and phenyl groups can improve the hydrophobicity of polysaccharide microspheres.

[0004] Research on the modification of polysaccharide microspheres has shown its potential and diversity in fields such as bioseparation technology. Although the modified polysaccharide microspheres have wider applications, the characterization methods of the modified polysaccharide microsphere structures are very limited, especially after the functional groups containing ether and phenyl groups are coupled to the polysaccharide microspheres, the phenyl functional groups are often very difficult to quantify. These obstacles limit the application potential of polysaccharide microspheres. Based on this, it is necessary to find a quantitative test method for phenyl functional groups to assist in the front-end structural design of polysaccharide microspheres. Summary of the invention

[0005] Based on this, the first aspect of the present application provides a method for detecting the content of functional groups coupled to polysaccharide microspheres, and its technical scheme is as follows:

[0006] A method for detecting the phenyl content of polysaccharide microsphere coupling comprises the following steps:

[0007] The polysaccharide microspheres coupled with the functional groups and the first acid agent are mixed to break the ether bonds in the functional groups to obtain a dissociation solution, wherein the structure of the functional groups is shown in Formula I: Formula I, wherein L1 is selected from a single bond or C 1-3 Alkylene, L2 are each independently selected from a single bond or C 1-6 Alkylene, R is selected from -OH, C 1-3 alkyl, , halogen, n is 0, 1, 2, 3, 4 or 5;

[0008] Mixing the dissociation solution and the first solvent to obtain a sample to be tested;

[0009] The standard product and the second acid are mixed to obtain a mixed solution, and the mixed solution is diluted with a second solvent to prepare standard solutions of different concentrations. The structural formula of the standard product is shown in Formula II: Formula II, wherein R1 is selected from -OH, C 1-3 alkyl, , halogen, R2 is selected from -H, halogen or -OH, n1 is 0, 1, 2, 3, 4 or 5;

[0010] The absorbance values ​​of the standard solution and the sample to be tested at wavelength λ of different concentrations are tested, a standard curve is drawn according to the concentration and absorbance values ​​of the standard solution of different concentrations, and the concentration of phenyl in the sample to be tested is calculated according to the standard curve.

[0011] Compared with the traditional solution, this application has the following beneficial effects:

[0012] The present application discloses a method for quantitatively detecting phenyl functional groups after coupling functional groups containing ether groups and phenyl groups on polysaccharide microspheres. The method first breaks the ether bond under acidic conditions provided by a first acid agent to obtain a dissociation solution containing phenyl groups, and then measures its absorbance value and brings it into the standard curve to obtain the concentration of phenyl groups. The above method can overcome the problem of difficulty in quantitative detection of phenyl functional groups on polysaccharide microspheres, structural characterization of polysaccharide microspheres, and assist in the front-end structural design of polysaccharide microspheres. The test method is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0014] Figure 1 It is a schematic diagram of the process of detecting the phenyl content of polysaccharide microsphere coupling;

[0015] Figure 2 The standard curve equation fitted by Example 1;

[0016] Figure 3 This is the standard curve equation fitted in Example 5. DETAILED DESCRIPTION

[0017] The present application will be further described in detail below in conjunction with specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thoroughly understood.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0019] the term

[0020] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0021] In the present application, "plurality", "multiple", "multiple times", "plurality", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0022] In this application, "optionally", "optional" and "optional" refer to optional, that is, to any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0023] In this application, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0024] In the present application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution within the above numerical interval is deemed to be continuous and includes the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), and every numerical value between the two numerical endpoints.

[0025] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0026] In the present application, the percentage content, unless otherwise specified, refers to mass percentage for solid-liquid mixing and solid-solid mixing, and volume percentage for liquid-liquid mixing.

[0027] In this application, when it comes to percentage concentration, unless otherwise specified, it refers to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0028] In the present application, %(w / w) and wt% both refer to weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass volume percentage.

[0029] The first aspect of the present application provides a method for detecting the phenyl content of polysaccharide microspheres. In one embodiment, see Figure 1 The method for detecting the phenyl content of polysaccharide microsphere coupling comprises the following steps:

[0030] S10, mixing polysaccharide microspheres coupled with functional groups and a first acid agent to break the ether bond in the functional groups to obtain a dissociation solution, wherein the structure of the functional groups is shown in Formula I: Formula I, wherein L1 is selected from a single bond or C 1-3 Alkylene, L2 are each independently selected from a single bond or C 1-6 Alkylene, R is selected from -OH, C 1-3 alkyl, , halogen, n is 0, 1, 2, 3, 4 or 5;

[0031] Optionally, the polysaccharide microspheres are selected from at least one of agarose microspheres, dextran microspheres, chitosan microspheres, mannan microspheres and mixed sugar matrix microspheres.

[0032] Understandably, " " indicates the connection between the functional group and the polysaccharide microspheres. The functional group includes an ether group and a phenyl group, and the phenyl group is connected to the polysaccharide microspheres through the ether group. After the polysaccharide microspheres coupled with the functional group and the first acid agent are mixed, under the acidic conditions provided by the first acid agent, the ether group of the functional group is broken, and the dissociation solution contains a phenyl group.

[0033] The ether group (COC) is relatively stable because the oxygen atom is connected to two carbon atoms. Although it is a non-linear configuration, the ether bond is relatively stable to alkali, oxidants and reductants due to its weak polarity. However, in some acidic conditions, the ether bond may break. Optionally, the first acid agent is selected from at least one of hydroiodic acid, hydrobromic acid, hydrochloric acid and sulfuric acid. Under the above acid agents, the concentration of the acid agent is controlled, and the ether bond may break. Generally speaking, for ether bonds that are difficult to break, concentrated hydroiodic acid or hydrobromic acid is generally used; for ether bonds that are easier to break, hydrochloric acid or sulfuric acid can be used.

[0034] Taking the first acid agent as hydroiodic acid as an example, the ether bond breaks in the following two ways, where A represents the polysaccharide microspheres:

[0035] The first case: When the structural formula of the functional group is as shown in Formula I, and L1 is a single bond, the principle of ether bond cleavage is as follows:

[0036]

[0037] Since the oxygen atom connected to the benzene ring does not share a pair of electrons and is conjugated with the π electrons of the benzene ring, the delocalization of electrons reduces the electron cloud density on the oxygen, increases the electron cloud density between Ph-O, and increases the strength of the bond, making it more difficult to break. At this time, it is difficult for S to occur on the carbon atom of the benzene ring. N Reaction, whether the reaction is to press S N 1 or S N 2, the result is the formation of iodoethane and phenol, even if an excess of concentrated HI is used, the product is phenol rather than iodobenzene.

[0038] In the second case, when the structural formula of the functional group is as shown in Formula II, and L1 is selected from C 1-3 The principle of alkylene and ether bond breaking is as follows:

[0039]

[0040] At this time, S N The reaction is more likely to occur at the L1 carbon atom connected to O for similar reasons to the reason why it is more likely to occur at the allylic carbon atom.N 1 or press S N 2, the ether bond cleavage occurs preferentially at the L1 carbon atom connected to O, generating phenylalkyl iodide and alcohol.

[0041] Optionally, the reaction time after mixing the polysaccharide microspheres coupled with the functional groups and the first acid agent is 1 to 2 hours. For example, the reaction time is 1 hour, 1.5 hours, or 2 hours. The mixing time is the time between the dissociation of the ether bond. Under the action of the first acid agent for the above time, the ether bond in the functional group is broken, and the dissociation solution contains phenyl.

[0042] It is understandable that before mixing the polysaccharide microspheres coupled with functional groups and the first acid agent, a step of drying the polysaccharide microspheres coupled with functional groups is also included. The moisture contained in the polysaccharide microspheres is the main source of error in the traditional wet dissociation test. Therefore, it is necessary to fully dry the polysaccharide microspheres. Since the water content of the polysaccharide microspheres is very high, it is difficult to control the degree of water removal by removing water by methods such as filtration. Water can be fully removed by drying or vacuum drying. The drying method of the polysaccharide microspheres needs to be selected according to the properties of the functional groups. The easily oxidizable functional groups need to be dried by vacuum drying, and if the pore size of the polysaccharide microspheres is small, the drying process needs to be longer. In some examples, the polysaccharide microspheres coupled with functional groups can be dried using a blast drying oven. The drying temperature is 40~80℃, preferably 60~70℃.

[0043] When the first acid is used to break the ether bond, the sugar chain of the polysaccharide microspheres will also be broken. However, whether the sugar chain is broken or not will not affect the subsequent UV absorbance value. Therefore, the problem of sugar chain breakage can be ignored in this test, and the insoluble matter produced by the sugar chain breakage can be filtered and removed after dissociation. In some examples, after the ether bond in the functional group is broken, the reaction solution is filtered to remove the insoluble matter and retain the sample clear solution as the dissociation solution.

[0044] S20, mixing the dissociation solution and the first solvent to obtain a sample to be tested;

[0045] Optionally, the first solvent is selected from methanol.

[0046] In some examples, the dissociation solution is diluted to 10 mL with the first solvent, mixed thoroughly and allowed to stand for 2 h to 4 h to obtain a sample to be tested.

[0047] S30, mixing the standard product and the second acid to obtain a mixed solution, and diluting the mixed solution with the second solvent to prepare standard solutions of different concentrations, wherein the structural formula of the standard product is shown in Formula II: Formula II, wherein R1 is selected from -OH, C 1-3 alkyl, , halogen, R2 is selected from -H, halogen or -OH, n1 is 0, 1, 2, 3, 4 or 5;

[0048] Among them, the selection of standard substances can be selected according to the cleavage of the ether bond.

[0049] In some examples, L1 is a single bond. In this case, the Ph-O bond is strong and difficult to break, and the ether bond breaks in the first case described above, and phenol is contained in the dissociation solution. Therefore, in the standard, R2 is -OH.

[0050] In some other examples, the L1 is C 1-3 Alkylene, at this time, the ether bond cleavage of the second case mentioned above occurs, that is, the ether bond cleavage occurs preferentially on the L1 carbon atom connected to O, the dissociation liquid contains alkylbenzene, and the R2 is selected from -H or halogen.

[0051] In this embodiment, a second acid agent is added to the standard solution so that the acidity of the standard solution is equivalent to the acidity of the sample to be tested.

[0052] Optionally, the second acid agent is selected from hydrochloric acid or sulfuric acid, wherein the hydrochloric acid can be provided by dilute hydrochloric acid, and the sulfuric acid can be provided by dilute sulfuric acid.

[0053] Optionally, the concentration of the second acid in the mixed solution of the standard and the second acid is 0.2M~1.2M.

[0054] The mixture of the standard and the second acid is diluted to different concentrations using the second solvent. In some examples, the concentration range of the standard in the standard solutions of different concentrations is 1.0 mg / mL to 3.0 mg / mL. Further optionally, the concentration range of the standard is 2.0 mg / mL to 2.5 mg / mL.

[0055] Optionally, the number of standard solutions with different concentrations is at least 6.

[0056] Alternatively, the first solvent may be selected from methanol.

[0057] S40, testing the absorbance values ​​of the standard solution and the sample to be tested at wavelength λ at different concentrations, drawing a standard curve according to the concentration and absorbance values ​​of the standard solution at different concentrations, and calculating the concentration of phenyl in the sample to be tested according to the standard curve.

[0058] Optionally, the wavelength λ is any value in the range of 260 nm to 270 nm. Further optionally, the wavelength λ is any value in the range of 265 nm to 270 nm.

[0059] Optionally, the sampling volume is 2 mL. The absorbance value is measured under a UV-visible spectrophotometer. According to the concentration of the standard solution of different concentrations and the measured absorbance value, a standard curve is made and a curve equation is fitted. According to the curve equation, the concentration of phenyl in the sample to be tested can be calculated, and then the content of phenyl can be obtained, that is, the content of phenyl in the polysaccharide microspheres coupled with functional groups can be obtained.

[0060] The above embodiment discloses a method for quantitatively detecting phenyl functional groups after coupling functional groups containing ether groups and phenyl groups on polysaccharide microspheres. The ether bond is first broken by the acidic conditions provided by the first acid agent to obtain a dissociation solution containing phenyl groups, and then its absorbance is measured and brought into the standard curve to obtain the concentration of phenyl groups. The above method can overcome the problem of difficulty in quantitative detection of phenyl functional groups on polysaccharide microspheres, perform structural characterization of polysaccharide microspheres, assist in the front-end structural design of polysaccharide microspheres, and the testing method is simple.

[0061] In addition, the above detection method has the following advantages: (1) The required reagents are few, and the required reagents, inorganic acid, methanol, and standard products are all common reagents; (2) The operation method is simple and convenient, with high accuracy and sensitivity. The linear equation R fitted in the standard curve is 2 The value is above 3 nines; (3) The method of measuring ultraviolet absorption is used to cleverly avoid the interference of irrelevant groups in the measurement.

[0062] The following is further described in conjunction with specific embodiments and comparative examples. The raw materials involved in the following specific embodiments and comparative examples, unless otherwise specified, can all be commercially available, the instruments used, unless otherwise specified, can all be commercially available, and the processes involved, unless otherwise specified, are all routinely selected by those skilled in the art.

[0063] Example 1

[0064] This embodiment provides a method for detecting the phenyl content in polysaccharide microspheres coupled with functional groups, and the steps are as follows:

[0065] Preparation of the sample to be tested: Dry the polysaccharide microspheres 1 coupled with functional groups in a forced air drying oven at 70° C. for 48 h. The structure of the polysaccharide microspheres 1 coupled with functional groups is ; Take 45.2 mg of the dried sample and accurately weigh it, then add 1 mL of concentrated sulfuric acid and fully dissociate it for 1.5 hours. At this time, the ether bond in the functional group is broken; the dissociation solution is made up to 10 mL with methanol, fully mixed and allowed to stand for 4 hours; filter out the insoluble matter and retain the sample clear liquid, which is the sample to be tested.

[0066] Standard curve drawing: Use benzyl alcohol as the standard, add a certain amount of hydrochloric acid, add a certain amount of methanol, and keep the concentration of hydrochloric acid at 1.0M, and keep the concentration of benzyl alcohol at 2.0mg / mL. Continue to add methanol to dilute, and get 7 concentration gradient standard solutions. Take 2mL of each into a UV-visible spectrophotometer and test its absorbance at a wavelength of 265nm. Make a standard curve based on the concentration and absorbance of the standard and fit the curve equation. The standard curve and the fitted straight line equation are shown in Figure 2 ;

[0067] Sample determination: 2 mL of the sample to be tested was added to a UV-visible spectrophotometer, and the absorbance value A was measured at a wavelength of 265 nm to be 0.670; the concentration of phenyl groups was obtained by substituting it into the standard curve equation, and the phenyl content in the polysaccharide microspheres coupled with functional groups was further calculated to be 743 umol / g.

[0068] Example 2

[0069] This embodiment provides a method for detecting the phenyl content in polysaccharide microspheres coupled with functional groups, and the steps are as follows:

[0070] Preparation of the sample to be tested: Dry the polysaccharide microspheres 2 coupled with functional groups in a vacuum drying oven at 40°C for 48 hours, wherein the structure of the polysaccharide microspheres 2 coupled with functional groups is ; Take 49.5 mg of the dried sample and accurately weigh it, then add 1 mL of concentrated hydrochloric acid and fully dissociate it for 1.5 hours. At this time, the ether bond in the functional group is broken; the dissociation solution after dissociation is made up to 10 mL with methanol, fully mixed and allowed to stand for 4 hours; filter out the insoluble matter and retain the sample clear liquid, which is the sample to be tested.

[0071] Sample determination: 2 mL of the sample to be tested was added to a UV-visible spectrophotometer, and the absorbance value A was measured at a wavelength of 265 nm to be 0.613; the concentration of phenyl groups was obtained by substituting the standard curve equation in Example 1, and the phenyl content in the polysaccharide microspheres coupled with functional groups was further calculated to be 715 umol / g.

[0072] Example 3

[0073] This embodiment provides a method for detecting the phenyl content in polysaccharide microspheres coupled with functional groups, and the steps are as follows:

[0074] Preparation of the sample to be tested: The polysaccharide microspheres 3 coupled with functional groups were dried in a vacuum drying oven at 40°C for 36 hours. The structure of the polysaccharide microspheres 3 coupled with functional groups is ; Take 48.0 mg of the dried sample and accurately weigh it, then add 1 mL of concentrated hydrobromic acid and fully dissociate it for 1.5 hours. At this time, the ether bond in the functional group is broken; the dissociation solution after dissociation is made up to 10 mL with methanol, fully mixed and allowed to stand for 4 hours; filter out the insoluble matter and retain the sample clear liquid, which is the sample to be tested.

[0075] Sample determination: 2 mL of the sample to be tested was added to a UV-visible spectrophotometer, and the absorbance value A was measured at a wavelength of 265 nm to be 0.186; the concentration of phenyl groups was obtained by substituting the standard curve equation in Example 1, and the phenyl content in the polysaccharide microspheres coupled with functional groups was further calculated to be 198 umol / g.

[0076] Example 4

[0077] This embodiment provides a method for detecting the phenyl content in polysaccharide microspheres coupled with functional groups, and the steps are as follows:

[0078] Preparation of the sample to be tested: The polysaccharide microspheres 4 coupled with functional groups were dried in a forced air drying oven at 60°C for 24 hours. The structure of the polysaccharide microspheres 4 coupled with functional groups is ; Take 50.3 mg of the dried sample and accurately weigh it, then add 1 mL of concentrated sulfuric acid and fully dissociate it for 1.5 hours. At this time, the ether bond in the functional group is broken; the dissociation solution is made up to 10 mL with methanol, fully mixed and allowed to stand for 4 hours; filter out the insoluble matter and retain the sample clear liquid, which is the sample to be tested.

[0079] Sample determination: 2 mL of the sample to be tested was added to a UV-visible spectrophotometer, and the absorbance value A was measured at a wavelength of 265 nm to be 0.663; the concentration of phenyl groups was obtained by substituting the standard curve equation in Example 1, and the phenyl content in the polysaccharide microspheres coupled with functional groups was further calculated to be 731 umol / g.

[0080] Example 5

[0081] This embodiment provides a method for detecting the phenyl content in polysaccharide microspheres coupled with functional groups, and the steps are as follows:

[0082] Preparation of the sample to be tested: The polysaccharide microspheres 5 coupled with functional groups were dried in a forced air drying oven at 60°C for 36 hours, wherein the structure of the polysaccharide microspheres 5 coupled with functional groups is ; Take 35.5 mg of the dried sample and accurately weigh it, then add 1 mL of concentrated hydrochloric acid and fully dissociate it for 2.0 hours. At this time, the ether bond in the functional group is broken; the dissociation solution is made up to 10 mL with methanol, mixed thoroughly and allowed to stand for 4 hours; filter out the insoluble matter and retain the sample clear liquid, which is the sample to be tested.

[0083] Standard curve determination: Use phenol as the standard, add a certain amount of hydrochloric acid, add a certain amount of methanol, and keep the concentration of hydrochloric acid at 1.0M, and keep the concentration of benzyl alcohol at 2.0mg / mL. Continue to add methanol to dilute, and get 7 concentration gradient standard solutions. Take 2mL of each into a UV-visible spectrophotometer and test its absorbance at a wavelength of 270nm. Make a standard curve based on the concentration and absorbance of the standard and fit the curve equation. The standard curve and the fitted straight line equation are shown in Figure 3 ;

[0084] Sample determination: Take 2 mL of the sample clear solution into the UV spectrometer cuvette, measure the absorbance value A at a wavelength of 270 nm to be 0.449, substitute it into the standard curve equation to obtain the concentration of phenyl, and further calculate that the phenyl content in the polysaccharide microspheres coupled with functional groups is 422 umol / g.

[0085] In the above embodiments, after the polysaccharide microspheres coupled with functional groups are fully dried, an appropriate amount is placed in a first acid environment to break the ether bonds in the functional groups, and the dissociation solution is diluted and its ultraviolet absorption is measured, and the calculation is performed by comparing with the standard curve to complete the detection of the phenyl content of the polysaccharide microspheres coupled with functional groups.

[0086] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0087] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A method for detecting the phenyl content of polysaccharide microspheres coupled, characterized in that: The following steps are involved: The polysaccharide microspheres coupled with the functional groups and the first acid agent are mixed to break the ether bonds in the functional groups to obtain a dissociation solution, wherein the structure of the functional groups is shown in Formula I: Formula I, wherein L1 is selected from a single bond or C 1-3 Alkylene, L2 is selected from a single bond or C 1-6 Alkylene, R is selected from -OH, C 1-3 alkyl, , halogen, n is 0, 1, 2, 3, 4 or 5; Mixing the dissociation solution and the first solvent to obtain a sample to be tested; The standard product and the second acid are mixed to obtain a mixed solution, and the mixed solution is diluted with a second solvent to prepare standard solutions of different concentrations. The structural formula of the standard product is shown in Formula II: Formula II, wherein when L1 is a single bond, R2 is -OH, and when L1 is C 1-3 In the case of alkylene, R2 is selected from -H or halogen, R1 is selected from -OH, C 1-3 alkyl, , halogen, n1 is 0, 1, 2, 3, 4 or 5, the second solvent is the same as the first solvent, and both are selected from methanol; The absorbance values ​​of the standard solution and the sample to be tested at wavelength λ of different concentrations are tested, a standard curve is drawn according to the concentration and absorbance values ​​of the standard solution of different concentrations, and the concentration of phenyl in the sample to be tested is calculated according to the standard curve.

2. The method for detecting the phenyl content of polysaccharide microsphere coupling according to claim 1, characterized in that: The structure of the polysaccharide microspheres coupled with functional groups is , , , or , wherein A represents polysaccharide microspheres.

3. The method for detecting the phenyl content of polysaccharide microsphere coupling according to claim 2, characterized in that: When the structure of the polysaccharide microspheres coupled with functional groups is , , or When , the standard substance is benzyl alcohol; When the structure of the polysaccharide microspheres coupled with functional groups is When , the standard substance is phenol.

4. The method for detecting the phenyl content of polysaccharide microsphere coupling according to any one of claims 1 to 3, characterized in that: The first acid agent is selected from at least one of hydroiodic acid, hydrobromic acid, hydrochloric acid and sulfuric acid.

5. The method for detecting the phenyl content of polysaccharide microsphere coupling according to claim 4, characterized in that: The reaction time after mixing the polysaccharide microspheres coupled with functional groups and the first acid agent is 1 h to 2 h.

6. The method for detecting the phenyl content of polysaccharide microsphere coupling according to any one of claims 1 to 3 and 5, characterized in that: The second acid agent is selected from hydrochloric acid or sulfuric acid.

7. The method for detecting the phenyl content of polysaccharide microsphere coupling according to claim 6, characterized in that: The concentration of the second acid in the mixed solution of the standard and the second acid is 0.2M-1.2M.

8. The method for detecting the phenyl content of polysaccharide microsphere coupling according to any one of claims 1 to 3 and 5, characterized in that: The wavelength λ is selected from any value in the range of 260 nm to 270 nm.

9. The method for detecting the phenyl content of polysaccharide microsphere coupling according to any one of claims 1 to 3 and 5, characterized in that: The polysaccharide microspheres are selected from at least one of agarose microspheres, dextran microspheres, chitosan microspheres, mannan microspheres and mixed sugar matrix microspheres.

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