A capillary electrophoresis chiral separation method based on lactose-isomerized lactose chiral selector

Abstract

The present invention belongs to the field of drug analysis, and specifically discloses a capillary electrophoresis chiral separation method based on lactose-isomerized lactose chiral selector. First, lactose and isomerized lactose are dissolved in a phosphate buffer to obtain an electrophoresis buffer containing a lactose-isomerized lactose chiral selector, and the capillary electrophoresis system is applied to the separation of phenylalanine enantiomers, which can achieve baseline separation of the two configurations. The present invention is simple to operate, environmentally friendly and economical, and has high repeatability, and establishes a new, fast and efficient capillary electrophoresis chiral separation method based on lactose-isomerized lactose chiral selector.

Description

Technical Field

[0001] The invention relates to a capillary electrophoresis chiral separation method based on a lactose-isomerized lactose chiral selector, and belongs to the technical field of drug analysis. Background Art

[0002] Chiral separation has important practical significance because a pair of enantiomers of chiral molecules exhibit different physiological activities and pharmacological toxicity. Lactose is one of the most commonly used pharmaceutical excipients. Lactulose is widely used in clinical medicine, health products and food additives, but there are few reports on the application of lactose-lactulose in chiral separation. Among the many chiral separation methods, high-performance capillary electrophoresis (HPCE) technology does not require the high-pressure pump transport system of HPLC, a large amount of organic reagents and expensive chromatographic columns. It can use micro-liter reagents to achieve efficient and rapid separation of the analyzed samples. Therefore, capillary electrophoresis is more environmentally friendly and economical. In the process of constructing the HPCE chiral separation system, the selection of chiral selectors is the key factor in determining the efficiency of chiral separation. However, there are few reports on the use of lactose-lactulose as a chiral selector for HPCE chiral separation.

[0003] In view of the above-mentioned defects, the present invention aims to create a capillary electrophoresis chiral separation method based on lactose-lactulose as a chiral selector, so as to make it more valuable for industrial use. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to broaden the scope of chiral selectors that can be used currently, and to provide a capillary electrophoresis chiral separation method based on lactose-lactulose chiral selectors. Lactose-lactulose is selected as a chiral selector and added to an electrophoresis buffer to achieve baseline separation of phenylalanine enantiomers. The present invention is a capillary electrophoresis chiral separation method based on lactose-lactulose chiral selectors, and the specific technical scheme is:

[0005] (1) firstly preparing a phosphate electrophoresis buffer, adjusting it to different pH values, adding lactose and lactulose to obtain an electrophoresis buffer containing a lactose-lactulose additive;

[0006] Phosphate buffer can maintain the stability of the pH value and ionic strength of the electrophoresis environment. During the electrophoresis process, positive and negative ions move under the action of the electric field, which will cause the instability of the electrophoresis environment. The buffer can maintain the stability of the pH value through its buffering effect. At the same time, the ions in the buffer can also maintain the ionic strength of the electrophoresis fluid to ensure the smooth progress of the electrophoresis process. In capillary electrophoresis, the ionic strength and pH value of the buffer will affect the migration speed of the sample. By adjusting the composition and concentration of the buffer, the migration speed of the sample can be controlled, thereby optimizing the separation effect. Using phosphate buffer can achieve better separation effects.

[0007] Sugar is a chiral molecule that contains multiple chiral centers and can interact with substrate molecules. Chiral separation can be achieved based on the difference in the forces between it and the two enantiomers of the chiral molecule. Lactose and lactulose are both disaccharides with multiple chiral carbon atoms in their structures, so chiral molecules can be separated.

[0008] (2) The above-obtained electrophoresis buffer containing the lactose-lactulose chiral additive is applied to the chiral separation of phenylalanine enantiomers. The chiral molecules are dissolved in an aqueous solution to prepare a sample solution. A phosphate buffer containing the lactose-lactulose additive is used as the electrophoresis buffer, and the capillary column is equilibrated with the electrophoresis buffer for 10 minutes before injection to separate the chiral molecules. The constructed capillary electrophoresis chiral separation system can achieve baseline separation of phenylalanine enantiomers.

[0009] There are differences in the interactions between the chiral additives lactose and lactulose and the two enantiomers of phenylalanine, which causes the two enantiomers of phenylalanine to migrate at different speeds during capillary electrophoresis, thereby achieving chiral separation of phenylalanine.

[0010] Furthermore, the concentration of lactose in step (1) is 20 mg / mL.

[0011] Furthermore, the concentration of lactulose in step (1) is 20 mg / mL.

[0012] Furthermore, in the capillary electrophoresis detection method constructed in step (2), the capillary electrophoresis separation voltage is 10-20 kV; the phosphate buffer concentration is 10-30 mM; the injection volume condition is 5 kPa, 1-6 s; and the pH value of the electrophoresis buffer is 7-8.

[0013] Furthermore, the capillary electrophoresis separation voltage of the capillary electrophoresis chiral separation system constructed in step (2) is 15 kV.

[0014] Furthermore, the pH value of the electrophoresis buffer of the capillary electrophoresis chiral separation system constructed in step (2) is 7.4.

[0015] Furthermore, the injection volume conditions of the capillary electrophoresis chiral separation system constructed in step (2) are 5 kPa, 3 s.

[0016] Furthermore, the concentration of the phosphate buffer in the capillary electrophoresis chiral separation system constructed in step (2) is 20 mM.

[0017] By means of the above scheme, the present invention achieves the following beneficial effects: the capillary electrophoresis chiral separation method based on the lactose-lactulose chiral selector provided by the present invention is simple to operate, further expands the application of lactose-lactulose as a chiral selector in chiral separation, and there are differences in the interaction between the lactose-lactulose chiral additive and the two enantiomers of phenylalanine, thereby causing the two enantiomers of phenylalanine to migrate at different speeds during capillary electrophoresis, thereby achieving the chiral separation of phenylalanine. Few literature reports use lactose-lactulose as a chiral additive for capillary electrophoresis chiral separation. Compared with traditional gel electrophoresis, high-efficiency capillary electrophoresis can be operated under high voltage conditions without generating thermal effects due to the use of thin inner diameter capillaries. Compared with high-performance liquid chromatography, a major feature of high-efficiency capillary electrophoresis is rapid analysis. In addition, the separation can be improved by adjusting the pH, voltage, temperature and other conditions of the buffer solution during the operation. The high-pressure pump transport system, large amounts of organic reagents and expensive chromatographic columns of high-performance liquid chromatography are not required. High-efficiency capillary electrophoresis can use micro-liter reagents to achieve rapid separation of a variety of samples, so capillary electrophoresis is more environmentally friendly and economical. In addition, high-efficiency capillary electrophoresis uses an external voltage as the driving force of electroosmotic flow, eliminating the influence of the radial diffusion of the parabolic flow type in pressure-driven liquid chromatography on the final chromatographic peak column efficiency, and has a higher separation column efficiency.

[0018] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show a certain embodiment of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 : Reproducibility of chiral separation of phenylalanine enantiomers by capillary electrophoresis.

[0021] Figure 2 :The effect of separation voltage on the separation of phenylalanine enantiomers. Separation voltage a, 10 kV; b, 15 kV; c, 20 kV.

[0022] Figure 3 :The effect of electrophoresis buffer pH on the separation of phenylalanine enantiomers. The buffer pH values ​​are a, 7; b, 7.4; c, 8.

[0023] Figure 4 :The effect of injection volume on the separation of phenylalanine enantiomers. Injection volume a, 5 kPa, 1 s; b, 5 kPa, 3 s; c, 5 kPa, 6 s.

[0024] Figure 5 : Effect of buffer concentration on the separation of phenylalanine enantiomers. Buffer concentrations a, 10 mM; b, 20 mM; c, 30 mM. DETAILED DESCRIPTION

[0025] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0026] A capillary electrophoresis chiral separation method based on lactose chiral selector, the method steps are as follows:

[0027] (1) preparing a phosphate electrophoresis buffer, adjusting the pH value to different values ​​with phosphoric acid, adding lactose and lactulose chiral selectors, and obtaining an electrophoresis buffer containing a lactose-lactulose additive;

[0028] (3) Applying the electrophoresis buffer containing the chiral selector described in step (1) to the chiral separation of phenylalanine enantiomers. The constructed capillary electrophoresis chiral separation system can achieve baseline separation of phenylalanine enantiomers.

[0029] Example 1

[0030] The quartz capillary was rinsed with pure water for 5 min, 1 mol / L sodium hydroxide for 5 min, pure water for 5 min, 0.1 mol / L hydrochloric acid for 5 min, and pure water for 5 min. Phosphate buffer containing lactose-lactulose additives was used as the electrophoresis buffer, and the lactose concentration in the electrophoresis buffer was 20 mg / mL and the lactulose concentration was 20 mg / mL. The capillary column was applied to the chiral separation of phenylalanine. The electroosmotic mobility and electrophoretic mobility were adjusted by optimizing the pH value of the phosphate electrophoresis buffer (pH 7-8) to improve the separation efficiency. At the same time, the electrophoretic separation conditions including voltage (10-20 kV), phosphate electrophoresis buffer concentration (10-30 mM), and injection volume (5 kPa, 1-6 s) were optimized to obtain the best electrophoresis conditions for baseline separation of phenylalanine enantiomers. The analysis of phenylalanine enantiomers by this electrophoresis system showed high separation repeatability ( Figure 1 ).

[0031] Example 2

[0032] According to the method of Example 1, the preferred value of the separation voltage was confirmed. Since the increase of the operating voltage will cause the increase of the electroosmotic flow, it will inhibit the retention of phenylalanine in the capillary column, change the separation column efficiency, and further affect the separation degree. Therefore, the effect of voltage change on its separation performance was further investigated in this experiment. Figure 2 It can be seen from the optimization results that with the increase of voltage, the retention time of the enantiomers of phenylalanine gradually decreases. This is because the voltage increases, the electroosmotic flow in the column increases, and the retention of phenylalanine decreases. Therefore, its peak time gradually shortens. With the increase of voltage, the column efficiency of the peak tends to increase gradually. When the voltage is 10kV, the electroosmotic flow is small, the retention time is long, the separation selectivity is strong, but the column efficiency is low. When the voltage is 20kV, the column efficiency increases, but the separation degree decreases. Finally, 15kV is determined to be the optimal voltage value.

[0033] During the capillary electrophoresis operation, the voltage not only directly affects the migration speed and separation efficiency of the molecules, but also determines the column efficiency of the peak. Since the increase in the operating voltage will cause the increase in electroosmotic flow, it will further affect the retention time of phenylalanine and affect the separation effect. Therefore, the optimized voltage is set.

[0034] Example 3

[0035] According to the method of Example 1, the preferred value of the pH value of the electrophoresis buffer is determined. Figure 3 The optimal results show that the retention time of phenylalanine enantiomers decreases with the increase of pH value. When the pH value is 7.4, the system has the best separation performance.

[0036] Adjusting the pH value of the buffer system to a range suitable for sample properties and separation efficiency is an important factor in determining separation efficiency. The pH value can affect the dissociation and electrophoretic speed of the sample, thereby affecting selectivity and sensitivity. Within the optimized pH range, the electroosmotic flow increases with increasing pH, so the optimized pH is set.

[0037] Example 4

[0038] According to the method of Example 1, the preferred value of the injection amount was confirmed. This example investigates the effect of the injection amount on the separation of phenylalanine enantiomers. Figure 4 The optimal test results show that as the injection volume increases, the peak area of ​​the chromatographic peak increases. Since a higher sample volume will cause peak tailing, the optimal value of the injection volume is finally determined to be 5kPa, 3s.

[0039] The injection volume will affect the separation efficiency. When the injection volume is too small, the sample is difficult to detect. If the injection volume is too large, it will cause the chromatographic peak to tail, the column efficiency to decrease, and the separation degree to decrease. Therefore, the optimal injection volume is set.

[0040] Example 5

[0041] According to the method of Example 1, the preferred value of the electrophoresis buffer concentration was determined. Figure 5 It can be seen from the results of the optimal value that with the increase of the electrophoresis buffer concentration, the retention time of the phenylalanine molecule gradually increases. This is because the increase in the electrophoresis buffer concentration will cause the electroosmotic flow to decrease, and the retention time of the phenylalanine enantiomers will increase. Figure 5 As shown, taking the retention time and separation efficiency into consideration, the separation ability of the capillary column is optimal when the electrophoresis buffer with the optimum concentration of 20 mM is used.

[0042] The concentration of buffer salt directly affects the ionic strength of the electrophoresis medium, thereby affecting the Zeta potential, and the change in Zeta potential will affect the electroosmotic flow. As the buffer concentration increases, the ionic strength increases, the thickness of the double layer decreases, the Zeta potential decreases, the electroosmotic flow decreases, and the retention time of the sample increases. If the electrolyte concentration is too high, the peak shape of the sample components will expand due to the thermal effect, and the separation effect will become worse. Therefore, the concentration of the electrophoresis buffer is optimized.

[0043] It can be seen from the above examples that capillary electrophoresis technology provides a fast, efficient and fast analysis platform for the separation of phenylalanine enantiomers. It is simple to operate, and further expands the application of lactose-lactulose as a chiral selector in chiral separation. There are differences in the interaction between the lactose-lactulose chiral additive and the two enantiomers of phenylalanine, thereby causing the two enantiomers of phenylalanine to have different migration speeds during capillary electrophoresis, and then the chiral separation of phenylalanine can be achieved. There are few literature reports that lactose-lactulose is used as a chiral additive for capillary electrophoresis chiral separation. Compared with traditional gel electrophoresis, high-efficiency capillary electrophoresis can be operated under high voltage conditions without producing thermal effects due to the use of thin inner diameter capillaries. Compared with high performance liquid chromatography, a major feature of high-efficiency capillary electrophoresis is rapid analysis. In addition, the separation can be improved by adjusting the pH, voltage, temperature and other conditions of the buffer solution during the operation. The high-pressure pump transport system, large amounts of organic reagents and expensive chromatographic columns of high-performance liquid chromatography are not required. High-efficiency capillary electrophoresis can use micro-liter reagents to achieve rapid separation of a variety of samples, so capillary electrophoresis is more environmentally friendly and economical. In addition, high-efficiency capillary electrophoresis uses an external voltage as the driving force of electroosmotic flow, eliminating the influence of the radial diffusion of the parabolic flow type in pressure-driven liquid chromatography on the final chromatographic peak column efficiency, and has a higher separation column efficiency.

[0044] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A capillary electrophoresis chiral separation method based on lactose-isomerized lactose chiral selector, characterized in that The specific separation steps are: (1) preparing a phosphate electrophoresis buffer, adding lactose and lactulose to obtain an electrophoresis buffer containing a lactose-lactulose additive; (2) The electrophoresis buffer containing the chiral selector obtained above is applied to the chiral separation of phenylalanine by capillary electrophoresis, and the baseline separation of phenylalanine enantiomers is achieved using the constructed capillary electrophoresis chiral separation system, wherein the capillary electrophoresis separation voltage is 10-20 kV; the phosphate buffer concentration is 10-30 mM; the injection volume condition is 5 kPa, 1-6 s; and the pH value of the electrophoresis buffer is 7-8.

2. The capillary electrophoresis chiral separation method based on lactose-lactulose chiral selector according to claim 1, characterized in that: The concentration of lactose in the electrophoresis buffer containing the lactose-lactulose additive in step (1) is 20 mg / mL.

3. The capillary electrophoresis chiral separation method based on lactose-lactulose chiral selector according to claim 1, characterized in that: The concentration of lactulose in the electrophoresis buffer containing the lactose-lactulose additive in step (1) is 20 mg / mL.

4. The capillary electrophoresis chiral separation method based on lactose-lactulose chiral selector according to claim 1, characterized in that: The capillary electrophoresis separation voltage of the capillary electrophoresis chiral separation system constructed in the step (2) is 15 kV.

5. The capillary electrophoresis chiral separation method based on lactose-lactulose chiral selector according to claim 1, characterized in that: The pH value of the electrophoresis buffer of the capillary electrophoresis chiral separation system constructed in step (2) is 7.

4.

6. The capillary electrophoresis chiral separation method based on lactose-lactulose chiral selector according to claim 1, characterized in that: The injection volume conditions of the capillary electrophoresis chiral separation system constructed in step (2) are 5 kPa, 3 s.

7. The capillary electrophoresis chiral separation method based on lactose-lactulose chiral selector according to claim 1, characterized in that: The concentration of the phosphate buffer in the capillary electrophoresis chiral separation system constructed in step (2) is 20 mM.

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