Solution polarity determination method based on liquid chromatography

Abstract

The invention relates to the technical field of detection, in particular to a solution polarity determination method based on liquid chromatography. According to the method, the high performance liquid chromatography is used for detecting the solution, the determination of the polarity of the solution can be completed in a short time, the detection result is accurate and reliable, and the repeatability is good. Meanwhile, sample preparation and instrument operation processes are relatively simple and easy to master and implement, and the detection working efficiency can be effectively improved. According to the invention, a standard substance is used as a contrast, and the detection quality can be effectively controlled and the accuracy and reliability of the result can be ensured through comparison of a standard curve method and multiple determination results. Meanwhile, a standard substance comparison method can be suitable for polarity detection of various solutions including blood, drug original solutions, leaching solutions, diluents, wines and the like, and has wide applicability.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and particularly relates to a method for determining the polarity of a solution based on liquid chromatography. Background Art

[0002] The detection of solution polarity is of great significance in many fields such as chemistry, medicine, and biology. Traditional detection methods have some limitations, such as insufficient accuracy, complex operation, and long time consumption. Therefore, it is particularly necessary to develop a high-efficiency, accurate, and widely applicable solution polarity detection method.

[0003] Currently, commonly used solution polarity detection methods include infrared spectroscopy, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, etc. Infrared spectroscopy requires a complex sample preparation process and is not sensitive enough to the response of polarity changes. Nuclear magnetic resonance spectroscopy equipment is expensive and the operation is complex, which is not suitable for rapid detection. Fluorescence spectroscopy requires the selection of a suitable fluorescence probe, and the fluorescence signal is easily interfered by environmental factors. In addition, these methods often require a large amount of samples during the detection process, and have high requirements for the purity and stability of the samples, making it difficult to achieve high-throughput and rapid detection.

[0004] In summary, traditional solution polarity detection methods have certain limitations in terms of accuracy, operation simplicity, detection speed, etc., and it is difficult to meet the high requirements for solution polarity detection in modern chemistry, medicine, biology and other fields. Therefore, it has important practical significance to develop a high-efficiency, accurate, and widely applicable solution polarity detection method. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for determining the polarity of a solution based on liquid chromatography. This method can quickly and accurately determine the polarity of various solutions through high-performance liquid chromatography, and has the advantages of simple operation, good repeatability, and wide application range. To solve the above technical problems, the technical solutions provided by the present invention are as follows:

[0006] A method for determining the polarity of a solution based on liquid chromatography, comprising the following steps:

[0007] S1. Sample pretreatment;

[0008] S2. Set a standard as a control group

[0009] Prepare a standard working solution, and the standard working solution has the same parameters as the sample;

[0010] S3. Use a high-performance liquid chromatograph to detect the sample;

[0011] The high-performance liquid chromatograph is also equipped with a diode array detector and a differential refractive index detector, and the standard sample is scanned separately in series by the differential refractive index detector and the diode array detector;

[0012] S4. Sample analysis

[0013] Calculate the polarity of the standard working solution, and draw two standard curves for differential detection and ultraviolet detection respectively, with the polarity of the standard working solution as the abscissa and the sum of peak areas as the ordinate;

[0014] For the sample solution, calculate the sum of the peak areas of the scanning spectra of the differential detector and the diode array detector respectively. Calculate the differential polarity and ultraviolet polarity of the test solution according to the standard curve. The diode array detector scans to generate a 3D spectrum, and the differential detector scans to generate a 2D spectrum. Analyze the polarity of the sample based on the total peak area of the 2D spectrum generated by the differential detector scanning, and perform sample characteristic analysis on the sample characteristic peaks using the 3D spectrum generated by the diode array detector scanning;

[0015] S5. Repeat the experiment to improve precision.

[0016] As an improvement, the method for sample pretreatment in step S1 includes high-speed centrifugation, microfiltration membrane or ultrasound.

[0017] As an improvement, in the high-performance liquid chromatography column analyzer in step S3, the chromatographic column is selected as: CAPECELL PAK MGS C18 column, 4.6mm×250mm, 5μm;

[0018] The mobile phase is methanol and water, with volume ratios of 3:7 respectively, the constant flow rate is 1.0 mL / min, the column temperature is 35°C, and the injection volume is 10 μl.

[0019] As an improvement, the scanning wavelength range of the diode array detector in step S3 is: 190 - 800 nm, and the temperature of the differential detector is 35°C

[0020] As an improvement, the standard substance selected in step S2 is n-butanol, and the theoretical polarity of the n-butanol molecule is 1.636 D.

[0021] As an improvement, the calculation formula for the standard working solution in step S4 is as follows:

[0022]

[0023] Among them, μ--the polarity of the standard substance working solution, unit Debye;

[0024] m--the mass of the standard substance working solution (grams);

[0025] M--the molar mass of the standard substance (grams / mole);

[0026] N---is Avogadro's constant 6.02×10²³;

[0027] μ---Polarity of the standard substance molecule (1.636 D).

[0028] 7. A method for measuring the polarity of a solution based on liquid chromatography according to claim 5, wherein the calculation formula for the polarity of the sample solution in step S4 is as follows:

[0029]

[0030] In the formula: μ measured---Polarity of the sample, in Debye D;

[0031] S measured---Sum of the peak areas of the measured sample chromatogram;

[0032] S standard—Sum of the peak areas of the standard sample chromatogram;

[0033] μ standard---Polarity of the standard substance (μ standard substance = Nm / M 1.636 D), the mass unit of the standard substance is grams, the molar mass unit of the standard substance is grams / mole, and the Avogadro constant is 6.02×1023.

[0034] As an improvement, in step S2, methanol is used as the diluent for the standard product to prepare standard product working solutions with different concentrations.

[0035] As an improvement, the high-performance liquid chromatography column analyzer in step S3 is pre-equilibrated for more than 1 h before use, and the sample solution analysis is started after the detector signal is stable.

[0036] As an improvement, the samples include blood, drug stock solution, extract, diluent, and liquor.

[0037] The present invention sums up the different retention peak areas generated by different polar molecules on the chromatographic column at different retention times for non-electrolyte or weak electrolyte solutions within the normal distribution range in the chromatographic column, and then compares with the peak area of the standard substance. The polarity of the solution is quantitatively calculated through the mathematical model of this method to obtain the polarity of the solution to be analyzed.

[0038] The present invention uses a tandem mode of a differential detector and a diode array detector to perform quantitative and qualitative dual-detection analysis on the solution sample. The different retention peak areas of different components in the solution on the differential detector are summed up and compared with the retention peak area of the standard substance for quantitative calculation. The 3D map generated by the diode array detector within the scanning wavelength range is used for qualitative analysis of the characteristic peaks of the sample to be measured.

[0039] The advantages of the present invention are:

[0040] 1. The present invention uses high performance liquid chromatography to detect solutions, which can complete the determination of solution polarity in a relatively short time, and the detection results are accurate and reliable with good repeatability. At the same time, the sample preparation and instrument operation processes are relatively simple, easy to master and implement, and can effectively improve the detection work efficiency.

[0041] 2. The present invention sets a reference substance as a control. By comparing the standard curve method and the results of multiple determinations, it can effectively control the detection quality and ensure the accuracy and reliability of the results. At the same time, the reference substance control method can be applied to the polarity detection of various solutions, including blood, drug stock solutions, extracts, diluents, wines, etc., and has wide applicability.

[0042] In summary, the present invention provides a method for detecting solution polarity that is efficient, accurate, and has a wide range of applications, with significant technical advantages and practical application values. It can be widely applied to the solution polarity analysis work in the fields of chemistry, medicine, biology, etc. Detailed implementation manners

[0043] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Usually, the components of the embodiments of the present invention described and illustrated herein can be arranged and designed in various different configurations.

[0044] Embodiment 1

[0045] This embodiment discloses a method for determining solution polarity based on liquid chromatography, including the following steps:

[0046] S1. Sample pretreatment

[0047] Take a certain amount of blood sample and perform pretreatment by high-speed centrifugation.

[0048] Place the blood sample in a high-speed centrifuge and centrifuge at a speed of 3500 r / min for 10 minutes to separate the supernatant, and then filter it through a 0.45 μm syringe-type aqueous microporous filter membrane to obtain the sample to be tested.

[0049] S2. Set a reference substance as a control group

[0050] Select n-butanol as the reference substance, and the theoretical polarity of n-butanol molecules is 1.636 D. Use methanol as the diluent to prepare reference substance working solutions with different concentrations, and the standard working solutions have the same parameters as the samples.

[0051] S3. Use a high performance liquid chromatograph to detect the sample

[0052] The high-performance liquid chromatograph used is equipped with a diode array detector and a differential refractive index detector. The chromatographic column selected is a CAPECELL PAK MGS C18 column, 4.6 mm × 250 mm, 5 μm. The mobile phase is methanol and water, with volume ratios of 3:7 respectively, and the constant flow rate is 1.0 mL / min. The column temperature is controlled at 35 °C, and the injection volume is 10 μl.

[0053] The scanning wavelength range of the diode array detector is set to 190 - 800 nm, and the temperature of the differential refractive index detector is set to 35 °C. The standard samples are scanned separately in series with the differential refractive index detector and the diode array detector.

[0054] Before use, pre-equilibrate for more than 1 h according to the above parameters, and start the analysis of the sample solution after the detector signal is stable.

[0055] S4. Sample analysis

[0056] Inject the standard series working solutions into the high-performance liquid chromatograph respectively, and record all detector signals within the scanning time. The standard samples are scanned separately in series with the differential refractive index detector and the diode array detector.

[0057] Calculate the polarity of the standard working solution, and the calculation formula is as follows:

[0058]

[0059] Among them, μ - the polarity of the standard substance working solution, unit Debye;

[0060] m - the mass of the standard substance working solution (grams);

[0061] M - the molar mass of the standard substance (grams / mole);

[0062] N--- is Avogadro's constant 6.02×10 2 3;

[0063] μ--- the molecular polarity of the standard substance (1.636 D).

[0064] Using the polarity of the standard working solution as the abscissa and the sum of the peak areas as the ordinate, draw two standard curves for differential detection and ultraviolet detection respectively.

[0065] Inject the sample solution into the high-performance liquid chromatograph for scanning, calculate the sum of the peak areas of the scanning spectra of the differential refractive index detector and the diode array detector respectively, calculate the differential polarity and ultraviolet polarity of the test solution according to the standard curve, use the differential polarity as the basis for quantitative calculation, and use the ultraviolet polarity as the basis for sample characteristic analysis.

[0066] The calculation formula for the polarity of the sample solution is as follows:

[0067]

[0068] Where: μ measured --- the polarity of the sample, in Debye (D);

[0069] S measured --- the sum of the peak areas of the measured sample spectrum;

[0070] S standard --- the sum of the peak areas of the standard sample spectrum;

[0071] μ standard --- the polarity of the reference substance (μ reference substance = N × 1.636 D), the mass unit of m reference substance is grams, the molar mass unit of M reference substance is grams per mole, and the Avogadro constant is 6.02 × 10 23 .

[0072] Draw the following table according to the test results:

[0073] Table 1 Concentration, Polarity, and Sum of Peak Areas of Reference Substances

[0074] 1 2 3 4 5 6 7 Concentration Sum of peak areas Polarity

[0075] S5. Repeated Tests

[0076] To improve the precision, conduct multiple repeated tests on the same sample to ensure the reliability of the results. The calculated results are expressed as the arithmetic mean of two independent determinations obtained under repeatability conditions, and the results are retained to two significant figures. The absolute difference between the two independent determinations obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.

[0077] Example 2

[0078] This example discloses a method for determining the polarity of a solution based on liquid chromatography, including the following steps:

[0079] S1. Sample Pretreatment

[0080] Take the original drug solution sample and perform pretreatment using the microporous membrane method. Filter the sample through a 0.45 μm syringe-type aqueous microporous membrane to obtain the sample to be tested.

[0081] S2. Set the reference as a control group

[0082] S3. Detect the sample using a high-performance liquid chromatograph

[0083] S4. Sample Analysis

[0084] S5. Repeated Tests

[0085] Steps S2 - S5 in this example are the same as those in Example 1.

[0086] Example 3

[0087] This embodiment discloses a method for measuring the polarity of a solution based on liquid chromatography, comprising the following steps:

[0088] S1. Sample pretreatment

[0089] Take a liquor sample and perform pretreatment by ultrasonic method. Place the liquor sample in an ultrasonic instrument and ultrasonically treat it for 10 minutes to fully mix the sample evenly, and then filter it through a 0.45μm syringe-type aqueous microporous filter membrane to obtain the sample to be measured.

[0090] S2. Set a standard as a control group

[0091] S3. Use a high-performance liquid chromatograph to detect the sample

[0092] S4. Sample analysis

[0093] S5. Repeat the experiment

[0094] In this embodiment, steps S2 - S5 are the same as those in Embodiment 1.

[0095] The method for measuring the polarity of a solution based on liquid chromatography of the present invention has good applicability and reliability, and can meet the polarity detection requirements of different types of solutions. In practical applications, appropriate pretreatment methods and instrument conditions can be selected according to the properties of specific samples and detection requirements to obtain accurate detection results.

[0096] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. A method for determining solution polarity based on liquid chromatography, characterized in that: The steps include: S1. Sample pretreatment; S2. Set up standard sample as control group Prepare standard working solution, the parameters of which are the same as those of the sample; S3. Use high performance liquid chromatography to test the sample; The high performance liquid chromatograph is also provided with a diode array detector and a parallax detector, and the standard samples are scanned respectively by using the parallax detector and the diode array detector in series; S4. Sample analysis Calculate the polarity of the standard working solution, and use the polarity of the standard working solution as the abscissa and the sum of the peak areas as the ordinate to draw two standard curves for differential detection and ultraviolet detection respectively; For the sample solution, the sum of the peak areas of the differential detector and the diode array detector scanning spectrum is calculated respectively, the differential polarity and ultraviolet polarity of the test solution are calculated according to the standard curve, the diode array detector scans to generate a 3D spectrum, and the differential detector scans to generate a 2D spectrum. The sum of the peak areas of the 2D spectrum scanned by the differential detector is used as the basis for quantitative calculation, the polarity of the sample is analyzed, and the characteristic peaks of the sample are analyzed by the 3D spectrum scanned by the diode array detector; S5. Repeat the experiment to improve precision.

2. The method for determining solution polarity based on liquid chromatography according to claim 1, characterized in that: The sample pretreatment methods in step S1 include high-speed centrifugation, microporous membrane filtration or ultrasonic treatment.

3. The method for determining solution polarity based on liquid chromatography according to claim 1, characterized in that: In the high-speed liquid chromatography column analyzer described in step S3, the chromatographic column selected is: CAPECELL PAK MGS C 18 column, 4.6 mm × 250 mm, 5 μm; The mobile phase was methanol and water in a volume ratio of 3:7, the constant flow rate was 1.0 mL / min, the column temperature was 35 °C, and the injection volume was 10 μl.

4. The method for determining solution polarity based on liquid chromatography according to claim 1, characterized in that: The scanning wavelength range of the diode array detector in step S3 is: 190-800nm, and the temperature of the differential detector is 35°C.

5. The method for determining solution polarity based on liquid chromatography according to claim 1, characterized in that: The standard substance in step S2 is n-butanol, and the theoretical polarity of n-butanol molecule is 1.636D.

6. The method for determining solution polarity based on liquid chromatography according to claim 5, characterized in that: The calculation formula of the standard working solution in step S4 is as follows: Wherein, μ is the polarity of the working solution of the standard substance, in Debye units; m--mass of standard substance working solution (g); M--molar mass of standard substance (g / mol); N---Avogadro constant 6.02×10 23 ; μ---Molecular polarity of standard substance (1.636D).

7. The method for determining solution polarity based on liquid chromatography according to claim 5, characterized in that: The calculation formula for the polarity of the sample solution in step S4 is as follows: Where: μ 实测 --- Polarity of the sample, in Debye D; S 实测 ---The sum of the peak areas of the measured sample spectra; S 标准 —The sum of the peak areas of the standard sample spectra; μ 标准 ---Polarity of standard substances The mass unit of m standard substance is gram, the molar mass unit of M standard substance is gram / mole, and the Avogadro constant is 6.02×10 23 .

8. The method for determining solution polarity based on liquid chromatography according to claim 5, characterized in that: In step S2, the standard sample is diluted with methanol to prepare standard sample working solutions of different concentrations.

9. The method for determining solution polarity based on liquid chromatography according to claim 3, characterized in that: The high-speed liquid chromatography column analyzer in step S3 is subjected to a balancing treatment for more than 1 hour before use, and the sample liquid analysis is started after the detector signal is stable.

10. The method for determining solution polarity based on liquid chromatography according to claim 1, characterized in that: The samples include blood, original drug solutions, extracts, diluents, and alcohol.

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