CN114062518A - Method for separating and measuring tartaric acid optical isomer content - Google Patents
Method for separating and measuring tartaric acid optical isomer content Download PDFInfo
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- CN114062518A CN114062518A CN202010742393.9A CN202010742393A CN114062518A CN 114062518 A CN114062518 A CN 114062518A CN 202010742393 A CN202010742393 A CN 202010742393A CN 114062518 A CN114062518 A CN 114062518A
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- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 235000002906 tartaric acid Nutrition 0.000 title claims abstract description 57
- 239000011975 tartaric acid Substances 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 30
- 230000003287 optical effect Effects 0.000 title claims abstract description 16
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 38
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 38
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims abstract description 32
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims abstract description 32
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 16
- 239000007983 Tris buffer Substances 0.000 claims abstract description 13
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 11
- 238000010829 isocratic elution Methods 0.000 claims abstract description 4
- 239000000523 sample Substances 0.000 claims description 51
- 239000012488 sample solution Substances 0.000 claims description 35
- 239000007788 liquid Substances 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 26
- 238000000926 separation method Methods 0.000 claims description 23
- 238000005303 weighing Methods 0.000 claims description 21
- 238000001514 detection method Methods 0.000 claims description 19
- 238000007865 diluting Methods 0.000 claims description 13
- 239000003960 organic solvent Substances 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 2
- 239000012046 mixed solvent Substances 0.000 claims description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 abstract description 80
- 229960001367 tartaric acid Drugs 0.000 abstract description 53
- 229960001270 d- tartaric acid Drugs 0.000 abstract description 26
- 239000000126 substance Substances 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 3
- FEWJPZIEWOKRBE-LWMBPPNESA-N levotartaric acid Chemical compound OC(=O)[C@@H](O)[C@H](O)C(O)=O FEWJPZIEWOKRBE-LWMBPPNESA-N 0.000 description 34
- 238000002474 experimental method Methods 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 7
- 239000012535 impurity Substances 0.000 description 7
- 230000003595 spectral effect Effects 0.000 description 7
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- FEWJPZIEWOKRBE-XIXRPRMCSA-N Mesotartaric acid Chemical compound OC(=O)[C@@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-XIXRPRMCSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- SXFBQAMLJMDXOD-VNQWSFCKSA-N C([C@H](O)[C@@H](O)C(=O)O)(=O)O.C([C@H](O)[C@@H](O)C(=O)O)(=O)O Chemical compound C([C@H](O)[C@@H](O)C(=O)O)(=O)O.C([C@H](O)[C@@H](O)C(=O)O)(=O)O SXFBQAMLJMDXOD-VNQWSFCKSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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Abstract
The invention relates to the technical field of chemical analysis, in particular to a method for separating and determining tartaric acid optical isomer content. The invention provides a method for separating and measuring tartaric acid optical isomer content, which is carried out by utilizing a high performance liquid chromatography, and the analysis conditions are as follows: amylose-tris [3, 5-dimethylcarbamate ] derivative is used as a stationary phase; n-hexane, isopropanol, tert-butanol and trifluoroacetic acid are taken as mobile phases, isocratic elution is carried out, L-tartaric acid and D-tartaric acid can be well resolved simply and rapidly, and the contents of the two substances are determined.
Description
Technical Field
The invention relates to the technical field of chemical analysis, in particular to a method for separating and determining tartaric acid optical isomer content.
Background
Tartaric acid exists in three optical isomers: levotartaric acid (D-tartaric acid), dextrotartaric acid (L-tartaric acid) and meso-tartaric acid (meso-tartaric acid). Equimolar levotartaric acid and dextrotartaric acid are mixed to form racemic tartaric acid. In the field of medicine, the control of the impurity content of raw materials is also one of the key steps for controlling the quality of medicines. In order to improve the production quality of medicines, the relative contents of L-tartaric acid and D-tartaric acid in a tartaric acid sample need to be analyzed and detected, and at present, a relatively simple and mature tartaric acid optical isomer separation and detection method is not seen for a while.
In the prior art, patent CN109738560A discloses a method for determining the content of optical isomers of tartaric acid, which comprises derivatizing tartaric acid to be detected, and detecting with a high performance liquid chromatograph combined with an ultraviolet detector. The method has complex detection process and high cost.
Therefore, it is highly desirable to develop a method for separating L-tartaric acid from D-tartaric acid and determining the contents thereof simply, rapidly and conveniently.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide a method for separating and determining the content of optical isomers of tartaric acid, which can simply and quickly carry out good resolution on L-tartaric acid and D-tartaric acid by adopting high performance liquid chromatography and determine the content of the two substances.
The invention provides a method for separating and measuring tartaric acid optical isomer content. According to the embodiment of the invention, a sample to be detected is detected by using high performance liquid chromatography to obtain a chromatogram map; and
obtaining the content of tartaric acid optical isomer based on the chromatogram;
wherein the chromatographic conditions of the high performance liquid chromatography are as follows:
amylose-tris [3, 5-dimethylcarbamate ] derivative is used as a stationary phase;
taking a mixed solvent of a first organic solvent and trifluoroacetic acid as a mobile phase;
wherein the first organic solvent is n-hexane, isopropanol and tert-butanol;
the elution mode is isocratic elution.
By adopting the method, the L-tartaric acid and the D-tartaric acid in the tartaric acid can be quickly and efficiently separated and the content of the L-tartaric acid and the D-tartaric acid can be measured, so that the quality of the tartaric acid can be effectively controlled. The detection method has the advantages of simple detection process, high accuracy and convenient operation.
The method for separating and determining the content of the tartaric acid optical isomers, provided by the embodiment of the invention, can further have at least one of the following additional technical characteristics:
according to an embodiment of the present invention, the method further comprises treating a sample to be tested with the mobile phase and a second organic solvent, wherein the second organic solvent is ethanol.
According to an embodiment of the present invention, the volume ratio of the mobile phase and the second organic solvent is 1: 1.
According to the embodiment of the invention, the volume ratio of n-hexane, isopropanol, tert-butanol and trifluoroacetic acid in the mobile phase is 830:170:10: 2-880: 120:10: 2. In the mobile phase, the volume ratio of n-hexane, isopropanol, tert-butanol and trifluoroacetic acid is not in the range, so that the separation degree of a main peak and an impurity peak is small, and the main peak and the impurity peak are not separated.
According to a preferable embodiment of the invention, the volume ratio of n-hexane, isopropanol, tert-butanol and trifluoroacetic acid in the mobile phase is 850:150:10: 2-880: 120:10: 2.
According to the embodiment of the invention, the chromatographic column in the high performance liquid chromatography is CHIRALPAK AD-H chromatographic column, and the column temperature of the chromatographic column is 20-30 ℃, preferably 25 ℃.
According to an embodiment of the invention, the flow rate of the mobile phase is 0.7-0.9 ml/min, preferably 0.7-0.8 ml/min.
In the high performance liquid chromatography provided by the invention, the separation degree of a main peak and an impurity peak in an obtained chromatogram can be ensured to reach a standard only when the column temperature of a chromatographic column and/or the flow rate of a mobile phase are/is in the range, and the main peak and the impurity peak can be separated.
According to an embodiment of the present invention, the detection wavelength in the high performance liquid chromatography is 223 nm.
According to an embodiment of the invention, the method comprises the steps of:
(1) preparing a sample solution: weighing a proper amount of tartaric acid sample, dissolving the sample by using the second organic solvent to prepare a sample with the concentration of 10mg/ml, and then diluting by using the mobile phase to prepare a solution containing 5mg of the sample per 1 ml;
(2) and (2) injecting 20 mu l of the sample solution obtained in the step (1) into a high performance liquid chromatograph, recording a chromatogram, and completing the separation and detection of the tartaric acid sample.
The method for separating and determining the content of the tartaric acid optical isomer can be realized according to the following method:
chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H4.6X 250mm, 5 μm;
mobile phase: mixed liquid of n-hexane, isopropanol, tert-butanol and trifluoroacetic acid, wherein the volume ratio of the mixed liquid is 830:170:10: 2-880: 120:10:2, and the mixed liquid is eluted at equal degree;
column temperature of the chromatographic column: 20-30 ℃;
flow rate of mobile phase: 0.7-0.9 ml/min;
detection wavelength: 223nm
Sample introduction amount: 20 μ l
1) Weighing a proper amount of tartaric acid sample, dissolving with ethanol to prepare a solution containing 10mg of the sample per 1ml, and diluting with a mobile phase to prepare a solution containing 5mg of the sample per 1 ml;
2) and (3) injecting 20 mu l of the sample solution obtained in the step 1) into a high performance liquid chromatograph, and recording a chromatogram.
Normal phase chromatography typically employs a polar stationary phase and a non-polar mobile phase. In normal phase chromatography, the sample molecules and the groups of the carrier matrix generate specific polar interaction, the polar sample molecules generating strong polar interaction with the stationary phase are difficult to elute and have longer retention time in the column, and conversely, the molecules with weaker polarity or nonpolar interaction with the stationary phase are easy to elute, so the retention time in the column is shorter. The normal phase chromatography achieves the separation purpose just according to the polarity difference of the solvent.
The inventor creatively discovers that the method for separating and determining the content of the tartaric acid optical isomer, which is provided by the invention, has good separation effect, and can effectively separate the chromatographic peaks in the sample in a short time, thereby more effectively controlling the quality of the tartaric acid raw material.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a high performance liquid chromatogram of a sample obtained according to example 1 of the present invention;
FIG. 2 shows a high performance liquid chromatogram of a sample obtained according to example 2 of the present invention;
FIG. 3 shows a high performance liquid chromatogram of a sample obtained according to example 3 of the present invention;
FIG. 4 shows a high performance liquid chromatogram of a sample obtained according to example 4 of the present invention;
FIG. 5 shows a high performance liquid chromatogram of a sample obtained according to example 5 of the present invention;
FIG. 6 shows a high performance liquid chromatogram of a sample obtained according to example 6 of the present invention;
FIG. 7 shows a high performance liquid chromatogram of a sample obtained according to example 7 of the present invention;
FIG. 8 shows a high performance liquid chromatogram of a sample obtained according to comparative example 1 of the present invention;
FIG. 9 shows a high performance liquid chromatogram of a sample obtained according to comparative example 2 of the present invention;
FIG. 10 shows a high performance liquid chromatogram of a sample obtained according to comparative example 3 of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Samples of tartaric acid were obtained from TRC (Toronto Research Chemicals) in the examples of the invention.
Example 1
1. Chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H4.6X 250mm, 5 μm
Stationary phase: amylose-tris [3, 5-dimethylcarbamate ] derivatives
Mobile phase: weighing 850ml of n-hexane, 150ml of isopropanol, 10ml of tert-butanol and 2ml of trifluoroacetic acid, mixing uniformly, and eluting at equal speed
Column temperature of the chromatographic column: 25 deg.C
Flow rate of mobile phase: 0.8ml/min
Detection wavelength: 223nm
Sample introduction amount: 20 μ L
2. Experimental procedure
1) Weighing a proper amount of tartaric acid sample, dissolving with ethanol to prepare 10mg of tartaric acid sample solution per 1ml, and diluting with mobile phase to obtain 5mg of tartaric acid sample solution per 1 ml;
2) and (3) injecting 20 mu l of the sample solution obtained in the step 1) into a high performance liquid chromatograph, and recording a chromatogram. The experimental result in the attached figure 1 shows that the peak 1 is D-tartaric acid, the peak 2 is L-tartaric acid, and it can be seen that under the condition, the main peak of L-tartaric acid can be well separated from D-tartaric acid, the main peak of L-tartaric acid is about 11.6min, and the separation degree between various spectral peaks can meet the requirement (the separation degree is more than or equal to 0.8).
Example 2
1. Chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H4.6X 250mm, 5 μm
Stationary phase: amylose-tris [3, 5-dimethylcarbamate ] derivatives
Mobile phase: measuring 830ml of n-hexane, 170ml of isopropanol, 10ml of tert-butanol and 2ml of trifluoroacetic acid, mixing uniformly, and eluting at equal speed
Column temperature of the chromatographic column: 25 deg.C
Flow rate of mobile phase: 0.8ml/min
Detection wavelength: 223nm
Sample introduction amount: 20 μ L
2. Experimental procedure
1) Weighing a proper amount of tartaric acid sample, dissolving with ethanol to prepare 10mg of tartaric acid sample solution per 1ml, and diluting with mobile phase to obtain 5mg of tartaric acid sample solution per 1 ml;
2) and (3) injecting 20 mu l of the sample solution obtained in the step 1) into a high performance liquid chromatograph, and recording a chromatogram. The experimental result in the attached figure 2 shows that the peak 1 is D-tartaric acid, the peak 2 is L-tartaric acid, and it can be seen that under the condition, the main peak of L-tartaric acid can be well separated from D-tartaric acid, the main peak of L-tartaric acid is about 8min, and the separation degree between the various spectral peaks can meet the requirement (the separation degree is more than or equal to 0.8).
Example 3
1. Chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H4.6X 250mm, 5 μm
Stationary phase: amylose-tris [3, 5-dimethylcarbamate ] derivatives
Mobile phase: weighing 880ml of n-hexane, 120ml of isopropanol, 10ml of tert-butanol and 2ml of trifluoroacetic acid, mixing uniformly, and performing isocratic elution
Column temperature of the chromatographic column: 25 deg.C
Flow rate of mobile phase: 0.8ml/min
Detection wavelength: 223nm
Sample introduction amount: 20 μ L
2. Experimental procedure
1) Weighing a proper amount of tartaric acid sample, dissolving with ethanol to prepare 10mg of tartaric acid sample solution per 1ml, and diluting with mobile phase to obtain 5mg of tartaric acid sample solution per 1 ml;
2) and (3) injecting 20 mu l of the sample solution obtained in the step 1) into a high performance liquid chromatograph, and recording a chromatogram. The experimental results in FIG. 3 show that peak 1 is D-tartaric acid and peak 2 is L-tartaric acid. Under the condition, the main peak of the L-tartaric acid can be well separated from the D-tartaric acid, the main peak of the L-tartaric acid is about 13min, and the separation degree between various spectral peaks can meet the requirement (the separation degree is more than or equal to 0.8).
Example 4
1. Chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H4.6X 250mm, 5 μm
Stationary phase: amylose-tris [3, 5-dimethylcarbamate ] derivatives
Mobile phase: weighing 850ml of n-hexane, 150ml of isopropanol, 10ml of tert-butanol and 2ml of trifluoroacetic acid, mixing uniformly, and eluting at equal speed
Column temperature of the chromatographic column: 20 deg.C
Flow rate of mobile phase: 0.8ml/min
Detection wavelength: 223nm
Sample introduction amount: 20 μ L
2. Experimental procedure
1) Weighing a proper amount of tartaric acid sample, dissolving with ethanol to prepare 10mg of tartaric acid sample solution per 1ml, and diluting with mobile phase to obtain 5mg of tartaric acid sample solution per 1 ml;
2) and (3) injecting 20 mu l of the sample solution obtained in the step 1) into a high performance liquid chromatograph, and recording a chromatogram. The experimental results in FIG. 4 show that peak 1 is D-tartaric acid and peak 2 is L-tartaric acid. Under the condition, the main peak of the L-tartaric acid can be well separated from the D-tartaric acid, the main peak of the L-tartaric acid is about 12min, and the separation degree between various spectral peaks can meet the requirement (the separation degree is more than or equal to 0.8).
Example 5
1. Chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H4.6X 250mm, 5 μm
Stationary phase: amylose-tris [3, 5-dimethylcarbamate ] derivatives
Mobile phase: weighing 850ml of n-hexane, 150ml of isopropanol, 10ml of tert-butanol and 2ml of trifluoroacetic acid, mixing uniformly, and eluting at equal speed
Column temperature of the chromatographic column: 30 deg.C
Flow rate of mobile phase: 0.8ml/min
Detection wavelength: 223nm
Sample introduction amount: 20 μ L
2. Experimental procedure
1) Weighing a proper amount of tartaric acid sample, dissolving with ethanol to prepare 10mg of tartaric acid sample solution per 1ml, and diluting with mobile phase to obtain 5mg of tartaric acid sample solution per 1 ml;
2) and (3) injecting 20 mu l of the sample solution obtained in the step 1) into a high performance liquid chromatograph, and recording a chromatogram. The experimental results in FIG. 5 show that peak 1 is D-tartaric acid and peak 2 is L-tartaric acid. Under the condition, the main peak of the L-tartaric acid can be well separated from the D-tartaric acid, the main peak of the L-tartaric acid is about 10min, and the separation degree between various spectral peaks can meet the requirement (the separation degree is more than or equal to 0.8).
Example 6
1. Chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H4.6X 250mm, 5 μm
Stationary phase: amylose-tris [3, 5-dimethylcarbamate ] derivatives
Mobile phase: weighing 850ml of n-hexane, 150ml of isopropanol, 10ml of tert-butanol and 2ml of trifluoroacetic acid, mixing uniformly, and eluting at equal speed
Column temperature of the chromatographic column: 25 deg.C
Flow rate of mobile phase: 0.7ml/min
Detection wavelength: 223nm
Sample introduction amount: 20 μ L
2. Experimental procedure
1) Weighing a proper amount of tartaric acid sample, dissolving with ethanol to prepare 10mg of tartaric acid sample solution per 1ml, and diluting with mobile phase to obtain 5mg of tartaric acid sample solution per 1 ml;
2) and (3) injecting 20 mu l of the sample solution obtained in the step 1) into a high performance liquid chromatograph, and recording a chromatogram. The experimental results in FIG. 6 show that peak 1 is D-tartaric acid and peak 2 is L-tartaric acid. Under the condition, the main peak of the L-tartaric acid can be well separated from the D-tartaric acid, the main peak of the L-tartaric acid is about 13min, and the separation degree between various spectral peaks can meet the requirement (the separation degree is more than or equal to 0.8).
Example 7
1. Chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H4.6X 250mm, 5 μm
Stationary phase: amylose-tris [3, 5-dimethylcarbamate ] derivatives
Mobile phase: weighing 850ml of n-hexane, 150ml of isopropanol, 10ml of tert-butanol and 2ml of trifluoroacetic acid, mixing uniformly, and eluting at equal speed
Column temperature of the chromatographic column: 25 deg.C
Flow rate of mobile phase: 0.9ml/min
Detection wavelength: 223nm
Sample introduction amount: 20 μ L
2. Experimental procedure
1) Weighing a proper amount of tartaric acid sample, dissolving with ethanol to prepare 10mg of tartaric acid sample solution per 1ml, and diluting with mobile phase to obtain 5mg of tartaric acid sample solution per 1 ml;
2) and (3) injecting 20 mu l of the sample solution obtained in the step 1) into a high performance liquid chromatograph, and recording a chromatogram. The experimental results in FIG. 7 show that peak 1 is D-tartaric acid and peak 2 is L-tartaric acid. Under the condition, the main peak of the L-tartaric acid can be well separated from the D-tartaric acid, the main peak of the L-tartaric acid is about 10min, and the separation degree between various spectral peaks can meet the requirement (the separation degree is more than or equal to 0.8).
Comparative example 1
1. Chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H4.6X 250mm, 5 μm
Stationary phase: amylose-tris [3, 5-dimethylcarbamate ] derivatives
Mobile phase: weighing 850ml of n-hexane and 150ml of isopropanol, mixing uniformly, and eluting at equal rate
Column temperature of the chromatographic column: 25 deg.C
Flow rate of mobile phase: 0.8ml/min
Detection wavelength: 223nm
Sample introduction amount: 20 μ L
2. Experimental procedure
1) Weighing a proper amount of tartaric acid sample, dissolving with ethanol to prepare 10mg of tartaric acid sample solution per 1ml, and diluting with mobile phase to obtain 5mg of tartaric acid sample solution per 1 ml;
2) and (3) injecting 20 mu l of the sample solution obtained in the step 1) into a high performance liquid chromatograph, and recording a chromatogram. The experimental results in FIG. 8 show that the peak No. 5 is D-tartaric acid and the peak No. 6 is L-tartaric acid, and it can be seen that the main peak of L-tartaric acid is poor in shape under the condition.
Comparative example 2
1. Chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H4.6X 250mm, 5 μm
Stationary phase: amylose-tris [3, 5-dimethylcarbamate ] derivatives
Mobile phase: measuring 830ml of n-hexane and 170ml of isopropanol, mixing uniformly, and eluting at equal speed
Column temperature of the chromatographic column: 25 deg.C
Flow rate of mobile phase: 0.8ml/min
Detection wavelength: 223nm
Sample introduction amount: 20 μ L
2. Experimental procedure
1) Weighing a proper amount of tartaric acid sample, dissolving with ethanol to prepare 10mg of tartaric acid sample solution per 1ml, and diluting with mobile phase to obtain 5mg of tartaric acid sample solution per 1 ml;
2) and (3) injecting 20 mu l of the sample solution obtained in the step 1) into a high performance liquid chromatograph, and recording a chromatogram. The experimental result in figure 9 shows that the peak 2 is D-tartaric acid, and the peak 3 is L-tartaric acid, and it can be seen that the main peak of L-tartaric acid and the impurity peak of D-tartaric acid are not separated under the condition, and the peak shape is asymmetric, the separation degree is very poor, and the peak shape is not good.
Comparative example 3
1. Chromatographic conditions are as follows:
a chromatographic column: CHIRALPAK AD-H4.6X 250mm, 5 μm
Stationary phase: amylose-tris [3, 5-dimethylcarbamate ] derivatives
Mobile phase: weighing 880ml of n-hexane and 120ml of isopropanol, mixing uniformly, and eluting at equal rate
Column temperature of the chromatographic column: 25 deg.C
Flow rate of mobile phase: 0.8ml/min
Detection wavelength: 223nm
Sample introduction amount: 20 μ L
2. Experimental procedure
1) Weighing a proper amount of tartaric acid sample, dissolving with ethanol to prepare 10mg of tartaric acid sample solution per 1ml, and diluting with mobile phase to obtain 5mg of tartaric acid sample solution per 1 ml;
2) and (3) injecting 20 mu l of the sample solution obtained in the step 1) into a high performance liquid chromatograph, and recording a chromatogram. The experimental results in FIG. 10 show that peak No. 7 is D-tartaric acid and peak No. 8 is L-tartaric acid. Under the condition, the main peak of the L-tartaric acid and the impurity peak of the D-tartaric acid can not be separated, the separation degree is extremely poor, the peak shapes are asymmetric, and the tailing phenomenon exists.
The comparative example shows that if tert-butanol and trifluoroacetic acid are not present in the mobile phase, even if the ratio of n-hexane to isopropanol in the chromatographic conditions employed is in the range of 830:170 to 880:120, the resulting chromatogram does not possess the desired peak shape.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (8)
1. A method for separating and determining tartaric acid optical isomer content is characterized in that a sample to be detected is detected by using a high performance liquid chromatography to obtain a chromatogram; and
obtaining the content of tartaric acid optical isomer based on the chromatogram;
wherein the chromatographic conditions of the high performance liquid chromatography are as follows:
amylose-tris [3, 5-dimethylcarbamate ] derivative is used as a stationary phase;
taking a mixed solvent of a first organic solvent and trifluoroacetic acid as a mobile phase;
wherein the first organic solvent is n-hexane, isopropanol and tert-butanol;
the elution mode is isocratic elution.
2. The method of claim 1, further comprising treating a sample to be tested with the mobile phase and a second organic solvent, wherein the second organic solvent is ethanol.
3. The method according to claim 2, wherein the volume ratio of the mobile phase to the second organic solvent is 1: 1.
4. The method according to claim 1 or 2, wherein the volume ratio of n-hexane, isopropanol, tert-butanol and trifluoroacetic acid in the mobile phase is 830:170:10: 2-880: 120:10:2, preferably 850:150:10: 2-880: 120:10: 2.
5. The method according to claim 1, wherein the chromatographic column in the high performance liquid chromatography is CHIRALPAK AD-H chromatographic column, and the column temperature of the chromatographic column is 20-30 ℃, preferably 25 ℃.
6. The method according to claim 1, wherein the flow rate of the mobile phase is 0.7 to 0.9ml/min, preferably 0.7 to 0.8 ml/min.
7. The method of claim 1, wherein the detection wavelength in the high performance liquid chromatography is 223 nm.
8. A method according to any one of claims 1 to 7, characterized in that the method comprises the steps of:
(1) preparing a sample solution: weighing a tartaric acid sample, dissolving the sample by using the second organic solvent to prepare a solution with the concentration of 10mg/ml, and diluting by using the mobile phase to prepare a solution containing 5mg of the sample per 1 ml;
(2) and (2) injecting 20 mu l of the sample solution obtained in the step (1) into a high performance liquid chromatograph, recording a chromatogram, and completing the separation and detection of the tartaric acid sample.
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