High performance liquid chromatography detection method of vitamin C fermentation liquor

Abstract

The invention relates to the technical field of chemical analysis and detection, in particular to a high performance liquid chromatography detection method of vitamin C fermentation liquor. According to the method disclosed by the invention, the detection of the raw material sorbitol and the product sorbose in the one-step fermentation process of the vitamin C and the detection of the raw material sorbose and the product L-gulonic acid in the two-step fermentation process of the vitamin C are realized, the complete separation of the sorbitol and the sorbose as well as the sorbose and the L-gulonic acid in a liquid chromatogram can be achieved, and meanwhile, the sorbitol, the sorbose and the L-gulonic acid are quantitatively detected. The linearity, the precision, the repeatability, the stability, the recovery rate, the detection limit and the quantitation limit of the method for measuring the content of the sorbitol, the sorbose and the L-gulonic acid all meet the requirements of GB / T27404-2008 Laboratory Quality Control Standard, and it is proved that the content measuring method is scientific and effective. The method disclosed by the invention is simple to operate, the detection time of each batch of samples is only 12 minutes, and three substances in two-step fermentation are simultaneously detected by one liquid phase, so that machines are saved, and meanwhile, the detection efficiency is greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis and detection, and particularly relates to a high performance liquid chromatography detection method for vitamin C fermentation broth. Background Art

[0002] At present, chemical titration is still used to detect the intermediates sorbose and L-gulonic acid in vitamin C fermentation broth. In the one-step fermentation of vitamin C, the glycerol copper titration method for total reducing sugars is used to detect the sorbose content. Since the total reducing sugars are measured, the titration method will include other non-sorbose reducing sugars in the fermentation broth in the sorbose content, resulting in a falsely high titration result; and during the entire titration process, titration needs to be carried out while boiling, and there are many potential safety hazards in the detection environment and operating conditions, and there are also large operation errors among different personnel.

[0003] To detect the content of sorbitol in the one-step fermentation broth, currently, the alcohol content at 0 hour is detected using an Abbe refractometer. During the fermentation process, since both sorbitol and sorbose have refractive indices, it is impossible to accurately measure the sorbitol content using an Abbe refractometer; or the existing liquid phase method is used to detect the content of sorbitol, but currently, according to the liquid phase method for detecting sorbitol in the national standard, the detection time for one sample is 40 minutes, and the detection timeliness is poor, and accurate data cannot be provided in a timely manner, which cannot meet the production requirements.

[0004] To detect the residual sorbose content in the two-step fermentation, the currently used anthrone colorimetric method requires accurate timing of a water bath for 10 minutes and precise control of the temperature at 38 °C, and these strict control conditions all bring relatively large errors to the detection.

[0005] To detect the content of L-gulonic acid in the two-step fermentation, currently, after a boiling water bath for 25 minutes, the method of titrating vitamin C with iodine is used, and the content of L-gulonic acid is deduced by substituting the coefficient; this method has high requirements for accurate timing, water bath pot, test tube and personnel operation, and there will be relatively large experimental errors.

[0006] The currently used titration method has large operation errors among different personnel, and the titration result of the sorbose content in the one-step fermentation broth is on the high side. The liquid phase method for detecting the raw material sorbitol takes a long time and cannot provide data in a timely manner to support production. Therefore, based on the existing conditions, the existing methods are improved and developed. Summary of the Invention

[0007] The purpose of the present invention is to provide a high performance liquid chromatography detection method for vitamin C fermentation broth, so as to solve the problem that when detecting the target substances in vitamin C fermentation broth, different detection means need to be used for the one-step fermentation broth and the two-step fermentation broth, resulting in low detection efficiency and safety risks.

[0008] To achieve the above object, the present invention adopts the following technical solution: A high performance liquid chromatography detection method for vitamin C fermentation broth, wherein the vitamin C fermentation broth is the first-stage fermentation broth and the second-stage fermentation broth in the process of preparing vitamin C by secondary fermentation with sorbitol as the raw material;

[0009] The detection targets in the vitamin C first-stage fermentation broth include sorbitol and sorbose;

[0010] The detection targets in the vitamin C second-stage fermentation broth include sorbose and L-gulonic acid;

[0011] The high performance liquid chromatography detection method includes:

[0012] Chromatographic column: Sulfonated polystyrene divinylbenzene copolymer hydrogen ion type resin chromatographic column;

[0013] Mobile phase: Sulfuric acid solution;

[0014] Detector: Differential refractive index detector;

[0015] The high performance liquid chromatography detection method is simultaneously applicable to the detection of targets in the first-stage fermentation broth and the second-stage fermentation broth.

[0016] Further, the model of the chromatographic column is Carbomix H-NP300*7.8mm, 8μm; the cross-linking degree is 12%; the chromatographic column temperature is 45-55°C.

[0017] Further, the concentration of sulfuric acid in the mobile phase is 5-50 mmol / L; the flow rate of the mobile phase is 0.5-0.7 mL / min.

[0018] Further, the detection temperature of the differential refractive index detector is 35-55°C, and the model is RID-20A.

[0019] Further, the detection of vitamin C fermentation broth includes the following steps:

[0020] S1. Preparation of the sample solution to be measured

[0021] Take the sample to be measured and place it in a volumetric flask, add diluent to dissolve it completely, and dilute and shake well to obtain the sample solution to be measured;

[0022] S2. Reference solution

[0023] Take the reference standard and place it in a volumetric flask, add diluent to dissolve it completely, and dilute and shake well to obtain the reference solution;

[0024] S3. Content determination

[0025] Separate the test sample solution S1 and the reference solution S2, and inject them into a high-performance liquid chromatograph to record the chromatogram; calculate the contents of sorbitol, sorbose, and L-gulonic acid in the sample as the intermediate of vitamin C fermentation broth by the external standard method based on the peak areas respectively.

[0026] Further, in S1 - S2, the diluent is ultrapure water with a conductivity of not less than 18.2 megohms.

[0027] Further, in S1, the test sample is the first-step fermentation broth or the second-step fermentation broth of vitamin C fermentation broth.

[0028] Further, in S2, the reference substances are the standards of sorbitol, sorbose, and L-gulonic acid, and their purities are all ≥ 99%.

[0029] Further, in S1 - S2, the concentration ranges of each solute in the test sample solution in S1 and the reference solution in S2 are both 0.625 - 10 g / L respectively.

[0030] Further, for sorbitol, sorbose, and L-gulonic acid in the vitamin C fermentation broth, when the concentration is between 0.625 - 10 g / L, they all have a good linear relationship, and their linearities reach 1, 0.9999, and 0.9998 respectively; the recoveries can all reach 99%, the detection limit is 0.05 g / L, and the quantification limit is 0.15 g / L.

[0031] Advantages of the present invention:

[0032] 1. Under the detection conditions provided by the method of the present invention, sorbitol, sorbose, and L-gulonic acid can be separated to the limit, and the resolution can meet the requirements of qualitative and quantitative analysis;

[0033] 2. The chromatographic conditions designed by the method of the present invention cause less damage to the chromatographic column used, the chromatographic column has a longer service life under normal use, and the detection cost is low;

[0034] 3. The sample pretreatment step of the method of the present invention is simple. Sorbitol, sorbose, and L-gulonic acid only need to be diluted and fixed with water, the detection time is short, and it has high sensitivity; it can better realize the determination of the contents of sorbitol, sorbose, and L-gulonic acid, which is beneficial to meeting the quality control requirements in the production process;

[0035] 4. The method of the present invention can be simultaneously applied to the detection of the first-step fermentation broth and the second-step fermentation broth of vitamin C. In the production process of vitamin C, one liquid phase can be reduced in use, and there are no more dangerous sources such as high-temperature boiling with an induction cooker and titration while boiling on an electric furnace at the detection site, reducing the personal risk of operators. Description of the Drawings

[0036] Figure 1It is the chromatogram of the mixed solution of sorbitol, sorbose, and L-gulonic acid reference substances in the method of the present invention;

[0037] Figure 2 It is the chromatogram of the one-step fermentation broth sample in Example 1 of the method of the present invention;

[0038] Figure 3 It is the chromatogram of the two-step fermentation broth sample in Example 1 of the method of the present invention;

[0039] Figure 4 It is the fitting standard curve graph of L-gulonic acid in the method of the present invention;

[0040] Figure 5 It is the fitting standard curve graph of sorbose in the method of the present invention;

[0041] Figure 6 It is the fitting standard curve graph of sorbitol in the method of the present invention. Detailed implementation manners

[0042] Next, the technical solution of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention.

[0043] Example 1

[0044] 1. Chromatographic conditions

[0045] Perform high-performance liquid chromatography analysis under the following chromatographic conditions to determine the contents of sorbitol, sorbose, and L-gulonic acid:

[0046] Chromatographic column: Carbomix H-NP 300 * 7.8 mm, 8 μm, sulfonated polystyrene divinylbenzene copolymer hydrogen ion type resin chromatographic column, cross-linking degree 12%;

[0047] Mobile phase: 0.005 mol / L sulfuric acid solution;

[0048] Flow rate: 0.6 mL / min;

[0049] Column temperature: 50 °C;

[0050] Detector: differential refractive index detector, RID-20A, temperature 40 °C;

[0051] Injection volume: 10 μL.

[0052] 2. Preparation of the mobile phase

[0053] Measure 0.27 mL of analytical grade concentrated sulfuric acid, add 1000 mL of ultrapure water, mix well, filter with a 0.45 μm water-based filter membrane, perform ultrasonic degassing, and store it in the mobile phase bottle.

[0054] 3. Preparation of the reference substance solution

[0055] Accurately weigh 0.25 g of the standards of sorbitol, sorbose, and L-gulonic acid respectively, accurate to 0.0001 g, place them in a 50 mL volumetric flask, dissolve with a diluent, dilute to the mark, and shake well to obtain a reference solution.

[0056] 4. Preparation of the solution of the vitamin C fermentation broth to be tested

[0057] Accurately pipette 1.00 mL of the fermentation broth (the first-step fermentation broth and the second-step fermentation broth) respectively into a 50 mL volumetric flask, dissolve thoroughly with a diluent, dilute to the mark, shake well, take a part and transfer it to a centrifuge tube, centrifuge at 10000 r / min for 5 min, and filter through a 0.45 μm aqueous filter membrane to obtain the solution of the sample to be tested.

[0058] 5. Determination

[0059] Pipette 10 μL of the solution of the sample to be tested (the first-step fermentation broth solution and the second-step fermentation broth solution) and the reference solution respectively into a high-performance liquid chromatograph, record the chromatogram; the resolution of L-gulonic acid and sorbose is 2.92, the resolution of sorbose and sorbitol is 1.84, and the theoretical plate numbers of L-gulonic acid, sorbose, and sorbitol are 4373, 4930, and 5183 respectively.

[0060] 6. Calculation

[0061] The peak areas of sorbitol, sorbose, and L-gulonic acid in the mixed reference are 694473, 712837, and 659910 respectively. The concentrations of each standard in the mixed reference solution are all 5 g / L. The peak areas of sorbitol and sorbose in the first-step fermentation broth are 639665 and 983052 respectively; the peak areas of sorbose and L-gulonic acid in the second-step fermentation broth are 473267 and 429034 respectively.

[0062] Calculation example:

[0063]

[0064] Finally, it is calculated that the content of sorbitol in the first-step fermentation broth is 230.27 g / L, and the content of sorbose is 344.77 g / L; the content of sorbose in the second-step fermentation broth is 165.98 g / L, and the content of L-gulonic acid is 162.54 g / L.

[0065] Example 2

[0066] To verify the feasibility and accuracy of the above Example 1, a linear experiment was carried out. The experimental concentrations were 0.625 g / L, 1.25 g / L, 2.5 g / L, 5 g / L, and 10 g / L of the mixed standard solution of L-gulonic acid, sorbose, and sorbitol. Using the same chromatographic conditions as in Example 1, the measurement results are shown in Table 1.

[0067] Table 1 Data Sheet of Linear Experiment Results

[0068]

[0069] Using the data in Table 1 to fit the standard curve to obtain Table 2:

[0070] Table 2 Data Sheet of the Fitted Standard Curve

[0071]

[0072] It can be seen from Table 2 and Figure 2-4 that the linearity of the three compounds is good.

[0073] Example 3

[0074] To verify the feasibility and accuracy of the above Example 1, a precision test was carried out. The test used a mixed standard solution of L-gulonic acid, sorbose, and sorbitol prepared at a concentration of 10 g / L. The sample was injected continuously 6 times under the same chromatographic conditions as in Example 1. The measurement results are shown in Table 3.

[0075] Table 3 Data Sheet of Precision Test Results

[0076]

[0077] It can be seen from Table 3 that the RSD values of the peak areas are all less than 2%, indicating good instrument stability.

[0078] The present invention is not limited to the above best implementation manner. Anyone can obtain various other forms of products under the inspiration of the present invention. However, no matter what changes are made in its shape or structure, as long as it has a technical solution identical or similar to the present application, it falls within the protection scope of the present invention.

[0079] Example 4

[0080] To verify the feasibility and accuracy of the above Example 1, the detection limit and quantification limit were tested. The test used a mixed standard solution of L-gulonic acid, sorbose, and sorbitol prepared at concentrations of 0.625 g / L respectively. The sample was injected continuously 6 times under the same chromatographic conditions as in Example 1. According to the calculation formula, detection limit = 3 * SD value / slope, quantification limit = 3 * detection limit. The measurement results are shown in Table 4.

[0081] Table 4 Data Sheet of Detection Limit and Quantification Limit Test Results

[0082]

[0083]

[0084] Example 5

[0085] To verify the feasibility and accuracy of the above Example 1, a spike recovery test was conducted. The sorbitol content in the diluted one-step fermentation broth in Example 1 was 4.6054 g / L, and the sorbose content was 6.8954 g / L; the sorbose content in the diluted two-step fermentation broth in Example 1 was 3.3196 g / L, and the L-gulonic acid content was 3.2508 g / L.

[0086] 1 mL of the diluted one-step fermentation broth and 1 mL of the diluted two-step fermentation broth in Example 1 were respectively taken, 1 mL of a 10 g / L mixed standard reference solution was added to each, and after mixing evenly, liquid phase detection was carried out under exactly the same chromatographic conditions as in Example 1.

[0087] Calculation 1:

[0088]

[0089] Calculation 2:

[0090]

[0091] Calculation 3:

[0092] Recovery concentration = Final measured concentration of the sample - Concentration of the basic sample after spiking. Calculation 4:

[0093]

[0094] Table 5 Statistical report of spike recovery test data

[0095]

[0096]

Claims

1. A high performance liquid chromatography (HPLC) detection method for vitamin C fermentation broth, characterized in that: The vitamin C fermentation broth is the primary fermentation broth and the secondary fermentation broth in the process of preparing vitamin C by secondary fermentation using sorbitol as the raw material; The detection targets in the vitamin C primary fermentation broth include sorbitol and sorbose; The detection targets in the vitamin C secondary fermentation broth include sorbose and L-gulonic acid; The HPLC detection method includes: Chromatographic column: Sulfonated polystyrene divinylbenzene copolymer hydrogen ion type resin chromatographic column; Mobile phase: Sulfuric acid solution; Detector: Differential refractive index detector; The HPLC detection method is applicable to the detection of targets in both the primary fermentation broth and the secondary fermentation broth.

2. The high performance liquid chromatography detection method of a vitamin C fermentation broth according to claim 1, characterized in that: The model of the chromatographic column is Carbomix H-NP300*7.8mm, 8μm; cross-linking degree 12%; the column temperature is 45 - 55°C.

3. The high performance liquid chromatography detection method for a vitamin C fermentation broth according to claim 1, wherein: The concentration of sulfuric acid in the mobile phase is 5 - 50 mmol / L; the flow rate of the mobile phase is 0.5 - 0.7 mL / min.

4. The high performance liquid chromatography detection method for a vitamin C fermentation broth according to claim 1, wherein: The detection temperature of the differential refractive index detector is 35 - 55°C, and the model is RID-20A.

5. The high performance liquid chromatography detection method for the vitamin C fermentation broth according to any one of claims 1-4, characterized in that The detection of vitamin C fermentation broth includes the following steps: S1. Preparation of the sample solution to be tested Take the sample to be tested and place it in a volumetric flask, add diluent to dissolve it fully, and dilute and shake well to obtain the sample solution to be tested; S2. Reference solution Take the reference standard and place it in a volumetric flask, add diluent to dissolve it fully, and dilute and shake well to obtain the reference solution; S3. Content determination Respectively pipette the sample solution to be tested in S1 and the reference solution in S2 into the HPLC instrument, record the chromatogram; calculate the contents of sorbitol, sorbose, and L-gulonic acid, the intermediates of vitamin C fermentation broth in the sample by the external standard method based on the peak areas respectively.

6. The high performance liquid chromatography detection method for the vitamin C fermentation broth according to claim 5, characterized in that: In S1 - S2, the diluent is ultrapure water with a conductivity of not less than 18.2 megohms.

7. The high performance liquid chromatography detection method for the vitamin C fermentation broth according to claim 5, wherein: In S1, the sample to be tested is the primary fermentation broth or the secondary fermentation broth of vitamin C fermentation broth.

8. The high performance liquid chromatography detection method for the vitamin C fermentation broth according to claim 5, characterized in that: In S2, the reference standards are sorbitol, sorbose, and L-gulonic acid reference standards with a purity of ≥99%.

9. The high performance liquid chromatography detection method for the vitamin C fermentation broth according to claim 5, characterized in that: In S1 - S2, the concentration ranges of each solute in the sample solution to be tested in S1 and the reference solution in S2 are both 0.625 - 10 g / L respectively.

10. The high performance liquid chromatography detection method for the vitamin C fermentation broth according to claim 5, wherein: For sorbitol, sorbose, and L-gulonic acid in the vitamin C fermentation broth, when the concentration is between 0.625 - 10 g / L, they all have good linear relationships, with linearities reaching 1, 0.9999, and 0.9998 respectively, the recoveries can all reach 99%, the detection limit is 0.05 g / L, and the quantification limit is 0.15 g / L.

Images