CN112432906B - Chiral substance qualitative and quantitative analysis method based on circular dichroism spectrum technology - Google Patents

Chiral substance qualitative and quantitative analysis method based on circular dichroism spectrum technology Download PDF

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CN112432906B
CN112432906B CN202010978151.XA CN202010978151A CN112432906B CN 112432906 B CN112432906 B CN 112432906B CN 202010978151 A CN202010978151 A CN 202010978151A CN 112432906 B CN112432906 B CN 112432906B
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应见喜
赵玉芬
丁瑞文
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Abstract

The invention discloses a circular dichroism spectrum technology-based method for qualitatively and quantitatively analyzing a chiral substance, which comprises the following steps: 1) quickly establishing a first standard curve by using a circular dichroism spectrum technology to obtain a concentration difference value of a levorotatory compound and a dextrorotatory compound in a chiral substance; 2) establishing a second standard curve by using a high performance liquid chromatography technology to obtain the concentration and the value of a levorotatory compound and a dextrorotatory compound in the chiral substance; 3) finally, the concentrations of the levorotatory compound and the dextrorotatory compound in the chiral substance are obtained through simple calculation, and qualitative and quantitative analysis is carried out; the method has the advantages that the concentration of the enantiomer in the chiral substance can be rapidly determined without adding a chiral reagent, the in-situ qualitative and quantitative analysis is realized, the sample is recovered without pollution, the operation is simple and convenient, the cost is low, the calculation is simple, the chiral purity of each batch of raw material medicines can be rapidly analyzed particularly in the field of detection of chiral raw material medicines, and the method is suitable for popularization and utilization.

Description

Chiral substance qualitative and quantitative analysis method based on circular dichroism spectrum technology
Technical Field
The invention belongs to the technical field of chemical detection, and particularly relates to a chiral substance qualitative and quantitative analysis method based on a circular dichroism spectrum technology.
Background
There are a large number of chiral substances in nature, including many biological molecules and drugs in the body of a living being. The raw materials used for synthesizing protein in organisms are all L-type amino acids, the nucleosides for synthesizing nucleic acid are all D-type, and the corresponding raw materials with opposite chirality cannot be used for synthesizing protein or nucleic acid; in the field of medicine, as people have been intensively studying chiral drugs, different isomers, especially enantiomers of chiral drugs are gradually recognized to have different and even opposite pharmacological and toxicological effects. Therefore, the development of qualitative and quantitative analysis methods for chiral substances is undoubtedly of great significance.
Commonly used methods for qualitative and quantitative analysis of chiral substances are: high Performance Liquid Chromatography (HPLC), Mass Spectrometry (MS), Nuclear Magnetic Resonance (NMR), and the like; wherein, the HPLC method carries out qualitative and quantitative analysis on the chiral molecules by means of chiral chromatographic columns or pre-column derivatization and the like; the MS method is based on the formation of different complexes of chiral additives with different isomers of chiral molecules, different complexes having different ionization efficiencies or cleavage laws, thereby exhibiting differences in mass-to-charge ratios (m/z) in the mass spectra; NMR methods, similar to MS methods, also require the assistance of chiral reagents to achieve qualitative and quantitative analysis of chiral molecules.
However, the HPLC method requires a chiral chromatographic column or pre-column derivatization for analyzing chiral substances, the chiral chromatographic column is expensive and has low universality, and different chiral molecules require different elution procedures or different chiral chromatographic columns to complete qualitative and quantitative analysis; in addition, for chiral substances without chromophoric groups or reasons such as too large or too small polarity, pre-column derivatization is also needed, and the pre-column derivatization also has the problems of high cost and low universality; both the MS method and the NMR method need to add a proper chiral reagent into a chiral substance sample, and then find out a regularity result from a spectrogram, on one hand, instruments and the added chiral reagent are generally expensive, and still have the problems of high cost and poor universality, on the other hand, data processing in the later period needs corresponding professional knowledge to better analyze and obtain a result, and the problems of complicated experimental steps and inconvenient operation exist. Therefore, in recent years, many researchers have focused on the application of the HPLC-CD method to qualitative and quantitative analysis for detecting chiral substances.
The HPLC-CD method is characterized in that a circular dichroism spectrometer and a high performance liquid chromatograph are used together, circular dichroism spectrum signals and chromatographic peak areas are respectively measured, and the purity of enantiomers in chiral substances is obtained through calculation; however, the detection method in the prior art still has the problems that an additive is needed as an internal standard, an original sample is easily polluted, the operation is complicated, and the calculation is complex, particularly in the field of detection of chiral raw material medicines, the chiral purity of each batch of raw material medicines is often needed to be rapidly analyzed, and the detection method in the prior art is difficult to popularize and utilize.
Disclosure of Invention
The invention provides the qualitative and quantitative analysis method of the chiral substance based on the circular dichroism spectrum technology, which does not need an additive as an internal standard, has simple and convenient operation, low cost and simple calculation, can quickly analyze the chiral purity of each batch of raw material medicines particularly in the field of detection of chiral raw material medicines, and is suitable for popularization and utilization.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a qualitative and quantitative analysis method of chiral substances based on circular dichroism spectroscopy comprises the following steps:
1) preparing a series of target chiral compound standard solutions to be detected with different concentration gradients, measuring a first signal value of each target chiral compound standard solution to be detected through a circular dichroism spectrometer, and drawing a first linear standard relation curve graph of the first signal value and corresponding concentration to obtain a first fitting equation;
2) measuring a second signal value of each target chiral compound standard solution to be measured through a high performance liquid chromatograph, and drawing a second linear relation curve chart of the second signal value and corresponding concentration to obtain a second fitting equation; wherein the HPLC should avoid using chiral chromatographic column and mobile phase containing chiral additive;
3) preparing a sample to be detected with a certain concentration, wherein the certain concentration is in the concentration gradient range of the standard solution of the target compound to be detected in the step 1), measuring a first signal value of the target chiral compound in the sample to be detected by a circular dichroism spectrometer, and calculating the concentration delta c of the target chiral compound in the sample to be detected by using the first fitting equation in the step 1)CD；
4) Measuring a second signal value of the target chiral compound in the sample to be measured in the step 3) through a high performance liquid chromatograph, and calculating the concentration delta c of the target chiral compound in the sample to be measured through the second fitting equation in the step 2)LC；
5) The concentration of the levorotatory compound in the enantiomer of the target chiral compound to be determined is denoted as cLThe concentration of the dextrorotatory compound in the optical enantiomer of the target chiral compound to be measured is denoted as cDTwo binary first-order equations are obtained, which are respectively Delta cCD=|cL-cD|，ΔcLC=cL+cD；
6) Respectively calculating the concentration c of the levorotatory compound in the enantiomer of the target chiral compound to be detected according to the two linear equations of the step 5)LAnd concentration c of dextrorotatory compoundDJudging c) according to the first signal value of each target chiral compound standard solution to be detected in the step 1) and the first signal value of the target chiral compound in the sample to be detected in the step 3)LAnd cDThe size of (2).
The first signal value in the step 1) is an ellipticity corresponding to the highest point of the circular dichroism peak of each detected target chiral compound standard solution to be detected, a linear standard relation curve graph of the ellipticity corresponding to the highest point of the circular dichroism peak and the corresponding concentration is drawn, and a fitting equation of the ellipticity corresponding to the highest point of the circular dichroism peak and the concentration is obtained; the first signal value of the target chiral compound in the sample to be detected in the step 3) is the ellipticity at the wavelength corresponding to the highest point of the circular dichroism peak of the standard solution of the target chiral compound to be detected.
The second signal value in the step 2) is an HPLC signal peak area integral value of each detected target chiral compound standard solution to be detected, a linear standard relation curve graph of the signal peak area integral value and corresponding concentration is drawn, and a fitting equation of the signal peak area integral value and the concentration is obtained; the second signal value of the target chiral compound in the sample to be detected in the step 4) is an HPLC signal peak area integral value of the target chiral compound in the sample to be detected.
The high performance liquid chromatograph in the step 2) is further connected with an ultraviolet detector, the second signal value is the absorbance of each detected target chiral compound standard solution to be detected, a linear standard relation curve graph of the absorbance and the corresponding concentration is drawn, and a fitting equation of the absorbance and the concentration is obtained; and 4) the second signal value of the target chiral compound in the sample to be detected in the step 4) is the absorbance of the target compound to be detected of the sample to be detected.
In the step 3), the sample to be detected is: when the sample is solid, dissolving in a solvent to form a sample to be detected; when the sample is liquid, the concentration gradient range of the target chiral compound standard solution to be detected is prepared.
Compared with the prior art, the invention has the advantages that:
1. the method is characterized in that qualitative and quantitative analysis is carried out on the chiral substances based on a circular dichroism spectrum technology method, a first standard curve is quickly established by utilizing the circular dichroism spectrum technology to obtain the concentration difference value of a levorotatory compound and a dextrorotatory compound in the chiral substances, a second standard curve is established by utilizing the high performance liquid chromatography technology to obtain the concentration sum value of the levorotatory compound and the dextrorotatory compound in the chiral substances, and finally, the obtained calculation result of a binary linear equation can be utilized to carry out qualitative and quantitative analysis on the levorotatory compound and the dextrorotatory compound in the chiral substances; as mentioned above, in the prior art, additives are often required to be used as internal standards, and the content of enantiomers in chiral substances is determined by a complex calculation method, but the method of the invention can quickly determine the concentration of the enantiomers in the chiral substances without adding a chiral reagent, thereby realizing in-situ qualitative and quantitative analysis and recovering samples without pollution.
2. According to the invention, the chiral substances are qualitatively and quantitatively analyzed based on the circular dichroism spectrum technology method, and for a pure chiral molecular sample, a standard curve of the circular dichroism spectrum signal and the concentration of a target chiral compound standard solution to be detected can be rapidly obtained, so that the chirality and the concentration of the sample can be qualitatively and quantitatively determined; for an enantiomer mixture sample, different components in the sample can be quantified by combining an achiral HPLC technology, and different enantiomers can be qualitatively and quantitatively analyzed by the method; the method adopted by the invention is rapid, simple and convenient, and simultaneously, the instrument adopted for detection has lower cost and better universality, is particularly suitable for the field of chiral bulk drugs, and can rapidly analyze the chiral purity of each batch of bulk drugs.
3. The method is based on the circular dichroism spectrum technology method to carry out qualitative and quantitative analysis on the chiral substances, and if achiral impurities exist in the sample, the achiral impurities do not have circular dichroism spectrum signals, so that the test result is not influenced; if chiral impurities exist in the sample, the chiral impurities still have no obvious influence on the test result as long as the position of the circular dichroism spectrum peak of the chiral impurities is different from that of the target chiral compound or the position of the high-speed liquid chromatography peak of the chiral impurities is different from that of the target chiral compound, so that the purpose of quantitative and qualitative analysis of the chiral substances is achieved.
Drawings
FIG. 1 is a circular dichroism spectrum of a standard solution of L-Leu of the present invention, which was measured at concentrations of 0.005M, 0.01M, 0.015M, 0.02M, and 0.025M, respectively;
FIG. 2 is a graph showing the linear relationship between the ellipticity θ of the circular dichroism spectrum peak signal and the concentration c at a wavelength of 200nm in the standard solution for L-Leu test in the present invention;
FIG. 3 is a graph showing the linear relationship between the peak area integral value of HPLC signal at a wavelength of 210nm and the concentration c for the standard solution for L-Leu tested in the present invention;
FIG. 4 is a circular dichroism spectrum of an L-Pro standard solution of the present invention tested at concentrations of 0.024M, 0.03M, 0.036M, 0.042M, and 0.048M, respectively;
FIG. 5 is a graph of the linear dependence of the ellipticity θ of the signal of the circular dichroism spectrum peak at a wavelength of 213nm on the concentration c of a test L-Pro standard solution of the present invention;
FIG. 6 is a graph of the linear relationship between the peak area integral value of HPLC signal at a wavelength of 210nm and the concentration c for the test L-Pro standard solution of the present invention.
Detailed Description
The signal detected by circular dichroism spectroscopy (ECD) is usually expressed by an ellipticity θ, which has the following relationship with the concentration c of an enantiomer in a chiral substance:
θ=3298•Δε•c•l
Figure 511816DEST_PATH_IMAGE001
c=θ/(3298•Δε•l)
wherein c is the concentration of enantiomer in the chiral substance, theta is the ellipticity, delta epsilon is the molar extinction coefficient, and l is the thickness of the medium; therefore, the ellipticity θ has a certain linear relationship with c, i.e., a corresponding first standard curve can be established by the concentration c of the enantiomer standard substance in the chiral substance and the ellipticity θ of the corresponding circular dichroism spectrum peak signal.
Enantiomers in chiral compounds, also called optical isomers, are stereoisomers that are not superimposable but physical and mirror images of each other; the enantiomers all have optical activity and can be divided into levorotatory compound L and dextrorotatory compound D, so that the concentration c of the levorotatory compoundLAnd levorotatory compound thetaLConcentration c of L-CompoundDAnd dextrorotatory compound thetaDThe following relationships are respectively provided:
cL=θLv (3298. DELTA. epsilon. l) and cD=θD/(3298•Δε•l)
The signal measured by the circular dichroism spectrometer is the signal reflected by the cancellation of the peak signals of the circular dichroism spectrums which are enantiomers mutually in the sample, so that the concentration delta c of the enantiomer mixture obtained by the circular dichroism spectrumCDCan be expressed by equation 1:
equation 1: Δ cCD=|cL-cD|
The achiral HPLC technology is an HPLC technology which does not use a chiral chromatographic column and does not contain a mobile phase of a chiral additive, and a corresponding second standard curve can be established by the concentration c of an enantiomer standard substance in a chiral substance and a corresponding achiral HPLC signal; the enantiomers of a chiral substance are perfectly superposed on an achiral chromatographic column, i.e. the concentration Δ c of the mixture of enantiomers is obtained by achiral HPLCLCCan be expressed by equation 2:
equation 2: Δ cLC=cL+cD
The concentrations c of the two enantiomeric components of the chiral compound can be solved by two quadratic equations of one unity of formula 1 and formula 2LAnd cDThe size of (2).
If the sample to be detected is a homochiral molecule, the Delta c can be calculated by the standard curve of the ECDCDWhen this is substituted into the formula 1, Δ cCDI.e. the concentration of the homochiral molecules; if the sample to be detected is the enantiomer mixture, performing ECD and HPLC detection respectively, and passing through the first standard curve and the second standard curve of ECDSecond standard curve of HPLC gave respectively Δ cCDAnd Δ cLCAnd combining the formula 1 and the formula 2 to calculate the concentration of each component of the enantiomer.
The invention is described in further detail below with reference to embodiments of the drawings.
(1) An experimental instrument: circular dichroism spectrometer (JASCOJ-1700), high performance liquid chromatography (Agilent 1260)
(2) Experimental materials: l-leucine (L-Leu), L-proline (L-Pro), D-leucine (D-Leu), D-proline (D-Pro), methanol, distilled water or deionized water
Example one
Determination of L-leucine (L-Leu) concentration by circular dichroism
Accurately weighing 26.23mg of L-leucine (L-Leu), and adding 2mL of distilled water or deionized water to serve as stock solution; then, the samples were sequentially diluted to standard solutions of 0.005M, 0.01M, 0.015M, 0.02M and 0.025M, and circular dichroism spectra of the respective L-Leu standard solutions were obtained by circular dichroism spectrum detection, and as shown in FIG. 1, a circular dichroism peak of a positive signal was observed at a wavelength of 200nm for L-Leu.
Respectively taking the ellipticity theta of the circular dichroism peak at the wavelength of 200nm and drawing a linear standard relation curve chart with the corresponding concentration c as shown in figure 2, thereby obtaining a fitting equation y =0.0002x-0.0005 of the ellipticity theta of the circular dichroism peak at the wavelength of 200nm and the concentration c, wherein R2=0.9993, indicating that the degree of linear correlation between θ and c is good.
Accurately weighing 1.05mg, 1.71mg and 2.36mg of L-Leu in a test tube, adding 1mL of distilled water or deionized water to obtain L-Leu solutions with the concentrations of 0.008 mol/L, 0.013 mol/L and 0.018 mol/L respectively, carrying out ECD detection to obtain the ellipticity theta of the circular dichroism peak at the wavelength of 200nm, substituting the ellipticity theta into a fitting equation y =0.0002x-0.0005 to obtain experimental values, and comparing the experimental values with theoretical values to obtain the results shown in Table 1.
TABLE 1 comparison of the experimental values with the theoretical values for L-Leu solutions of different concentrations
Figure 661037DEST_PATH_IMAGE002
Wherein Δ is the difference between the experimental value and the theoretical value, where Δ = | experimental value-theoretical value |/theoretical value 100%;
and (4) experimental conclusion: from the experimental results, the concentration of L-Leu can be rapidly and accurately measured by using the ECD technology, and the error is less than 10%; namely, if the sample to be detected is a pure chiral molecule, the content of the chiral molecule can be directly calculated through the standard curve of the ECD.
Example two
Determination of the concentration of the Components of the enantiomeric mixture of Leu
The enantiomers of Leu are two, namely, L-Leu, D-Leu; the absorption peaks of L-Leu and D-Leu on achiral chromatographic column are overlapped, and achiral HPLC determination is performed by using L-Leu standard solution of five concentrations in example one, wherein achiral HPLC technique is to plot the integral value of the peak area of the signal at 210nm as a linear relationship with the concentration without using chiral chromatographic column and mobile phase containing no chiral additive, as shown in FIG. 3, to obtain the fitting equation y =4E-5x-0.001, wherein R2=0.9991, indicating that the degree of linear correlation of the signal peak area integrated value with concentration is good.
Determination of the concentration of mixture a: respectively taking L-Leu and D-Leu with the concentration of 0.008M, mixing according to the volume ratio of 60:40 to obtain a mixture A, wherein the concentration of the L-Leu is marked as cL-LeuAnd the concentration of D-Leu is denoted as cD-Leu(ii) a Performing ECD detection on the mixture A to obtain the ellipticity theta of the circular dichroism spectrum peak at the wavelength of 200nm, substituting the ellipticity theta into a fitting equation y =0.0002x-0.0005, and calculating the concentration delta cCDThen, equation 1 is obtained: Δ cCD=|cL-Leu -cD-Leu |；
Detecting the mixture A by achiral HPLC to obtain the peak area integral value of the signal with the wavelength of 210nm, substituting the integral value into a fitting equation y =4E-5x-0.001, and calculating the concentration delta cLCThen, equation 2 is obtained: Δ cLC=cL-Leu +cD-Leu；
C is calculated by two linear equations of formula 1 and formula 2L-LeuAnd cD-LeuThe values of (a) and (b) are combined with a circular dichroism diagram of the L-Leu standard solution shown in figure 1, namely, the concentrations of the enantiomers L-Leu and D-Leu in the mixture A are respectively obtained;
determination of the concentration of mixture B: L-Leu and D-Leu, both at a concentration of 0.013M, were mixed at a volume ratio of 60:40 to give mixture B, and the remaining concentrations were determined to be the same as those of mixture A.
The results obtained by comparing the experimental values of the measured concentrations of L-Leu and D-Leu in mixture A and mixture B with the theoretical values are shown in Table 2.
TABLE 2 comparison of the experimental values with the theoretical values for the concentrations of L-Leu and D-Leu in mixture A and mixture B
Figure 197061DEST_PATH_IMAGE003
Where Δ is the difference between the experimental value and the theoretical value, and Δ = | experimental value-theoretical value |/theoretical value 100%.
And (4) experimental conclusion: from the above experimental results, it can be seen that the concentrations of the components of the enantiomeric mixture of Leu can be rapidly and accurately measured by using ECD technology in combination with achiral HPLC technology; respectively carrying out ECD and achiral HPLC detection if the sample to be detected is an enantiomer mixture, and respectively obtaining deltac through a first standard curve of ECD and a second standard curve of HPLCCDAnd Δ cLCAnd combining the formula 1 and the formula 2 to calculate the concentration of each component of the enantiomer.
EXAMPLE III
The hplc of the second embodiment is further connected to an ultraviolet detector, to obtain the absorbance at the wavelength of 210nm of the L-Leu standard solutions of the five concentrations in the first embodiment, and plot the linear relationship between the absorbance of each L-Leu standard solution and the corresponding concentration to obtain a fitting equation of the absorbance and the concentration, and the rest is the same as the first embodiment.
Example four
Determination of L-type proline (L-Pro) concentration by ECD technology
Accurately weighing 27.63mg of L-proline (L-Pro), and adding 2mL of distilled water or deionized water to serve as stock solution; after dilution sequentially to the standard solutions of 0.024M, 0.03M, 0.036M, 0.042M and 0.048M in concentration, respectively, the circular dichroism spectra of the respective L-Pro standard solutions were examined by circular dichroism spectroscopy, and as shown in FIG. 4, it was found that L-Pro exhibited a circular dichroism peak exhibiting a negative signal at a wavelength of 194nm and a circular dichroism peak exhibiting a positive signal at 213 nm.
Respectively taking the ellipticity theta of the circular dichroism peak at the wavelength of 213nm and drawing a linear standard relation curve graph with the corresponding concentration c, as shown in fig. 5, obtaining a fitting equation y =0.001x +2E-06 of the ellipticity theta of the circular dichroism peak at the wavelength of 213nm and the concentration c, wherein R2=0.9998, indicating that the degree of linear correlation of θ with c is good.
Accurately weighing 4.61mg, 2.76mg and 3.57mg of L-proline (L-Pro) in a test tube, adding 1mL of distilled water or deionized water to obtain L-Pro solutions with the concentrations of 0.04 mol/L, 0.025 mol/L and 0.032 mol/L respectively, carrying out ECD detection to obtain the ellipticity theta at 213nm, substituting the ellipticity theta into a fitting equation y =0.001x +2E-06 to obtain experimental values, and comparing the experimental values with theoretical values, wherein the results are shown in Table 3.
TABLE 3 comparison table of actual values and experimental values of L-Pro solutions of different concentrations
Figure 369416DEST_PATH_IMAGE004
Where Δ is the difference between the experimental value and the theoretical value, and Δ = | experimental value-theoretical value |/theoretical value 100%.
And (4) experimental conclusion: from the above experimental results, it can be known that the ECD technique can be used to rapidly and accurately measure the L-Pro concentration with an error of less than 10%.
EXAMPLE five
Determination of the concentration of the Components of the enantiomeric mixture of Pro
Pro enantiomers are of two kindsI.e., L-Pro and D-Pro; the absorption peaks of L-Pro and D-Pro on the achiral chromatographic column are overlapped, achiral HPLC measurement is carried out by using L-Pro solutions with five concentrations in example four, and the integrated value of the peak area of the signal peak of L-Pro at the wavelength of 210nm measured by HPLC is plotted with the concentration to obtain a standard curve chart, as shown in FIG. 6, and the fitting equation is y =8E-5x-0.0005, wherein R is2=0.9996, showing that the degree of linear correlation of the signal peak area integrated value with the concentration is good.
Determination of the concentration of mixture C: respectively taking L-Pro and D-Pro with the concentration of 0.025M, and mixing according to the volume ratio of 60:40 to obtain a mixture C, wherein the concentration of the L-Pro is recorded as CL-proAnd the concentration of D-Pro is denoted as cD-proThe remainder was determined in the same manner as the concentration of mixture A.
Determination of the concentration of mixture D: the mixture D was obtained by mixing L-Pro and D-Pro each having a concentration of 0.032M at a volume ratio of 60:40, and the rest was the same as the measurement of the concentration of the mixture A.
The results obtained by comparing the experimental values of the measured concentrations of L-Leu and D-Leu in mixture A and mixture B with the theoretical values are shown in Table 4.
TABLE 4 comparison of the experimental values with the theoretical values of the L-Pro and D-Pro concentrations in enantiomeric mixtures of Pro
Figure 383508DEST_PATH_IMAGE005
Where Δ is the difference between the experimental value and the theoretical value, and Δ = | experimental value-theoretical value |/theoretical value 100%.
And (4) experimental conclusion: from the above experimental results, it can be seen that the concentration of each component of the enantiomeric mixture of Pro can be rapidly and accurately measured using ECD techniques in combination with achiral HPLC techniques.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention are within the scope of the present invention, and the scope of the present invention is subject to the claims.

Claims (3)

1. A qualitative and quantitative analysis method of chiral substances based on circular dichroism spectroscopy is characterized by comprising the following steps:
1) preparing a series of target chiral compound standard solutions to be detected with different concentration gradients, measuring a first signal value of each target chiral compound standard solution to be detected through a circular dichroism spectrometer, wherein the first signal value is an ellipticity corresponding to the highest point of a circular dichroism peak of each target chiral compound standard solution to be detected, drawing a linear standard relation curve graph of the ellipticity corresponding to the highest point of the circular dichroism peak and the corresponding concentration, and obtaining a first fitting equation;
2) measuring a second signal value of each target chiral compound standard solution to be detected through a high performance liquid chromatograph, wherein the second signal value is a peak area integral value of an HPLC signal of each target chiral compound standard solution to be detected, and drawing a linear standard relation curve graph of the peak area integral value of the signal and the corresponding concentration to obtain a second fitting equation; wherein the HPLC should avoid using chiral chromatographic column and mobile phase containing chiral additive;
3) preparing a sample to be detected with a certain concentration, wherein the certain concentration is in the concentration gradient range of the standard solution of the target compound to be detected in the step 1), and measuring a first signal value of the target chiral compound in the sample to be detected by using a circular dichroism spectrometer, wherein the first signal value of the target chiral compound in the sample to be detected is the ellipticity of the wavelength corresponding to the highest point of the circular dichroism peak of the standard solution of the target chiral compound to be detected; calculating the concentration delta cCD of the target chiral compound in the sample to be detected according to the first fitting equation in the step 1);
4) measuring a second signal value of the target chiral compound in the sample to be measured in the step 3) by using a high performance liquid chromatograph, wherein the second signal value of the target chiral compound in the sample to be measured is an HPLC signal peak area integral value of the target chiral compound in the sample to be measured; calculating to obtain the concentration delta cLC of the target chiral compound in the sample to be detected according to the second fitting equation in the step 2);
5) recording the concentration of a levorotatory compound in an enantiomer of a target chiral compound to be detected as cL and the concentration of a dextrorotatory compound in an optical enantiomer of the target chiral compound to be detected as cD to obtain two binary first-order equations, wherein the two binary first-order equations are respectively delta cCD ═ cL-cD |, and delta cLC ═ cL + cD;
6) respectively calculating the concentration cL of the levorotatory compound and the concentration cD of the dextrorotatory compound in the enantiomer of the target chiral compound to be detected according to the two linear equations of the step 5), and judging the magnitudes of cL and cD according to the first signal value of each target chiral compound standard solution to be detected in the step 1) and the first signal value of the target chiral compound in the sample to be detected in the step 3).
2. The method according to claim 1, wherein the HPLC in step 2) is further connected to an UV detector, the second signal value is the absorbance of each detected target chiral compound standard solution, a linear standard relationship curve of absorbance and corresponding concentration is plotted, and a fitting equation of absorbance and concentration is obtained; and 4) the second signal value of the target chiral compound in the sample to be detected in the step 4) is the absorbance of the target compound to be detected of the sample to be detected.
3. The method for qualitative and quantitative analysis of chiral substance based on circular dichroism spectroscopy as claimed in claim 1, wherein in step 3), the sample to be tested is: when the sample is solid, dissolving in a solvent to form a sample to be detected; when the sample is liquid, the concentration gradient range of the target chiral compound standard solution to be detected is prepared.
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