Method, equipment and medium for measuring and calculating mass difference of cations with equal molar concentration

Abstract

The invention belongs to the field of physical testing, and particularly relates to a method and equipment for measuring and calculating the mass difference of cations with equal molar concentration, a medium and a program product. The method comprises the following steps: acquiring the masses of a first cation solution, a second cation solution, a first cation to-be-detected substance solution and a second cation to-be-detected substance solution which are equal in molar concentration; obtaining a first difference value based on the mass of the first cation solution and the mass of the first cation solution to be detected, obtaining a second difference value based on the mass of the second cation solution and the mass of the second cation solution to be detected, and obtaining the cation mass difference based on the first difference value and the second difference value. The cation mass difference with the equal molar concentration is proposed for the first time to replace the necessity of a standard substance in the field of protein content measurement and calculation, and the technical obstacle that the standard substance is difficult to obtain is avoided.

Description

Technical Field

[0001] The present invention relates to the field of physical testing, and tests or analyzes materials by measuring the chemical or physical properties of the materials; more specifically, it relates to a method, device, medium and program product for calculating the mass difference of cations with equimolar concentration. Background Art

[0002] A reference material (RM) is a substance or material that has been determined to have one or more sufficiently homogeneous characteristic values. It plays the role of a "measuring tool" in the field of analytical measurement, and has the characteristics of accuracy, homogeneity, stability of characteristic quantity values, and transitivity of quantity values. It is a physical form of measurement standard.

[0003] Reference materials are divided into primary reference materials and secondary reference materials. Among them, primary reference materials are mainly used to calibrate reference materials of a lower level than itself, calibrate high-accuracy measuring instruments, and research and evaluate standard methods. Their characteristic value accuracies are relatively high, and the requirements for homogeneity and stability are also more stringent. Secondary reference materials are mainly used to meet some general detection and analysis needs, as well as the general requirements of the social industry. It can be directly used as a working reference material for on-site method research and evaluation, and daily analytical measurements with lower requirements. Classified by technical characteristics: it can be divided into chemical composition reference materials, physical property and physical-chemical property measurement reference materials, and engineering technology property measurement reference materials.

[0004] By comparing a reference material with a substance or material to be measured, the characteristic value of the substance or material to be measured can be measured.

[0005] Methods for detecting protein content include the Kjeldahl method, the Lowry method, the BCA method, the HPLC method, and the amino acid composition method. These measurement methods often rely on the acquisition of reference materials, and the protein content is obtained by comparing with reference materials. As can be known from the introduction of the above reference materials, reference materials are divided into different grades, and high-grade reference materials are difficult to obtain, while low-grade reference materials cannot meet the requirements of accurate measurement. Therefore, in the field of protein measurement, there is an urgent need for a measurement and calculation method that does not rely on reference materials. Summary of the Invention

[0006] In view of the above problems, the inventor proposes a method to replace the reference material used in protein content calculation, and calculates the protein content based on the mass difference of cations with equimolar concentration.

[0007] The present application (in the first aspect) discloses a method for calculating the mass difference of cations with equimolar concentration, the method comprising:

[0008] Obtain the masses of the first cation solution, the second cation solution, the first cation-protein solution, and the second cation-protein solution with equal molar concentrations;

[0009] Obtain a first difference based on the mass of the first cation solution and the mass of the first cation-protein solution, and obtain a second difference based on the mass of the second cation solution and the mass of the second cation-protein solution;

[0010] Obtain the cation mass difference based on the first difference and the second difference.

[0011] Furthermore, the cation mass difference is the difference between the sum of the masses of the two cation solutions and the mass of the cation-protein solution;

[0012] Optionally, the cation mass difference is expressed as:

[0013] m = ||M n0 - M np | - |M k0 - M kp ||

[0014] where m represents the cation mass difference; M n0 is the mass of the first cation solution, M np is the mass of the first cation-protein solution, M k0 is the mass of the second cation solution, M kp is the mass of the second cation-protein solution.

[0015] Optionally, the cation mass difference is twice the unilateral cation mass difference and is expressed as:

[0016]

[0017] where m s represents the unilateral cation mass difference of the protein layer double electric layer; M n0 is the mass of the first cation solution, M np is the mass of the first cation-protein solution, M k0 is the mass of the second cation solution, M kp is the mass of the second cation-protein solution.

[0018] Furthermore, the protein concentrations of the first cation solution and the second cation solution are zero; the protein concentrations of the first cation-protein solution and the second cation-protein solution are equal;

[0019] Optionally, the specific steps for obtaining the masses of the first cation solution, the second cation solution, the first cation-protein solution, and the second cation-protein solution are as follows:

[0020] Step 1: Obtain the target protein solution;

[0021] Step 2: Use the congeners of the first cation and the second cation to obtain an SEC system with two mobile phases having equal molar concentrations. Add an equal amount of the target protein solution to the SEC systems of the two mobile phases respectively, and collect the effluent during the target protein peak period to obtain the first cation protein solution and the second cation protein solution; Do not add samples to the SEC systems of the two mobile phases, and collect the effluent during the target protein peak period to obtain the first cation solution and the second cation solution;

[0022] Step 3: Weigh the first cation solution, the second cation solution, the first cation protein solution, and the second cation protein solution respectively to obtain their respective masses;

[0023] Optionally, the SEC system is an SEC-HPLC system;

[0024] Optionally, the target protein peak period is determined through preliminary experiments. After adding the target protein solution to the SEC-HPLC system of the first mobile phase, a chromatogram is obtained, and the target protein peak period is determined according to the protein retention time on the chromatogram;

[0025] Optionally, add an equal amount of the target protein solution to the SEC systems of the two mobile phases respectively, obtain chromatograms under the two mobile phases, and determine the target protein peak period according to the protein retention time on the chromatograms under the two mobile phases;

[0026] Optionally, the detection wavelength of the SEC-HPLC system sets corresponding detection wavelengths according to the two mobile phases;

[0027] Optionally, the target protein includes a specified protein;

[0028] Optionally, the target protein includes multiple proteins;

[0029] Optionally, use a high-precision balance for the weighing.

[0030] Furthermore, the method further includes: obtaining the masses of the first cation solution, the second cation solution, the first cation protein solution, and the second cation protein solution with at least two molar concentrations; calculating to obtain at least

[0031] two mass differences of cations with different molar concentrations;

[0032]

[0033] Optionally, the protein concentrations of the two cation protein solutions with different molar concentrations are equal;

[0034] The first cation and the second cation are sodium ions and potassium ions respectively; ​

[0035] Optionally, the masses of the first cation solution, the second cation solution, the first cation protein solution, and the second cation protein solution are the average values obtained by collecting and weighing multiple times;

[0036] The isomeric compounds of the first cation and the second cation are Na2SO4 and K2SO4 respectively;

[0037] Optionally, the isomeric compounds of the first cation and the second cation are NaCl and KCl respectively;

[0038] Optionally, the isomeric compounds of the first cation and the second cation are NaF and KF respectively;

[0039] Optionally, the isomeric compounds of the first cation and the second cation are NaNO3 and KNO3 respectively. A method for calculating the protein mass based on the mass difference of cations with equimolar concentration, the method includes:

[0040] Using the method described in any one of the above to obtain at least two molar concentrations and their corresponding cation mass differences,

[0041] Solving for the protein mass based on the mapping relationship, where the mapping relationship is that the cation mass difference is determined by the molar concentration and the protein properties and protein mass in the cation protein solution corresponding to the molar concentration. Further, the mapping relationship is that different molar concentrations and their corresponding cation mass differences are mapped to the protein mass through the average

[0042] Protein charge constant, protein ion displacement ability constant, applicable type constant, intercept parameter, and conversion parameter.

[0043] Further, the mapping relationship is expressed as:

[0044]

[0045] Where, m p Represents the protein mass, m i Represents the cation mass difference at the i-th molar concentration, C i ≠0 represents the i-th molar concentration, a≠0 represents the average protein charge constant, n represents the protein ion displacement ability constant, b represents the cation mass difference when the molar concentration is equal to the average protein charge constant, f represents the conversion coefficient between the ion mass difference and its corresponding actual mass, and p represents the applicable type constant;

[0046] Optionally, when the applicable type constant is 3, the mapping relationship is expressed as:

[0047]

[0048] Optionally, when the applicable type constant is 2, the conversion process of the mapping relationship is expressed as:

[0049]

[0050] Optionally, the slope parameter is expressed as: k = b·f p , where k represents the slope parameter, b represents the intercept parameter, f represents the conversion parameter, and p represents the applicable type parameter;

[0051] Optionally, the applicable type constant determination layer obtains the applicable type constant based on the tightness of the molecular structure of the protein;

[0052] Optionally, four molar concentrations and their corresponding cation mass differences are obtained, and the protein mass is solved when the protein average charge constant and the protein ion displacement ability constant are unknown;

[0053] Optionally, five molar concentrations and their corresponding cation mass differences are obtained, and the protein mass is solved when the protein average charge constant and the protein ion displacement ability constant are unknown.

[0054] The second aspect of the present application discloses a protein content calculation system based on the cation mass difference of equimolar concentration in SEC-HPLC, including:

[0055] An acquisition module 201: used to acquire the masses of a first cation solution, a second cation solution, a first cation protein solution, and a second cation protein solution with equal molar concentrations;

[0056] A calculation module 202: used to obtain a first difference based on the mass of the first cation solution and the mass of the first cation protein solution, obtain a second difference based on the mass of the second cation solution and the mass of the second cation protein solution, and obtain a cation mass difference based on the first difference and the second difference.

[0057] The third aspect of the present application discloses a computer device, which includes: a memory and a processor; the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, it is used to execute the steps of the above method.

[0058] The fourth aspect of the present application discloses a computer-readable storage medium, on which a computer program is stored, and when the computer program is executed by a processor, it implements the steps of the above method.

[0059] The fifth aspect of the present application discloses a computer program product, including a computer program, and when the computer program is executed by a processor, it implements the steps of the above method.

[0060] The present application has the following beneficial effects:

[0061] (1) By deeply analyzing the spatial position, charge force, etc. of the analyte (such as protein) in the mobile phase solution and the free charged ions in the solution, the present application proposes that the cation mass difference measured under the equimolar concentration cation solution reflects the properties and mass of the protein;

[0062] (2) The equimolar concentration cation mass difference first proposed by the present application replaces the necessity of standard substances in the field of protein content measurement, bypassing the technical obstacle of difficult acquisition of standard substances;

[0063] (3) In the present application, the error can be reduced by collecting and measuring the mass of the effluent at a set of molar concentrations multiple times and taking the average value, further overcoming the problem that the error of standard substances is difficult to eliminate;

[0064] (4) The cation mass difference of the present application reflects the properties of substances with charge adsorption ability in the measurement field. Correspondingly, in the wide application scenarios of SEC-HPLC, the method of cation mass difference proposed by the present application has the potential to replace standard substances in the field of analysis of substances with charge on the surface that require comparison with standard substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0066] Figure 1 is a schematic flowchart of the method provided in the first aspect of the embodiment of the present invention;

[0067] Figure 2 is a schematic diagram of the program product provided in the second aspect of the embodiment of the present invention;

[0068] Figure 3 is a schematic diagram of the computer device provided in the embodiment of the present invention;

[0069] Figure 4 is a schematic diagram of the architecture of the exemplary computing device provided in the embodiment of the present invention;

[0070] Figure 5 is a schematic diagram of the storage medium provided in the embodiment of the present invention;

[0071] Figure 6 is the protein peak appearance time of two different protein solutions measured by SEC-HPLC provided in the embodiment of the present invention;

[0072] Figure 7 It is a schematic diagram of the hydration layer and double electric layer structure of a protein solution provided by an embodiment of the present invention;

[0073] Figure 8 It is a schematic diagram of the double electric layer structure of another protein solution provided by an embodiment of the present invention;

[0074] Figure 9 It is a schematic diagram for calculating the protein content provided by an embodiment of the present invention, where P in the figure represents protein;

[0075] Figure 10 It is a schematic diagram of the compliance of the measured cation mass difference with the salt molar concentration, the salt molar concentration with the mathematical model, and the cation mass difference with the mathematical model provided by an embodiment of the present invention;

[0076] Figure 11 It is a schematic diagram for the derivation of the conversion coefficient f provided by an embodiment of the present invention;

[0077] Figure 12 It is a schematic diagram of the result of calculating the protein concentration according to the weighing result provided by an embodiment of the present invention;

[0078] Figure 13 It is the protein peak appearance time of two different protein solutions measured by SEC-HPLC provided by an embodiment of the present invention;

[0079] Figure 14 It is a schematic diagram of the compliance of the measured cation mass difference with the salt molar concentration, the salt molar concentration with the mathematical model, and the cation mass difference with the mathematical model of another kind provided by an embodiment of the present invention;

[0080] Figure 15 It is a schematic diagram of the result of calculating the protein concentration according to the weighing result of another kind provided by an embodiment of the present invention. Detailed implementation manners

[0081] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0082] In some of the processes described in the specification, claims, and above-mentioned drawings of the present invention, a plurality of operations appear in a specific order. However, it should be clearly understood that these operations can be executed not in the order in which they appear herein or in parallel. The serial numbers of the operations, such as S101, S102, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. Additionally, these processes may include more or fewer operations, and these operations can be executed sequentially or in parallel. It should be noted that the descriptions such as "first" and "second" in this article are used to distinguish different messages, devices, modules, etc., do not represent a sequence, and do not limit that "first" and "second" are of different types.

[0083] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0084] Figure 1 It is a schematic flowchart of a method for calculating the protein content based on an SEC-HPLC equimolar concentration sodium-potassium scale provided by an embodiment of the present invention. Specifically, the method includes the following steps:

[0085] S101: Obtain the masses of the first cation solution, the second cation solution, the first cation protein solution, and the second cation protein solution with equal molar concentrations;

[0086] S102: Obtain a first difference based on the mass of the first cation solution and the mass of the first cation protein solution, and obtain a second difference based on the mass of the second cation solution and the mass of the second cation protein solution.

[0087] S103: Obtain the cation mass difference based on the first difference and the second difference.

[0088] The present application makes the following important improvements to the prior art, so that the process of protein content detection no longer needs to rely on comparing with the chromatogram of the reference substance. Here, only the protein content detection is taken as an example, which does not mean that the cation mass difference can only be used for the detection of protein content.

[0089] I. Preliminary experiment to determine the time period when the target protein peak appears

[0090] In some embodiments, a sodium sulfate solution is prepared as the mobile phase. After the sample is pretreated, it is loaded into the SEC-HPLC instrument, and the start and end times for collecting the effluent in the subsequent experiment are determined according to the time when the target protein peak appears in the chromatogram.

[0091] In some embodiments, a potassium sulfate solution is prepared as the mobile phase. After the sample is pretreated, it is loaded into the SEC-HPLC instrument, and the start and end times for collecting the effluent during the subsequent experiment are determined according to the time when the target protein peak appears in the chromatogram.

[0092] As Figure 6 shown, the collection period for all proteins determined by the preliminary experiment is a 6-minute interval from the 5th minute to the 11th minute.

[0093] In some embodiments, it is desired to measure the start and end times for collecting the target protein. As Figure 6 shown, if it is desired to measure the content of the main peak protein, the collection period is determined to be from the 8.5th minute to the 11th minute.

[0094] II. Experimental collection of effluent weighing calculation (using a high-precision balance for weighing)

[0095] 2.1 Prepare the samples to be tested, sodium salts, and potassium salts;

[0096] 2.2 Prepare the mobile phase: Prepare the mobile phase using sodium sulfate solution and potassium sulfate solution with equal molar concentrations respectively. For example, first prepare a 0.2 M sodium sulfate solution and a potassium sulfate solution;

[0097] 2.3 Prepare the sample for loading

[0098] If the sample to be tested is solid, it is diluted with physiological saline, or it can also be diluted with water or a solution of the mobile phase to obtain a liquid sample of the sample;

[0099] If the sample is liquid, the buffer system can be replaced or other pretreatment operations can be carried out. For example, the protein solution of the sample to be tested is diluted with physiological saline to about 20 mg / ml.

[0100] This step (2.3) can adopt the sample processing methods of the existing technologies in the field.

[0101] Only for facilitating the calculation of the protein content in the corresponding sample based on the calculated protein mass finally, it is necessary to record the mass of the initial sample to be tested.

[0102] 2.4 SEC-HPLC system collection and weighing (core step)

[0103] 2.4.1 Collect the first group

[0104] Load the sample into the SEC-HPLC system with a sodium sulfate solution mobile phase for detection and collect the effluent from the 5th minute to the 11th minute to obtain the effluent of the sodium salt protein solution; pay attention to the treatment of the liquid droplets at the tube head during collection;

[0105] Specific step example: Inject 100 μL of sample, collect all protein peaks, and precisely collect the protein effluent within a 6-minute period (all);

[0106] Replace the mobile phase with a precisely prepared 0.2 M K2SO4 solution (the salt needs to be weighed to a constant weight), conduct the experiment again, and collect the effluent of the potassium salt protein solution; Note: When changing the buffer system, repeat the above steps of sample addition - collection;

[0107] Meanwhile, it is necessary to collect the effluent of the sodium salt solution in the SEC - HPLC system with a mobile phase of sodium sulfate solution without sample addition within a 6 - minute period;

[0108] Collect the effluent of the potassium salt solution in the SEC - HPLC system with a mobile phase of sodium sulfate solution without sample addition within a 6 - minute period;

[0109] Through the above steps, we have collected the effluent of the sodium salt protein solution, potassium salt protein solution, sodium salt solution, and potassium salt solution at a molar concentration of 0.2 M;

[0110] Use a high - precision balance to weigh and obtain the masses of the effluent of the sodium salt protein solution, potassium salt protein solution, sodium salt solution, and potassium salt solution respectively.

[0111] In some embodiments, the high - precision balance used is the Sartorius MSA125P balance from Germany (one in a hundred thousand).

[0112] 2.4.2 Collect the above four solutions at different molar concentrations and weigh them

[0113] Replace the mobile phase with solutions of other precisely prepared molar concentrations, such as 0.4 M, 0.3 M, 0.1 M, and 0.05 M Na2SO4 solutions (the salt needs to be weighed to a constant weight) and K2SO4 solutions, and repeat the experiment.

[0114] The usage specifications of the SEC - HPLC system in the above collection process are consistent with the conventional operations. Only a brief description is provided here, and for the parts not involved, please refer to the conventional operations in the field.

[0115] In some embodiments, the condition guarantees for the SEC - HPLC system detected by the scheme include:

[0116] ① Conduct HPLC calibration: Ensure that the flow rate: RSD < 2%; injection volume: RSD < 2%;

[0117] ② Calibrate the high - precision balance used for weighing; Standard operating procedures are required during weighing;

[0118] ③ The chromatographic column used in the liquid phase method is a TSK-G3000SW gel chromatographic column; the detection wavelengths used for the sodium sulfate solution and the potassium sulfate solution are 280 nm and 214 nm respectively (as Figure 6 shown);

[0119] ④ Flow rate: 1 mL / min; column temperature: 25 °C; sample cell temperature: 10 °C; sample loading volume: 100 μL.

[0120] In some embodiments, in order to reduce the weighing error, the above experiments are carried out multiple times for weighing, and the average value of the weights collected multiple times is used as the mass of the effluent of the sodium salt protein solution, the mass of the effluent of the potassium salt protein solution, the mass of the effluent of the sodium salt solution, and the mass of the effluent of the potassium salt solution (the total mass of the collected liquid in Table 1). In a specific embodiment, the data obtained by weighing the finally collected liquid in the experiment are shown in Table 1:

[0121] Table 1 Experimental data collection of sodium-potassium solutions with equimolar concentration of Sample A

[0122]

[0123] Table note: The total weight of the collected liquid (mg) is the mass (or average mass) of the effluents of the four solutions; P_na represents the total mass of the liquid collected within the protein peak presentation time after adding the test sample to the SEC-HPLC system with Na2SO4 mobile phase, that is, the mass of the effluent of the sodium salt protein solution; P_K represents the total mass of the liquid collected within the protein peak presentation time after adding the test sample to the SEC-HPLC system with K2SO4 mobile phase, the mass of the effluent of the potassium salt protein solution; Na2SO4 represents the total mass of the liquid collected within the protein peak presentation time of the SEC-HPLC system with Na2SO4 mobile phase without adding a sample, that is, the mass of the effluent of the sodium salt solution; K2SO4 represents the total mass of the liquid collected within the protein peak presentation time of the SEC-HPLC system with K2SO4 mobile phase without adding a sample, that is, the mass of the effluent of the potassium salt solution, and the differences in the table are all absolute values of the differences.

[0124] III. Calculation of cation mass difference

[0125] This part is the core principle of the measurement of the cation mass difference. Based on the following explanation of the cation mass difference, those skilled in the art should understand that the cation mass difference obtained in this application is not limited to protein content measurement. Substances with charged surfaces, such as nucleic acids, and SEC-HPLC can also be used in the molecular weight determination and purity analysis of nucleic acids (such as DNA and RNA) to replace the application of standard substances.

[0126] Among them, when selecting ions, sodium ions and potassium ions are common ions in the field of protein testing, and other ions such as lithium ions can also be used.

[0127] Figure 9 Shows the steps of ion mass difference calculation, outlined as follows: Figure 9 The difference between A and Figure 9 C gives Figure 9 E; Figure 9 The difference between B and Figure 9 D gives Figure 9 F; Figure 9 The difference between E and Figure 9 F gives Figure 9 G. The principle of this calculation step involves the physicochemical structure of chemical substances (taking proteins as an example), which is the creative labor achievement of the applicant. To enable those in the field to understand Figure 9 the natural science laws contained in the calculation process shown, this part makes the following explanations for Figure 9 the calculation process with specific examples:

[0128] First of all, those in the field can know that proteins have a hydration layer / hydration sphere and a double electric layer / double electric sphere structure. Figure 7 is a schematic diagram of the hydration layer (Bulk Water) and double electric layer structure of a protein solution provided by an embodiment of the present invention; most globular proteins have many polar groups on their surfaces, which are easy to adsorb water molecules to form a hydration layer. It not only makes the protein soluble in water but also isolates the protein, making it not easy to precipitate. Generally, each gram of protein can adsorb 0.3 to 0.5 grams of water. The hydration layer is closely related to the biological function of proteins. In the absence of water, almost all biological macromolecules are inactive. The hydration layer is particularly important for the stability of the protein structure and the function of specific sites. Research shows that the water molecules in the hydration layer are ordered and also dynamically changing. Rigid water molecules hinder the function of proteins, while mobile and less rigid water structures are beneficial to their activity; proteins are amphoteric electrolytes, and the dissociable groups in the molecule are mainly side chain groups, including terminal amino groups and carboxyl groups. Proteins also have an isoelectric point, that is, the pH value at which the net charge carried is zero. The isoelectric points of most proteins are slightly acidic to neutral, about 5. The isoelectric point of proteins is mainly determined by the side chain groups in the peptide chain. Most dissociable groups in proteins are distributed on the molecular surface, but there can also be a small number of groups buried inside the molecule, which do not contribute to the isoelectric point. The pK values of dissociable groups in proteins are close to those of the corresponding amino acids but are not exactly the same. When proteins are at the isoelectric point, the net charge is zero, so there is no repulsion of the same kind of charges, so they are unstable, have the smallest solubility, and are easy to aggregate and precipitate. The dissociable groups in proteins can combine with ions in the solution, so the isoelectric point is actually related to the ion composition of the solution. In the absence of other salts, the pH at which the number of protons dissociated by protein proton donors is equal to the number of protons combined by proton acceptors is called the plasma point. Strictly speaking, the plasma point of proteins is a constant; when the dissociable groups on the protein molecular surface carry the same charge, they can form a stable double electric layer with the surrounding counterions ( Figure 7The +- shown (indicating charge) increases the stability of the protein. This is also one of the reasons why proteins can form stable colloids. Due to the existence of the hydration layer and the double electric layer of the protein, the hydration sphere (inside the hydration layer) has a density similar to that of the surrounding water. The volume of the protein including that within the double electric layer changes with the change of the external salt ion concentration; in the double electric layer, the salt ion concentration changes with the change of the external salt ion concentration and the interaction with the protein charge.

[0129] As Figure 9 shown, first, the mass difference of the sodium salt solution caused by the protein (including the mass difference of part of the sodium ions, anions, and the water displaced by the ions) is calculated based on the mass difference between the flowing phase of the sodium sulfate solution of the sample and the effluent of the sodium sulfate solution. The mass difference of the potassium salt solution caused by the protein (including the mass difference of part of the potassium ions, anions, and the water displaced by the ions) is calculated based on the difference between the flowing phase of the potassium sulfate solution of the sample and the potassium sulfate solution, that is Figure 9 The difference between A and Figure 9 C is Figure 9 E: This is because: when protein is added to the sodium sulfate solution, compared with the sodium sulfate solution, the volume of the protein displaces the sodium sulfate solution. For example, when an iron ball is put into a basin full of water, the volume of the iron ball will displace an equal volume of water molecules causing the water to overflow. However, this is because the iron ball replaces the original volume of water, and the weight of the current water basin will change. The volume of the protein displacing the sodium sulfate solution in this application is similar; the volume of the protein displaces the original volume of the sodium sulfate solution. At the same time, due to the existence of the double electric layer, free sodium ions and sulfate ions in the sodium sulfate solution will be adsorbed on the double electric layer of the protein, which is equivalent to displacing the water molecules that should have been in this position. As Figure 9 shown in E, the anions outside the double electric layer adsorb sodium ions and displace water molecules and the sulfate ions corresponding to the free sodium ions (for simplicity, the sulfate ions are not marked on the figure). The anions inside the double electric layer displace sodium ions (charge repulsion, for simplicity, to represent the sulfate ions), and adsorb water molecules (hydration layer); similarly, for the potassium sulfate solution, Figure 9 The difference between B and Figure 9 D is Figure 9 F.

[0130] Furthermore, Figure 9 The difference between E and Figure 9 F is Figure 9 G: Since Figure 9 E and Figure 9The anion concentrations in F are consistent (equal molar concentration of potassium sulfate solution and sodium sulfate solution, based on the same structure of the protein double electric layer, the free anion concentrations are the same). Therefore, after subtraction, the mass of the protein and the mass of the anions (sulfate ions) are eliminated, and the resulting difference is essentially the mass difference of the cations and the mass difference of water. For simplicity, it is expressed as the mass difference of the cations. The essence of this mass difference is the difference in the adsorbed or displaced sodium ions and potassium ions determined by the structure and properties of the protein: outside the double electric sphere, the mass difference is the mass difference of the adsorbed sodium ions and potassium ions (including the mass difference of the displaced water), and inside the double electric sphere, the mass difference is the mass difference of the displaced sodium ions and potassium ions. In this way, the double-ion mass difference we measured is essentially the mass difference of the cations (sodium and potassium ions) caused by the same amount of protein (including the mass difference of the water displaced by the ions), and from Figure 9 G, it can be obtained that this cation difference is essentially twice the mass difference of the sodium and potassium ions on either side of the double electric layer, because the directions of the inner and outer sides of the double electric layer are opposite (the difference between K ions and sodium ions on the inner side of the double electric layer, and the mass difference between sodium ions and potassium ions on the outer side of the double electric layer, and the absolute values of the two differences are equal);

[0131] Therefore, the unilateral cation mass difference caused by the protein is expressed as:

[0132]

[0133] where m s represents the unilateral cation mass difference; M n0 is the mass of the first cation solution, M np is the mass of the first cation-protein solution, M k0 is the mass of the second cation solution, M kp is the mass of the second cation-protein solution.

[0134] When the salt molar concentration changes, the adsorbed and displaced salts (sodium salts, potassium salts) inside and outside the double electric layer are different. As Figure 10 shown in A, the relationship curve between the salt molar concentration and the sodium-potassium mass difference obtained by the final weighing is similar to a parabola.

[0135] By measuring based on different molar concentrations, the protein mass in the sample can be inversely deduced through this ion mass difference determined by the properties and structure of the protein.

[0136] Taking the TEST1 molar concentration in Table 1 as 0.4M as an example:

[0137] The mass of the collected liquid of Na2SO4 is 6.28804mg, the mass of the collected liquid of P_na is 6.28131mg, and the mass difference is 6.28804 - 6.28131 = 0.00673mg.

[0138] The mass of the liquid collected for K2SO4 is 6.31077 mg, and the mass of the liquid collected for P_K is 6.29111 mg. The mass difference is 6.31077 - 6.29111 = 0.01966 mg.

[0139] Finally, by further taking the difference, the mass difference between sodium and potassium ions and the mass difference of water molecules are obtained as 0.01966 - 0.00673 = 0.1293 mg;

[0140] The Figure 1 mass differences under 5 different equimolar concentrations are solved to obtain Table 2:

[0141] Table 2 Cation mass differences under 5 different equimolar concentrations

[0142]

[0143] To further demonstrate that the measured cation mass difference truly reflects the characteristics of substances (such as proteins, nucleic acids, etc.) in the solution, we take proteins as an example to demonstrate the application scenarios of the cation mass difference.

[0144] Those skilled in the art should understand that the following are examples rather than the only application scenarios.

[0145] IV. Parameter Solving

[0146] The mass difference between sodium and potassium ions (Na + -K + +△mH2O) finally calculated in Table 2 conforms to the following mathematical model ( Figure 10 shown in A):

[0147]

[0148] where a≠0, C i ≠0, n∈[-11, 11]; C i represents the salt molar concentration of the solution, a represents the average charge concentration constant of the protein, and this constant represents the ability of the protein to adsorb Na ions on the double electric layer as described above; n represents the relationship between the ions on the double electric layer of the protein and the salt ions displaced by the corresponding space of the double electric layer; a and n can be determined for a specific target protein because these two values are related to the function and structure of the protein itself. For example, for protein HAS: a = 0.167; n = 0.398.

[0149] Among them, the average charge concentration constant a can be understood as the interaction between the protein and the surrounding charge concentration, and this value is related to the protein structure. For example, if a protein has a charge at a certain point, the charge intensity is fixed. That is to say, for a determined protein, that is, the spatial structure of the protein is determined, then the charge intensity on the protein surface is fixed, and a measures the average number of charges on the protein surface.

[0150] In a sense, the molar concentration characterizes the distance between charges; by changing the molar concentration, the values of a and n can be calculated.

[0151] The value of b represents the difference in cation mass and water mass corresponding to 1-fold protein mass (concentration), and k represents the change in cation mass and water mass caused by 1-fold protein mass (concentration). For simplicity, it is stated that k represents the change in cation mass caused by 1-fold protein mass, and b represents the difference in cation mass corresponding to 1-fold protein mass;

[0152] Since the available data for a and n in the prior art in this application is missing, this application maintains a lookup table for a and n of proteins (shown in Table 3). When initially measuring a new protein, at least four different molar concentrations are required to obtain the four parameters k, b, a, and n in formula (1); however, since a and n are determined by the properties of a specific target protein and are not affected by the protein content in the sample, after their values are obtained, in subsequent measurements of the protein content of the same protein or the protein component content of the same drug, it is not necessary to repeat the solution of a and n. The protein mass of the sample can be measured and calculated more quickly by the difference in cation mass at two different molar concentrations;

[0153] Table 3 Protein Constant Table

[0154] Protein Name a n HAS 0.167 0.398 G-HAS 0.203 1.641

[0155] For the a and n tables of proteins not yet disclosed in this application; at least four sets of equimolar concentrations are required for weighing the differences in the masses of two cations obtained during the initial measurement to obtain four unknown parameters; during subsequent measurements, the weighing steps can be simplified based on the already calculated values of a and n, and only two sets of weighings are required for parameter solution, and only k and b are solved.

[0156] Figure 10 As shown in B, the relationship between different salt molar concentrations and Factor is presented. Factor is the factor after k in formula (1) and is expressed as:

[0157]

[0158] Since the values of a and n are determined for a specific protein, it can be seen that Figure 10 B and Figure 10 the trend of the distribution of measurement points in A is consistent;

[0159] Figure 10The C display shows the relationship between Factor and the cation mass difference; that is, converting formula (1) into a regression line for Factor, expressed as formula (3). It can be seen that the measured mass difference and Factor conform to a linear relationship; based on this linear relationship, the parameters k and b can be solved for.

[0160] |m i | = k·Factor + b (3)

[0161] The value of k represents the slope of the degree of conformity between the mass difference |m i | and the mathematical model (formula (3)), and the value of b represents the intercept of the degree of conformity between the mass difference |m i | and the mathematical model (formula (3)).

[0162] In formula (1), |m i | is the mass difference between sodium and potassium ions and the mass difference between water molecules at different molar concentrations calculated through experimental measurements in Table 2.

[0163] As mentioned above, when a and n are unknown, at least four different molar concentrations C i and m i are required to solve for the parameters k, b, a, and n; as Figure 10 shown, based on the measured mass difference m i (where i = 1, 2,..., 5) represents the mass difference m measured at 5 different molar concentrations. i , substituting it into the mathematical model and solving gives: k = 0.000334, b = 0.000335; (when the number of measurement groups is more than the number of parameters, the optimal solution is obtained through model fitting optimization, and the commonly used optimization method is least squares optimization).

[0164] V. Calculate the protein mass in the sample based on the parameters

[0165] Regarding the calculation result of Table 2 (Na + -K + +△m H2O ) as a virtual ion "∑△", the protein is suspended in the solution composed of this virtual ion with equal density. The mass of the virtual ion displaced by the protein and the mass difference of the virtual ion within the double layer ([[]] Figure 9 shown in G) is twice the mass of the virtual ion "∑△".

[0166] As shown in Figure 11, by normalizing the volume of the virtual ion, it can be obtained that the value of k is the cube of the virtual ion mass (b) converted from the actual mass of the protein and the conversion coefficient (f); the virtual ion mass here is the mass difference between sodium and potassium ions and the mass difference between water molecules obtained in Table 2; that is, the value of b is: the virtual ion mass converted from the actual mass of the drug protein in the mobile phase.

[0167] Figure 11 A schematic k (mathematical model of the volume of the mold when the protein structure is relatively compact); Figure 11 B schematic b; Figure 11 C schematic m p , that is Figure 11 A represents the composition factor of k under 1-fold protein mass, including the second or third power of b and f, Figure 11 B represents the corresponding cation mass difference under 1-fold protein mass; Figure 11 C is 1-fold protein mass converted by f in b and k;

[0168] m p = b·f, where m p represents 1-fold protein mass, and f is the conversion coefficient that maps the cation mass difference corresponding to 1-fold protein mass to 1-fold protein mass; the conversion coefficient f can be calculated from k and b:

[0169]

[0170] It can also be expressed as: k = b·f 3 ;

[0171] |m i | and b have the dimension of mass unit gram (g), C i and a have the dimension of molar concentration unit (mol / L), and f and n are conversion and adjustment coefficients without dimension;

[0172] Then the target protein mass m p has multiple representation forms:

[0173] or or m p = b·f or

[0174] Thus, the target protein mass m in the drug of the preparation sample is obtained p , therefore, the protein content is obtained by multiplying the ratio of the target protein mass to the drug mass or drug volume by 100%. Figure 12 Shows the protein content results measured based on five different molar concentrations.

[0175] In some embodiments, the protein content obtained by comparing with a reference substance by the current method is 0.002 g, and the protein content calculated by the method of the present application is 0.000335 g / mL × 6 mL( Figure 12 ), that is, the protein content is 0.002008 g (the decimal places of the present application retain multiple decimal places of weighing, and the weighing error is reduced by taking the average value through multiple measurements).

[0176] Thus, it can be seen that the present method is practical and can obtain good results.

[0177] The Figure 11 relationship shown and the expression of the target protein content are sorted out to obtain the following conversion formula:

[0178]

[0179] where, m i represents the mass difference of ions at the i-th molar concentration, i = 1, 2, …, T; T represents the experimental results of a total of T groups of different molar concentrations;

[0180] a≠0, Ci≠0, k≠0, n∈[-11, 11], if Ci = a, |Ci - a| / (Ci - a) = 1

[0181] In some embodiments, the calculation process is represented by the sorted formula as: After sorting, the measured m i and the relationship with the target protein mass m p are shown as follows:

[0182]

[0183] After conversion, it is:

[0184]

[0185] a≠0, Ci≠0, k≠0, n∈[-11, 11], if Ci = a, |Ci - a| / (Ci - a) = 1.

[0186] In some embodiments, to avoid the unsolvable situation caused by Ci = a during the solution process, five groups of cation mass differences are measured during the test for the above solution.

[0187] VI. Experimental Calibration

[0188] The above method of the present application has undergone rigorous theoretical derivation and its measurement effect has been verified by experimental results. And it has been tested by the inventor through multiple repeated experiments. The theoretical value of k is greater than 0, but in some embodiments, the k calculated by the above steps is less than 0, so that the accurate protein mass cannot be obtained by the above steps. After analyzing the physicochemical structures of these special proteins, the inventor believes that the proteins with double electric spheres applicable to the above calculation process have a relatively large volume, which conforms to the theory of protein displacing water, and it is a situation dominated by the three-dimensional volume conversion coefficient. Therefore, the conversion coefficient is obtained through volume normalization, and the conversion coefficient has a cubic relationship: k = b·f 3 ;

[0189] When the structure of the protein is relatively loose, the conversion coefficient is dominated by the square at this time, and the mathematical model at this time is actually an area mathematical model; the above-mentioned is a volume mathematical model:

[0190] When k calculated based on the volume mathematical model is less than 0, the mathematical model for updating parameters is the area mathematical model:

[0191]

[0192] The calculation method of protein mass is:

[0193]

[0194] The experiment verified the effectiveness of the calculation in this corrected case.

[0195] If the protein structure is relatively compact, it occupies a relatively small space. Then, the distance between the double electric sphere of the protein and the surface layer of the protein structure is relatively far, which can be understood as a structure with a small core and a relatively large double electric sphere. In this case, due to the relatively large double electric sphere, it is in line with the theoretical assumption to adopt the volume mathematical model, and this theoretical assumption has been verified by experimental data.

[0196] However, the situation where k < 0 obtained based on the volume mathematical model is as follows: the structure of the protein is relatively loose, and its electric double layer is very close to the protein itself. That is to say, almost all of the double electric sphere is a relatively large protein core. Therefore, in the above calculation process, it is no longer the large double electric sphere that plays a dominant role. Through the analysis of the structure of this loose protein and its double electric sphere structure, we corrected and obtained the area mathematical model of the loose protein.

[0197] That is to say, when k obtained by calculating based on the volume model is a positive value, the calculation is based on the volume displacement; the conversion coefficient is also converted based on the volume;

[0198] When k obtained by calculating based on the volume model is a positive value and K is negative, the displacement is calculated based on the square.

[0199] Since the k value currently adopted in this application first calculates the k value using the volume mathematical model, if the obtained k value is less than 0, then the area mathematical model is used to recalculate. Through the data collection of the protein combined with the modeling analysis and prediction of the protein performance structure, it is possible to realize the classification judgment of applying the area model or the volume model for protein content determination according to the protein structure.

[0200] So far, this application has constructed a complete method for solving the protein content based on the cation mass; and the accuracy and effectiveness have been repeatedly verified by experiments.

[0201] In another embodiment, the collection period of the protein peak obtained through preliminary experiments is: 5 - 12 min( Figure 13 as shown); the measured masses are shown in Table 4:

[0202] Table 4 Experimental data collection of sodium-potassium solution with equimolar concentration of sample B

[0203]

[0204] The relationships between the cation mass difference and molar concentration, salt molar concentration and mathematical model, and factor and mass difference calculated based on the masses in Table 4 are as Figure 14 shown:

[0205] The relevant constants of Protein G-HAS are: a = 0.203; n = 1.641;

[0206] The calculated values of k and b are as Figure 15 shown, being 0.001277 and 0.000130 respectively;

[0207] Figure 15 shown as the calculation results of the software product of the calculation method: the protein mass is 0.001951 g; the protein concentration is: 0.0002779 g / mL, which is close to the protein mass of 0.002 g measured by other methods, and the decimal places of this application are more precise.

[0208] Figure 3 is a schematic diagram of a computer device provided by an embodiment of the present invention, as Figure 3 shown, the device may include: one or more processors, and one or more memories; wherein, computer-readable code is stored in the memory, and when the computer-readable code is run by the one or more processors, the above-mentioned method can be executed.

[0209] The processor in this embodiment may be an integrated circuit chip with signal processing capabilities. The above-mentioned processor may be a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA) or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components. It can implement or execute the various methods, operations and logic block diagrams disclosed in the embodiments of the present disclosure. The general-purpose processor may be a microprocessor or the processor may also be any conventional processor, etc., which may be of the X86 architecture or the ARM architecture.

[0210] In general, the various exemplary embodiments of the present disclosure may be implemented in hardware or dedicated circuits, software, firmware, logic, or any combination thereof. Some aspects may be implemented in hardware, while other aspects may be implemented in firmware or software that can be executed by a controller, microprocessor, or other computing device. When aspects of the embodiments of the present disclosure are illustrated or described as block diagrams, flowcharts, or using some other graphical representation, it will be understood that the blocks, devices, systems, techniques, or methods described herein may be implemented as non-limiting examples in hardware, software, firmware, dedicated circuits or logic, general hardware or a controller or other computing device, or some combination thereof.

[0211] For example, the method or apparatus according to an embodiment of the present disclosure may also be implemented by means of Figure 4 the architecture of the computing device 3000 shown. As Figure 4 shown, the computing device 3000 may include a bus 3010, one or more CPUs 3020, a read-only memory (ROM) 3030, a random access memory (RAM) 3040, a communication port 3050 connected to a network, input / output components 3060, a hard disk 3070, and so on. The storage devices in the computing device 3000, such as the ROM 3030 or the hard disk 3070, may store various data or files used for the processing and / or communication of the method provided by the present disclosure, as well as the program instructions executed by the CPU. The computing device 3000 may also include a user interface 3080. Of course, Figure 4 the architecture shown is only exemplary, and when implementing different devices, one or more components shown in the Figure 4 computing device may be omitted according to actual needs.

[0212] An embodiment of the present invention also provides a computer-readable storage medium, such as Figure 5As shown, it is a schematic diagram of a storage medium provided by an embodiment of the present invention. Computer-readable instructions 4010 are stored on the computer storage medium 4020. When the computer-readable instructions 4010 are run by a processor, the method according to the embodiments of the present disclosure described with reference to the above drawings can be executed. The computer-readable storage medium in the embodiments of the present disclosure may be a volatile memory or a non-volatile memory, or may include both volatile and non-volatile memories. The non-volatile memory may be a read-only memory (ROM), a programmable read-only memory (PROM), an erasable programmable read-only memory (EPROM), an electrically erasable programmable read-only memory (EEPROM), or a flash memory. The volatile memory may be a random access memory (RAM), which is used as an external cache. By way of example but not limitation, many forms of RAM are available, such as static random access memory (SRAM), dynamic random access memory (DRAM), synchronous dynamic random access memory (SDRAM), double data rate synchronous dynamic random access memory (DDR SDRAM), enhanced synchronous dynamic random access memory (ESDRAM), synchronous link dynamic random access memory (SLDRAM), and direct memory bus random access memory (DR RAM). It should be noted that the memory for the methods described herein is intended to include, but is not limited to, these and any other suitable types of memory. It should be noted that the memory for the methods described herein is intended to include, but is not limited to, these and any other suitable types of memory.

[0213] Embodiments of the present disclosure also provide a computer program product or a computer program, which when executed by a processor implements the steps of the above method, such as Figure 2 As shown, the computer program product or the computer program includes:

[0214] An acquisition module 201: configured to acquire the masses of a first cation solution, a second cation solution, a first cation protein solution, and a second cation protein solution with equal molar concentrations;

[0215] A calculation module 202: configured to obtain a first difference based on the mass of the first cation solution and the mass of the first cation protein solution, obtain a second difference based on the mass of the second cation solution and the mass of the second cation protein solution, and obtain a cation mass difference based on the first difference and the second difference.

[0216] It should be noted that the flowcharts and block diagrams in the accompanying drawings illustrate the possible architectures, functions, and operations of systems, methods, and computer program products according to various embodiments of the present disclosure. In this regard, each block in the flowchart or block diagram may represent a module, a program segment, or a part of code, and the module, program segment, or part of code contains one or more executable instructions for implementing the specified logical function. It should also be noted that in some alternative implementations, the functions marked in the blocks may occur in a different order than that marked in the accompanying drawings. For example, two consecutive blocks shown may actually be executed substantially in parallel, and they may sometimes be executed in the reverse order, depending on the functions involved. It should also be noted that each block in the block diagram and / or flowchart, and the combination of blocks in the block diagram and / or flowchart, can be implemented by a dedicated hardware-based system for performing the specified functions or operations, or can be implemented by a combination of dedicated hardware and computer instructions.

[0217] Generally speaking, various example embodiments of the present disclosure can be implemented in hardware or dedicated circuits, software, firmware, logic, or any combination thereof. Some aspects can be implemented in hardware, while other aspects can be implemented in firmware or software that can be executed by a controller, a microprocessor, or other computing devices. When aspects of the embodiments of the present disclosure are illustrated or described as block diagrams, flowcharts, or using some other graphical representation, it will be understood that the blocks, devices, systems, technologies, or methods described herein can be implemented as non-limiting examples in hardware, software, firmware, dedicated circuits or logic, general hardware or controllers or other computing devices, or some combination thereof.

[0218] Those skilled in the art can clearly understand that for the convenience and brevity of description, the specific working processes of the systems, devices, and units described above can refer to the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0219] In several embodiments provided in the present application, it should be understood that the disclosed systems, devices, and methods can be implemented in other ways. For example, the device embodiments described above are merely illustrative. For example, the division of the units is only a logical function division, and there may be other division methods in actual implementation. For example, multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the couplings or direct couplings or communication connections shown or discussed with each other can be indirect couplings or communication connections through some interfaces, devices, or units, and can be in electrical, mechanical, or other forms.

[0220] The unit described as a separation component may or may not be physically separated. The component shown as a unit may or may not be a physical unit, that is, it may be located in one place or distributed to multiple network units. Some or all of the units can be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0221] In addition, each functional unit in various embodiments of the present invention may be integrated in a processing unit, or each unit may exist physically alone, or two or more units may be integrated in one unit. The above integrated unit may be implemented in the form of hardware or in the form of a software functional unit.

[0222] The exemplary embodiments of the present disclosure described in detail above are merely illustrative and not restrictive. Those skilled in the art should understand that various modifications and combinations can be made to these embodiments or their features without departing from the principles and spirit of the present disclosure, and such modifications should fall within the scope of the present disclosure.

Claims

1. A method for calculating the mass difference of cations with equal molar concentrations, characterized in that: The method comprises: Obtaining the masses of the first cation solution, the second cation solution, the first cation test substance solution, and the second cation test substance solution having equal molar concentrations; A first difference is obtained based on the mass of the first cation solution and the mass of the first cation sample solution, a second difference is obtained based on the mass of the second cation solution and the mass of the second cation sample solution, and a cation mass difference is obtained based on the first difference and the second difference.

2. The method for calculating the mass difference of cations with equal molar concentrations according to claim 1, wherein: The cation mass difference is expressed as: m=||M n0 -M np |-|M k0 -M kp || Where m represents the cation mass difference; M n0 is the mass of the first cation solution, M np is the mass of the first cationic analyte solution, M k0 is the mass of the second cation solution, M kp is the mass of the second cationic analyte solution; Optionally, the first cationic test substance solution is a first cationic protein solution, and the second cationic test substance solution is a second cationic protein solution; the cationic mass difference is the difference between the masses of the two cationic solutions and the mass of the cationic protein solution; Optionally, half of the cation mass difference is the cation mass difference on one side of the double electrical layer; Optionally, the one-sided cation mass difference is expressed as: Among them, m s M represents the mass difference of cations on one side of the protein double layer; n0 is the mass of the first cation solution, M np is the mass of the first cationic protein solution, M k0 is the mass of the second cation solution, M kp is the mass of the second cationic protein solution.

3. The method for calculating the mass difference of cations with equal molar concentrations according to claim 2, wherein: The protein concentrations of the first cationic solution and the second cationic solution are zero; the protein concentrations of the first cationic protein solution and the second cationic protein solution are equal; Optionally, the specific steps of obtaining the masses of the first cationic solution, the second cationic solution, the first cationic protein solution, and the second cationic protein solution having equal molar concentrations are: Step 1: Obtain target protein solution; Step 2: using the same root compound of the first cation and the second cation to obtain a SEC system with two mobile phases of equal molar concentration, adding equal amounts of the target protein solution to the SEC system of the two mobile phases, respectively, and collecting the effluent within the target protein peak period to obtain a first cationic protein solution and a second cationic protein solution; Without adding a sample to the SEC system of the two mobile phases, the effluent in the target protein peak period is collected to obtain a first cationic solution and a second cationic solution; Step 3: Weigh the first cationic solution, the second cationic solution, the first cationic protein solution, and the second cationic protein solution to obtain respective masses; Optionally, the SEC system is a SEC-HPLC system; Optionally, the target protein peak time period is determined by preliminary experiments, the target protein solution is added to the SEC-HPLC system of the first mobile phase to obtain a chromatogram, and the target protein peak time period is determined according to the protein fixed retention time on the chromatogram; Optionally, equal amounts of the target protein solution are added to the SEC system of the two mobile phases, respectively, to obtain chromatograms under the two mobile phases, and the target protein peak time period is determined according to the protein fixed retention time on the chromatograms under the two mobile phases; Optionally, the detection wavelength of the SEC-HPLC system is set to a corresponding detection wavelength according to the two mobile phases; Optionally, the target protein includes a specified protein; Optionally, the target protein includes multiple proteins; Optionally, a high-precision balance is used for the weighing.

4. The method for calculating the mass difference of cations with equal molar concentrations according to claim 2, wherein: The method further comprises: obtaining the masses of the first cation solution, the second cation solution, the first cation protein solution, and the second cation protein solution of at least two molar concentrations; calculating the cation mass difference of at least two molar concentrations; Optionally, the protein concentrations of the two molar concentrations of the cationic protein solutions are equal; Optionally, the first cation and the second cation are sodium ion and potassium ion respectively; Optionally, the first cation and the second cation are sodium ion and lithium ion respectively; Optionally, the first cation and the second cation are potassium ion and lithium ion respectively; Optionally, the masses of the first cationic solution, the second cationic solution, the first cationic protein solution, and the second cationic protein solution are average values ​​of multiple collections and weighings; The homologous compounds of the first cation and the second cation are Na2SO4 and K2SO4 respectively; Optionally, the homologous compounds of the first cation and the second cation are NaCl and KCl respectively; Optionally, the homologous compounds of the first cation and the second cation are NaF and KF respectively; Optionally, the homologous compounds of the first cation and the second cation are NaNO3 and KNO3 respectively.

5. A method for calculating protein mass based on the mass difference of cations with equal molar concentrations, characterized in that: The method comprises: Using the method described in any one of claims 1 to 4 to obtain at least two molar concentrations and their corresponding cation mass differences, The protein mass is obtained based on the mapping relationship, wherein the cationic mass difference is determined by the molar concentration and the protein properties and protein mass in the cationic protein solution of the corresponding molar concentration.

6. The method for calculating protein content based on equimolar concentration cation mass difference according to claim 5, characterized in that: The mapping relationship is that different molar concentrations and their corresponding cation mass differences are mapped through protein average charge constant, protein ion-dispelling capacity constant, applicable type constant, intercept parameter, and conversion parameter to obtain protein mass; Optionally, a set of equations is constructed based on the measured cation mass difference and the mapping relationship, and the protein mass is obtained by solving the equations.

7. The method for calculating protein content based on equimolar concentration cation mass difference according to claim 6, characterized in that: The mapping relationship is expressed as: Among them, m p Indicates protein mass, m i represents the mass difference of the cation with the i-th molar concentration, C i ≠0 indicates the i-th molar concentration, a≠0 indicates the average charge constant of the protein, n indicates the protein's ability to exclude ions constant, b indicates the cation mass difference when the molar concentration is equal to the protein's average charge constant, f indicates the conversion coefficient between the ion mass difference and its corresponding actual protein mass, and p indicates the applicable type constant; Optionally, when the applicable type constant is 3, the mapping relationship is expressed as: Optionally, when the applicable type constant is 2, the mapping relationship is expressed as: Optionally, the slope parameter is expressed as: k = b·f p , where k represents the slope parameter, b represents the intercept parameter, f represents the conversion parameter, and p represents the applicable type parameter; Optionally, the applicable type constant judgment layer obtains the applicable type constant based on the compactness of the molecular structure of the protein; Optionally, obtaining at least four molar concentrations and their corresponding cation mass differences, and solving for the protein mass when the protein average charge constant and the protein ion-repelling ability constant are unknown; Optionally, at least five molar concentrations and their corresponding cation mass differences are obtained, and when the average charge constant of the protein and the protein's ion-repelling ability constant are unknown, the protein mass is obtained by solving them.

8. A computer device, characterized in that: The device comprises: a memory and a processor; the memory is used to store a computer program; the processor executes the computer program to implement the steps of the method according to any one of claims 1 to 7.

9. A computer-readable storage medium, characterized in that: A computer program is stored thereon, and when the computer program is executed by a processor, the steps of the method according to any one of claims 1 to 7 are implemented.

10. A computer program product, comprising a computer program, characterized in that When the computer program is executed by a processor, the steps of the method described in any one of claims 1 to 7 are implemented.

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