A high performance liquid chromatography analysis method for tannic acid component content

Abstract

The present application relates to the technical field of spectral analysis, and specifically to a high performance liquid chromatography analysis method for tannic acid component content, the method comprising: obtaining the chromatographic peak symmetry weights of the corresponding moments of each element according to the symmetry of the distribution of each element in the chromatographic peak standard vector; constructing a standard chromatographic characteristic vector and a chromatographic characteristic vector to be analyzed, and adjusting the element values ​​of the chromatographic characteristic vector to be analyzed; determining the chromatographic peak aliasing interference intensity at each moment; calculating the absorbance correction weight at each moment; based on the absorbance at each moment and its symmetric moment in the chromatographic peak analysis vector and combined with the absorbance correction weight, correcting each absorbance to determine the tannic acid feed peak area; obtaining a fitting function based on the concentration of standard tannic acid solutions of different concentrations and the tannic acid standard peak area, and obtaining the tannic acid concentration in the feed extract. The present application can eliminate the chromatographic peak aliasing interference phenomenon caused by other substances and improve the measurement accuracy of tannic acid.

Description

Technical Field

[0001] The present application relates to the technical field of spectral analysis, and in particular to a high performance liquid chromatography analysis method for tannic acid component content. Background Art

[0002] Liquid chromatography is an efficient, fast and accurate method for analyzing chemical components. Due to its high efficiency, speed and accuracy, liquid chromatography is used in feed quality testing in feed companies.

[0003] The component analysis method of liquid chromatography is to pass the object to be tested through the stationary phase and the mobile phase to form a mixed phase. The stationary phase has different fixation abilities for different components, which causes the object to be tested to separate in the mixed phase. This in turn causes the liquid flowing out of the mixed phase at different times to contain different substances. At this time, the liquid chromatogram is obtained by detecting the absorbance of the liquid. The detection time represents the different material components, and the peak value and peak width of the absorbance represent the material content, thereby completing the measurement of the object. Summary of the invention

[0004] In order to solve the above technical problems, the present application provides a high performance liquid chromatography analysis method for the content of tannic acid components to solve the existing problems.

[0005] The high performance liquid chromatography analysis method for the content of tannic acid components in the present application adopts the following technical scheme:

[0006] An embodiment of the present application provides a high performance liquid chromatography analysis method for tannic acid component content, the method comprising the following steps:

[0007] Obtain feed chromatograms of feed sample extracts at different wavelengths and standard chromatograms of standard tannic acid solutions at different wavelengths, and obtain analysis vectors of each chromatographic peak and standard vectors of each chromatographic peak, wherein each element in the vector is the absorbance of the solution at the corresponding moment;

[0008] According to the symmetry of the distribution of each element in the chromatographic peak standard vector, the chromatographic peak symmetry weight of each element at the corresponding time is obtained;

[0009] Based on the differences of the standard vectors of the chromatographic peaks of all adjacent bands and the differences of the chromatographic analysis vectors, a standard chromatographic feature vector and a chromatographic feature vector to be analyzed are constructed, and the element values ​​of the chromatographic feature vector to be analyzed are adjusted;

[0010] Based on the difference between the standard chromatographic characteristic vector and the adjusted chromatographic characteristic vector to be analyzed, determining the chromatographic peak aliasing interference intensity at the corresponding time of each element in the chromatographic peak analysis vector;

[0011] Based on the difference in chromatographic peak aliasing interference intensity at each moment and its symmetric moment in the chromatographic peak analysis vector, combined with the chromatographic peak symmetry weight, determine the absorbance correction weight at each moment;

[0012] Based on the absorbance at each moment and its symmetrical moment in the chromatographic peak analysis vector and combined with the absorbance correction weight, each absorbance is corrected to determine the tannic acid feed peak area;

[0013] The fitting function was obtained based on the concentration of standard tannic acid solutions with different concentrations and the standard peak area of ​​tannic acid, and the tannic acid concentration in the feed extract was obtained by combining the peak area of ​​tannic acid feed.

[0014] Optionally, the method for acquiring the standard vector of each chromatographic peak and the analysis vector of each chromatographic peak is:

[0015] Analyze the sum of the elements of each vector corresponding to each standard chromatogram, record the vector with the largest sum of elements as the optimal band vector, and record the corresponding wavelength as the optimal wavelength; take the time when the peak of the optimal band vector is located as the standard retention time, select two time points symmetrical about the time when the peak is located, and the time period composed of them is used as the standard detection time period T, and all elements of each standard chromatogram in the detection time period are used to form the standard vector of each chromatographic peak;

[0016] For each feed chromatogram, obtain the mean vector corresponding to all vectors, calculate the time difference between the corresponding time of each extreme point in the mean vector and the standard retention time, and take the time corresponding to the minimum time difference as the retention time of the feed chromatogram , the detection period of feed chromatogram is , all elements in each feed chromatogram during the detection period are combined into analysis vectors for each chromatographic peak.

[0017] Optionally, the method for obtaining the chromatographic peak symmetry weight is:

[0018] For each element in the standard vector of the chromatographic peak of each band, the difference between each element and the element symmetrical with respect to the standard retention time is analyzed and recorded as the absorbance difference, and the sum of each element and the element symmetrical with respect to the standard retention time is calculated and recorded as the absorbance sum value;

[0019] The ratio of the absorbance difference to the absorbance sum value is analyzed, and the difference between the value 1 and the ratio is determined as the chromatographic peak symmetry weight of each element at the corresponding time.

[0020] Optionally, the method for constructing the standard chromatogram characteristic vector and the chromatogram characteristic vector to be analyzed is:

[0021] The weighted average of all elements in the standard vector of each chromatographic peak is analyzed and recorded as the standard absorbance of each band, wherein the symmetric weight of the chromatographic peak of each element is used as the weighted weight of each element; the difference between the standard absorbance of the latter band and the previous band in all adjacent bands is used to construct the standard chromatographic characteristic vector;

[0022] The difference between the elements at the same position in the chromatographic peak analysis vectors of the latter band and the previous band in all adjacent bands is used to form the chromatographic feature vector to be analyzed at the time corresponding to each same position.

[0023] Optionally, the step of adjusting the element values ​​of the chromatographic feature vector to be analyzed further comprises:

[0024] The ratio of the element mean of the standard chromatographic characteristic vector to the element mean of the chromatographic characteristic vector to be analyzed at each moment is recorded as the absorption intensity ratio at each moment, and the absorption intensity ratio at each moment is multiplied by the standard chromatographic characteristic vector to obtain the chromatographic characteristic vector to be analyzed at each moment after the element value is adjusted.

[0025] Optionally, the method for determining the chromatographic peak aliasing interference intensity is:

[0026] The distance between the standard chromatographic characteristic vector and the chromatographic characteristic vector to be analyzed at each moment after the element value is adjusted is determined as the chromatographic peak aliasing interference intensity at each moment.

[0027] Optionally, the symmetric moments of each moment in the chromatographic peak analysis vector are moments at which each moment is symmetric with respect to the retention time of the feed chromatogram.

[0028] Optionally, the method for determining the absorbance correction weight at each moment is:

[0029] The difference between the chromatographic peak aliasing interference intensity at each moment and the symmetric moment in the chromatographic peak analysis vector is determined as the aliasing weight difference at each moment;

[0030] If the aliasing weight difference is positive, the absorbance correction weight is equal to the product of the aliasing weight difference and the chromatographic peak symmetry weight;

[0031] If the aliasing weight difference is negative, the absorbance correction weight at the corresponding moment is not calculated.

[0032] Optionally, the correcting each absorbance further comprises:

[0033] If the aliasing weight difference is positive, the absorbance at each moment after correction is the sum of the absorbance at its symmetrical moment multiplied by the absorbance correction weight and the absorbance at each moment before correction multiplied by 1 minus the absorbance correction weight;

[0034] If the aliasing weight difference is negative, the absorbance at the corresponding moment remains unchanged.

[0035] Optionally, the tannic acid feed peak area is the sum of elements of the corrected chromatographic peak analysis vector corresponding to the optimal wavelength, and the tannin peak standard area is the sum of elements of the chromatographic peak standard vector.

[0036] This application has at least the following beneficial effects:

[0037] This application aims to solve the problem of inaccurate tannic acid determination caused by interference from other substances when using liquid chromatography to determine the content of tannic acid components in feed, including:

[0038] The retention time of tannic acid is determined, and the chromatogram data within the existence time of tannic acid is intercepted to obtain the tannic acid chromatographic peak data vector; the chromatographic peak symmetry weight is calculated for the chromatographic peak standard vector to characterize the low-quality data part of the tannic acid chromatographic peak data with "tailing peak" or "fronting peak" phenomenon;

[0039] The standard absorbance is calculated by the chromatographic peak standard vector and the chromatographic peak symmetry weight, and then the standard chromatographic characteristic vector is calculated; the chromatographic characteristic vector to be analyzed for the chromatographic peak analysis vector; the two absorbance-light wavelength characteristic vectors are compared to obtain the chromatographic peak aliasing interference intensity, which characterizes the intensity of chromatographic peak aliasing interference at different times;

[0040] Finally, the chromatographic peak symmetry weight and the chromatographic peak aliasing interference intensity were combined to obtain the tannic acid feed peak area, which was used as the characteristic value for quantitative analysis of tannic acid content.

[0041] Finally, the peak area was used as the characteristic value and the traditional regression equation analysis method was used to determine the tannic acid content.

[0042] Among them, the chromatographic peak symmetry weight can eliminate the interference of low-quality data and obtain more accurate measurement results; the chromatographic peak aliasing interference intensity can eliminate the chromatographic peak aliasing interference caused by other substances in the feed and solve the problem of inaccurate tannic acid measurement. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions and advantages in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0044] Figure 1 A flow chart of the steps of a high performance liquid chromatography analysis method for tannic acid component content provided in the present application;

[0045] Figure 2 Schematic diagram of chromatographic peak aliasing interference provided for this application. DETAILED DESCRIPTION

[0046] In order to further explain the technical means and effects adopted by the present application to achieve the predetermined invention purpose, the following is a detailed description of the high performance liquid chromatography analysis method for the content of tannic acid components proposed in the present application, its specific implementation, structure, characteristics and effects, in conjunction with the accompanying drawings and preferred embodiments. In the following description, different "one embodiment" or "another embodiment" does not necessarily refer to the same embodiment. In addition, specific features, structures or characteristics in one or more embodiments may be combined in any suitable form.

[0047] Unless otherwise defined, terms such as "comprises", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that a circuit structure, article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such articles or devices. In the absence of further restrictions, an element defined by the statement "comprising a ..." does not exclude the presence of other identical elements in the article or device comprising the element. In addition, the term "and\or" used herein includes any and all combinations of one or more related listed items. All technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present application belongs.

[0048] The specific scheme of the high performance liquid chromatography analysis method for tannic acid component content provided by the present application is described in detail below in conjunction with the accompanying drawings.

[0049] An embodiment of the present application provides a high performance liquid chromatography analysis method for the content of tannic acid components. For details, please refer to Figure 1 , the method comprises the following steps:

[0050] Step 1: Obtain feed chromatograms of feed sample extracts at different bands and standard chromatograms of standard tannic acid solutions at different bands, and obtain analysis vectors of each chromatographic peak and standard vectors of each chromatographic peak, wherein each element in the vector is the absorbance of the solution at the corresponding moment.

[0051] Tannic acid, also known as galloylglucose, is a polymer of gallic acid and glucose connected by ester bonds. The connection between ester bonds and glucose is complex. Different connection positions and different amounts of gallic acid and glucose will form different types of tannic acid. Therefore, during the tannic acid detection process, it is necessary to determine the tannic acid component and content in the test substance.

[0052] Optionally, a U3000 liquid chromatograph is used as a detection instrument. Unlike a traditional chromatogram, this detector does not capture the absorbance of a specific wavelength along the time axis, but captures a series of absorbance values ​​in the wavelength range of 190~400nm (nanometers) along the time axis (the accuracy can be set, set to 1nm in this embodiment).

[0053] First, different tannic acid standards are obtained as samples; the types of tannic acid samples are the types of tannic acid commonly found in feed, and 1-0-galloylglucose standard is taken as an example here;

[0054] The sample was dissolved in methanol solution, and then deionized water was added to prepare a 250 μg / mL stock solution. The stock solution was measured on the instrument to obtain N standard chromatograms;

[0055] Secondly, the feed sample is weighed and mixed with the methanol solution, and the tannic acid in the feed is extracted through multiple centrifugation and the supernatant is taken to obtain the feed sample extract; the feed sample extract is re-dissolved with the methanol solution, and the feed is measured on a machine to obtain N feed chromatograms.

[0056] The parameters for the on-machine measurement in this embodiment are set as follows: measurement time 30 minutes, measurement compensation range 200~300nm, the number of chromatograms measured is N, corresponding to N vectors, N=100, where the nth vector represents data with a wavelength of 199+n nanometers, and the length of each vector in this embodiment is M=18000, and the mth element represents the absorbance of the sample at the mth moment. It should be noted that the setting of the above specific parameters during the measurement process is set by the implementer according to the actual application scenario, and this application does not impose any special restrictions on this.

[0057] Among them, it should be noted that the type of tannic acid standard product is the corresponding tannic acid component that needs to be measured. If the content of a certain tannic acid component in the feed needs to be measured, the standard product corresponding to a certain tannic acid should be selected as the sample.

[0058] The data was further preprocessed to obtain the peak data of tannic acid. First, the retention time and detection period of the standard tannic acid solution were analyzed. The specific process is as follows:

[0059] In a chromatogram, the retention time is the corresponding time of the sample peak on the x-axis. The retention time of the same substance in the chromatogram is the same. The retention time is a basic indicator of the chromatogram. This embodiment specifies the detection period for the chromatogram based on the retention time, which is convenient for extracting the chromatogram peak data of the chromatogram;

[0060] The N standard chromatograms obtained correspond to N standard data vectors, and the element sum is calculated for each standard data vector. The standard data vector with the largest element sum is recorded as the optimal band vector. The main absorption wavelength of tannic acid is 199+A nanometers. This wavelength band is used as the measurement band, and A is the wavelength corresponding to the optimal band vector, which is recorded as the optimal wavelength.

[0061] For the optimal band vector, the technicians marked the peak time of the chromatographic peak of tannic acid and recorded the time as the standard retention time Tmid; at the same time, two times were marked as t1 and t2, and in particular, the time t1 and the time t2 were symmetrical about the standard retention time Tmid. The chromatographic peak representing tannic acid appeared between t1 and t2. For the convenience of analysis and expression, the time between t1 and t2 was recorded as the detection period. Therefore, the existence time of tannic acid in this embodiment was recorded as ;

[0062] It should be noted that the implementation of the selection of time t1 and time t2 is based on the actual situation of the changes in the chromatographic peaks at both ends of the peak. In this implementation, the time corresponding to the point in the chromatographic peak on the left side of the peak moment that is closest to the peak moment and has the largest slope is taken as time t1, and the time on the right side of the peak moment that is symmetrical to time t1 is taken as time t2.

[0063] The N feed chromatograms obtained correspond to N feed data vectors. The mean vector of the N vectors is calculated, and then the mean vector is calculated using the mean filtering algorithm, and the output is the filtered mean vector. Further, the filtered mean vector is used as input, and the extreme point detection algorithm is used for calculation, and the extreme point position of the filtered mean vector is output; the time difference between the time corresponding to each extreme point and the standard retention time is calculated, and the extreme point position with the smallest time difference is selected, and its time is used as the retention time of the feed chromatogram. , the detection period of the feed chromatogram is To time .

[0064] Among them, mean filtering algorithm and extreme point detection algorithm are commonly used technologies in the field of data processing.

[0065] Further, the tannic acid chromatographic peak data vector is obtained, including the chromatographic peak standard vector and the chromatographic peak analysis vector, specifically:

[0066] For the nth standard data vector, the data from time t1 to t2 are intercepted to form a data vector with a length of K, which is recorded as the chromatographic peak standard vector of the nth wavelength band;

[0067] For the nth feed data vector, the data from time t3 to t4 is intercepted to form a data vector with a length of K, which is recorded as the chromatographic peak analysis vector of the nth wavelength band.

[0068] Step 2: Obtain the chromatographic peak symmetry weight of each element at the corresponding moment according to the symmetry of the distribution of each element in the chromatographic peak standard vector.

[0069] In the liquid chromatograph, since some components in the feed sample extract are not fully separated when passing through the mixed phase of the liquid chromatograph, the chromatogram peak image of tannic acid is overlapped with the chromatogram peak images of other substances, so the feed chromatogram needs to be corrected.

[0070] When performing chromatogram correction, it is necessary to select the data portion with higher quality in the chromatogram, through which a more accurate chromatogram correction result can be obtained. The embodiment of the present application will construct the chromatogram peak symmetry weight, and the specific process is as follows:

[0071] In the chromatogram, the y-axis represents absorbance. The absorbance of tannic acid first increases and then decreases during the detection period, which means that the content of tannic acid in the detector first increases and then decreases. Ideally, since the adsorption result of the fixed phase on tannic acid molecules in the liquid chromatograph is random, the change in the number of tannic acid molecules released in the mixed phase over time should conform to the Gaussian distribution, that is, ideally, the shape of the chromatographic peak of tannic acid should be a symmetrical figure.

[0072] In the actual detection process, due to factors such as equipment aging and unreasonable equipment parameter debugging, it is difficult to achieve ideal detection conditions. Therefore, the width of the back and front parts of the chromatographic peak will be different in the chromatogram. This phenomenon is called "tailing peak" or "front peak". This part of the data will affect the quantitative analysis of tannic acid and is a low-quality data part. When correcting the chromatogram, this part of the data should be given a lower reference weight. Therefore, the chromatographic peak symmetry weight is set to characterize this part of the data.

[0073] In this embodiment, the chromatographic peak standard vector of the nth wavelength band is taken as an example for detailed description. For the chromatographic peak standard vector of the nth wavelength band, considering that the standard data is obtained through tannic acid samples and is not interfered by the chromatographic peak aliasing of other substances in the feed, the asymmetry of this part of the data is caused by factors such as equipment aging and unreasonable equipment parameter debugging. Therefore, this embodiment will calculate the degree of symmetry according to each element in the chromatographic peak standard vector of the nth wavelength band. The lower the degree of symmetry, the lower the corresponding chromatographic peak symmetry weight;

[0074] For each element in the chromatographic peak standard vector of each band, the difference between each element and the element symmetrical with respect to the standard retention time is analyzed and recorded as the absorbance difference, and the sum of each element and the element symmetrical with respect to the standard retention time is calculated and recorded as the absorbance sum value; the ratio of the absorbance difference to the absorbance sum value is analyzed, and the difference between the value 1 and the ratio is determined as the chromatographic peak symmetry weight of each element at the corresponding time.

[0075] Specifically, in this embodiment, taking the kth element in the chromatographic peak standard vector of the nth wavelength band as an example, the calculation method is: for the chromatographic peak standard vector of the nth wavelength band, the symmetric position of the kth element is element K-k+1, that is, the symmetric moment of the kth moment is the K-k+1th moment. Further, the ratio of the absolute value of the difference between the two elements to the sum of the two elements is analyzed, and then the difference between the value 1 and the ratio is used as the chromatographic peak symmetry weight of the kth element;

[0076] Among them, the obtained ratio is always less than 1, so the numerical relationship of the weight can be adjusted by subtracting the ratio from the value 1; the smaller the final chromatographic peak symmetry weight, the more asymmetric the data representing the kth element position is, and this part is the data part with poor quality in the chromatogram.

[0077] Step 3: Based on the differences in the standard vectors of the chromatographic peaks of all adjacent bands and the differences in the chromatographic analysis vectors, a standard chromatographic feature vector and a chromatographic feature vector to be analyzed are constructed, and the element values ​​of the chromatographic feature vector to be analyzed are adjusted.

[0078] In the chromatographic peak analysis vector, when the chromatographic peak of tannic acid is close to the chromatographic peaks of other substances, chromatographic peak aliasing will occur. The tannic acid data of the aliased part will be interfered and it is difficult to be used as data for measuring the content of tannic acid components. Therefore, it is necessary to calculate the chromatographic peak aliasing interference intensity for each element in the chromatographic peak analysis vector, and screen out the data part with weak chromatographic peak aliasing interference to determine the content of tannic acid components.

[0079] Since different substances have different functional groups, they have different absorption capabilities for light of different wavelengths, that is, different substances have corresponding spectral characteristics; corresponding to the standard chromatogram, the absorbance variation law of tannic acid in the detector for light of different wavelengths at the same time is the same;

[0080] When chromatographic peak aliasing occurs in the feed chromatogram, the absorbance-light wavelength absorption law of the part not interfered by aliasing is the same as the absorbance-light wavelength absorption law in the standard chromatogram; the part interfered by aliasing is affected by other substances, and its absorption law of light of different wavelengths is determined by multiple substances including tannic acid, and its light absorption law changes, and the more severe the change, the more severe the chromatographic peak aliasing phenomenon of this part.

[0081] Therefore, the change law of absorbance-light wavelength is first calculated through the standard chromatogram. When calculating the law, the symmetry weight of the chromatographic peak should be taken into account, and the data part with high credibility should be selected to calculate the change law; further judge the data at different times in the feed chromatogram. The more inconsistent the time is with the obtained change law, the greater the chromatographic peak aliasing interference intensity of the data is, and the data at this time should not be used to determine the tannic acid component content.

[0082] Optionally, calculate the element mean value of the chromatographic peak standard vector of the nth wavelength band. During the calculation of the mean value, use the chromatographic peak symmetry weight of each element of the chromatographic peak standard vector of the nth wavelength band as the weight for calculating the mean value, and record the mean value as the standard absorbance of the nth wavelength band.

[0083] Among them, when calculating the standard absorbance, using the chromatographic peak symmetry weight as the weight value can partially eliminate the chromatogram data error caused by "tailing peak" or "front-extended peak".

[0084] Furthermore, when n < N, take the difference between the standard absorbances of the nth wavelength band and the (n - 1)th wavelength band as the nth element of the standard chromatographic feature vector. Correspondingly, obtain each element in the standard chromatographic feature vector, and form the standard chromatographic feature vector, which is used to characterize the law of the absorbance of tannic acid changing with the light wavelength characteristics. When the number of tannic acid molecules in the detector increases, the absorbance will increase as a whole, but the change law characterized in the feature vector will not change and remains relatively stable.

[0085] Furthermore, for the kth element of the N chromatographic peak analysis vectors, when n < N, take the difference between the kth elements of the nth wavelength band and the (n - 1)th wavelength band, and record it as the absorbance difference. Among the N chromatographic peak analysis vectors, all the absorbance differences obtained from the kth elements of the chromatographic peak analysis vectors corresponding to the subsequent wavelength and the previous wavelength of all adjacent wavelengths form the chromatographic feature vector to be analyzed of the kth element.

[0086] Furthermore, calculate the chromatographic peak aliasing interference intensity:

[0087] In this embodiment, the ratio of the element mean value of the standard chromatographic feature vector to the element mean value of the chromatographic feature vector to be analyzed at each moment is recorded as the absorption intensity ratio at each moment. Taking the kth element in the chromatographic peak analysis vector as an example, for the convenience of description, record the element mean value of the standard chromatographic feature vector as the standard absorbance intensity, and record the element mean value of the chromatographic feature vector to be analyzed corresponding to the kth element in the chromatographic peak analysis vector at a certain moment as the feed absorbance intensity at the kth moment; record the ratio of the standard absorbance intensity to the feed absorbance intensity at the kth moment as the absorption intensity ratio;

[0088] Furthermore, multiply the absorption intensity ratio by the standard chromatographic feature vector to obtain the chromatographic feature vector to be analyzed with adjusted element values, and complete the adjustment of the element values of the chromatographic feature vector to be analyzed.

[0089] Fourth step: Based on the difference between the standard chromatographic feature vector and the adjusted chromatographic feature vector to be analyzed, determine the chromatographic peak aliasing interference intensity at each moment corresponding to each element in the chromatographic peak analysis vector.

[0090] The distance between the standard chromatographic feature vector and the chromatographic feature vector to be analyzed at each moment after the element value is adjusted is determined as the chromatographic peak aliasing interference intensity at each moment. Specifically, in this embodiment, the Manhattan distance between the standard chromatographic feature vector and the chromatographic feature vector to be analyzed after the element value is adjusted for the kth element is calculated, and recorded as the chromatographic peak aliasing interference intensity at the moment corresponding to the kth element in the chromatographic peak analysis vector.

[0091] It should be understood that the greater the chromatographic peak aliasing interference intensity is, the more serious the chromatographic peak aliasing interference received by the feed chromatogram at the kth moment is, and the chromatographic data at that moment should not be used to obtain the final tannic acid content.

[0092] It should be noted that the elements in the chromatographic peak analysis vector correspond to the time one by one, each element corresponds to a time, and the value of each element is the absorbance of the feed sample extract at the corresponding time.

[0093] Step 5: Based on the difference in chromatographic peak aliasing interference intensity between each moment and its symmetric moment in the chromatographic peak analysis vector, combined with the chromatographic peak symmetry weight, determine the absorbance correction weight at each moment.

[0094] When analyzing the content of a substance through a liquid chromatograph, it is usually done by measuring the substance concentration and the chromatographic peak area of ​​the target detection object, constructing a regression curve through the two, and completing the quantitative analysis of the substance through the relationship between the regression curve reflecting the chromatographic peak area and the substance content.

[0095] In this embodiment, when chromatographic peaks are aliased due to interference from other substances in the feed, the chromatographic peak area in the chromatographic peak of tannic acid is greater than the actual value, so the peak area of ​​tannic acid needs to be corrected by the chromatographic peak aliasing interference intensity.

[0096] The schematic diagram of chromatographic peak aliasing interference is as follows Figure 2 As shown, the ordinate AU is the unit of absorbance, the abscissa is the unit of time, a is the standard solution chromatogram of 250 μg / mL stock solution, number 1 in a is the tannic acid chromatographic peak, b is the feed chromatogram of the feed sample extract, number 2 in b is the part where the chromatographic peak aliasing interference occurs, and in the absence of interference, the chromatographic peak of tannic acid should be as shown by the dotted line.

[0097] When correcting the tannic acid chromatographic peak, since the chromatographic peak of the interfering substance is usually located on one side of the tannic acid chromatographic peak, and as the time difference between the peak moments of the two chromatographic peaks increases, the intensity of the aliasing interference decreases sharply; therefore, when the interfering substance causes aliasing interference to the tannic acid chromatographic peak, the interference intensity is usually greater only on one side of the chromatographic peak, while the interference intensity on the other side is usually smaller or almost non-existent; therefore, for the time position where the aliasing interference intensity of the chromatographic peak is higher, the tannic acid chromatographic peak data at its symmetrical time can be used as a reference for data correction.

[0098] Step 6: Based on the absorbance at each moment and its symmetrical moment in the chromatographic peak analysis vector and combined with the absorbance correction weight, each absorbance is corrected to determine the tannic acid feed peak area.

[0099] In this embodiment, the tannic acid peak area will be analyzed in the detection period of the chromatographic peak standard vector and the chromatographic peak analysis vector. Further, for the chromatographic peak standard vector, there is no chromatographic peak aliasing interference caused by other substances in the feed, so the chromatographic peak standard vector is element-summed and recorded as the tannic acid standard peak area; at the same time, for the chromatographic peak analysis vector, considering that tannic acid has the strongest absorption capacity for light waves with a wavelength of A+199 nanometers, this embodiment will determine the tannic acid feed peak area based on each element in the chromatographic peak analysis vector of the Ath wavelength band, but considering the existence of interference, the specific acquisition process of the non-tannic acid feed peak area in the chromatographic peak analysis vector is as follows:

[0100] First, the aliasing interference intensities of K chromatographic peaks are normalized and recorded as aliasing interference weights. For the kth moment, the symmetric moment is K-k+1, and the aliasing interference weight of the former is subtracted from the aliasing interference weight of the latter, which is recorded as the aliasing weight difference. If the aliasing weight difference is positive, it means that the aliasing interference at moment k is stronger than that at moment K-k+1, and the data at moment k needs to be corrected. If the aliasing weight difference is negative, there is no need to correct the data at moment k, that is, the absorbance data at this moment remains unchanged, and there is no absorbance correction weight.

[0101] When the data at time k needs to be corrected, that is, the aliasing weight difference at time k is positive, the absorbance correction weight at time k is calculated to correct the absorbance data at time k. The absorbance correction weight is equal to the aliasing weight difference multiplied by the chromatographic peak symmetry weight. The larger the aliasing weight difference, the greater the impact of aliasing interference at time k, and the more symmetrical time data should be used for data correction; the larger the chromatographic peak symmetry weight, the better the data symmetry of the two symmetrical times when measuring the chromatographic peak, and the more the data at time k can be corrected with the symmetrical time data.

[0102] Finally, the corrected data at time k is equal to the data at time K-k+1 multiplied by the absorbance correction weight, plus the data before correction at time k multiplied by 1 minus the absorbance correction weight; correction operations are performed on each element in the chromatographic peak analysis vector of the Ath wavelength band at time K to achieve the correction of the chromatographic peak analysis vector.

[0103] After the chromatographic peak analysis vector of the Ath wavelength band is corrected according to the above process of this embodiment, the element sum of the corrected chromatographic peak analysis vector of the Ath wavelength band is calculated and recorded as the tannic acid feed peak area.

[0104] Step 7: Obtain a fitting function based on the concentration of standard tannic acid solutions of different concentrations and the standard peak area of ​​tannic acid, and obtain the tannic acid concentration in the feed extract by combining the peak area of ​​tannic acid feed.

[0105] First, the 250 μg / mL stock solution is gradiently diluted, that is, the standard tannic acid solution is diluted to multiple concentrations, optionally, diluted to concentrations of 0.5, 2, 5, 10, 15, 20, 30, and 50 μg / mL, respectively. The diluted stock solutions are measured on the machine respectively, and the measurement band is 199+A. The other measurement parameters are kept consistent with the first step, and the corresponding chromatograms can be obtained respectively.

[0106] Then, the above process and method of this embodiment are used to calculate the standard peak area of ​​tannic acid for each chromatogram.

[0107] Finally, for a stock solution with a concentration of B, it corresponds to a tannic acid standard peak area of ​​C, forming a coordinate data (B, C), and for stock solutions with multiple concentrations, there are multiple coordinate data, forming a quantitative data set. The quantitative data set is fitted to obtain a fitting function, which is used to characterize the corresponding relationship between concentration and area.

[0108] Specifically, in this embodiment, the quantitative data set is used as input, and the least squares method is used for fitting. The fitting equation adopts a quadratic polynomial based on experience, and the output is the fitted quadratic polynomial, which is recorded as the linear regression equation of tannic acid content: The least squares fitting method is a commonly used technology in the field of data processing. The specific fitting process is a prior art and will not be described in detail in this embodiment.

[0109] Preferably, in this embodiment, the tannic acid feed peak area is finally used as the input of the linear regression equation of tannic acid content, and the output function value is the tannic acid concentration in the feed extract. The total mass of tannic acid in the extract is then calculated based on the volume of the extract. Finally, the mass fraction of tannic acid in the feed is obtained by dividing the total mass of tannic acid by the mass of the selected feed sample to complete the content determination.

[0110] It is understood that references to "one embodiment" or "some embodiments" etc. described in the specification of the present application mean that one or more embodiments of the present application include specific features, structures or characteristics described in conjunction with the embodiment. Therefore, if "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. appear in different places in this specification, they do not necessarily refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "including", "comprising", "having" and their variations all mean "including but not limited to", unless otherwise specifically emphasized in other ways.

[0111] It should be noted that the sequence of the above-mentioned embodiments of the present application is only for description and does not represent the advantages and disadvantages of the embodiments. And the above-mentioned specific embodiments of this specification are described. In addition, the process depicted in the accompanying drawings does not necessarily require the specific order or continuous order shown to achieve the desired results. In some embodiments, multi-tasking and parallel processing are also possible or may be advantageous. At the same time, the size of the sequence number of each step in the embodiment does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments in this specification.

[0112] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application, and should all be included in the protection scope of the present application.

Claims

1. A high performance liquid chromatography analysis method for tannic acid component content, characterized in that: The method comprises the following steps: Obtain feed chromatograms of feed sample extracts at different wavelengths and standard chromatograms of standard tannic acid solutions at different wavelengths, and obtain analysis vectors of each chromatographic peak and standard vectors of each chromatographic peak, wherein each element in the vector is the absorbance of the solution at the corresponding moment; According to the symmetry of the distribution of each element in the chromatographic peak standard vector, the chromatographic peak symmetry weight of each element at the corresponding time is obtained; Based on the differences of the standard vectors of the chromatographic peaks of all adjacent bands and the differences of the chromatographic analysis vectors, a standard chromatographic characteristic vector and a chromatographic characteristic vector to be analyzed are constructed, and the element values ​​of the chromatographic characteristic vector to be analyzed are adjusted; Based on the difference between the standard chromatographic characteristic vector and the adjusted chromatographic characteristic vector to be analyzed, determining the chromatographic peak aliasing interference intensity at the corresponding time of each element in the chromatographic peak analysis vector; Based on the difference in chromatographic peak aliasing interference intensity at each moment and its symmetric moment in the chromatographic peak analysis vector, combined with the chromatographic peak symmetry weight, determine the absorbance correction weight at each moment; Based on the absorbance at each moment and its symmetrical moment in the chromatographic peak analysis vector and combined with the absorbance correction weight, each absorbance is corrected to determine the tannic acid feed peak area; A fitting function is obtained based on the concentration of standard tannic acid solutions with different concentrations and the standard peak area of ​​tannic acid, and the concentration of tannic acid in the feed extract is obtained by combining the peak area of ​​tannic acid feed; The construction method of the standard chromatogram characteristic vector and the chromatogram characteristic vector to be analyzed is: The weighted average value of all elements in the standard vector of each chromatographic peak is analyzed and recorded as the standard absorbance of each band, wherein the chromatographic peak symmetry weight of each element is used as the weighted weight of each element; the difference between the standard absorbance of the latter band and the previous band in all adjacent bands is used to construct the standard chromatographic characteristic vector; The difference between the elements at the same position in the chromatographic peak analysis vectors of the latter band and the previous band in all adjacent bands is used to form the chromatographic characteristic vector to be analyzed at the time corresponding to each same position; The step of adjusting the element values ​​of the chromatographic characteristic vector to be analyzed further comprises: The ratio of the element mean of the standard chromatographic characteristic vector to the element mean of the chromatographic characteristic vector to be analyzed at each moment is recorded as the absorption intensity ratio at each moment, and the absorption intensity ratio at each moment is multiplied by the standard chromatographic characteristic vector to obtain the chromatographic characteristic vector to be analyzed at each moment after the element value is adjusted.

2. The high performance liquid chromatography analysis method for tannic acid content according to claim 1, characterized in that: The method for obtaining the standard vector of each chromatographic peak and the analysis vector of each chromatographic peak is: Analyze the sum of the elements of each vector corresponding to each standard chromatogram, record the vector with the largest sum of elements as the optimal band vector, and record the corresponding wavelength as the optimal wavelength; take the time when the peak of the optimal band vector is located as the standard retention time, select two time points symmetrical about the time when the peak is located, and the time period composed of them is used as the standard detection time period T, and all elements of each standard chromatogram in the detection time period are used to form the standard vector of each chromatographic peak; For each feed chromatogram, obtain the mean vector corresponding to all vectors, calculate the time difference between the corresponding time of each extreme point in the mean vector and the standard retention time, and take the time corresponding to the minimum time difference as the retention time of the feed chromatogram , the detection period of feed chromatogram is , all elements in each feed chromatogram during the detection period are combined into analysis vectors for each chromatographic peak.

3. The high performance liquid chromatography analysis method for tannic acid content according to claim 2, characterized in that: The method for obtaining the chromatographic peak symmetry weight is: For each element in the standard vector of the chromatographic peak of each band, the difference between each element and the element symmetrical with respect to the standard retention time is analyzed and recorded as the absorbance difference, and the sum of each element and the element symmetrical with respect to the standard retention time is calculated and recorded as the absorbance sum value; The ratio of the absorbance difference to the absorbance sum value is analyzed, and the difference between the value 1 and the ratio is determined as the chromatographic peak symmetry weight of each element at the corresponding time.

4. The high performance liquid chromatography analysis method for tannic acid content according to claim 1, characterized in that: The method for determining the chromatographic peak aliasing interference intensity is: The distance between the standard chromatographic characteristic vector and the chromatographic characteristic vector to be analyzed at each moment after the element value is adjusted is determined as the chromatographic peak aliasing interference intensity at each moment.

5. The high performance liquid chromatography analysis method for tannic acid content according to claim 2, characterized in that: The symmetric moments of each moment in the chromatographic peak analysis vector are moments at which each moment is symmetric with respect to the retention time of the feed chromatogram.

6. The high performance liquid chromatography analysis method for tannic acid content according to claim 5, characterized in that: The method for determining the absorbance correction weight at each moment is: The difference between the chromatographic peak aliasing interference intensity at each moment and the symmetric moment in the chromatographic peak analysis vector is determined as the aliasing weight difference at each moment; If the aliasing weight difference is positive, the absorbance correction weight is equal to the product of the aliasing weight difference and the chromatographic peak symmetry weight; If the aliasing weight difference is negative, the absorbance correction weight at the corresponding moment is not calculated.

7. The high performance liquid chromatography analysis method for tannic acid content according to claim 6, characterized in that: The correction of each absorbance further comprises: If the aliasing weight difference is positive, the absorbance at each moment after correction is the sum of the absorbance at its symmetrical moment multiplied by the absorbance correction weight and the absorbance at each moment before correction multiplied by 1 minus the absorbance correction weight; If the aliasing weight difference is negative, the absorbance at the corresponding moment remains unchanged.

8. The high performance liquid chromatography analysis method for tannic acid content according to claim 2, characterized in that: The tannic acid feed peak area is the element sum of the chromatographic peak analysis vector after correction corresponding to the optimal wavelength, and the tannic acid standard peak area is the element sum of the chromatographic peak standard vector.

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