Application of indantrione color reagent in determination of polyamide hydrolysis product and method for determining content of polyamide hydrolysis product

Abstract

The application relates to the field of polyamide material analysis, and discloses application of an indantrione color developing reagent in determination of polyamide hydrolysis products and a method for determining the content of the polyamide hydrolysis products, wherein the indantrione color developing reagent contains an indantrione-ethylene glycol solution and an acetic acid-sodium acetate buffer solution. The application provided in the application adopts ethylene glycol as a single solvent of the indantrione, effectively inhibits oxidative degradation of the indantrione under high-temperature color developing conditions, and enables uniform color development of oligomers. The application does not add antioxidants, avoids interference with color developing reactions, is accurate and reliable in determination, is simple in preparation due to the single solvent, has a long storage time under light shielding at 4 DEG C, and can not only accurately determine monomers of polyamides and dimers to heptamers of the monomers, but also accurately determine various polyamide hydrolysis products, and has a good linear relationship.

Description

Technical Field

[0001] This invention relates to the field of polyamide material analysis, specifically to the application of a ninhydrin colorimetric reagent in the determination of polyamide hydrolysis products and a method for determining the content of polyamide hydrolysis products. Background Technology

[0002] The ninhydrin colorimetric method utilizes the principle of the specific reaction between ninhydrin and α-ketoamino compounds to generate Ruhrmann violet (e.g., Figure 8 As shown in the figure, this is a classic analytical technique for the quantitative determination of amino substances at a wavelength of 570 nm. Due to its high sensitivity and ease of operation, this method has been widely used in biochemical analysis and polymer material characterization. In the degradation study of polyamide materials, the accurate quantification of its hydrolysis products is of great significance for assessing the degree of hydrolysis, and the ninhydrin colorimetric method provides a reliable analytical approach for the determination of this indicator.

[0003] However, this technology faces a key technical bottleneck in practical applications: ninhydrin molecules are prone to oxidative degradation under high-temperature colorimetric reaction conditions, leading to a significant reduction in colorimetric efficiency. This results in a systematic deviation between the absorbance measurement and the concentration of the analyte amino group, severely affecting the accuracy and reproducibility of quantitative analysis. Therefore, improving the chemical stability of ninhydrin during the determination process has become the core issue in optimizing this analytical method.

[0004] To address the issue of insufficient stability of ninhydrin, existing technologies have proposed several improvement schemes. One approach is to inhibit ninhydrin oxidation by adding antioxidants (such as vitamin C). However, studies have found that antioxidants interfere with the normal colorimetric reaction between ninhydrin and amino groups, leading to distorted measurement results. Another approach uses a mixed solvent system to enhance ninhydrin stability, such as a mixture of n-propanol, n-butanol, and ethylene glycol. However, this method has limitations, including complex procedures, difficulty in precisely controlling solvent ratios, and the inability to effectively measure water-soluble amino substances due to the slight water solubility of n-butanol. More importantly, ninhydrin reagents prepared using this mixed solvent system have a short shelf life, as reported in the literature, making it difficult to meet the needs of routine laboratory batch determinations and long-term stable use.

[0005] Furthermore, existing technologies have very limited ability to detect oligomers (dimers to heptamers) in polyamide hydrolysis products. Theoretically, ninhydrin can detect amino-containing compounds, but for oligomers with greater steric hindrance and lower reactivity, existing systems often suffer from incomplete color development and poor linearity, making accurate quantification difficult. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of poor high-temperature stability of ninhydrin colorimetric reagents, interference of antioxidants with the colorimetric reaction, poor detection ability of oligomers in polyamide hydrolysis products, and short reagent shelf life in existing technologies. This invention provides an application of ninhydrin colorimetric reagent in the determination of polyamide hydrolysis products and a method for determining the content of polyamide hydrolysis products. This ninhydrin colorimetric reagent has the characteristics of high stability, no interference, simple operation, and long shelf life. It can accurately determine not only the monomers of polyamide, but also the dimers, trimers, and even heptamers of the monomers, exhibiting good linearity in the concentration range below 1 mM.

[0007] To achieve the above objectives, a first aspect of the present invention provides the application of a ninhydrin colorimetric reagent in the determination of polyamide hydrolysis products, wherein the ninhydrin colorimetric reagent contains a ninhydrin-ethylene glycol solution and an acetate-sodium acetate buffer solution.

[0008] A second aspect of the present invention provides a method for determining the content of polyamide hydrolysis products, comprising the following steps: S1. Preparation of ninhydrin colorimetric reagent: Dissolve hydrated ninhydrin in ethylene glycol to prepare ninhydrin-ethylene glycol solution, and mix the ninhydrin-ethylene glycol solution with acetate-sodium acetate buffer to obtain ninhydrin colorimetric reagent; S2. The test sample containing polyamide hydrolysis products is mixed with the ninhydrin colorimetric reagent and heated to react. After cooling, the absorbance of the product of the heated reaction is measured at a wavelength of 560-580 nm. The content of polyamide hydrolysis products in the test sample is calculated based on the absorbance and the standard curve of polyamide hydrolysis products.

[0009] Through the above technical solution, the application provided by this invention uses ethylene glycol as a single solvent for ninhydrin, effectively inhibiting the oxidative degradation of ninhydrin under high-temperature colorimetric conditions, while also enabling uniform color development of oligomers. This invention does not add antioxidants, avoiding interference with the colorimetric reaction, ensuring accurate and reliable determination; it uses a single solvent, making preparation simple; the reagent has a long storage time at 4°C in the dark; furthermore, this invention can accurately determine not only the monomers of polyamides, but also the dimers to heptamers of the monomers, with good linearity. Attached Figure Description

[0010] Figure 1 This is the standard curve of 6-aminohexanoic acid in Example 1; Figure 2 This is the standard curve of the 6-aminohexanoic acid dimer in Example 4; Figure 3 This is the standard curve of the 6-aminohexanoic acid trimer in Example 5; Figure 4The images show the color changes of ninhydrin solutions prepared in different solvent systems (methanol, ethanol, n-propanol, isopropanol, n-butanol, ethylene glycol, acetonitrile, and dimethyl sulfoxide) in Comparative Example 6 after being left at room temperature for 24 hours. Figure 5 The full-wavelength scan spectra of ninhydrin solutions prepared in different solvent systems in Comparative Example 6 after being placed at room temperature for 24 h. Figure 6 These are comparative photographs of the colorimetric reaction of ninhydrin with 6-aminohexanoic acid in Comparative Example 7, using ethanol, n-butanol, and ethylene glycol as solvents, respectively. Figure 7 The full-wavelength scan spectra of the colorimetric reaction of ninhydrin with 6-aminohexanoic acid in Comparative Example 7, using ethanol, n-butanol, and ethylene glycol as solvents, respectively. Figure 8 This is a structural diagram illustrating the principle of the ninhydrin colorimetric reaction of the present invention; Figure 9 This is the standard curve of the 6-aminohexanoic acid heptamer in Example 8; Figure 10 This is the standard curve of the 5-aminovaleric acid heptamer in Example 9; Figure 11 This is the standard curve of hexamethylenediamine in Example 10. Detailed Implementation

[0011] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0012] The first aspect of this invention provides the application of a ninhydrin colorimetric reagent in the determination of polyamide hydrolysis products; the ninhydrin colorimetric reagent contains a ninhydrin-ethylene glycol solution and an acetate-sodium acetate buffer solution.

[0013] The application provided by this invention uses ethylene glycol as the single solvent for ninhydrin, effectively inhibiting its oxidative degradation under high-temperature colorimetric conditions, while also enabling uniform color development of oligomers. This invention does not add antioxidants, avoiding interference with the colorimetric reaction, ensuring accurate and reliable measurements; it uses a single solvent, making preparation simple; and the colorimetric reagent has a long storage time at 4°C in the dark. Furthermore, this invention can accurately determine not only 6-aminohexanoic acid monomers but also dimers to heptamers, with good linearity.

[0014] According to the present invention, preferably, the polyamide hydrolysis product contains polyamide monomers and / or oligomers of said monomers. The inventors have found that applying this method to the analysis of polyamide hydrolysis products can simultaneously meet the detection requirements for both polyamide monomers and oligomers of said monomers, thus having a wide range of applications.

[0015] More preferably, the monomer of the polyamide is selected from at least one of 6-aminohexanoic acid, γ-aminobutyric acid, 5-aminovaleric acid, and hexamethylenediamine; the oligomer of the monomer is selected from at least one of the monomer dimer, monomer trimer, monomer tetramer, monomer pentamer, monomer hexamer, and monomer heptamer. The inventors have found that limiting the monomer type and oligomer degree of polymerization within the above-mentioned ranges can cover the hydrolysis products of various common polyamides (such as polyamide 6, polyamide 5, and polyamide 66), further broadening the applicability of the method.

[0016] More preferably, the polyamide is polyamide 6, the monomer of polyamide 6 is 6-aminohexanoic acid, and the oligomer of 6-aminohexanoic acid is selected from at least one of 6-aminohexanoic acid dimer, 6-aminohexanoic acid trimer, 6-aminohexanoic acid tetramer, 6-aminohexanoic acid pentamer, 6-aminohexanoic acid hexamer, and 6-aminohexanoic acid heptamer. The inventors unexpectedly discovered in their research that when ethylene glycol is used as a single solvent, not only is the high-temperature stability of ninhydrin significantly improved, allowing for accurate and reliable quantitative detection of the monomer 6-aminohexanoic acid in polyamide 6, but more surprisingly, oligomers (dimers to heptamers) that were previously difficult to detect due to large steric hindrance and low reactivity can all achieve uniform and complete colorimetric reactions, and possess a good linear relationship (R0). 2 >0.99).

[0017] According to the present invention, preferably, in the ninhydrin-ethylene glycol solution, the amount of ninhydrin used relative to 100 mL of ethylene glycol is 0.5-0.7 g, specifically 0.5, 0.55, 0.6, 0.65, 0.7, or any value between the above two values. The inventors have found that controlling the amount of ninhydrin within this preferred range results in a sufficient colorimetric reaction and good linearity, making the determination results more accurate and reliable.

[0018] According to the present invention, preferably, the pH of the acetate-sodium acetate buffer solution is 4.2-4.8, specifically 4.2, 4.3, 4.5, 4.6, 4.8, or any value between the above two values. The inventors have found that controlling the pH of the buffer solution within this preferred range provides a suitable acidic environment for the colorimetric reaction, further resulting in higher colorimetric efficiency, more stable absorbance, and more accurate and reliable measurement results.

[0019] According to the present invention, preferably, in the ninhydrin colorimetric reagent, the volume ratio of the ninhydrin-ethylene glycol solution to the acetate-sodium acetate buffer solution is 8-15:1, specifically 8:1, 10:1, 12:1, 14:1, 15:1, or any value between the above two values. When the volume ratio of the ninhydrin-ethylene glycol solution to the acetate-sodium acetate buffer solution is within this preferred range, the colorimetric reaction is most sensitive and stable. If the buffer ratio is too high, the increased acidity of the system will inhibit the colorimetric reaction; if the buffer ratio is too low, the stable pH environment required for the reaction cannot be effectively maintained, resulting in decreased absorbance and a worsened linear relationship. The inventors have found in their research that using this preferred volume ratio results in good sensitivity of the colorimetric reaction, and the linear correlation coefficient of the standard curve can stably reach R0. 2 >0.999 (6-aminohexanoic acid) and R 2 >0.99 (oligomers), the precision and accuracy of the measurement results are significantly improved.

[0020] A second aspect of the present invention provides a method for determining the content of polyamide hydrolysis products, the method comprising the following steps: S1. Preparation of ninhydrin colorimetric reagent: Dissolve hydrated ninhydrin in ethylene glycol to prepare ninhydrin-ethylene glycol solution, and mix the ninhydrin-ethylene glycol solution with acetate-sodium acetate buffer to obtain ninhydrin colorimetric reagent; S2. The test sample containing polyamide hydrolysis products is mixed with the ninhydrin colorimetric reagent and heated to react. After cooling, the absorbance of the product of the heated reaction is measured at a wavelength of 560-580 nm. The content of polyamide hydrolysis products in the test sample is calculated based on the absorbance and the standard curve of polyamide hydrolysis products.

[0021] This invention, through the above steps of preparing ninhydrin colorimetric reagent and performing heating reaction and absorbance measurement, enables simple and accurate quantitative analysis of polyamide hydrolysis products. In this method, the oxidative degradation of ninhydrin is effectively inhibited, the colorimetric reaction is sufficiently stable, and a good linear relationship is observed between absorbance and hydrolysis product concentration, demonstrating high precision and reliability.

[0022] According to the present invention, preferably, the polyamide hydrolysis product contains polyamide monomers and / or oligomers of said monomers. The inventors have found that applying this method to the analysis of polyamide hydrolysis products can simultaneously meet the detection requirements for both polyamide monomers and oligomers of said monomers, thus having a wide range of applications.

[0023] More preferably, the monomer of the polyamide is selected from at least one of 6-aminohexanoic acid, γ-aminobutyric acid, 5-aminovaleric acid, and hexamethylenediamine; the oligomer of the monomer is selected from at least one of the monomer dimer, monomer trimer, monomer tetramer, monomer pentamer, monomer hexamer, and monomer heptamer. The inventors have found that limiting the monomer type and oligomer degree of polymerization within the above-mentioned ranges can cover the hydrolysis products of various common polyamides (such as polyamide 6, polyamide 5, and polyamide 66), further broadening the applicability of the method.

[0024] More preferably, the polyamide is polyamide 6, the monomer of polyamide 6 is 6-aminohexanoic acid, and the oligomer of 6-aminohexanoic acid is selected from at least one of 6-aminohexanoic acid dimer, 6-aminohexanoic acid trimer, 6-aminohexanoic acid tetramer, 6-aminohexanoic acid pentamer, 6-aminohexanoic acid hexamer, and 6-aminohexanoic acid heptamer. The inventors unexpectedly discovered in their research that when ethylene glycol is used as a single solvent, not only is the high-temperature stability of ninhydrin significantly improved, allowing for accurate and reliable quantitative detection of the monomer 6-aminohexanoic acid in polyamide 6, but more surprisingly, oligomers (dimers to heptamers) that were previously difficult to detect due to large steric hindrance and low reactivity can all achieve uniform and complete colorimetric reactions, and possess a good linear relationship (R0). 2 >0.99).

[0025] According to the present invention, preferably, in the ninhydrin-ethylene glycol solution, the amount of ninhydrin used relative to 100 mL of ethylene glycol is 0.5-0.7 g, specifically 0.5, 0.55, 0.6, 0.65, 0.7 g, or any value between the above two values. The inventors have found that controlling the amount of ninhydrin within this preferred range results in a sufficient colorimetric reaction, good linearity, and accurate and reliable measurement results.

[0026] According to the present invention, preferably, the pH of the acetate-sodium acetate buffer solution is 4.2-4.8, specifically 4.2, 4.3, 4.5, 4.6, 4.8, or any value between the above two values. The inventors have found that controlling the pH of the buffer solution within this preferred range provides a suitable acidic environment for the colorimetric reaction, resulting in higher colorimetric efficiency, more stable absorbance, and more accurate and reliable measurement results.

[0027] According to the present invention, preferably, in the ninhydrin colorimetric reagent, the volume ratio of the ninhydrin-ethylene glycol solution to the acetate-sodium acetate buffer solution is 8-15:1, specifically 8:1, 10:1, 12:1, 14:1, 15:1, or any value between the above two values. The inventors have found that controlling this volume ratio within the preferred range results in a sensitive and stable colorimetric reaction; if the buffer ratio is too high or too low, it will lead to a decrease in absorbance and a deterioration in linearity.

[0028] In the application of this invention, the heating temperature is 90-100℃, specifically 90℃, 92℃, 96℃, 99℃, 100℃, or any value between these two. The heating time is 20-30 min, specifically 20 min, 22 min, 24 min, 26 min, 30 min, or any value between these two. The inventors have found that within this preferred temperature and time range, the colorimetric reaction between ninhydrin and the amino compound can proceed fully, while the oxidative degradation of ninhydrin itself is effectively inhibited. If the heating temperature is too low or the time is too short, the colorimetric reaction is incomplete, and the absorbance is low. If the heating temperature is too high or the time is too long, the oxidative degradation of ninhydrin is aggravated, and the linear relationship deteriorates. By adopting the above preferred conditions, the colorimetric reaction can be more complete, and the detection results of 6-aminohexanoic acid and its oligomers can be more accurate and reliable.

[0029] According to the present invention, preferably, in step S2, the process of plotting the standard curve of the polyamide hydrolysis product includes: (1) Prepare multiple standard solutions of polyamide hydrolysis products of different concentrations; (2) Mix the standard solution with the ninhydrin colorimetric reagent and carry out the heating reaction under the same conditions as in step S2. After cooling, measure the absorbance of the product of the heating reaction at a wavelength of 560-580 nm. Plot a standard curve with the concentration of the polyamide hydrolysis product standard solution as the abscissa and the absorbance as the ordinate.

[0030] The inventors discovered that by using the above method to plot a standard curve, the correlation between absorbance and the concentration of polyamide hydrolysis products can be accurately established. The standard curve plotted by this method exhibits good linearity, ensuring the accuracy and reliability of the quantitative results.

[0031] According to the present invention, preferably, the concentration of the polyamide hydrolysis product standard solution is not higher than 1 mM, specifically it can be 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM, or any value between the above two values. The inventors have found that controlling the concentration of the standard solution within this preferred range maintains a good linear relationship between absorbance and concentration, ensuring the reliability of the standard curve.

[0032] According to the present invention, preferably, the linear correlation coefficient R of the standard curve of the polyamide hydrolysis product is... 2 ≥0.99. The linear correlation coefficient R0 of the standard curve for the 6-aminohexanoic acid in the standard curve of the polyamide hydrolysis products is ≥0.99. 2 The linear correlation coefficient R of the standard curve of the 6-aminohexanoic acid oligomer is ≥0.999. 2≥0.99. This indicates a good linear relationship between absorbance and the concentration of polyamide hydrolysis products. Using this standard curve for quantification can ensure the accuracy and reliability of the measurement results.

[0033] According to a particularly preferred embodiment of the present invention, a method for determining the content of polyamide hydrolysis products is provided, wherein the amount of ninhydrin in the ninhydrin-ethylene glycol solution is 0.5-0.7 g / 100 mL ethylene glycol, the pH of the acetate-sodium acetate buffer solution is 4.2-4.8, and the volume ratio of the ninhydrin-ethylene glycol solution to the acetate-sodium acetate buffer solution is 8-15:1.

[0034] The method includes the following steps: S1. Dissolve hydrated ninhydrin in ethylene glycol to prepare a ninhydrin-ethylene glycol solution, and then mix it with acetate-sodium acetate buffer at the volume ratio described above to obtain the ninhydrin colorimetric reagent. S2. Mix the test sample containing polyamide hydrolysis products with the ninhydrin colorimetric reagent, heat and react at 90-100℃ for 20-30 min, and measure the absorbance at 560-580 nm wavelength after cooling. S3. Prepare a series of standard solutions of polyamide hydrolysis products with a concentration not higher than 1 mM. Perform colorimetric reaction and absorbance measurement under the same conditions as in step S2. Plot a standard curve with the concentration of the standard solution as the abscissa and the absorbance as the ordinate. Substitute the absorbance of the sample to be tested into the standard curve to calculate the content of hydrolysis products.

[0035] Through the above-described preferred embodiments, the preferred conditions work synergistically to more effectively inhibit the oxidative degradation of ninhydrin, making the colorimetric reaction more complete and stable, and showing a good linear relationship between absorbance and hydrolysis product concentration, thereby further improving the accuracy and reliability of the polyamide hydrolysis product content determination results.

[0036] The present invention will be described in detail below through examples. In the following examples, polyamide and polyamide oligomer were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; ninhydrin was purchased from Shanghai Maclean Biotechnology Co., Ltd.; ethylene glycol was purchased from Sangon Biotech (Shanghai) Co., Ltd.; and the remaining raw materials and reagents were all commercially available products.

[0037] Example 1: Preparation of ninhydrin colorimetric reagent and determination of 6-aminohexanoic acid (1) Preparation of ninhydrin colorimetric reagent: Weigh 0.6 g of ninhydrin hydrate and dissolve it in 105 mL of ethylene glycol to prepare a ninhydrin-ethylene glycol solution. Separately prepare an acetate-sodium acetate buffer solution with a pH of 4.54: weigh anhydrous sodium acetate to prepare a 0.2 mol / L sodium acetate solution, and measure glacial acetic acid to prepare a 0.2 mol / L acetic acid solution. Mix the sodium acetate solution and acetic acid solution in a certain ratio and adjust the pH to 4.54. Mix the above ninhydrin-ethylene glycol solution with the pH 4.54 acetate-sodium acetate buffer solution at a volume ratio of 35:3 and stir until homogeneous to obtain the ninhydrin colorimetric reagent.

[0038] (2) Establishment of the standard curve for 6-aminohexanoic acid: Accurately weigh 6-aminohexanoic acid standard, dissolve it in distilled water, and prepare a 10 mM standard stock solution. Dilute with distilled water to prepare a series of standard working solutions with concentrations of 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM.

[0039] Take 2 mL of each concentration standard solution, add 3 mL of the above colorimetric reagent, shake well, and heat in a 95℃ water bath for 25 min, then cool in an ice-water bath. Zero the instrument using a blank control (distilled water instead of the standard solution) and measure the absorbance at 570 nm. Plot a standard curve with concentration on the x-axis and absorbance on the y-axis. The results are shown below. Figure 1 As shown.

[0040] The regression equation is: Y = 2.411X + 0.02961, R0 2 = 0.9996. The results show that, below 1 mM, the concentration of 6-aminocaproic acid exhibits a good linear relationship with the absorbance value.

[0041] (3) Determination of 6-aminohexanoic acid in the hydrolysis products of polyamide 6: Take an appropriate amount of the hydrolysate of polyamide 6 to be tested, centrifuge or filter it, and collect the supernatant. Dilute it appropriately with distilled water to bring the sample concentration to within 1 mM. Accurately measure 2 mL of the diluted sample solution, add 3 mL of colorimetric reagent, shake well, heat in a 95℃ water bath for 25 min, cool in an ice-water bath, and measure the absorbance at 570 nm. Substitute the absorbance value into the standard curve equation to calculate the concentration, and convert it to the content in the original hydrolysate according to the dilution factor. Perform three parallel determinations. The relative standard deviation (RSD) should be <5%, and the spiked recovery rate should be between 95% and 105%.

[0042] Example 2 The preparation of ninhydrin colorimetric reagent, the establishment of a standard curve for the monomer of polyamide 6 (6-aminohexanoic acid), and the determination of its content were carried out according to the method in Example 1. The difference was that 6-aminohexanoic acid was replaced with the monomer of polyamide 4 (γ-aminobutyric acid), a standard curve for γ-aminobutyric acid was established, and the γ-aminobutyric acid in the hydrolysis products of polyamide 4 was determined.

[0043] The regression equation is: Y = 2.396X - 0.04052, R0 2 = 0.9964. The results show that, below 1 mM, the concentration of γ-aminobutyric acid (GABA) exhibits a good linear relationship with the absorbance value.

[0044] Example 3 The preparation of ninhydrin colorimetric reagent, the establishment of a standard curve for the monomer of polyamide 6 (6-aminohexanoic acid), and the determination of its content were carried out according to the method in Example 1. The difference was that 6-aminohexanoic acid was replaced with the monomer of polyamide 5 (5-aminovaleric acid). A standard curve for 5-aminovaleric acid was established, and the 5-aminovaleric acid in the hydrolysis product of polyamide 5 was determined.

[0045] The regression equation is: Y = 2.401X - 0.03997, R0 2 = 0.9982. The results show that, below 1 mM, the concentration of 5-aminovaleric acid exhibits a good linear relationship with the absorbance value.

[0046] Example 4: Colorimetric effect of ninhydrin at a dosage of 0.5 g / 100 mL ethylene glycol The ninhydrin colorimetric reagent was prepared according to the method in Example 1, except that the amount of ninhydrin was adjusted to 0.5 g (relative to 100 mL ethylene glycol), the pH of the buffer solution was adjusted to 4.2, and the volume ratio of the ninhydrin-ethylene glycol solution to the acetate-sodium acetate buffer solution was adjusted to 15:1. The absorbance of 6-aminohexanoic acid (0.5 mM) was determined according to the method in Example 1. The results showed that the absorbance was 0.987, the linear correlation coefficient of the standard curve was 0.9994, and the spiked recovery rate was between 95% and 105%. This indicates that accurate and reliable determination results can still be obtained when the amount of ninhydrin is 0.5 g / 100 mL ethylene glycol.

[0047] Example 5: Colorimetric effect of ninhydrin at a dosage of 0.7 g / 100 mL ethylene glycol The ninhydrin colorimetric reagent was prepared according to the method in Example 1, except that the amount of ninhydrin was adjusted to 0.7 g (relative to 100 mL ethylene glycol), the pH of the buffer solution was adjusted to 4.8, and the volume ratio of the ninhydrin-ethylene glycol solution to the acetate-sodium acetate buffer solution was adjusted to 8:1. The absorbance of 6-aminohexanoic acid (0.5 mM) was determined according to the method in Example 1. The results showed that the absorbance was 1.061, the linear correlation coefficient of the standard curve was 0.9995, and the spiked recovery rate was between 95% and 105%. This indicates that accurate and reliable determination results can still be obtained when the amount of ninhydrin is 0.7 g / 100 mL ethylene glycol.

[0048] Example 66: Establishment and determination of the standard curve for 6-aminohexanoic acid dimer (1) Establishment of the standard curve for the dimer: Accurately weigh 0.2443 g of 6-aminohexanoic acid dimer standard and dilute to 100 mL with distilled water to prepare a 10 mM standard stock solution. Dilute with distilled water to prepare a series of standard working solutions with concentrations of 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM. Take 2 mL of each concentration standard solution, add 3 mL of the colorimetric reagent prepared in Example 1, shake well, heat in a 95°C water bath for 25 min, cool in an ice-water bath, and measure the absorbance at 570 nm. Plot a standard curve with concentration on the x-axis and absorbance on the y-axis. The results are shown below. Figure 2 As shown.

[0049] The regression equation is: Y = 2.378X + 0.04421, R0 2 = 0.9939. The results indicate that below 1 mM, the concentration of 6-aminohexanoic acid dimer exhibits a good linear relationship with absorbance values ​​(R0). 2 >0.99).

[0050] (2) Determination of 6-aminohexanoic acid dimer in polyamide 6 hydrolysis products: Take an appropriate amount of the hydrolysate of the polyamide 6 to be tested, and perform sample pretreatment, colorimetric reaction, and absorbance measurement according to the method in Example 1. Substitute the measured absorbance value into the above standard curve equation to calculate the concentration of 6-aminohexanoic acid dimer in the sample. Perform three parallel determinations, with a relative standard deviation (RSD) <5% and a spiked recovery rate between 95% and 105%.

[0051] Example 76: Establishment of the standard curve for aminohexanoic acid trimer Accurately weigh 6-aminohexanoic acid trimer standard and dilute to 100 mL with distilled water to prepare a 10 mM standard stock solution. Dilute with distilled water to prepare a series of standard working solutions with concentrations of 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM. Take 2 mL of each concentration standard solution, add 3 mL of the colorimetric reagent prepared in Example 1, shake well, heat in a 95°C water bath for 25 min, cool in an ice-water bath, and measure the absorbance at 570 nm. Plot a standard curve with concentration on the x-axis and absorbance on the y-axis. The results are as follows: Figure 3 As shown.

[0052] The regression equation is: Y = 2.392X - 0.03397, R0 2 = 0.9952. The results indicate that below 1 mM, the concentration of 6-aminohexanoic acid trimer exhibits a good linear relationship with the absorbance value (R0). 2 >0.99).

[0053] Example 86: Establishment of the standard curve for aminohexanoic acid heptamer Accurately weigh 6-aminoheptameric acid heptamer standard and dilute to 100 mL with distilled water to prepare a 10 mM standard stock solution. Dilute with distilled water to prepare a series of standard working solutions with concentrations of 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM. Take 2 mL of each concentration standard solution, add 3 mL of the colorimetric reagent prepared in Example 1, shake well, heat in a 95°C water bath for 25 min, cool in an ice-water bath, and measure the absorbance at 570 nm. Plot a standard curve with concentration on the x-axis and absorbance on the y-axis. The results are as follows: Figure 9 As shown.

[0054] The regression equation is: Y = 2.319X - 0.03544, R0 2 = 0.9951. The results show that below 1 mM, the concentration of 6-aminohexanoic acid heptamer exhibits a good linear relationship with the absorbance value (R0). 2 >0.99).

[0055] Example 95: Establishment of the standard curve for 5-aminovaleric acid heptamer Accurately weigh 5-aminovaleric acid heptameric standard and dilute to 100 mL with distilled water to prepare a 10 mM standard stock solution. Dilute with distilled water to prepare a series of standard working solutions with concentrations of 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM. Take 2 mL of each concentration standard solution, add 3 mL of the colorimetric reagent prepared in Example 1, shake well, heat in a 95°C water bath for 25 min, cool in an ice-water bath, and measure the absorbance at 570 nm. Plot a standard curve with concentration on the x-axis and absorbance on the y-axis. The results are as follows: Figure 10 As shown.

[0056] The regression equation is: Y = 2.118X - 0.01591, R0 2 = 0.9991. The results indicate that below 1 mM, the concentration of 5-aminovaleric acid heptamer shows a good linear relationship with the absorbance value (R0). 2 >0.99).

[0057] Example 10: Establishment of the hexamethylenediamine standard curve Accurately weigh hexamethylenediamine (monomer of polyamide 66) standard and dilute to 100 mL with distilled water to prepare a 10 mM standard stock solution. Dilute with distilled water to prepare a series of standard working solutions with concentrations of 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM. Take 2 mL of each concentration standard solution, add 3 mL of the colorimetric reagent prepared in Example 1, shake well, heat in a 95°C water bath for 25 min, cool in an ice-water bath, and measure the absorbance at 570 nm. Plot a standard curve with concentration on the x-axis and absorbance on the y-axis. The results are as follows: Figure 11 As shown.

[0058] The regression equation is: Y = 1.341X + 0.2654, R0 2 = 0.9949. The results show that below 1 mM, the concentration of hexamethylenediamine exhibits a good linear relationship with the absorbance value (R0). 2 >0.99).

[0059] Example 11 The absorbance of 6-aminocaproic acid (0.5 mM) was determined according to the method in Example 1, with the difference being that the heating time was set to 20 min, 25 min, and 30 min, respectively. The results showed that the absorbance was 1.031 at 20 min, 1.042 at 25 min, and 1.058 at 30 min, all three achieving good color development. The linear correlation coefficients of the standard curves were all greater than 0.999. This indicates that accurate and reliable measurement results can be obtained when the heating time is within the range of 20-30 min.

[0060] Example 12 Storage stability test of ninhydrin colorimetric reagent The ninhydrin colorimetric reagent prepared in Example 1 was aliquoted into brown bottles and stored in a refrigerator at 4°C, protected from light. The reagent was removed at 0, 30, 60, 90, and 100 days of storage, and the standard curve for 6-aminocaproic acid was determined according to the method in Example 1. The results showed that after 100 days of storage, the reagent remained colorless and transparent, and the standard curve exhibited good linearity (R0). 2The slope of the standard curve equation changed by less than 5% compared to the freshly prepared reagent (>0.999), indicating that the reagent can be stored for more than 100 days under light-protected conditions at 4°C.

[0061] Comparative Example 1: Experimental Study of Systems Without Organic Solvents A ninhydrin solution (water + buffer only) was prepared according to the method in Example 1, using distilled water instead of ethylene glycol as the solvent. The standard curve for 6-aminocaproic acid was determined according to the method in Example 1. The results showed that ninhydrin rapidly underwent oxidative degradation during heating at 95°C, resulting in a distinctly brown solution. The colorimetric reaction was almost impossible, the absorbance value was extremely low, and no effective linear relationship could be established.

[0062] Comparative Example 2: Ethanol Solvent System Comparison Experiment Using ethanol instead of ethylene glycol as the solvent, a ninhydrin-ethanol solution was prepared according to the method in Example 1, and then mixed with an acetate-sodium acetate buffer solution at pH 4.54 at a volume ratio of 35:3 to prepare a colorimetric reagent. The absorbance of 6-aminohexanoic acid (0.5 mM) was measured according to the method in Example 1. The results showed that the solution was pale yellow after the colorimetric reaction, with no obvious blue color, and the absorbance at 570 nm was only 0.22, which did not meet the requirements for quantitative analysis.

[0063] Comparative Example 3: Comparative Experiment with n-Butanol Solvent System Using n-butanol instead of ethylene glycol as the solvent, a ninhydrin-n-butanol solution was prepared according to the method in Example 1, and then mixed with an acetate-sodium acetate buffer solution at pH 4.54 at a volume ratio of 35:3 to prepare a colorimetric reagent. The absorbance of 6-aminohexanoic acid (0.5 mM) was measured according to the method in Example 1. The results showed that the solution exhibited obvious stratification after the colorimetric reaction (a light blue upper layer and a colorless lower layer), and the absorbance at 570 nm was only 0.17, which could not meet the requirements for accurate quantification.

[0064] Comparative Example 4: Comparative Experiment of Mixed Solvent Systems The ninhydrin reagent was prepared according to the mixed solvent system described in the journal article "Diagnosis of Nitrogen Nutrition in Rice—II. Determination of Amino Nitrogen—Ninhydrin Method": 1.1 g of ninhydrin was weighed, dissolved in 15 mL of n-propanol, then 30 mL of n-butanol and 60 mL of ethylene glycol were added, mixed well, and 9 mL of pH 5.4 acetate buffer was added. The standard curve of 6-aminohexanoic acid dimer was determined according to the method in Example 4. The results showed that the reagent prepared with this mixed solvent system did not show complete color development when determining the dimer, and the solution exhibited slight stratification. The linearity of the standard curve was significantly worse than that of this invention, and it could not meet the requirements for accurate quantification.

[0065] Comparative experiment of 5DMSO single solvent system A ninhydrin-DMSO solution was prepared using dimethyl sulfoxide (DMSO) instead of ethylene glycol as the sole solvent, following the method in Example 1. This solution was then mixed with a pH 4.54 acetate-sodium acetate buffer at a volume ratio of 35:3 to prepare a colorimetric reagent. The standard curve for the 6-aminohexanoic acid dimer was determined according to the method in Example 4. The results showed that the DMSO system had a high background absorbance (the blank control had an absorbance >0.2 at 570 nm), significant interference, and the dimer did not develop color, making it impossible to establish a linear relationship.

[0066] Comparative Example 6: Stability Comparison Test of Different Solvent Systems at Room Temperature Ninhydrin solutions (0.57 g / 100 mL solvent) were prepared according to the method in Example 1 using methanol, ethanol, n-propanol, isopropanol, n-butanol, ethylene glycol, acetonitrile, and dimethyl sulfoxide, respectively. 5 mL of each solution was placed in a transparent glass bottle and left at room temperature for 24 h. The color change of the solution was observed, and the results are as follows. Figure 4 As shown.

[0067] from Figure 4 It can be seen that the ninhydrin solutions prepared with methanol, ethanol, n-propanol, isopropanol, and n-butanol all turned light brown after standing at room temperature for 24 hours; the solution prepared with acetonitrile turned light brown; the solution prepared with dimethyl sulfoxide turned dark brown; while the solution prepared with ethylene glycol remained colorless and transparent.

[0068] Simultaneously, a full-wavelength scan was performed on the above eight solvent systems, and the results are as follows: Figure 5 As shown. From Figure 5 It can be seen that at 570 nm, the absorbance is the lowest and the initial interference is the least when ethylene glycol is used as the solvent.

[0069] Experimental results showed that, except for ethylene glycol, ninhydrin solutions prepared with other organic solvents all exhibited varying degrees of color change after being left at room temperature for 24 hours, indicating that ninhydrin had undergone oxidative deterioration. In contrast, the ninhydrin solution prepared with ethylene glycol remained colorless and transparent after 24 hours, demonstrating that ethylene glycol effectively inhibits the room-temperature oxidation of ninhydrin and significantly improves its storage stability.

[0070] Comparative Example 7: Comparison of color development effects of different solvent systems Ninhydrin solutions (0.57 g / 100 mL solvent) were prepared according to the method in Example 1 using ethanol, n-butanol, and ethylene glycol, respectively. Colorimetric reactions were then performed with 2 mM 6-aminohexanoic acid standard solution: 2 mL of standard solution was added to 3 mL of the colorimetric reagent, shaken well, heated in a 95°C water bath for 25 min, cooled in an ice-water bath, and the colorimetric phenomena were observed. The photographs after the colorimetric reactions are shown below. Figure 6 As shown, the full-wavelength scan results are as follows: Figure 7 As shown.

[0071] from Figure 6 It can be seen that: when ethanol is used as the solvent, the solution is pale yellow with no obvious blue color; when n-butanol is used as the solvent, obvious layering occurs (the upper layer is light blue and the lower layer is colorless); when ethylene glycol is used as the solvent, the solution is uniformly dark blue with no layering.

[0072] from Figure 7 The full-wavelength scan results show that, with ethylene glycol as the solvent, the absorbance value in the 500-600 nm range is significantly higher than that of the ethanol and n-butanol systems, especially at the characteristic absorption peak of 570 nm, where the absorbance value of the ethylene glycol system is the highest. The absorbance value at 570 nm was measured, and the comparison results are shown in Table 1.

[0073] Table 1

[0074] The results showed that when ethylene glycol was used as the solvent alone, the colorimetric reaction proceeded uniformly and stably, with complete color development and the highest absorbance value. While some color development was observed when n-butanol was used as the solvent, layering occurred. Ethanol failed to effectively achieve the colorimetric reaction. This comparative experiment fully demonstrates the necessity and superiority of using ethylene glycol as the solvent in this invention.

[0075] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. The application of ninhydrin colorimetric reagent in the determination of polyamide hydrolysis products, characterized in that, The ninhydrin colorimetric reagent contains ninhydrin-ethylene glycol solution and acetate-sodium acetate buffer.

2. The application according to claim 1, characterized in that, The polyamide hydrolysis product contains polyamide monomers and / or oligomers of the monomers; Preferably, the monomer of the polyamide is selected from at least one of 6-aminohexanoic acid, γ-aminobutyric acid, 5-aminovaleric acid, and hexamethylenediamine; Preferably, the oligomer of the monomer is selected from at least one of the monomer dimer, monomer trimer, monomer tetramer, monomer pentamer, monomer hexamer and monomer heptamer; Preferably, the polyamide is polyamide 6, the monomer of the polyamide 6 is 6-aminohexanoic acid, and the oligomer of the 6-aminohexanoic acid is selected from at least one of 6-aminohexanoic acid dimer, 6-aminohexanoic acid trimer, 6-aminohexanoic acid tetramer, 6-aminohexanoic acid pentamer, 6-aminohexanoic acid hexamer and 6-aminohexanoic acid heptamer.

3. The application according to claim 1, characterized in that, In the ninhydrin-ethylene glycol solution, the amount of ninhydrin used is 0.5-0.7 g relative to 100 mL of ethylene glycol; Preferably, the pH of the acetate-sodium acetate buffer solution is 4.2-4.8; Preferably, in the ninhydrin colorimetric reagent, the volume ratio of the ninhydrin-ethylene glycol solution to the acetate-sodium acetate buffer solution is 8-15:

1.

4. A method for determining the content of polyamide hydrolysis products, characterized in that, The method includes the following steps: S1. Preparation of ninhydrin colorimetric reagent: Dissolve hydrated ninhydrin in ethylene glycol to prepare ninhydrin-ethylene glycol solution, and mix the ninhydrin-ethylene glycol solution with acetate-sodium acetate buffer to obtain ninhydrin colorimetric reagent; S2. The test sample containing polyamide hydrolysis products is mixed with the ninhydrin colorimetric reagent and heated to react. After cooling, the absorbance of the product of the heated reaction is measured at a wavelength of 560-580 nm. The content of polyamide hydrolysis products in the test sample is calculated based on the absorbance and the standard curve of polyamide hydrolysis products.

5. The method according to claim 4, characterized in that, The polyamide hydrolysis product contains polyamide monomers and / or oligomers of the monomers; Preferably, the monomer of the polyamide is selected from at least one of 6-aminohexanoic acid, γ-aminobutyric acid, 5-aminovaleric acid, and hexamethylenediamine; Preferably, the oligomer of the monomer is selected from at least one of the monomer dimer, monomer trimer, monomer tetramer, monomer pentamer, monomer hexamer and monomer heptamer; Preferably, the polyamide is polyamide 6, the monomer of the polyamide 6 is 6-aminohexanoic acid, and the oligomer of the 6-aminohexanoic acid is selected from at least one of 6-aminohexanoic acid dimer, 6-aminohexanoic acid trimer, 6-aminohexanoic acid tetramer, 6-aminohexanoic acid pentamer, 6-aminohexanoic acid hexamer and 6-aminohexanoic acid heptamer.

6. The method according to claim 4, characterized in that, In the ninhydrin-ethylene glycol solution, the amount of ninhydrin used is 0.5-0.7 g relative to 100 mL of ethylene glycol; Preferably, the pH of the acetate-sodium acetate buffer solution is 4.2-4.8; Preferably, in the ninhydrin colorimetric reagent, the volume ratio of the ninhydrin-ethylene glycol solution to the acetate-sodium acetate buffer solution is 8-15:

1.

7. The method according to any one of claims 4 to 6, characterized in that, In step S2, the conditions for the heating reaction include: a temperature of 90-100°C and a time of 20-30 min.

8. The method according to any one of claims 4 to 6, characterized in that, In step S2, the process of plotting the standard curve of the polyamide hydrolysis products includes: (1) Prepare multiple standard solutions of polyamide hydrolysis products of different concentrations; (2) Mix the standard solution with the ninhydrin colorimetric reagent and carry out the heating reaction under the same conditions as in step S2. After cooling, measure the absorbance of the product of the heating reaction at a wavelength of 560-580 nm. Plot a standard curve with the concentration of the polyamide hydrolysis product standard solution as the abscissa and the absorbance as the ordinate.

9. The method according to claim 8, characterized in that, The concentration of the standard solution of the polyamide hydrolysis product is not higher than 1 mM.

10. The method according to claim 8, characterized in that, The linear correlation coefficient R of the standard curve of the polyamide hydrolysis products 2 ≥0.99.

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