Method for simultaneous determination of 5-hydroxymethylfurfural and bis-(5-formylfurfuryl) ether content

Abstract

The application discloses a method for simultaneously determining the content of 5-hydroxymethylfurfural and bis-(5-formylfurfuryl) ether, which comprises the following steps: sampling 5-hydroxymethylfurfural crude product, preparing a sample solution to be measured by using methanol as a solvent; performing high performance liquid chromatography analysis on the sample solution to be measured under chromatographic conditions, recording the chromatographic peak area of 5-hydroxymethylfurfural and bis-(5-formylfurfuryl) ether respectively, substituting the standard curve equation of 5-hydroxymethylfurfural and bis-(5-formylfurfuryl) ether, and calculating the content of 5-hydroxymethylfurfural and bis-(5-formylfurfuryl) ether respectively. The retention time of HMF and OBMF is 4.9 min and 7.76 min under the chromatographic conditions of the application, the resolution R of the method is greater than 1.5, the impurities have no interference, and the content of HMF and OBMF in HMF crude product can be accurately determined simultaneously.

Description

Technical Field

[0001] This invention belongs to the field of furan-based bio-based content determination technology, and more specifically, relates to a method for simultaneously determining the content of 5-hydroxymethylfurfural (HMF) and bis-(5-formylfurfural) ether (OBMF) in crude 5-hydroxymethylfurfural. Background Technology

[0002] 5-Hydroxymethylfurfural is a bio-based intermediate with an aromatic furan structure. It is a high-tech, high-potential, clean and environmentally friendly platform compound that is expected to lead the chemical industry from the "benzene era" to a cleaner and more environmentally friendly "furan era".

[0003] Bis-(5-formylfurfural) ether is a bio-based intermediate with an aromatic furan structure. It is a high-tech, high-potential, clean, and environmentally friendly platform compound. Bis-(5-formylfurfural) ether exists in various plants, and has been isolated from hawthorn, honeysuckle, Ganoderma lucidum, and Ligusticum chuanxiong. Pharmacological experiments have shown that it has antiviral, antioxidant, and tuberculosis-inhibiting effects. However, research on its synthesis is limited. Larousse C. et al. reported the catalytic dehydration of 5-hydroxymethylfurfural in DMSO at high temperature (155°C) to produce bis-(5-formylfurfural) ether. Other reports describe the preparation of bis-(5-formylfurfural) ether from sucrose via a simple three-step reaction: acid dehydration, halogenation, hydrolysis, and room-temperature dehydration.

[0004] In the crude product of 5-hydroxymethylfurfural, due to the presence of acid and water, its etherification product, namely bis-(5-formylfurfural) ether, is readily generated. Therefore, bis-(5-formylfurfural) ether is the most important byproduct generated during the synthesis and storage of crude 5-hydroxymethylfurfural. Determining the content of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether in crude 5-hydroxymethylfurfural helps to understand the quality changes of 5-hydroxymethylfurfural and provides a theoretical basis for separating the two. Summary of the Invention

[0005] Technical issues

[0006] This invention is designed to solve the above-mentioned problems in the prior art. The purpose of this invention is to provide a method for simultaneously determining the content of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether. This method can simultaneously and accurately determine the content of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether in crude 5-hydroxymethylfurfural products. The operation is simple and quick, and the results are accurate.

[0007] Technical solution

[0008] To achieve the above objectives, according to the present invention, a method for simultaneously determining the content of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether is provided, the method comprising:

[0009] A sample of crude 5-hydroxymethylfurfural was taken, and a sample solution was prepared using methanol as a solvent.

[0010] Under the following chromatographic conditions, the above-mentioned sample solution was analyzed by high performance liquid chromatography, and the peak areas of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether were recorded respectively.

[0011] The peak areas of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether were substituted into the standard curve equations for 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether, respectively, to calculate the contents of each.

[0012] The chromatographic conditions are as follows:

[0013] Chromatographic column: C18 column;

[0014] Mobile phase: methanol / water, gradient elution;

[0015] Detection wavelength: 260-300nm.

[0016] Beneficial effects

[0017] The method for simultaneously determining HMF and OBMF content of this invention is simple and easy to operate. Under the chromatographic conditions of this invention, the retention times of HMF and OBMF were determined to be 4.9 min and 7.76 min, respectively. Comparing the retention times of the standard and sample chromatograms confirms that the sample was indeed measuring HMF and OBMF. Calculations show a resolution R > 1.5, indicating no interference from impurities, demonstrating high method feasibility. This method can simultaneously and accurately determine the content of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether in crude 5-hydroxymethylfurfural. Attached Figure Description

[0018] The accompanying drawings are provided to further illustrate the technical solutions of the present invention and constitute a part of this specification. They are used together with the embodiments of the present invention to explain the present invention and do not constitute a limitation thereof.

[0019] In the attached diagram:

[0020] Figure 1 The standard curve of OBMF obtained in the embodiments of the present invention;

[0021] Figure 2 The standard curve of HMF obtained in the embodiments of the present invention;

[0022] Figure 3The above is the HPLC chromatogram of the OBMF standard obtained in the embodiments of the present invention;

[0023] Figure 4 The above is the HPLC chromatogram of the HMF standard obtained in the embodiments of the present invention;

[0024] Figure 5 The HPLC chromatograms of HMF and OBMF in the test sample solution prepared from crude 5-hydroxymethylfurfural are shown.

[0025] Figure 6 This is a graph showing the trend of OBMF content over time obtained from HPLC analysis of crude HMF at different storage temperatures.

[0026] Figure 7 The HPLC chromatograms obtained from the HPLC analysis of the HMF crude product sample solution in Comparative Example 1 are shown below.

[0027] Figure 8 The HPLC chromatograms obtained from the HPLC analysis of the HMF crude product sample solution in Comparative Example 2 are shown below. Detailed Implementation

[0028] The method for simultaneously determining the contents of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether according to the present invention will be described in more detail below to aid in understanding the present invention.

[0029] According to the present invention, a method for simultaneously determining the content of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether is provided, the method comprising:

[0030] A sample of crude 5-hydroxymethylfurfural was taken, and a sample solution was prepared using methanol as a solvent.

[0031] Under the following chromatographic conditions, the above-mentioned sample solution was analyzed by high performance liquid chromatography, and the peak areas of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether were recorded respectively.

[0032] The peak areas of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether were substituted into the standard curve equations for 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether, respectively, to calculate the contents of each.

[0033] The chromatographic conditions are as follows:

[0034] Chromatographic column: C18 column;

[0035] Mobile phase: methanol / water, gradient elution;

[0036] Detection wavelength: 260-300nm.

[0037] According to one embodiment of the present invention, the crude 5-hydroxymethylfurfural product is a crude 5-hydroxymethylfurfural product that is easily generated during the synthesis and storage of 5-hydroxymethylfurfural due to the presence of acid and water, which readily produces its etherification product, namely bis-(5-formylfurfural) ether.

[0038] According to one embodiment of the present invention, when preparing the sample solution to be tested, for example, crude HMF is weighed, methanol is added as a solvent, and after ultrasonic dissolution, the solution is diluted to a volumetric flask of 100 ml. The solution is then ultrasonically dissolved again, cooled to room temperature, and stored for later use. After filtration through a 0.45 μm filter membrane, it can be analyzed by HPLC. The ultrasonic dissolution time can be 1 to 60 s, preferably 10 to 30 s; the mass of the crude HMF can be 5 mg to 25 mg, preferably 10 mg to 15 mg.

[0039] According to one embodiment of the present invention, the above-mentioned sample solution was analyzed by high-performance liquid chromatography under the following chromatographic conditions, and the peak areas of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether were recorded respectively. The chromatographic conditions are as follows:

[0040] Chromatographic column: C18 column;

[0041] Mobile phase: methanol / water, gradient elution;

[0042] Detection wavelength: 260-300nm.

[0043] The C18 column, also known as an octadecylsilane-bonded silica column, is, for example, an Agilent ZORBAX Extend column. C18, 4.6x250mm, 5μm HPLC column, or other equivalent C18 columns.

[0044] The mobile phase can be methanol / water, and the gradient elution method can be as follows:

[0045] Time / min Methanol / volume% Water / volume% 0min 5 95 10min 100 0 15min 5 95 17min 5 95

[0046] When performing high performance liquid chromatography (HPLC) detection, the flow rate can be 0.4–2 mL / min, specifically, it can be 1 mL / min.

[0047] When performing high performance liquid chromatography (HPLC) detection, the injection volume can be 5 μL to 20 μL, specifically 10 μL.

[0048] When performing high performance liquid chromatography (HPLC) detection, the column temperature can be between 25℃ and 60℃, specifically 40℃.

[0049] When performing high performance liquid chromatography (HPLC) detection, the detection wavelength can be 260–300 nm, specifically 278 nm.

[0050] According to one embodiment of the present invention, the chromatographic peak areas of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether are substituted into the standard curve equations of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether, respectively, to calculate the content of each. The standard curve equations of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether are obtained as follows: 5-hydroxymethylfurfural standard and bis-(5-formylfurfural) ether standard are accurately weighed, and a series of HMF standard solutions and OBMF standard solutions of different concentrations are prepared using methanol as solvent. Under the same chromatographic conditions, high-performance liquid chromatography (HPLC) is performed, and the peak areas are recorded. A standard curve is plotted with the peak area as the ordinate and the standard solution concentration as the abscissa to obtain the regression equation.

[0051] Example

[0052] The method for simultaneously determining the content of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether according to the present invention will be described in more detail below through examples, but the scope of protection of the present invention is not limited to these examples.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are commercially available.

[0054] 1. Experimental Section

[0055] 1.1 Raw materials and pharmaceuticals

[0056] HMF standard (HPLC purity > 99.8%, Zhejiang Tangneng Technology Co., Ltd.), crude HMF (obtained under different storage conditions); methanol (AR, Shanghai Titan Technology Co., Ltd.); methanol (chromatographic grade, Shanghai Titan Technology Co., Ltd.); OBMF standard (HPLC purity > 99.5%, Zhejiang Tangneng Technology Co., Ltd.); purified water (Wahaha purified water);

[0057] 1.2 Experimental Instruments and Equipment

[0058] High-performance liquid chromatograph: Agilent 1260 (including online degasser, quaternary pump, autosampler, column oven, UV detector, and chromatography workstation); Column: ZORBAX Extend C18, 4.6x250mm, 5μm (Agilent); analytical balance 0.00001g (Mettler-Toledo Instruments Ltd.); ultrasonic cleaner (Shanghai Kedao Ultrasonic Instruments Co., Ltd.);

[0059] 1.3 Method Principles

[0060] This invention uses high performance liquid chromatography to analyze the trend of HMF and OBMF content in crude HMF products at different storage temperatures over time. The stored samples are taken at specific sampling times and analyzed.

[0061] Weigh approximately 0.01 g of each of the above samples, record the accurate mass, dissolve in methanol and dilute to a volumetric flask of 100 ml, sonicate to aid dissolution, cool to room temperature to prepare the sample solution to be tested, filter through a 0.45 μm filter membrane, and perform analysis by HPLC.

[0062] 1.4 Chromatographic conditions

[0063] Mobile phase: 0 min, methanol:water = 5:95; 10 min, methanol:water = 100:0; 15 min, methanol:water = 5:95, followed by gradient elution with a running time of 2 min, wherein the ratio of methanol to water is by volume.

[0064] Detection wavelength: 278nm;

[0065] Flow rate: 1.0 mL / min;

[0066] Injection volume: 10 μL;

[0067] Column temperature: 40℃;

[0068] 1.5 Plotting the standard curves of OBMF and HMF

[0069] 1.5.1 Plotting the Standard Curve of OBMF

[0070] (1) Accurately weigh 0.01444 g of OBMF, dissolve it in methanol and make up to 100 mL in a volumetric flask, and use sonication to dissolve it to prepare a standard stock solution of 0.1444 g / L.

[0071] (2) Take 6.25 mL, 12.5 mL, 25 mL, and 50 mL of the above stock solution and dilute to 100 mL volumetric flasks to prepare five standards with increasing concentrations: 0.009 g / L, 0.0180 g / L, 0.0361 g / L, 0.0722 g / L, and 0.1444 g / L. Perform analysis under the aforementioned chromatographic conditions, injecting each sample twice and recording the peak area of ​​the OBMF. Figure 3 As shown, Figure 3 This is the HPLC chromatogram of the OBMF standard.

[0072] (3) Plot a standard curve with peak area Y (mAu) as the ordinate and standard concentration X (g / L) as the abscissa, and perform regression processing.

[0073] Figure 1 This is the standard curve for OBMF.

[0074] according to Figure 1 The standard curve is: Y = 67621.3542X - 10.3899, and the correlation coefficient R0 is... 2 =0.99999. This indicates that the peak area of ​​OBMF is linearly related to the sample concentration in the concentration range of 0.009 g / L to 0.1444 g / L.

[0075] 1.5.2 Plotting the Standard Curve of HMF

[0076] (1) Accurately weigh 0.01312 g of HMF, dissolve it in methanol and dilute it to 100 mL in a volumetric flask, and use sonication to dissolve it to prepare a standard stock solution of 0.1312 g / L.

[0077] (2) Take 6.25 mL, 12.5 mL, 25 mL, and 50 mL of the above stock solution and dilute to 100 mL volumetric flasks to prepare five standards with increasing concentrations: 0.0082 g / L, 0.0164 g / L, 0.0328 g / L, 0.0656 g / L, and 0.1312 g / L. Perform analysis under the aforementioned chromatographic conditions, injecting each sample twice, recording and calculating the average peak area of ​​the HMF. Figure 4 As shown, Figure 4 This is the HPLC chromatogram of the HMF standard.

[0078] (3) Plot a standard curve with peak area Y (mAu) as the ordinate and standard concentration X (g / L) as the abscissa, and perform regression processing.

[0079] Figure 2 This is the standard curve for HMF.

[0080] according to Figure 2 The standard curve is: Y = 71507.8513X + 14.4614, and the correlation coefficient R0 is... 2 =0.99997. This indicates that within the concentration range of 0.0082 g / L to 0.1312 g / L, the chromatographic peak area is linearly related to the sample amount.

[0081] 1.6 Precision Test

[0082] 1.6.1 Precision test of OBMF determination (n=6)

[0083] To prepare a 1 wt% OBMF methanol solution, weigh approximately 1 g of OBMF, record the accurate mass, dissolve it in methanol, and dilute to a 100 ml volumetric flask. Shake well to prepare the sample solution. Filter the solution through a 0.45 μm filter membrane and perform repeated measurements 6 times using HPLC. Calculate the OBMF content and the relative standard deviation (RSD) of OBMF.

[0084]

[0085] As can be seen from the table above, the OBMF content in the sample remained basically unchanged, with an RSD value of 0.0539%, which is less than 1%, indicating that the precision of OBMF determination using this method is high.

[0086] 1.6.2 Precision test of HMF determination (n=6)

[0087] To prepare a 0.5 wt% HMF methanol solution, weigh approximately 0.5 g of HMF, record the accurate mass, dissolve it in methanol, and dilute to a 100 ml volumetric flask. Shake well to prepare the sample solution to be tested. Filter the solution through a 0.45 μm filter membrane and perform repeated measurements 6 times using HPLC. After conversion, the HMF content and the relative standard deviation (RSD) of HMF are obtained.

[0088]

[0089] As can be seen from the table above, the HMF content in the sample remained basically unchanged, with an RSD value of 0.0690%, which is less than 1%, indicating that the precision of HMF determination using this method is high.

[0090] 1.7 Spike Recovery Experiment

[0091] 1.7.1 OBMF sample spiked recovery test (n=3)

[0092] 1) To prepare a 0.5 g / kg OBMF sample, add 0.5 g of pure OBMF to 999.5 g of methanol, sonicate to aid dissolution, and cool to room temperature to prepare sample A;

[0093] 2) Take 100g of sample A, add 0.05g of pure OBMF, sonicate to dissolve, cool to room temperature to prepare sample B, take 5g of sample B into a 10ml volumetric flask, dissolve in methanol and dilute to the mark, and then perform HPLC analysis to calculate the OBMF content in sample B, i.e., the OBMF measured value. OBMF recovery rate = (OBMF measured value - OBMF content in sample) / OBMF spiked amount × 100%, repeat three times.

[0094] 3) Take 100g of sample A, add 0.1g of pure OBMF, sonicate to dissolve, cool to room temperature to prepare sample C, take 5g of sample C into a 10ml volumetric flask, dissolve in methanol and dilute to the mark, and then perform HPLC analysis to calculate the OBMF content in sample C, i.e., the OBMF measured value. OBMF recovery rate = (OBMF measured value - OBMF content in sample) / OBMF spiked amount × 100%, repeat three times.

[0095] 4) Take 100g of sample A, add 0.2g of pure OBMF, sonicate to dissolve, cool to room temperature to prepare sample D, take 5g of sample D into a 10ml volumetric flask, dissolve in methanol and dilute to the mark, and then perform HPLC analysis to calculate the OBMF content in sample D, i.e., the OBMF measured value. OBMF recovery rate = (OBMF measured value - OBMF content in sample) / OBMF spiked amount × 100%, repeat three times.

[0096]

[0097] As can be seen from the table above, in the OBMF recovery test, the relative standard deviation of OBMF was less than 1%; and the measured recovery rate was between 90% and 110%, proving that this method has high accuracy.

[0098] 1.7.2 HMF sample spiked recovery test (n=3)

[0099] 1) To prepare a 0.5 g / kg HMF sample, add 0.5 g of pure HMF to 999.5 g of methanol, sonicate to aid dissolution, and cool to room temperature to prepare sample A;

[0100] 2) Take 100g of sample A, add 0.05g of pure HMF, sonicate to dissolve, cool to room temperature to prepare sample B, take 5g of sample B into a 10ml volumetric flask, dissolve in methanol and dilute to the mark, and then perform HPLC analysis to calculate the HMF content in sample B, i.e., the HMF determination value. HMF recovery rate = (HMF determination value - HMF content in sample) / HMF spiked amount × 100%, repeat three times.

[0101] 3) Take 100g of sample A, add 0.1g of pure HMF, sonicate to dissolve, cool to room temperature to prepare sample C, take 5g of sample C into a 10ml volumetric flask, dissolve in methanol and dilute to the mark, and then perform HPLC analysis to calculate the HMF content in sample C, i.e., the HMF determination value. HMF recovery rate = (HMF determination value - HMF content in sample) / HMF spiked amount × 100%, repeat three times.

[0102] 4) Take 100g of sample A, add 0.2g of pure HMF, sonicate to dissolve, cool to room temperature to prepare sample D. Take 5g of sample D into a 10ml volumetric flask, dissolve in methanol and dilute to the mark. Analyze by HPLC and calculate the HMF content in sample D, i.e., the HMF determination value. HMF recovery rate = (HMF determination value - HMF content in sample) / HMF spiked amount × 100%. Repeat three times.

[0103]

[0104] As can be seen from the table above, in the HMF recovery test, the relative standard deviation of HMF was less than 1%; and the measured recovery rate was between 90% and 110%, proving that this method has high accuracy.

[0105] 1.8 Determination of HMF and OBMF content in samples

[0106] Following the methods in steps 1.3 and 1.4, approximately 0.01 g of the crude HMF sample was weighed and its accurate mass was recorded. The sample was dissolved in methanol and diluted to a 100 ml volumetric flask. The solution was sonicated to aid dissolution, cooled to room temperature, and the solution was then filtered through a 0.45 μm filter membrane and analyzed by high-performance liquid chromatography (HPLC). Figure 5 The spectrum.

[0107] Substituting the obtained OBMF chromatographic peak area (peak area 619.21) and HMF chromatographic peak area (peak area 7504.02) into the OBMF standard curve equation and HMF standard curve equation in step 1.5 respectively, we obtained that the HMF content is 0.1047 g / L and the OBMF content is 0.0093 g / L.

[0108] 1.9 Determination of OBMF content in samples

[0109] Following the methods in steps 1.3 and 1.4, HMF crude product samples at different storage temperatures were processed and subjected to high-performance liquid chromatography (HPLC). The obtained chromatographic peak areas were substituted into the OBMF standard curve equation in step 1.5 to obtain the OBMF content in the HMF crude product.

[0110] 2 Results Analysis

[0111] 2.1 Feasibility Analysis of Experimental Methods

[0112] The OBMF in the sample was determined under the above chromatographic conditions. The chromatograms of the standard and the sample are shown below. Figure 3 and Figure 5 .

[0113] Figure 3 This is the HPLC chromatogram of OBMF standard.

[0114] Figure 5 This is the HPLC chromatogram of the OBMF sample.

[0115] Under the above chromatographic conditions, the retention time of OBMF was determined to be 7.76 min. Comparing the retention times of the standard and sample chromatograms confirms that the sample was indeed OBMF. Calculations showed a resolution R > 1.5, indicating no interference from impurities, demonstrating the high feasibility of the method.

[0116] 2.2 Analysis of Experimental Results on OBMF Content in Crude HMF Products at Different Storage Temperatures

[0117] Figure 6 This is a graph showing the trend of OBMF content in crude HMF products at different storage temperatures.

[0118] Depend on Figure 6 It can be seen that, under the same storage time, the OBMF content shows a significant upward trend with the increase of storage temperature; when the storage temperature is above 0℃, the OBMF content tends to increase over time, and this trend is closely related to the storage temperature. Specifically, the OBMF content increases as the corresponding storage temperature increases; when the storage temperature is less than or equal to 0℃, the OBMF content does not change significantly over time and remains at a low level. Therefore, 0℃ and below is a better storage temperature for crude HMF products.

[0119] Comparative Example 1

[0120] Weigh approximately 0.01 g of crude HMF sample, record the accurate mass, dissolve in methanol and dilute to a 100 ml volumetric flask, sonicate to aid dissolution, cool to room temperature to prepare the test sample solution, filter through a 0.45 μm filter membrane, and perform HPLC analysis under the following chromatographic conditions:

[0121] Mobile phase: a mixture of methanol and water in a ratio of 5:95 (volume ratio);

[0122] Elution; isocratic elution;

[0123] Detection wavelength: 278nm;

[0124] Flow rate: 1.0 mL / min;

[0125] Injection volume: 10 μL;

[0126] Column temperature: 40℃;

[0127] The obtained spectrum is as follows Figure 7 As shown, the HMF peak (RT = 8.366 min) is a split peak and the OBMF peak is not separated.

[0128] Comparative Example 2

[0129] Weigh approximately 0.01 g of crude HMF sample, record the accurate mass, dissolve in methanol and dilute to a 100 ml volumetric flask, sonicate to aid dissolution, cool to room temperature to prepare the test sample solution, filter through a 0.45 μm filter membrane, and perform HPLC analysis under the following chromatographic conditions:

[0130] Mobile phase: methanol

[0131] Elution; isocratic elution;

[0132] Detection wavelength: 278nm;

[0133] Flow rate: 1.0 mL / min;

[0134] Injection volume: 10 μL;

[0135] Column temperature: 40℃;

[0136] The obtained spectrum is as follows Figure 8 As shown, the HMF peak (RT = 2.55 min) is affected by extraneous peaks, indicating insufficient separation.

[0137] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for simultaneously determining the content of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether, the method comprising: A sample of crude 5-hydroxymethylfurfural was taken, and a sample solution was prepared using methanol as a solvent. Under chromatographic conditions, the above-mentioned sample solution was analyzed by high performance liquid chromatography, and the chromatographic peak areas of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether were recorded respectively. The peak areas of 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether were substituted into the standard curve equations for 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether, respectively, to calculate the contents of each. The chromatographic conditions are as follows: Chromatographic column: C18 column; Mobile phase: methanol / water, gradient elution; the gradient elution is as follows: 0 min, methanol:water = 5:95; 10 min, methanol:water = 100:0; 15 min, methanol:water = 5:95; 17 min, methanol:water = 5:95; where the ratio of methanol to water is by volume. Detection wavelength: 260–300 nm.

2. The method according to claim 1, characterized in that, The C18 column is an Agilent ZORBAX Extend80Å C18, 4.6 x 250 mm, 5 µm HPLC column, or other equivalent C18 columns.

3. The method according to claim 1, characterized in that, in When performing high performance liquid chromatography (HPLC) detection, the flow rate is 0.4–2 mL / min, the injection volume is 5 μL–20 μL, and the column temperature is 25℃–60℃.

4. The method according to claim 1, characterized in that, The chromatographic conditions are as follows: Chromatographic column: C18 column; Mobile phase: 0 min, methanol:water = 5:95; 10 min, methanol:water = 100:0; 15 min, methanol:water = 5:95; 17 min, methanol:water = 5:95; where the ratio of methanol to water is by volume. Detection wavelength: 278nm; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Column temperature: 40℃.

5. The method according to claim 1, characterized in that, in When preparing the sample solution to be tested, weigh crude 5-hydroxymethylfurfural (HMF), add methanol as solvent, and after ultrasonic dissolution, dilute to a volumetric flask of 100 ml. After ultrasonic dissolution, cool to room temperature and store for later use. The ultrasonic dissolution time is 1 to 60 seconds, and the mass of crude HMF is 5 mg to 25 mg.

6. The method according to claim 1, characterized in that, The standard curve equations for 5-hydroxymethylfurfural and bis-(5-formylfurfural) ether were obtained as follows: 5-hydroxymethylfurfural standard and bis-(5-formylfurfural) ether standard were accurately weighed, and a series of HMF and OBMF standard solutions of different concentrations were prepared using methanol as solvent. Under the chromatographic conditions, high-performance liquid chromatography (HPLC) was performed, and the peak areas were recorded. A standard curve was plotted with the peak area as the ordinate and the standard solution concentration as the abscissa to obtain the regression equation.

7. The method according to claim 4, characterized in that, The retention time of the 5-hydroxymethylfurfural was 4.9 min; the retention time of the bis-(5-formylfurfural) ether was 7.76 min.

8. The method according to claim 4, characterized in that, The standard curve equation for 5-hydroxymethylfurfural is Y = 71507.8513X + 14.4614; the standard curve equation for bis-(5-formylfurfural) ether is Y = 67621.3542X - 10.3899.

Images