Method for separating and determining 4, 6-diamino resorcinol dihydrochloride and impurities thereof by high performance liquid chromatography

Abstract

The invention discloses a method for separating and determining 4, 6-diamino resorcinol dihydrochloride and impurities thereof by high performance liquid chromatography, gradient elution is carried out by adopting a mixed solution of a mobile phase A and a mobile phase B, and the volume ratio of the mobile phase A to the mobile phase B is increased from 10%: 90% to 60%: 40% within 0-10 min; the balance is 60%: 40% in 10-15 min; then adjusting to 10%: 90% after 1 min; the balance is 10%: 90% in 16-20 min; a mobile phase A is an ammonium acetate aqueous solution, a mobile phase B is acetonitrile, and a chromatographic column is a Diol-HILIC chromatographic column. According to the method, 4, 6-diamino resorcinol dihydrochloride and the main impurity 4-amino resorcinol hydrochloride can be effectively separated by selecting a proper chromatographic column and a mobile phase, and meanwhile, the method is high in specificity, high in sensitivity, good in separation degree, good in repeatability and good in precision.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a method for separating and determining 4,6-diaminoresorcinol dihydrochloride and its impurities by a high performance liquid chromatography method. Background Art

[0002] The development of the aerospace and safety protection industries has put forward higher requirements for the strength, modulus, heat resistance and flame retardancy of materials, which has greatly promoted the development of high-performance polymer materials. Polybenzoxazole (PBO) fiber is far superior to aromatic polyamide fiber (also known as aramid fiber) in heat resistance and flame retardancy, and is one of the best-performing fiber products among the commercialized man-made organic fibers.

[0003] The tensile strength of PBO fiber can reach more than 5.8GPa, which is about twice that of aramid II fiber and 1.5 times that of carbon fiber; the tensile modulus of PBO fiber can reach more than 280GPa, which is about 2.5 times that of aramid II fiber. PBO fiber with a diameter of 1mm can withstand a weight of 460kg and has high specific strength and specific modulus. Polybenzoxazole also has good flame retardant properties and thermal stability, and is known as the "king of fibers".

[0004] 4,6-Diaminoresorcinol dihydrochloride is an important monomer raw material for the synthesis of polybenzoxazole, and its quality directly affects the performance of the material. The CAS number of 4,6-Diaminoresorcinol dihydrochloride is 16523-31-2, a white crystalline solid with a molecular formula of C 6 H 10 Cl 2 N 2 O 2 , the relative molecular weight is 213.06, and the structural formula is as follows: .

[0005] 4-Aminoresorcinol hydrochloride is the main impurity of 4,6-diaminoresorcinol dihydrochloride, with CAS number 34781-86-7 and molecular formula C 6 H 8 ClNO 2 , the relative molecular weight is 161.59, and the structural formula is as follows: .

[0006] In addition, 4,6-diaminoresorcinol dihydrochloride is very easy to oxidize and deteriorate. Impurities in the monomer will seriously affect the normal progress of the polymerization reaction, causing unpredictable fluctuations in product quality, resulting in the inability of PBO fibers to achieve ideal tensile strength and tensile modulus. In addition, the preparation of PBO fibers has strict requirements on the purity and impurities of the product, which poses a challenge to the purity control of 4,6-diaminoresorcinol dihydrochloride.

[0007] So far, there is no literature report on the separation and determination of 4,6-diaminoresorcinol dihydrochloride. Summary of the invention

[0008] The purpose of the present invention is to solve the above problems and provide a method for separating and determining 4,6-diaminoresorcinol dihydrochloride and its impurities by high performance liquid chromatography to fill the gap in the art.

[0009] The technical solution for achieving the purpose of the present invention is: a method for separating and determining 4,6-diaminoresorcinol dihydrochloride and its impurities by high performance liquid chromatography, using a mixed solution of mobile phase A and mobile phase B as the mobile phase for gradient elution; the mobile phase A is an ammonium acetate aqueous solution; the mobile phase B is acetonitrile.

[0010] The concentration of the ammonium acetate aqueous solution is 5-50 mmol / L, preferably 10 mmol / L.

[0011] The gradient elution procedure is as follows: at 0-10 min, the volume ratio of mobile phase A and mobile phase B is increased from 10%:90% to 60%:40%; at 10-15 min, the volume ratio of mobile phase A and mobile phase B is balanced at 60%:40%; then it takes 1 min to adjust back to the initial volume ratio of 10%:90%; at 16-20 min, the volume ratio of mobile phase A and mobile phase B is balanced at 10%:90%.

[0012] See Table 1 for details.

[0013] Table 1 Elution time Mobile phase A Mobile phase B 0min 10% 90% 10.0min 60% 40% 15.0min 60% 40% 16.0min 10% 90% 20.0min 10% 90%

[0014] The chromatographic column used in the present invention is a Diol-HILIC chromatographic column (diol-based hydrophilic chromatographic column).

[0015] 4,6-Diaminoresorcinol dihydrochloride has strong polarity and salt-forming properties, so it will be free in conventional C18 columns and acidic systems, with weak retention, poor peak shape, tailing, and poor separation. After a large number of experiments, the applicant found that the use of Diol-HILIC columns and ammonium acetate buffer salt systems can effectively control the degree of ionization of target compounds, thereby enhancing the retention of polar compounds, significantly improving peak shape and separation, while ensuring the repeatability and reliability of the analysis results.

[0016] Other chromatographic conditions of the HPLC are as follows: Chromatographic column temperature: 20-40°C, preferably 30°C.

[0017] Mobile phase flow rate: 0.8-1.2 mL / min, preferably 1.0 mL / min.

[0018] Detection wavelength: 210-250 nm, preferably 210 nm.

[0019] Injection volume: 1 μL to 10 μL, preferably 5 μL.

[0020] Diluent: methanol or acetonitrile or water, preferably methanol.

[0021] The present invention has positive effects: the method of the present invention can effectively separate 4,6-diaminoresorcinol dihydrochloride from its main impurity 4-aminoresorcinol hydrochloride by selecting a suitable chromatographic column and a mobile phase, and the method of the present invention has strong specificity, high sensitivity, good separation, good repeatability and good precision. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the chromatogram of the blank solution in Example 1.

[0023] Figure 2 It is the chromatogram of the sample solution in Example 1.

[0024] Figure 3 It is the chromatogram of the impurity solution in Example 1.

[0025] Figure 4 It is the chromatogram of the sample solution in Comparative Example 1.

[0026] Figure 5 It is the chromatogram of the impurity solution in Comparative Example 1.

[0027] Figure 6 It is the chromatogram of the sample solution in Comparative Example 2.

[0028] Figure 7 It is the chromatogram of the impurity solution in Comparative Example 2.

[0029] Figure 8 This is the chromatogram of the specific experimental solution in Experimental Example 1.

[0030] Fig. 9 This is the chromatogram of the sensitivity test solution in Experimental Example 2.

[0031] Fig.10 The linearity and range experimental results of Experimental Example 3 are shown in FIG. DETAILED DESCRIPTION

[0032] (Example 1) 1. Instruments.

[0033] High performance liquid chromatograph: Agilent 1260 HPLC, DAD detector.

[0034] Chromatographic column: Diol-HILIC column (4.6 mm × 250 mm, 5 μm).

[0035] 2. Chromatographic conditions.

[0036] Chromatographic column temperature: 30℃.

[0037] Detection wavelength: 210nm.

[0038] Injection volume: 5 μL.

[0039] Mobile phase flow rate: 1.0 mL / min.

[0040] Diluent: methanol.

[0041] Mobile phase A: 10 mmol / L ammonium acetate aqueous solution.

[0042] Mobile phase B: acetonitrile.

[0043] The gradient elution conditions are shown in Table 1.

[0044] 3. Solution preparation.

[0045] 1. Blank solution: diluent.

[0046] 2. Sample solution: Weigh 100 mg of sample into a 100 mL volumetric flask, add diluent, make up to volume, shake well, and obtain a sample solution [concentration of 1 mg / mL].

[0047] 3. Impurity solution: Weigh 100 mg of impurity [4-aminoresorcinol hydrochloride standard] into a 100 mL volumetric flask, add diluent, make up to volume, shake well, and use it as the impurity solution [concentration of 1 mg / mL].

[0048] 4. Determination: Precisely measure 5 μL of blank solution, sample solution and impurity solution respectively, inject into chromatograph, perform HPLC analysis according to the above chromatographic conditions, record chromatogram, and the results are shown in Table 1. Figure 1 to Figure 3 .

[0049] Depend on Figure 1 to Figure 3 It can be seen that the retention time of the product is 9.097 min, the retention time of the impurity is 3.675 min, and the product and impurity can be effectively separated.

[0050] (Comparative Example 1) 1. Instruments.

[0051] High performance liquid chromatograph: same as in Example 1.

[0052] Chromatographic column: Extend-C18 (4.6 mm × 250 mm, 5 μm).

[0053] 2. Chromatographic conditions.

[0054] Same as Example 1.

[0055] 3. Experimental steps.

[0056] Accurately measure 5 μL of the sample solution and impurity solution in Example 1, inject them into the chromatograph, perform high performance liquid chromatography analysis according to the above chromatographic conditions, record the chromatogram, and the results are shown in Figure 4 and Figure 5 .

[0057] Depend on Figure 4 and Figure 5 It can be seen that the retention time of the product is 2.869min and 3.136min, and the retention time of the impurity is 6.624min. Although the product and impurity can be effectively separated, the product has two peaks and the peak shape is poor.

[0058] (Comparative Example 2) 1. Instruments.

[0059] Same as Example 1.

[0060] 2. Chromatographic conditions.

[0061] Mobile phase A was 0.1% phosphoric acid aqueous solution, and the rest was the same as in Example 1.

[0062] 3. Experimental steps.

[0063] Accurately measure 5 μL of the sample solution and impurity solution in Example 1, inject them into the chromatograph, perform high performance liquid chromatography analysis according to the above chromatographic conditions, record the chromatogram, and the results are shown in Figure 6 and Figure 7 .

[0064] Depend on Figure 6 and Figure 7 It can be seen that the retention time of the product is 9.576min, 9.763min and 14.556min, the retention time of the impurity is 9.807min, the product and the impurity cannot be effectively separated, the asymmetry factors of the product and the impurity are 3.64 and 3.70 respectively, the product has three peaks and the peak shapes of the product and the impurity are poor.

[0065] (Test Example 1, Specificity Experiment) This test example is a specific experiment of the detection method of the present invention.

[0066] Prepare specific experimental solution: weigh 100 mg of sample and transfer 1 mL of the impurity solution prepared in Example 1 to a 100 mL volumetric flask, add diluent, make up to volume, shake well, and obtain a specific experimental solution [sample concentration is 1 mg / mL, impurity concentration is 0.01 mg / mL].

[0067] Accurately measure 5 μL of the above-mentioned specific experimental solution, inject it into the chromatograph, perform high performance liquid chromatography analysis according to the chromatographic conditions of Example 1, record the chromatogram, and the results are shown in Figure 8 .

[0068] Depend on Figure 8 It can be seen that: at the retention time of the product peak and the impurity peak, there is no interference from the blank, the separation degree between the main peak and the impurity is 38.02, which meets the requirement of separation degree ≥ 1.5, and the asymmetry factor of the main peak is 1.08, indicating a good peak shape.

[0069] (Test Example 2, Sensitivity Test) This test example is a sensitivity experiment of the detection method of the present invention.

[0070] Prepare sensitivity test solution: transfer 1.0 mL of the sample solution prepared in Example 1 to a 100 mL volumetric flask, add diluent, adjust to volume, shake well, then transfer 1.0 mL of the above solution to a 20 mL volumetric flask, add diluent, adjust to volume, shake well, and obtain a sensitivity solution [sample concentration is 0.00050 mg / mL].

[0071] Accurately measure 5 μL of the above sensitivity test solution, inject it into the chromatograph, perform high performance liquid chromatography analysis according to the chromatographic conditions of Example 1, record the chromatogram, and the results are shown in Fig. 9 .

[0072] Depend on Fig. 9 It can be seen that when the sample concentration is 0.00050 mg / mL, the signal-to-noise ratio is 36.2, and the sensitivity is good.

[0073] (Test Example 3) This test example is a linearity test of the detection method of the present invention.

[0074] Referring to the preparation method of the sample solution in Example 1, a series of standard solutions with a concentration range of 0.00050 mg / mL to 2.0 mg / mL were prepared (0.00050 mg / mL, 0.0050 mg / mL, 0.010 mg / mL, 0.10 mg / mL, 0.50 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL).

[0075] Accurately measure 5 μL of each of the above linear solutions and inject them into the chromatograph. Perform HPLC analysis according to the chromatographic conditions of Example 1. Record the chromatogram. Draw a standard curve with concentration (in mg / mL) as the abscissa and peak area as the ordinate. Develop a linear regression equation and correlation coefficient. The results are shown in Tables 2 and Fig.10 .

[0076] Depend on Fig.10It can be seen that within the concentration range of 0.0005 mg / mL to 2.0 mg / mL, the linear relationship is good.

[0077] Table 2

[0078] (Test Example 4) This test example is a precision test of the detection method of the present invention.

[0079] Referring to the preparation method of the sample solution in Example 1, a 1 mg / mL precision test solution was prepared.

[0080] Accurately measure 5 μL of the precision experiment solution, inject it into the chromatograph, inject 6 injections continuously, perform high performance liquid chromatography analysis according to the chromatographic conditions of Example 1, record the retention time and peak area, and calculate the relative standard deviation. The results are shown in Table 3.

[0081] Table 3 1 needle 2-pin 3-pin 4-pin 5-pin 6-pin RSD Retention time / min 9.10 9.11 9.10 9.11 9.11 9.10 0.06% Peak area 115.565 115.935 116.323 115.524 115.452 116.217 0.33%

[0082] It can be seen from Table 3 that the detection method of the present invention has good precision.

[0083] (Test Example 5) This test example is a stability experiment of the detection method of the present invention.

[0084] Referring to the preparation method of the sample solution in Example 1, a 1 mg / mL stability test solution was prepared.

[0085] The stability test solutions were placed at room temperature for 0, 4h, 8h, 12h, 18h, and 24h, and 5μL of each solution was accurately measured and injected into the chromatograph. HPLC analysis was performed according to the chromatographic conditions of Example 1, the retention time and peak area were recorded, and the relative standard deviation was calculated. The results are shown in Table 4.

[0086] Table 4 0h 4h 8h 12h 18h 24h RSD Retention time / min 9.10 9.11 9.12 9.11 9.12 9.12 0.09% Peak area 115.565 116.145 116.191 116.545 116.855 117.012 0.46%

[0087] It can be seen from Table 4 that the product is stable in the solution for at least 24 hours and meets the requirement of RSD ≤ 2% of the main peak area, indicating that the detection method of the present invention has good stability.

Claims

1. A method for separating and determining 4,6-diaminoresorcinol dihydrochloride and its impurities by high performance liquid chromatography, characterized in that: A mixed solution of mobile phase A and mobile phase B is used as the mobile phase for gradient elution; the mobile phase A is an ammonium acetate aqueous solution; and the mobile phase B is acetonitrile.

2. The method for separating and determining 4,6-diaminoresorcinol dihydrochloride and its impurities by high performance liquid chromatography according to claim 1, characterized in that: The concentration of the ammonium acetate aqueous solution is 5-50 mmol / L.

3. The method for separating and determining 4,6-diaminoresorcinol dihydrochloride and its impurities by high performance liquid chromatography according to claim 1, characterized in that: The concentration of the ammonium acetate aqueous solution is 10 mmol / L.

4. The method for separating and determining 4,6-diaminoresorcinol dihydrochloride and its impurities by high performance liquid chromatography according to any one of claims 1 to 3, characterized in that: The chromatographic column used is Diol-HILIC column.

5. The method for separating and determining 4,6-diaminoresorcinol dihydrochloride and its impurities by high performance liquid chromatography according to any one of claims 1 to 3, characterized in that The gradient elution procedure is as follows: at 0-10 min, the volume ratio of mobile phase A and mobile phase B is increased from 10%:90% to 60%:40%; at 10-15 min, the volume ratio of mobile phase A and mobile phase B is balanced at 60%:40%; then it takes 1 min to adjust back to the initial volume ratio of 10%:90%; at 16-20 min, the volume ratio of mobile phase A and mobile phase B is balanced at 10%:90%.

6. The method for separating and determining 4,6-diaminoresorcinol dihydrochloride and its impurities by high performance liquid chromatography according to claim 4, characterized in that The gradient elution procedure is as follows: at 0-10 min, the volume ratio of mobile phase A and mobile phase B is increased from 10%:90% to 60%:40%; at 10-15 min, the volume ratio of mobile phase A and mobile phase B is balanced at 60%:40%; then it takes 1 min to adjust back to the initial volume ratio of 10%:90%; at 16-20 min, the volume ratio of mobile phase A and mobile phase B is balanced at 10%:90%.

7. The method for separating and determining 4,6-diaminoresorcinol dihydrochloride and its impurities by high performance liquid chromatography according to any one of claims 1 to 3, characterized in that Other chromatographic conditions of the high performance liquid chromatography are as follows: column temperature of the chromatographic column is 20-40° C.; mobile phase flow rate is 0.8-1.2 mL / min; detection wavelength is 210-250 nm; injection volume is 1 μL-10 μL.

8. The method for separating and determining 4,6-diaminoresorcinol dihydrochloride and its impurities by high performance liquid chromatography according to claim 4, characterized in that Other chromatographic conditions of the high performance liquid chromatography are as follows: column temperature of the chromatographic column is 20-40° C.; mobile phase flow rate is 0.8-1.2 mL / min; detection wavelength is 210-250 nm; injection volume is 1 μL-10 μL.

9. The method for separating and determining 4,6-diaminoresorcinol dihydrochloride and its impurities by high performance liquid chromatography according to claim 5, characterized in that Other chromatographic conditions of the high performance liquid chromatography are as follows: column temperature of the chromatographic column is 20-40° C.; mobile phase flow rate is 0.8-1.2 mL / min; detection wavelength is 210-250 nm; injection volume is 1 μL-10 μL.

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