Method for detecting beta-cyclodextrin

Abstract

The invention belongs to the technical field of chemical detection, and provides a method for detecting beta-cyclodextrin, which comprises the following steps: raising the temperature of beta-CD-containing feed liquid to be greater than or equal to 65 DEG C and less than 85 DEG C, inclusion phenolphthalein under a heat preservation condition, and then detecting the color development degree in an alkaline environment. According to the method, the detection range is greatly widened, tedious dilution is not needed, the operation is simplified, the efficiency is improved, and errors introduced in the dilution process are avoided; the method can be directly applied to detection of common high-concentration samples in industrial production.

Description

Technical Field

[0001] This invention belongs to the field of chemical detection technology, specifically relating to a method for detecting β-cyclodextrin based on spectral analysis. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] In enzymatic conversion processes primarily using β-cyclodextrin (β-CD), the feed solution before secondary crystallization typically needs to be heated to near boiling to dissolve the β-CD, followed by cooling for crystallization and purification. To precisely control the crystallization process and assess the yield, rapid and accurate detection of the β-CD concentration in the feed solution is necessary. However, due to the extremely low solubility of β-CD at room temperature, rapid cooling after sampling from the high-temperature feed solution can lead to β-CD precipitation, forming an unstable suspension. This presents a significant challenge for quantitative sampling and analysis.

[0004] Existing analytical methods for detecting β-CD include spectroscopic analysis, chromatography, and enzymatic methods. Among these methods, spectroscopic analysis has advantages such as low equipment cost, simple operation, good method stability, and good reproducibility, making it a commonly used method for β-CD concentration detection.

[0005] However, this technique has significant limitations for complex suspension systems containing other substrates with unknown β-CD concentrations, as described above. Its commonly used concentration range is low (e.g., 0.5-1 mg / mL), making it unsuitable for most applications. According to existing technical literature and publicly available data, the solubility of β-CD at room temperature using this method is approximately 1.8% (w / v or v / v). This characteristic severely limits its application in the direct detection of high-concentration β-CD samples. When detecting samples exceeding this range, a cumbersome dilution process is necessary, increasing operational complexity and introducing additional error risks. For example, in experiments using high-performance liquid chromatography to measure the β-CD concentration in the feed solution before secondary crystallization, the suspension can be shaken, and rapid sampling can be attempted during shaking intervals, followed by gradient dilution to obtain a homogeneous solution suitable for injection. However, in practical applications, this suspension is prone to stratification, and even brief settling can affect the representativeness of the sample. Furthermore, when the diluted sample is injected into the HPLC system, the instrument's sample chamber temperature is often below room temperature. β-CD precipitates at the low-temperature region before entering the temperature-controlled column (e.g., 40°C), clogging the column head, leading to increased column pressure and system alarms. The resulting concentration data cannot accurately reflect the original sample solution's condition. To mitigate this problem, the HPLC sample chamber temperature must be pre-determined, and dilution should be performed using a solvent no higher than this temperature. However, this not only increases the complexity and time cost of sample pretreatment but also, due to the narrow linear detection range of HPLC, makes it difficult to determine the dilution factor in one attempt, often requiring repeated trials. More importantly, the concentration of each sample taken from the continuously precipitating suspension may fluctuate significantly, resulting in a lack of reproducibility in the dilution results, further weakening the reliability and efficiency of the detection. Therefore, existing HPLC-based methods struggle to achieve rapid and accurate determination of β-CD content in high-concentration samples before recrystallization. Summary of the Invention

[0006] To address the problems in the prior art, the present invention provides a method for detecting β-CD, which increases the detectable concentration range to 15 wt% by changing the measurement conditions.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] A method for detecting β-CD content involves raising the temperature of a β-CD-containing solution to a temperature not lower than 65°C and lower than 85°C, encapsulating phenolphthalein under heat preservation conditions, and detecting the β-CD content in the suspension solution by reducing the color development of phenolphthalein in an alkaline environment.

[0009] Specifically, it includes the following steps: Prepare β-CD solutions with a weight concentration of 0.128-0.136 g / g at 65-85℃. Construct a standard curve at concentrations of 20%, 40%, 60%, 80%, and 100% of the maximum concentration. Take four portions of each concentration sample and add 3-4 portions of 0.01-0.014% (w / v) phenolphthalein solution. Add alkali to adjust the pH of the system to ≥8.2. After mixing, let stand for 2-30 minutes, and measure the absorbance. Fit a regression curve equation between the mass of β-CD and the absorbance value, and calculate the mass of β-CD in the sample solution.

[0010] The insulation conditions are 70-75℃.

[0011] The concentrations of the β-CD standard solution and the sample solution to be tested are not higher than 15 wt%.

[0012] To enable on-site instrument-free testing, colorimetric cards can be prepared using the colorimetric properties of β-CD at different concentration ranges, allowing for rapid on-site testing.

[0013] The present invention has the following advantages: (1) The upper limit of linear detection concentration of β-CD is increased from less than 1.8% to 15%, which greatly expands the detection range; (2) For high-concentration samples, direct measurement can be achieved without cumbersome dilution, simplifying the operation, improving efficiency, and avoiding errors introduced by the dilution process; (3) Using traditional ultraviolet spectrophotometry equipment has all the advantages of low cost, simple operation and good stability, which makes it more conducive to promotion and application; (4) This method can be directly applied to the detection of high-concentration samples commonly used in industrial production, which significantly expands the scope of application of the technology. Detailed Implementation

[0014] The present invention will be further described below with reference to the embodiments, but the present invention is not limited to the following embodiments.

[0015] Example 1: Establishment and Validation of the Detection Method 1. Effects of temperature and concentration To investigate the stability of the complex formed by high concentrations of β-CD and phenolphthalein (hereinafter referred to as β-CD complex) at different temperatures, the following method was used to screen for concentrations that could maintain a dissolved state at room temperature for a certain period of time: Weigh out a certain amount of β-CD and add 1 mL of water. Dissolve it completely at 70℃ and 80℃ respectively. Add 300 μL of 0.012% phenolphthalein ethanol solution and 500 μL of 0.04M Na2CO3 solution. Observe for how long at room temperature and whether crystals precipitate.

[0016] Table 1 Cooling crystallization time of β-CD complex The results showed that the β-CD complex dissolved at 80℃ precipitated within 30 minutes, while β-CD dissolved at 70℃ only showed white crystals after 2 hours. This method indicates that 70℃ is the optimal temperature and concentration for β-CD solubility detection.

[0017] 2. Range estimation To roughly estimate the detection upper limit of the method, the following method is used for visual estimation: Weigh out 0.14 g, 0.07 g, and 0.035 g of β-CD respectively and add 1 mL of water to prepare solutions of 14%, 7%, and 3.5%, respectively, and label them No. 1, No. 2, and No. 3. Heat the above solutions and water in a water bath to 70°C, add 300 μL of 0.012% phenolphthalein ethanol solution and 500 μL of 0.04 M Na₂CO₃ solution, and then add water to a final volume of 2 mL. Visually observe the color change.

[0018] The results showed that the sample without β-CD was a deep purplish-red, sample 1 was a light purplish-red, sample 2 was an even lighter purplish-red, and sample 3 was almost colorless with a few fine dust-like suspended particles. Visual inspection indicated that the upper limit of the detection concentration at 70℃ was approximately 15%.

[0019] 3. Effect of the ratio of β-CD to phenolphthalein on color development time Table 2 Effect of reaction system on color development time The concentration of phenolphthalein was found to have a significant impact on the appearance time of the colorimetric gradient (Table 2). Specifically, the colorimetric process of high-concentration phenolphthalein was relatively slow, with gradient differences not appearing until 3 hours later; while the colorimetric time window of low-concentration phenolphthalein was too narrow, which was not conducive to data acquisition. Therefore, the optimal phenolphthalein concentration with a moderate colorimetric time window and clear gradient performance was finally selected as the optimal condition for subsequent analysis.

[0020] 4. Effect of the ratio of β-CD to phenolphthalein on the linearity of content. Different detection systems were prepared according to the amounts added in Tables 3 and 4. 200 μL of each system was added to a 96-well plate at different time points during the reaction to detect OD. 550 Constructing β-CD quality and OD 550 The linear relationship. (0.012% phenolphthalein ethanol solution, 0.04M Na2CO3 solution) Table 3 150 μL Phenolphthalein System Table 4 200 μL Phenolphthalein System Table 5. Linear equations for 150 μL phenolphthalein solution at different time points. Table 6. Linear equations for 200 μL phenolphthalein solution at different time points. Note: Since the β-CD was prepared at high temperature during the test, conventional volumetric flasks were not suitable. Therefore, the concentration is expressed as wt% (w / v%), not w / v%. The β-CD mass fraction in the solution was calculated to be 0.1146 g / g, and the density was 1.144 g / mL. The weight concentrations of 100, 50, and 25 μL of solution added were 0.1146, 0.0578, and 0.0290 g / g, respectively, and a concentration-labeled curve was fitted based on this data.

[0021] The results are shown in Tables 5 and 6. When the amount of phenolphthalein added to the system is 150 and 200 μL, the correlation R within the reaction time of less than 30 min is shown. 2 All values ​​were above 0.85, indicating better reliability; while the addition amount of R was 200 μL. 2 Higher, with a reaction time of less than 30 minutes, R 2 All are above 0.95.

[0022] In the reaction, β-CD and phenolphthalein react directly. When the amount of β-CD is constant, the amount of phenolphthalein can affect the degree and speed of color development. If the amount of phenolphthalein is too small, it may be rapidly encapsulated and unable to develop color in an alkaline environment. If the amount of phenolphthalein is too large, the free phenolphthalein will be stable in an alkaline environment and unable to distinguish concentration gradients. Only with appropriate dosage can a colorimetric effect be observed within a suitable experimental time, allowing for its application in production.

[0023] 5. Effect of sodium carbonate dosage The volume of sodium carbonate solution was varied to 150, 200, 250, 300, 350, 400, and 450 μL according to the conditions in Table 4. After reacting for 30 min, the absorbance (OD) was measured. 550 The test results showed that solutions within the above dosage range all exhibited a pink color change, and R 2 All values ​​were above 0.90. This indicates that, with the above-mentioned amount of sodium carbonate added, sodium carbonate provided the necessary alkaline environment for phenolphthalein to develop color.

[0024] 6. Accuracy To verify the accuracy of the detection method, the following spiking recovery experiment was designed. Take 1 mL of the high-concentration test solution (the test solution is a high-concentration suspension sampled before crystallization in the production of β-CD), add 3 mL of distilled water to dilute, heat to 70℃ to completely dissolve, and divide into four equal portions. Add 10 mg, 20 mg, and 30 mg of β-CD standard to three portions respectively, and heat until completely dissolved; these are used as the spiking recovery samples. The remaining portion is used as the background sample. Using the above determination method, the absorbance of each of the four samples is measured, and the concentration is calculated by substituting the values ​​into the standard curve. The spiking recovery rate is then calculated.

[0025] For the creation of a standard curve: Prepare a β-CD solution with a weight concentration of 0.130 g / g at 70℃. Construct a standard curve at concentrations of 25%, 50%, 75%, and 100% of the maximum concentration of 0.130 g / g. Take 100 μL of each concentration sample, add 150 μL of 0.01% (w / v) phenolphthalein solution, and then add 400 μL of 0.04 M sodium carbonate solution to adjust the pH of the system to ≥8.2. After mixing and standing for 2 minutes, transfer 200 μL to a 96-well plate and read the A550 reading using a microplate reader.

[0026] A linear relationship between β-CD concentration and OD value was established. OD was measured using an Opentrons pipetting robot for automated pipetting. 550 .

[0027] For ease of calculation, the mass of 1 mL of pipette is estimated as 1 g; the blank is the sample with a β-CD concentration of 0 in the standard curve (the same applies below).

[0028] The linear regression equation for the standard curve obtained from the data is: y = -0.0355x - 0.1668 R 2 = 0.9106. The results in Table 7 are obtained using this data.

[0029] Table 7 Recovery Rate The above results show that the average recovery rate of this method is 87.95%, which indicates that the method meets the accuracy requirements. Since there is no simple, rapid and optimized screening method for high-concentration suspension systems of β-CD in this field, this method has practical value.

[0030] Example 2: The effect of different detection temperatures on detection A β-CD solution with a weight concentration of 0.134 g / g was prepared at 80℃. A standard curve was constructed at concentrations of 20%, 40%, 60%, 80%, and 100% of the maximum concentration of 0.134 g / g. For each concentration, 100 μL of the sample was taken, and 200 μL of 0.01% (w / v) phenolphthalein solution was added, followed by 400 μL of 0.04 M sodium carbonate solution, to adjust the pH of the system to ≥8.2. After mixing and standing for 2 minutes, 200 μL was transferred to a 96-well plate, and the A value was read using a microplate reader. 550 The readings show that the absolute value of the difference between the 0-point and the highest concentration reading is 2.7, which is significant. The R-squared value of the regression equation is... 2 The value is 0.99, indicating good linearity.

[0031] A β-CD solution with a weight concentration of 0.136 g / g was prepared at 75℃. A standard curve was constructed at concentrations of 20%, 40%, 60%, 80%, and 100% of the maximum concentration of 0.136 g / g. Four portions of each concentration sample were taken, and three portions of 0.014% (w / v) phenolphthalein solution were added, followed by nine portions of 0.04 M sodium carbonate solution, to adjust the pH of the system to ≥8.2. After mixing and standing for 30 minutes, 200 μL was transferred to a 96-well plate, and the A value was read using a microplate reader. 550 The readings show that the absolute value of the difference between the 0-point and the highest concentration reading is 3.0, which is significant. The R-squared value of the regression equation is... 2 The value is 0.88, indicating good linearity.

[0032] A β-CD solution with a weight concentration of 0.132 g / g was prepared at 65℃. A standard curve was constructed at concentrations of 20%, 40%, 60%, 80%, and 100% of the maximum concentration of 0.132 g / g. Four aliquots of each concentration were taken, and four aliquots of 0.012% (w / v) phenolphthalein solution were added. Alkali was then added to adjust the pH of the system to ≥8.2. After mixing and standing for 12 minutes, 200 μL was transferred to a 96-well plate, and the A value was read using a microplate reader. 550 The readings show that the absolute value of the difference between the 0-point and the highest concentration reading is 3.2, which is significant. The R-squared value of the regression equation is... 2 The value is 0.95, indicating good linearity.

[0033] A β-CD solution with a weight concentration of 0.128 g / g was prepared at 85℃. A standard curve was constructed at concentrations of 20%, 40%, 60%, 80%, and 100% of the maximum concentration of 0.128 g / g. Four portions of each concentration sample were taken, and four portions of 0.013% (w / v) phenolphthalein solution were added, followed by eight portions of 0.04 M sodium carbonate solution, to adjust the pH of the system to ≥8.2. After mixing and standing for 20 minutes, 200 μL was transferred to a 96-well plate, and the A value was read using a microplate reader. 550The readings show that the absolute value of the difference between the 0-point and the highest concentration reading is 3.5, which is significant. The R-squared value of the regression equation is... 2 The value is 0.92, indicating good linearity.

[0034] A β-CD solution with a weight concentration of 0.130 g / g was prepared at 70℃. A standard curve was constructed at concentrations of 20%, 40%, 60%, 80%, and 100% of the maximum concentration of 0.130 g / g. Four portions of each concentration sample were taken, and four portions of 0.011% (w / v) phenolphthalein solution were added, followed by eight portions of 0.04 M sodium carbonate solution, to adjust the pH of the system to ≥8.2. After mixing and standing for 7 minutes, 200 μL was transferred to a 96-well plate, and the A value was read using a microplate reader. 550 The readings show that the absolute value of the difference between the 0-point and the highest concentration reading is 2.3, which is significant. The R-squared value of the regression equation is... 2 The value is 0.85, indicating good linearity.

[0035] Example 3: Practical Application of the Detection Method For direct detection of high-concentration solutions, three 1mL portions of the solution were taken, completely dissolved under heating conditions, and their OD values ​​were measured respectively. 550 The value was then used to calculate the concentration.

[0036] To prepare the standard curve: Weigh 0.1200g of β-CD, add 1mL of distilled water, heat until completely dissolved, and construct the linear relationship between β-CD concentration and OD value according to the table below.

[0037] Table 8. Ratio of Standard Curve Table 9. Standard Curve Concentration and Absorbance Based on the data in Table 9, the linear regression equation for the standard curve is: y = -0.0064x - 1.146, R0 2 = 0.9037.

[0038] Table 10 Sample Concentration Detection The above experiments show that this method can directly measure the concentration of the feed solution at 94.4 mg / g, and has certain advantages for determining high concentrations of β-CD in the β-CD production process.

[0039] Compared to the previous method, the detection limit has been significantly increased. Operators can directly take samples from the reaction vessel, check for dissolution after simple heating, and dilute them 1-2 times if necessary before testing. The concentration data of β-CD in the reaction solution can be obtained within 30 minutes.

[0040] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A method for detecting β-CD content, characterized in that, Includes the following steps: The temperature of the feed solution containing β-CD was raised to 65°C or higher but less than 85°C, and phenolphthalein was encapsulated under heat preservation conditions. Then, the degree of color development in an alkaline environment was measured.

2. The method according to claim 1, characterized in that, The concentration of β-CD in the feed solution is not higher than 15 wt%.

3. The method according to claim 1, characterized in that, The degree of color development can be determined using a colorimetric card or absorbance value.

4. The method according to claim 1, characterized in that, Includes the following steps: Prepare β-CD solutions with a weight concentration of 0.128-0.136 g / g at 65-85℃. Construct a standard curve at concentrations of 20%, 40%, 60%, 80%, and 100% of the maximum concentration. Take four portions of each concentration sample and add 3-4 portions of 0.01-0.014% (w / v) phenolphthalein solution. Add alkali to adjust the pH of the system to ≥8.

2. After mixing, let stand for 2-30 minutes, and measure the absorbance. Fit a regression curve equation between the mass of β-CD and the absorbance value, and calculate the mass of β-CD in the sample solution.

5. The method according to claim 4, characterized in that, The insulation conditions are 70-75℃.

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