A method for separating galactose end group diastereomers by capillary electrochromatography

Abstract

The present invention relates to a method for separating galactose end group diastereomers by capillary electrochromatography, and belongs to the technical field of chromatographic analysis. The present invention forms a negatively charged complex with galactose by using borax electrophoresis buffer, which can improve the absorbance of galactose at ultraviolet 195nm, and realize direct detection of galactose without going through a complicated derivatization process or using other expensive detectors. At the same time, the charged complex can migrate in an electric field, and the complex formed by α-galactose and β-galactose is separated based on the difference in its electrophoretic mobility and interaction with the capillary silica gel monolithic column stationary phase, without the need to add organic reagents, and environmental protection and saving. The method for separating galactose end group diastereomers provided by the present invention is simple to operate and highly repeatable, and further expands the application of capillary electrochromatography technology in the field of galactose separation.

Description

Technical Field

[0001] The invention relates to a method for separating galactose end group diastereomers by capillary electrochromatography, belonging to the technical field of chromatographic analysis. Background Art

[0002] Galactose is a monosaccharide composed of six carbons and one aldehyde group. D-galactose is generally present in milk as a structural part of lactose. The lactose in milk is broken down into glucose and galactose by the human body and absorbed and utilized. Galactose is part of the lactose molecule, and the other half is glucose. Under the catalytic action of β-lactase, galactose can be obtained from the hydrolysis of lactose. Galactose has a mutarotation phenomenon. When galactose is dissolved in water, the interconversion between the ring and chain isomers occurs, so the optical rotation of the newly prepared monosaccharide solution will gradually change during the placement process, but after a certain period of time, the optical rotation will no longer change after the different end group isomers (α type and β type) reach equilibrium. The chromatographic analysis method commonly used to separate the α and β configurations of sugar molecules today is high performance liquid chromatography (HPLC), but this method is cumbersome to operate, consumes a large amount of reagents, and the chromatographic column is expensive, which has limitations in the detection of trace samples.

[0003] Capillary electrochromatography (CEC) technology combines the high selectivity of HPLC with the high efficiency of capillary electrophoresis. It can use micro-liters of reagents to achieve rapid separation of multiple samples, so CEC is more environmentally friendly and economical. Compared with traditional gel electrophoresis, CEC can be operated under high voltage conditions without generating thermal effects due to the use of thin inner diameter capillaries. Compared with HPLC, CEC has many advantages. A major feature of CEC is rapid analysis. Under the action of an external high electric field, the analysis time of the analyte is generally no more than 30 minutes. CEC can improve separation by adjusting buffer pH, voltage, temperature and other conditions during the operation, without the need for HPLC's high-pressure pump transport system and a large amount of organic reagents and expensive chromatographic columns.

[0004] Capillary electrochromatography is currently used in the following areas: small molecule drugs and related substances; chiral separation; traditional Chinese medicine and natural products; in vivo drug analysis; macromolecule and biological product analysis. However, few studies have used CEC for the analysis of galactose end group diastereomers.

[0005] In view of the above-mentioned defects, the present invention aims to create a method for separating galactose end group diastereomers by capillary electrochromatography to make it more valuable for industrial use. Summary of the invention

[0006] To solve the above technical problems, the purpose of the present invention is to provide a method for separating galactose end group diastereomers by capillary electrochromatography. By using borax electrophoresis buffer to form a negatively charged complex with galactose, the absorbance of galactose at ultraviolet 195nm can be increased, and direct detection of galactose can be achieved without going through a complicated derivatization process or using other expensive detectors. At the same time, the charged complex can migrate in an electric field, and the complex formed by α-galactose and β-galactose is separated based on the difference in their electrophoretic mobility and interaction with the capillary silica monolithic column stationary phase, without the need to add organic reagents, which is environmentally friendly and economical.

[0007] The present invention discloses a method for separating galactose end group diastereomers by capillary electrochromatography, wherein the specific separation method is as follows:

[0008] S1. Prepare borax electrophoresis buffer and adjust it to different pH values;

[0009] S2, dissolving the galactose sample in the borax electrophoresis buffer to prepare a galactose solution in which the α and β configurations reach a dynamic equilibrium at room temperature;

[0010] S3, using the above obtained galactose solution containing both α and β configurations as a sample to be analyzed;

[0011] S4. Preparation of capillary silica monolithic column:

[0012] The capillary column was pretreated with alkali and acid, polyethylene glycol, urea, acetic acid and tetramethoxysilane were mixed, stirred in an ice bath to form a sol, injected into the capillary column, placed in a 40°C water bath, further heated at 120°C for reaction, and after one week, the temperature was raised to 320°C for calcination to obtain a silica gel monolithic column;

[0013] The sample to be analyzed was passed through a silica gel monolithic column to achieve baseline separation of galactose end group diastereomers.

[0014] Furthermore, the pH value of the borax electrophoresis buffer in S1 is adjusted to 7.2-8.0.

[0015] Furthermore, the pH value of the electrophoresis buffer used to dissolve the galactose sample in S2 is 10.

[0016] Furthermore, the water bath time in the preparation process of the capillary silica monolithic column described in S4 is 40 hours, the further heating time is 3 hours, and the calcination time is 24 hours.

[0017] Furthermore, the capillary electrophoresis separation voltage used for the analyzed sample in S4 to pass through the silica gel monolithic column is 5 to 30 kV; the buffer concentration is 55 to 85 mM; the injection volume is 5 kPa, 3 to 9 s; and the buffer pH is 7.2 to 8.0.

[0018] Furthermore, the pH value of the capillary electrophoresis separation buffer used for passing the analyzed sample through the silica gel monolithic column described in S4 is 7.6.

[0019] Furthermore, the capillary electrophoresis separation voltage used for the analyzed sample in S4 to pass through the silica gel monolithic column is 20 kV.

[0020] Furthermore, the concentration of the buffer used for capillary electrophoresis separation of the analyzed sample through the silica gel monolithic column described in S4 is 75 mM.

[0021] First, a borax electrophoresis buffer was prepared and adjusted to different pH values ​​(pH 7.2-10); a borax buffer with a pH value of 10 was used to dissolve the galactose sample to obtain a galactose solution in which the α and β configurations reached a dynamic equilibrium; the galactose solution was used as the sample to be analyzed, a capillary silica monolithic column was prepared, and a capillary electrochromatography UV direct detection method was constructed. Borax was used as an electrophoresis buffer to increase the absorbance of galactose at UV 195nm, thereby achieving direct detection of galactose. Borax electrophoresis buffer forms a negatively charged complex with galactose under pH>7 conditions, which can migrate in an electric field. The electroosmotic mobility and electrophoretic mobility were adjusted by optimizing the buffer pH value (pH7.2-8), thereby improving the separation efficiency. In addition, the electrophoretic separation conditions including voltage (5-30kV), buffer concentration (55-85mM), and injection volume (5kPa, 3-9s) were optimized, thereby optimizing the separation of galactose end group diastereomers.

[0022] By means of the above scheme, the present invention has at least the following advantages:

[0023] The present invention uses borax electrophoresis buffer to form a negatively charged complex with galactose, which can increase the absorbance of galactose at ultraviolet 195nm, and realize direct detection of galactose without going through a complicated derivatization process or using other expensive detectors. At the same time, the charged complex can migrate in an electric field, and the complex formed by α-galactose and β-galactose is separated based on the difference in their electrophoretic mobility and interaction with the capillary silica monolithic column stationary phase, without adding organic reagents, and is environmentally friendly and economical. The method for separating galactose end group diastereomers provided by the present invention is simple to operate and highly repeatable, and further expands the application of capillary electrochromatography technology in the field of galactose separation.

[0024] 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

[0025] 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.

[0026] Figure 1 : Intra-day repeatability of separation of galactose anomers by capillary electrochromatography.

[0027] Figure 2 : Day-to-day repeatability of separation of galactose anomers by capillary electrochromatography.

[0028] Figure 3 :Effect of electrophoresis buffer pH on the separation of galactose end group isomers.

[0029] Among them, the buffer pH value is a, 7.2; b, 7.4; c, 7.6; d, 7.8; e, 8.

[0030] Figure 4 :Effect of separation voltage on the separation of galactose end group diastereomers.

[0031] Among them, the separation voltage is a, 5kV; b, 10kV; c, 15kV; d, 20kV; e, 25kV; f, 30kV.

[0032] Figure 5 :Effect of injection volume on the separation of galactose anomers.

[0033] Among them, the injection volume is a, 5kPa, 3s; b, 5kPa, 6s; c, 5kPa, 9s.

[0034] Figure 6 : Effect of buffer concentration on the separation of galactose anomers.

[0035] Among them, the buffer concentration is a, 55mM; b, 65mM; c, 75mM; d, 85mM. DETAILED DESCRIPTION

[0036] 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.

[0037] Example 1

[0038] 1. Preparation of galactose sample solution:

[0039] Prepare borax electrophoresis buffer: weigh 3.0183 g of anhydrous sodium tetraborate and dissolve it in ultrapure water. Use saturated sodium hydroxide solution to adjust the pH to 10 to prepare 200 mL of borax buffer with a concentration of 75 mM.

[0040] Purchased D-galactose (McLean, Shanghai, China) was dissolved in borax electrophoresis buffer (pH 10) to prepare a 20 mg / mL galactose solution at room temperature.

[0041] Galactose molecules undergo mutarotation in aqueous solution. Alkaline conditions can increase the mutarotation rate, promote the reversible interconversion between α and β forms, and ultimately achieve dynamic equilibrium.

[0042] The galactose solution containing the two configurations of α and β is further used as an analyzed sample and is directly separated and detected by capillary electrochromatography under ultraviolet.

[0043] 2. Capillary electrochromatography analysis:

[0044] Take a capillary column with an inner diameter of 75 μm and rinse it with water and 1 mol L-1 NaOH for 45 min respectively, then rinse it with water and 0.1 mol L -1 HCl was rinsed for 45 min each, and finally rinsed with water and methanol. Nitrogen was blown at 120 ° C for 3 h before use. Weigh 0.225 g urea and 0.22 g polyethylene glycol (PEG) respectively, dissolve in 5 mL 0.01 mol L-1 acetic acid solution, slowly add 0.9 mL tetramethoxysilane (TMOS), stir for 45 min under 0 ° C ice bath conditions, and then filter the obtained sol twice with a 0.22 μm microporous filter membrane, and then inject it into the capillary. After sealing both ends of the capillary, put it in a 40 ° C constant temperature water bath and age it for a certain period of time. Use an oven at 120 ° C to prepare the mesopores, and then use a programmed temperature rise for calcination. The prepared capillary silica monolithic column is used, and the running buffer is a borax electrophoresis buffer solution, which can increase the absorbance of galactose at ultraviolet 195 nm, and realize the direct ultraviolet detection of galactose. Borax electrophoresis buffer forms a negatively charged complex with galactose under the condition of pH>7, which can migrate in the electric field and achieve baseline separation of the α and β configurations based on the difference in electrophoretic mobility. By optimizing the pH value of the buffer (pH 7.2-8), the electroosmotic mobility and electrophoretic mobility are adjusted to improve the separation efficiency. At the same time, the electrophoretic separation conditions including voltage (5-30kV), buffer concentration (55-85mM), and injection volume (5kPa, 3-9s) are optimized to examine the effect of electrophoretic conditions on the separation of galactose end group diastereomers. The analysis of galactose end group diastereomers by this electrophoretic system shows a high separation repeatability. Figure 1 and Figure 2 As shown, the separation system has high intra-day and inter-day stability.

[0045] Example 2

[0046] According to the method of Example 1, the pH value of the electrophoresis buffer was optimized. Figure 3 It can be seen that as the pH value increases, the retention time of the galactose end group isomer increases. This is because as the pH value increases, it is more conducive to the complexation of borate and galactose molecules to form a negatively charged complex, and its apparent mobility decreases as the pH value increases, so the retention time increases. When the pH value is 7.6, the system has the best separation performance.

[0047] Example 3

[0048] According to the method of Example 1, the separation voltage was optimized. Since the increase of the operating voltage will cause the increase of electroosmotic flow, it will inhibit the retention of galactose molecules by 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 4 It can be seen that as the voltage increases, the retention time of the terminal diastereomer of galactose gradually decreases. This is because as the voltage increases, the electroosmotic flow in the column increases, and the retention of galactose decreases. Therefore, its peak time gradually shortens. When the voltage is 5kV, the electroosmotic flow is small, the retention time is long, and the separation selectivity is strong. 20kV is the optimal voltage value.

[0049] Example 4

[0050] According to the method of Example 1, the injection volume was optimized. This experiment investigated the effect of the injection volume on the separation of galactose end group diastereomers. Figure 5 It can be seen that the resolution decreases with the increase of injection volume. This is because a higher sample volume will cause peak tailing and reduce column efficiency, so the resolution of galactose end group isomers decreases with the increase of sample volume.

[0051] Example 5

[0052] According to the method of Example 1, the concentration of the buffer solution was explored. As shown in Table 6, the effect of the buffer concentration on the separation of galactose end group diastereomers was investigated. Figure 6 It can be seen that as the buffer concentration increases, the retention time of galactose molecules gradually increases. This is because the increase in buffer concentration will cause the electroosmotic flow to decrease, and the retention time of the galactose end group isomers will increase. Figure 6 As shown, when the buffer concentration is 75 mmol L-1, the separation degree is the largest. This is because when the buffer concentration is too small or too large, the change in the ionic strength of the solution will affect the electroosmotic flow in the column. When the optimal concentration of buffer is used, the separation ability of the silica gel column is optimal.

[0053] It can be seen from the above examples that capillary electrochromatography technology provides a fast, efficient and rapid analytical platform for the separation of galactose end group diastereomers.

[0054] 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 method for separating galactose end group diastereomers by capillary electrochromatography, characterized in that The specific separation method is: S1. Prepare borax electrophoresis buffer and adjust the pH value to 10; S2, dissolving the galactose sample in the borax electrophoresis buffer to prepare a galactose solution in which the α and β configurations reach a dynamic equilibrium at room temperature; S3, using the above obtained galactose solution containing both α and β configurations as a sample to be analyzed; S4. Preparation of capillary silica monolithic column: The capillary column was pretreated with alkali and acid, polyethylene glycol, urea, acetic acid and tetramethoxysilane were mixed, stirred in an ice bath to form a sol, injected into the capillary column, placed in a 40°C water bath for 40 hours, further heated at 120°C for 3 hours, and after being placed for one week, the temperature was raised to 320°C for calcination for 24 hours to obtain a silica gel monolithic column; The analyzed sample is passed through a silica gel monolithic column to achieve baseline separation of galactose end group diastereomers; The pH value of the capillary electrophoresis separation buffer used for the analyzed sample to pass through the silica gel monolithic column is 7.6; The capillary electrophoresis separation voltage used for the analyzed sample passing through the silica gel monolithic column is 20 kV; The concentration of the buffer solution used for capillary electrophoresis separation of the analyzed sample through the silica gel monolithic column is 75mM.

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