Determination method for detecting content of binary acid by high performance liquid chromatography

Abstract

The invention provides a determination method for detecting the content of binary acid by high performance liquid chromatography, which comprises the following steps: S1, weighing about 150mg of a test sample, dissolving with methanol, diluting to 50mL, and shaking uniformly to obtain a test solution; the test sample is any one or a combination of dodecanedioic acid fermentation liquor, liquid generated in a dodecanedioic acid extraction process and a dodecanedioic acid finished product; s2, detecting the test solution by adopting a high performance liquid chromatograph; the speed of the test solution passing through the chromatographic column is 0.8-1.2 ml / min; a chromatographic column adopted by the high performance liquid chromatograph is an octadecyl silane bonded silica gel column; a mobile phase adopted by the high performance liquid chromatograph is a water-methanol mixed solution; according to the determination method provided by the invention, the concentration and the peak area have a good linear relationship (r = 1.000) in an investigated concentration range, the average recovery rate is 100.1%, the operation is simple, the analysis time is short, and the accuracy is high.

Description

Technical Field

[0001] The invention relates to the field of chemical raw material detection, and in particular to a method for detecting the content of dibasic acid by high performance liquid chromatography. Background Art

[0002] In the chemical industry, dibasic acids are an important class of organic compounds, which are widely used in many fields such as plastics, synthetic fibers, surfactants and medicine. Among them, dodecanedioic acid, as a long-chain dibasic acid, has unique physical and chemical properties and broad application prospects, especially playing a key role in the synthesis of nylon materials. However, due to the complex production process of dodecanedioic acid, which involves multiple steps such as fermentation, extraction and purification, the accurate determination of its content and purity is crucial to ensure product quality and control production costs.

[0003] Traditional methods for determining the content of dibasic acids, such as titration and gravimetric methods, are simple to operate, but have disadvantages such as long sample analysis time, low sensitivity, poor accuracy, and long time consumption, which are difficult to meet the needs of rapid and accurate detection in modern industrial production. Therefore, it is particularly important to develop an efficient and accurate method for determining the content of dibasic acids. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for detecting the content of dibasic acid by high performance liquid chromatography, comprising the following steps: S1. Weigh about 150 mg of the test sample, dissolve it in methanol and dilute it to 50 mL, shake well to obtain the test solution; The test sample is any one or a combination of dodecanedioic acid fermentation broth, liquid produced during dodecanedioic acid extraction, and dodecanedioic acid finished product; S2, testing the test solution using a high performance liquid chromatograph; The speed at which the test solution passes through the chromatographic column is 0.8-1.2 ml / min; The chromatographic column used in the high performance liquid chromatograph is an octadecylsilane bonded silica gel column; The temperature of the chromatographic column is 25°C and the elution time is 10 minutes; The mobile phase used in the high performance liquid chromatograph is a water-methanol mixture; In the water-methanol mixture, the volume ratio of water to methanol is 190-210:790-810; The pH of the water-methanol mixture is adjusted to 3.8-4.2 with phosphoric acid.

[0005] Furthermore, the dibasic acid is dodecanedioic acid.

[0006] Moreover, the speed at which the test solution passes through the chromatographic column is 1.0 ml / min.

[0007] Moreover, the high performance liquid chromatograph adopts an ultraviolet detector.

[0008] Furthermore, the injection volume of the HPLC was 20 μl.

[0009] Moreover, the detection wavelength of the high performance liquid chromatograph is 220 nm.

[0010] Moreover, the temperature of the chromatographic column is 25° C., and the elution time is 10 minutes.

[0011] Furthermore, the phosphoric acid concentration was 4.4 mol / L.

[0012] Moreover, in the water-methanol mixture, methanol is 100% methanol.

[0013] Moreover, the concentration of the test solution is 3 mg / mL.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The method for determining the content of dibasic acid by high performance liquid chromatography provided by the present invention has a good linear relationship (r=1.000) between the concentration and the peak area within the concentration range investigated (diacid concentration range of 2.0 mg / ml to 4.6 mg / ml), an average recovery rate of 100.1%, simple operation, an elution time of only 10 minutes, a short analysis time and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the liquid chromatogram of the blank solution in Experiment 2; Figure 2 This is the liquid chromatogram of the control solution in Experiment 2; Figure 3 This is the liquid chromatogram of the test solution in Experiment 2; Figure 4 This is the linear graph in Experiment 2; Figure 5 It is the liquid chromatogram of Example 1; Figure 6 It is the liquid chromatogram of Comparative Example 1; Figure 7 It is the liquid chromatogram of Comparative Example 2; Figure 8 It is the liquid chromatogram of Comparative Example 3; Fig. 9 It is the liquid chromatogram of Comparative Example 4. DETAILED DESCRIPTION

[0016] Example 1 A method for determining the content of dibasic acid by high performance liquid chromatography comprises the following steps: S1. Accurately weigh about 150 mg of the test sample, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute it to the mark, shake well to obtain the test solution; S2, the test solution is detected by high performance liquid chromatography, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column; The mobile phase used in the high performance liquid chromatography method is a water-methanol mixture (the volume ratio of water to methanol is 200:800, and the pH is adjusted to 4.0 with 4.4 mol / L phosphoric acid).

[0017] The temperature of the chromatographic column is 25° C., and the elution time is 10 minutes.

[0018] Furthermore, the dibasic acid is dodecanedioic acid.

[0019] Moreover, in the step S2, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column.

[0020] Moreover, the speed at which the test solution passes through the chromatographic column is 0.8 ml / min.

[0021] Moreover, the detector adopted by the liquid chromatograph is an ultraviolet detector.

[0022] Furthermore, the injection volume of the HPLC method was 20 μl.

[0023] Furthermore, the detection wavelength of the high performance liquid chromatography is 220 nm.

[0024] Example 2 A method for determining the content of dibasic acid by high performance liquid chromatography comprises the following steps: S1. Accurately weigh about 150 mg of the test sample, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute it to the mark, shake well to obtain the test solution; S2, the test solution is detected by high performance liquid chromatography, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column; The mobile phase used in the high performance liquid chromatography method is a water-methanol mixture (the volume ratio of water to methanol is 200:800, and the pH is adjusted to 4.0 with 4.4 mol / L phosphoric acid).

[0025] The temperature of the chromatographic column is 25° C., and the elution time is 10 minutes.

[0026] Furthermore, the dibasic acid is dodecanedioic acid.

[0027] Moreover, in the step S2, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column.

[0028] Moreover, the speed at which the test solution passes through the chromatographic column is 1.2 ml / min.

[0029] Moreover, the detector adopted by the liquid chromatograph is an ultraviolet detector.

[0030] Furthermore, the injection volume of the HPLC method was 20 μl.

[0031] Furthermore, the detection wavelength of the high performance liquid chromatography is 220 nm.

[0032] Example 3 A method for determining the content of dibasic acid by high performance liquid chromatography comprises the following steps: S1. Accurately weigh about 150 mg of the test sample, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute it to the mark, shake well to obtain the test solution; S2, the test solution is detected by high performance liquid chromatography, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column; The mobile phase used in the high performance liquid chromatography method is a water-methanol mixture (the volume ratio of water to methanol is 200:800, and the pH is adjusted to 4.0 with 4.4 mol / L phosphoric acid).

[0033] The temperature of the chromatographic column is 20° C., and the elution time is 10 minutes.

[0034] Furthermore, the dibasic acid is dodecanedioic acid.

[0035] Moreover, in the step S2, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column.

[0036] Moreover, the speed at which the test solution passes through the chromatographic column is 1.0 ml / min.

[0037] Moreover, the detector adopted by the liquid chromatograph is an ultraviolet detector.

[0038] Furthermore, the injection volume of the HPLC method was 20 μl.

[0039] Furthermore, the detection wavelength of the high performance liquid chromatography is 220 nm.

[0040] Example 4 A method for determining the content of dibasic acid by high performance liquid chromatography comprises the following steps: S1. Accurately weigh about 150 mg of the test sample, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute it to the mark, shake well to obtain the test solution; S2, the test solution is detected by high performance liquid chromatography, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column; The mobile phase used in the high performance liquid chromatography method is a water-methanol mixture (the volume ratio of water to methanol is 200:800, and the pH is adjusted to 4.0 with 4.4 mol / L phosphoric acid).

[0041] The temperature of the chromatographic column is 30° C., and the elution time is 10 minutes.

[0042] Furthermore, the dibasic acid is dodecanedioic acid.

[0043] Moreover, in the step S2, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column.

[0044] Moreover, the speed at which the test solution passes through the chromatographic column is 1.0 ml / min.

[0045] Moreover, the detector adopted by the liquid chromatograph is an ultraviolet detector.

[0046] Furthermore, the injection volume of the HPLC method was 20 μl.

[0047] Furthermore, the detection wavelength of the high performance liquid chromatography is 220 nm.

[0048] Example 5 A method for determining the content of dibasic acid by high performance liquid chromatography comprises the following steps: S1. Accurately weigh about 150 mg of the test sample, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute it to the mark, shake well to obtain the test solution; S2, the test solution is detected by high performance liquid chromatography, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column; The mobile phase used in the high performance liquid chromatography method is a water-methanol mixture (the volume ratio of water to methanol is 200:800, and the pH is adjusted to 4.2 with 4.4 mol / L phosphoric acid).

[0049] The temperature of the chromatographic column is 25° C., and the elution time is 10 minutes.

[0050] Furthermore, the dibasic acid is dodecanedioic acid.

[0051] Moreover, in the step S2, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column.

[0052] Moreover, the speed at which the test solution passes through the chromatographic column is 1.0 ml / min.

[0053] Moreover, the detector adopted by the liquid chromatograph is an ultraviolet detector.

[0054] Furthermore, the injection volume of the HPLC method was 20 μl.

[0055] Furthermore, the detection wavelength of the high performance liquid chromatography is 220 nm.

[0056] Example 6 A method for determining the content of dibasic acid by high performance liquid chromatography comprises the following steps: S1. Accurately weigh about 150 mg of the test sample, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute it to the mark, shake well to obtain the test solution; S2, the test solution is detected by high performance liquid chromatography, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column; The mobile phase used in the high performance liquid chromatography method is a water-methanol mixture (the volume ratio of water to methanol is 200:800, and the pH is adjusted to 3.8 with 4.4 mol / L phosphoric acid).

[0057] The temperature of the chromatographic column is 25° C., and the elution time is 10 minutes.

[0058] Furthermore, the dibasic acid is dodecanedioic acid.

[0059] Moreover, in the step S2, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column.

[0060] Moreover, the speed at which the test solution passes through the chromatographic column is 1.0 ml / min.

[0061] Moreover, the detector adopted by the liquid chromatograph is an ultraviolet detector.

[0062] Furthermore, the injection volume of the HPLC method was 20 μl.

[0063] Furthermore, the detection wavelength of the high performance liquid chromatography is 220 nm.

[0064] Example 7 A method for determining the content of dibasic acid by high performance liquid chromatography comprises the following steps: S1. Accurately weigh about 150 mg of the test sample, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute it to the mark, shake well to obtain the test solution; S2, the test solution is detected by high performance liquid chromatography, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column; The mobile phase used in the high performance liquid chromatography method is a water-methanol mixture (the volume ratio of water to methanol is 210:790, and the pH is adjusted to 4.0 with 4.4 mol / L phosphoric acid).

[0065] The temperature of the chromatographic column is 25° C., and the elution time is 10 minutes.

[0066] Furthermore, the dibasic acid is dodecanedioic acid.

[0067] Moreover, in the step S2, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column.

[0068] Moreover, the speed at which the test solution passes through the chromatographic column is 1.0 ml / min.

[0069] Moreover, the detector adopted by the liquid chromatograph is an ultraviolet detector.

[0070] Furthermore, the injection volume of the HPLC method was 20 μl.

[0071] Furthermore, the detection wavelength of the high performance liquid chromatography is 220 nm.

[0072] Example 8 A method for determining the content of dibasic acid by high performance liquid chromatography comprises the following steps: S1. Accurately weigh about 150 mg of the test sample, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute it to the mark, shake well to obtain the test solution; S2, the test solution is detected by high performance liquid chromatography, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column; The mobile phase used in the high performance liquid chromatography method is a water-methanol mixture (the volume ratio of water to methanol is 190:810, and the pH is adjusted to 4.0 with 4.4 mol / L phosphoric acid).

[0073] The temperature of the chromatographic column is 25° C., and the elution time is 10 minutes.

[0074] Furthermore, the dibasic acid is dodecanedioic acid.

[0075] Moreover, in the step S2, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane bonded silica gel column.

[0076] Moreover, the speed at which the test solution passes through the chromatographic column is 1.0 ml / min.

[0077] Moreover, the detector adopted by the liquid chromatograph is an ultraviolet detector.

[0078] Furthermore, the injection volume of the HPLC method was 20 μl.

[0079] Furthermore, the detection wavelength of the high performance liquid chromatography is 220 nm.

[0080] Comparative Example 1 The difference between this comparative example and Example 3 is that the detector used in the liquid chromatograph is a PDA detector, and the detection wavelength range of the high performance liquid chromatography is 190-240 nm.

[0081] Comparative Example 2 The difference between this comparative example and Example 3 is that in step S2, the pH of the water-methanol mixture is adjusted to 3.0 by 4.4 mol / L phosphoric acid.

[0082] Comparative Example 3 The difference between this comparative example and Example 3 is that in step S2, the pH of the water-methanol mixture is adjusted to 5.0 with 4.4 mol / L phosphoric acid.

[0083] Comparative Example 4 The difference between this comparative example and Example 3 is that in step S2, the pH of the water-methanol mixture is adjusted to 6.0 with 4.4 mol / L phosphoric acid.

[0084] Experimental Section

[0085] Experiment 1 Preparation of test solution: Accurately weigh an appropriate amount of test sample and place it in a 50mL volumetric flask. Use methanol, ethanol, formamide, acetamide, chloroform, tetrahydrofuran, ether and other solvents to perform dissolution experiments on the dibasic acid. By comparison, it is found that methanol has the best solubility and is a commonly used reagent in laboratories and is easy to obtain.

[0086] Then the solubility of the dibasic acid was tested using 60% methanol, 70% methanol, 80% methanol, 90% methanol, and 100% methanol respectively. As the proportion of methanol continued to increase, the solubility increased, and 100% methanol had the best effect.

[0087] Experiment 2 1. Instruments and HPLC conditions instrument: Shimadzu LC-20AT liquid chromatograph, octadecylsilane bonded silica gel chromatographic column, column size 250 mm × 4.6 mm × 5 μm, Mettler-Toledo XPR205DR electronic balance; Chromatographic conditions: Detector: UV; Flow rate: 1.0 ml / min; Injection volume: 20 μl; Detection wavelength: 220 nm; Column temperature: initial temperature is 25°C, elution time: 10 minutes.

[0088] 2. Materials Phosphoric acid: AR grade, batch number S1102201001, provided by Tianjin Komiou Chemical Reagent Co., Ltd.; Methanol: chromatographically pure, batch number S1242401006, provided by SIGMA-ALDRICH.

[0089] 3. Measurement of test products Blank solution: methanol; Preparation of reference solution: Accurately weigh about 150 mg of dodecanedioic acid reference substance, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute to the scale, shake well, and prepare two parallel portions.

[0090] Preparation of test solution: Accurately weigh about 150 mg of dodecanedioic acid, place in a 50 mL volumetric flask, dissolve and dilute to the scale with methanol, shake well, the concentration is 3 mg / mL.

[0091] Accurately measure 20 μL of each of the above solutions and inject them into a high performance liquid chromatograph, record the peak area, and measure the content of the dibasic acid by the external standard method. The results are shown in Table 1.

[0092] Table 1 Determination results of dibasic acid

[0093] 4. Methodological Validation 4.1 Exclusivity The blank solution, control solution and test solution determined by the above method are shown in Table 1. Figure 1-Figure 3 , Figure 1 is the liquid chromatogram of the blank solution. Figure 2 is the liquid chromatogram of the reference solution. Figure 3 This is the liquid chromatogram of the test solution. It can be seen from the figure that the blank solution does not interfere with the determination of dibasic acid.

[0094] 4.2 Linearity Figure 4 Linear graph, from Figure 4 It can be seen that in the range of dibasic acid concentration from 2.0 mg / ml to 4.6 mg / ml, the linear relationship is good, the linear equation is: Y=193976.0039+505.2527, correlation coefficient: 1.000.

[0095] 4.3 Precision Six samples from the same batch were taken for parallel determination and injected into the liquid chromatograph respectively. The average content was 99.7% and RSD=0.1%. At two different times, six test solutions were prepared in parallel using different instruments and injected into the HPLC (n=12). The average result was 99.8% and RSD=0.1%.

[0096] 4.4 Accuracy 80% recovery solution: Accurately weigh about 75 mg of sample and place it in a 50 ml volumetric flask. Accurately add about 45 mg of reference substance, dissolve it in methanol and dilute to the scale. Shake well to make a solution with a concentration equivalent to 80% of the concentration of the reference solution.

[0097] 100% recovery solution: Accurately weigh about 75 mg of sample and place it in a 50 ml volumetric flask. Accurately add about 75 mg of reference substance, dissolve it in methanol and dilute to the scale. Shake well to make a solution with a concentration equivalent to 100% of the concentration of the reference solution.

[0098] 120% recovery solution: Accurately weigh about 75 mg of the sample and place it in a 50 ml volumetric flask, accurately add about 105 mg of the reference substance, dissolve it in methanol and dilute it to the mark, shake well, and make a solution with a concentration equivalent to 120% of the concentration of the reference solution. Prepare 3 copies of each concentration in parallel, perform the test, and calculate the recovery of the dibasic acid.

[0099] Table 2 Results of m-cresol recovery

[0100] 4.5 Durability Three chromatographic columns of the same specification but different brands were used; the flow rate varied by ±0.2 ml / min; the column temperature varied by ±5 °C; the mobile phase pH varied by ±0.2 (i.e., pH 3.8, pH 4.0, and pH 4.2); the control solution and the test solution were measured respectively under the changing proportion of the mobile phase (i.e., 210 ml of water and 790 ml of methanol were added during the preparation of the mobile phase; 190 ml of water and 810 ml of methanol were added). The system applicability and test results met the requirements, and the method was durable.

[0101] Experiment 3 The liquid chromatograms obtained from the test solutions of Example 1 and Comparative Examples 1-4 were compared respectively. Figure 5 is the liquid chromatogram of Example 1, Figure 6 The liquid chromatogram of Comparative Example 1, Figure 7 The liquid chromatogram of Comparative Example 2, Figure 8 The liquid chromatogram of Comparative Example 3, Fig. 9 It is the liquid chromatogram of Comparative Example 4.

[0102] from Figure 5 and Figure 3 From the comparison, it can be seen that there is no effect on the test results within the adjustment flow rate range, and the test method has good durability.

[0103] from Figure 6 It can be seen that the absorption value of dodecanedioic acid is the largest near the wavelength of 220nm, that is, the response is the highest, so the detection wavelength is selected as 220nm; from Figure 7 It can be seen that at pH = 3.0, the blank solution interferes with the test sample detection. Figure 8-9 It can be seen that at mobile phase pH = 5.0 and pH = 6.0, the response of dodecanedioic acid decreases, and the peak front becomes abnormal as the pH increases.

Claims

1. A method for determining the content of dibasic acid by high performance liquid chromatography, characterized in that: The steps include: S1. Weigh about 150 mg of the test sample, dissolve it in methanol and dilute it to 50 mL, shake well to obtain the test solution; The test sample is any one or a combination of dodecanedioic acid fermentation broth, liquid produced during dodecanedioic acid extraction, and dodecanedioic acid finished product; S2, testing the test solution using a high performance liquid chromatograph; The speed at which the test solution passes through the chromatographic column is 0.8-1.2 ml / min; The chromatographic column used in the high performance liquid chromatograph is an octadecylsilane bonded silica gel column; The temperature of the chromatographic column is 20-30°C, and the elution time is 10 minutes; The mobile phase used in the high performance liquid chromatograph is a water-methanol mixture; In the water-methanol mixture, the volume ratio of water to methanol is 190-210:790-810; The pH of the water-methanol mixture is adjusted to 3.8-4.2 with phosphoric acid.

2. The method for determining the content of dibasic acid by high performance liquid chromatography according to claim 1, characterized in that: The dibasic acid is dodecanedioic acid.

3. The method for determining the content of dibasic acid by high performance liquid chromatography according to claim 1, characterized in that: The speed at which the test solution passes through the chromatographic column is 1.0 ml / min.

4. The method for determining the content of dibasic acid by high performance liquid chromatography according to claim 1, characterized in that: The high performance liquid chromatograph adopts an ultraviolet detector.

5. The method for determining the content of dibasic acid by high performance liquid chromatography according to claim 1, characterized in that: The injection volume of the HPLC was 20 μl.

6. The method for determining the content of dibasic acid by high performance liquid chromatography according to claim 1, characterized in that: The detection wavelength of the high performance liquid chromatograph is 220 nm.

7. The method for determining the content of dibasic acid by high performance liquid chromatography according to claim 1, characterized in that: The temperature of the chromatographic column is 25° C., and the elution time is 10 minutes.

8. The method for determining the content of dibasic acid by high performance liquid chromatography according to claim 1, characterized in that: The concentration of the phosphoric acid is 4.4 mol / L.

9. The method for determining the content of dibasic acid by high performance liquid chromatography according to claim 1, characterized in that: In the water-methanol mixed liquid, methanol is 100% methanol.

10. The method for determining the content of dibasic acid by high performance liquid chromatography according to claim 1, characterized in that: The concentration of the test solution is 3 mg / mL.

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