High performance liquid chromatography detection method for mixture of D-mannitol and sodium gluconate

Abstract

The invention relates to the technical field of chemical analysis, in particular to a high performance liquid chromatography detection method for a mixture of D-mannitol and sodium gluconate. According to the method disclosed by the invention, by changing components of a mobile phase and diluting a sample in a proper proportion, complete separation of the D-mannitol and the sodium gluconate in a liquid chromatogram can be achieved, and meanwhile, the D-mannitol and the sodium gluconate are accurately and quantitatively detected. Linear, precision, repeatability, detection limit, quantitation limit and recovery rate experiments are carried out on the method for determining the content of D-mannitol and sodium gluconate, all the experiments meet the requirements of GB / T27404-2008 Laboratory Quality Control Standards, and it is proved that the method for determining the content is scientific and effective. The method disclosed by the invention is simple to operate, and by adopting the method disclosed by the invention, the detection time of each batch of samples is only 15 minutes, so that the detection efficiency is greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis, in particular to a high performance liquid chromatography detection method for a mixture of D-mannitol and sodium gluconate. Background Art

[0002] D-mannitol is an organic compound with the molecular formula C6H 14 O6, a colorless or white crystalline powder with a slightly sweet taste similar to sucrose, plays a certain role in medicine, food, feed, and life science research; it can be used as a sweetener, anti-sticking agent, nutritional supplement, quality improver, and moisturizer.

[0003] The chemical formula of sodium gluconate is C6H 11 NaO7 is widely used in industry. Sodium gluconate can be used as a high-efficiency chelating agent in the construction, textile printing and dyeing, metal surface treatment and water treatment industries, as well as a steel surface cleaner, glass bottle cleaner, and aluminum oxide coloring in the electroplating industry. In the concrete industry, it is used as a high-efficiency retarder and high-efficiency water reducer to delay the setting time of concrete. When the dosage is below 0.15%, the logarithm of the initial setting time is directly proportional to the added dosage, that is, if the added dosage is doubled, the initial setting time is delayed by ten times. This extends the working time from a few hours to several days without compromising the strength. This is an important advantage, especially in hot weather and when the concrete needs to be placed for a long time.

[0004] The production of D-mannitol is mainly achieved through two methods: microbial fermentation and chemical synthesis. Among them, microbial fermentation has gradually become the mainstream production method due to its advantages such as being green and environmentally friendly and having a wide range of raw material sources. In the actual production and fermentation process, in order to monitor fermentation indicators, it is necessary to accurately and quantitatively detect the concentration of D-mannitol. Sodium gluconate is a by-product of D-mannitol. It is difficult to simultaneously and effectively and accurately detect both sodium gluconate and D-mannitol using conventional detection methods. Therefore, it is particularly necessary to develop new detection methods. Summary of the Invention

[0005] The present invention aims to provide a high performance liquid chromatography (HPLC) method for detecting a mixture of D-mannitol and sodium gluconate, so as to solve the problem that the sodium gluconate byproduct produced during the fermentation of D-mannitol during the production process cannot be completely separated and the sodium gluconate cannot be accurately quantitatively detected.

[0006] To achieve the above object, the present invention adopts the following technical solution: a high performance liquid chromatography detection method for a mixture of D-mannitol and sodium gluconate, wherein the high performance liquid chromatography detection method comprises:

[0007] Chromatographic column: Sulfonated polystyrene divinylbenzene copolymer hydrogen ion resin column;

[0008] Mobile phase: a mixture of aqueous sulfuric acid and acetonitrile;

[0009] Detector: Differential refractive index detector.

[0010] Furthermore, the model of the chromatographic column is HPX-87H 300*7.8mm, 8μm; the cross-linking degree is 12%; and the chromatographic column temperature is 65-80°C.

[0011] Furthermore, the concentration of sulfuric acid in the mobile phase is 5-50 mmol / L; the volume ratio of aqueous sulfuric acid solution to acetonitrile is 80-85:15-20, and the flow rate of the mobile phase is 0.5-0.7 mL / min.

[0012] Furthermore, the detection temperature of the differential detector is 35-55° C., and the model is RID-20A.

[0013] Further, the detection of the D-mannitol and sodium gluconate mixture comprises the following steps:

[0014] S1. Preparation of sample solution to be tested

[0015] Place the sample to be tested in a volumetric flask, add diluent to fully dissolve it, and dilute and shake well to prepare the sample solution to be tested;

[0016] S2, reference solution

[0017] Place the standard in a volumetric flask, add diluent to fully dissolve it, and dilute and shake well to serve as the reference solution;

[0018] S3. Content determination

[0019] The test sample solution S1 and the reference solution S2 were respectively injected into the high performance liquid chromatograph, and the chromatograms were recorded; the contents of D-mannitol and sodium gluconate in the samples were calculated by the peak areas according to the external standard method.

[0020] Furthermore, in said S1-S2, the diluent is the mobile phase.

[0021] Furthermore, in the S2, the standard substances in the reference solution are D-mannitol and sodium gluconate, and the purity of both substances is ≥99%.

[0022] Furthermore, in the above-mentioned S1-S2, the concentrations of D-mannitol and sodium gluconate in the test sample solution in S1 and the reference solution in S2 are both 0.625-10 g / L.

[0023] Furthermore, the D-mannitol and sodium gluconate have a good linear relationship when the concentrations are between 0.625 and 10 g / L, and the linearity reaches 0.9999 and 0.9999, respectively; the detection limit reaches 0.05 g / L, the quantification limit is 0.15 g / L; and the recovery rate is above 99%.

[0024] Beneficial effects of the present invention:

[0025] 1. Under the detection conditions provided by the method of the present invention, D-mannitol standard and sodium gluconate can achieve ultimate separation, presenting a symmetrical Gaussian peak with good peak shape, and the theoretical number of plates can reach more than 3000, which can meet the qualitative and quantitative requirements;

[0026] 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 long life under normal use, and the detection cost is low;

[0027] 3. The sample pretreatment steps of the method of the present invention are simple. D-mannitol and sodium gluconate only need to be diluted with the mobile phase to a constant volume. The detection time is short and the sensitivity is high. The method can better realize the determination of the content of D-mannitol and sodium gluconate, which is conducive to meeting the needs of quality control in the production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a detection chromatogram of a reference solution after mixing sodium gluconate and D-mannitol standard substances of the present invention;

[0029] Figure 2 1 is a detection chromatogram of a sample containing sodium gluconate and D-mannitol in an embodiment of the present invention;

[0030] Figure 3 It is the standard curve of sodium gluconate in the embodiment of the present invention;

[0031] Figure 4 This is the standard curve of D-mannitol in the examples of the present invention. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0033] Example 1

[0034] 1. Chromatographic conditions

[0035] The contents of D-mannitol and sodium gluconate were determined by HPLC analysis under the following chromatographic conditions:

[0036] Chromatographic column: HPX-87H 300*7.8mm, 8μm, cross-linking degree 12%, sulfonated polystyrene divinylbenzene copolymer hydrogen ion resin column;

[0037] Mobile phase: a mixture of 10 mmol / L sulfuric acid solution and acetonitrile = 80:20;

[0038] Flow rate: 0.6 mL / min;

[0039] Column temperature: 70°C;

[0040] Detector: differential refractive index detector, RID-20A, temperature 40°C;

[0041] Injection volume: 10uL.

[0042] 2. Preparation of mobile phase

[0043] Measure 0.432 mL of high-grade concentrated sulfuric acid, add 800 mL of ultrapure water, add 200 mL of chromatography-grade acetonitrile, mix thoroughly, filter with a 0.45 μm organic filter membrane, degas ultrasonically, and store in a brown bottle.

[0044] 3. Preparation of Mixed Reference Solution

[0045] Accurately weigh 0.25 g of standard sodium gluconate and D-mannitol respectively, accurate to 0.0001 g, place them in a 50 mL volumetric flask, add diluent to dissolve, dilute to the scale, and shake well to serve as the reference solution.

[0046] 4. Preparation of the Chromatographic Separation Solution

[0047] Accurately measure 1.00 mL of the chromatographic separation sample in a 25 mL volumetric flask, add diluent to fully dissolve it, and dilute it to the scale. Shake well, take out a portion and transfer it to a centrifuge tube. Centrifuge at 10,000 r / min for 5 minutes. After passing through a 0.45 μm organic filter membrane, use it as the sample solution to be tested.

[0048] 5. Determination

[0049] 10 μL of the mixed reference solution and the sample solution to be tested were respectively injected into the high performance liquid chromatograph, and the chromatogram was recorded; the separation degree of the two peaks of D-mannitol and sodium gluconate in the chromatogram was 3.557, and the theoretical plate numbers were 8868 and 11445, respectively.

[0050] 6. Calculation

[0051] The peak areas of sodium gluconate and D-mannitol in the mixed reference solution were 576845 and 562185, respectively, and the concentration of each standard in the mixed reference solution was 5 g / L. The peak areas of sodium gluconate and D-mannitol in the chromatographically separated sample solution were 595861 and 554615, respectively.

[0052] Calculation example:

[0053]

[0054] The final calculated content of sodium gluconate in the chromatographic separation sample was 129.12 g / L, and the content of D-mannitol was 123.32 g / L.

[0055] Example 2

[0056] To verify the feasibility and accuracy of Example 1, a linear experiment was conducted. The experiment used mixed reference solutions of D-mannitol and sodium gluconate with concentrations of 0.625 g / L, 1.25 g / L, 2.5 g / L, 5 g / L and 10 g / L, and adopted the same chromatographic conditions as in Example 1. The measurement results are shown in Table 1.

[0057] Table 1 Linearity test results data table

[0058]

[0059] The data in Table 1 were used to fit the standard curve. Figure 2 and Figure 3 , the standard curve regression equations are: D-mannitol y=115375x+345.42, R 2 =0.9999, good linear relationship; sodium gluconate y = 120885x + 4924.5, R 2 =0.9999, good linear relationship.

[0060] Example 3

[0061] To verify the feasibility and accuracy of Example 1, precision and repeatability tests were conducted. The test used a mixed reference solution of D-mannitol and sodium gluconate with a concentration of 10 g / L, and the sample was injected 6 times continuously. The chromatographic conditions were exactly the same as those in Example 1. The measurement results are shown in Table 2.

[0062] Table 2 Precision test results data table

[0063]

[0064] As can be seen from Table 2, the RSD values of the peak areas are all less than 2%, indicating good instrument stability.

[0065] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, any technical solution that is the same or similar to that of the present application falls within the scope of protection of the present invention.

[0066] Example 4

[0067] To verify the feasibility and accuracy of Example 1, the detection limit and quantification limit were tested. The test used a mixed reference solution of sodium gluconate and D-mannitol with a concentration of 0.625 g / L, respectively. The sample was injected 6 times continuously. The chromatographic conditions were exactly the same as those in Example 1. The detection limit was calculated according to the formula: 3*SD value / slope, and the quantification limit was calculated as 3*detection limit. The measurement results are shown in Table 4.

[0068] Table 4 Detection limit and quantification limit test results

[0069]

[0070]

[0071] Example 5

[0072] In order to verify the feasibility and accuracy of the above Example 1, a spike recovery test was conducted.

[0073] 1 mL of the diluted chromatographic separation sample in Example 1 was taken respectively, and 1 mL of the 5 g / L mixed reference solution was added respectively. After mixing, the mixture was subjected to liquid phase detection using the same chromatographic conditions as in Example 1.

[0074] Calculation 1:

[0075]

[0076] Calculation 2:

[0077]

[0078] Calculation 3:

[0079] Recovery concentration = final measured sample concentration - basic sample concentration after spike 4:

[0080]

[0081] Table 5 Statistics of spike recovery test data

[0082]

Claims

1. High performance liquid chromatography detection method of D-mannitol and sodium gluconate mixture, characterized in that: The high performance liquid chromatography detection method comprises: Chromatographic column: Sulfonated polystyrene divinylbenzene copolymer hydrogen ion resin column; Mobile phase: a mixture of aqueous sulfuric acid and acetonitrile; Detector: Differential refractive index detector.

2. The high performance liquid chromatography (HPLC) method for detecting the mixture of D-mannitol and sodium gluconate according to claim 1, wherein: The model of the chromatographic column is HPX-87H 300*7.8mm, 8μm; the cross-linking degree is 12%; and the temperature of the chromatographic column is 65-80°C.

3. The high performance liquid chromatography detection method of the D-mannitol and sodium gluconate mixture according to claim 1, wherein: The sulfuric acid concentration in the mobile phase is 5-50 mmol / L; the volume ratio of the sulfuric acid aqueous solution to acetonitrile is 80-85:15-20, and the flow rate of the mobile phase is 0.5-0.7 mL / min.

4. The high performance liquid chromatography detection method of the D-mannitol and sodium gluconate mixture according to claim 1, wherein: The detection temperature of the differential detector is 35-55° C., and the model is RID-20A.

5. The high performance liquid chromatography detection method of the D-mannitol and sodium gluconate mixture according to any one of claims 1 to 4, characterized in that: The test for the mixture of D-mannitol and sodium gluconate includes the following steps: S1. Preparation of sample solution Place the sample to be tested in a volumetric flask, add diluent to fully dissolve it, and dilute and shake well to prepare the sample solution to be tested; S2, reference solution Place the standard in a volumetric flask, add diluent to fully dissolve it, and dilute and shake well to serve as the reference solution; S3. Content determination The test sample solution S1 and the reference solution S2 were respectively injected into the high performance liquid chromatograph, and the chromatograms were recorded; the contents of D-mannitol and sodium gluconate in the samples were calculated by the peak areas according to the external standard method.

6. The high performance liquid chromatography detection method of the D-mannitol and sodium gluconate mixture according to claim 5, wherein: In the above-mentioned S1-S2, the diluent is the mobile phase.

7. The high performance liquid chromatography detection method of the D-mannitol and sodium gluconate mixture according to claim 5, wherein: In the S2, the standard substances in the reference solution are D-mannitol and sodium gluconate, both of which have a purity of ≥99%.

8. The high performance liquid chromatography detection method of the D-mannitol and sodium gluconate mixture according to claim 5, wherein: In the above-mentioned S1-S2, the concentrations of D-mannitol and sodium gluconate in the test sample solution in S1 and the reference solution in S2 are both 0.625-10 g / L.

9. The high performance liquid chromatography detection method of the D-mannitol and sodium gluconate mixture according to claim 5, wherein: The D-mannitol and sodium gluconate have a good linear relationship when their concentrations are between 0.625 and 10 g / L, and their linearity reaches 0.9999 and 0.9999 respectively; the detection limit reaches 0.05 g / L, the quantification limit is 0.15 g / L; and the recovery rate is above 99%.

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