A method for detecting phenol content in bio-based fermentation broth

Abstract

The present invention discloses a method for detecting phenol content in a bio-based fermentation broth. The method is a high-performance liquid chromatography method using an ion exclusion chromatography column and an aqueous sulfuric acid solution as the mobile phase. The method is rapid, accurate, efficient, reproducible, simple to operate, and has a low detection limit. Furthermore, the entire detection process uses minimal solvent, and no toxic liquids such as methanol or acetonitrile are involved. The wastewater is easily disposable, making it environmentally friendly.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and particularly relates to a method for detecting phenol content in a bio-based fermentation broth. Background Art

[0002] Phenol is flammable, highly toxic, and harmful to human health. It has a strong corrosive effect on the skin and mucous membranes, can cause burns to the human body, and can also inhibit the central nervous system or damage liver and kidney function. It is also seriously harmful to the environment and can cause pollution to water bodies and the atmosphere.

[0003] Currently, there are several methods for detecting phenol, including bromometry, spectrophotometry, and high-performance liquid chromatography. However, bromometry has large detection errors and insufficient sensitivity; spectrophotometry is only applicable to the determination of phenol content in water, with a single detection target, a complex and tedious testing process, and large errors in the results. High-performance liquid chromatography, such as CN108872440A, discloses a method for measuring phenol content in water. This method requires the use of a fluorescence detector and toxic reagents such as acetonitrile and methanol. The samples tested are also relatively simple and do not involve bio-based materials.

[0004] In view of the problems existing in the prior art, the present invention realizes the accurate detection of phenol content in bio-based fermentation broth by high performance liquid chromatography by selecting appropriate chromatographic columns and mobile phase solvents in order to detect bio-based materials. Summary of the Invention

[0005] The present invention aims to provide a method for detecting the phenol content in a bio-based fermentation broth. The detection method of the present invention has the advantages of good separation, simple operation, high accuracy, and good reproducibility.

[0006] A method for detecting phenol content in a bio-based fermentation broth is provided. The method is a high performance liquid chromatography method. The chromatographic conditions include: using an ion exclusion chromatography column and using a sulfuric acid aqueous solution as the mobile phase.

[0007] Preferably, the bio-based fermentation liquid is a biomass fermentation liquid such as crop straw, wheat bran, corn cob, sugarcane bagasse, lignocellulose processing liquid, sugar liquid, etc.

[0008] Preferably, the ion exclusion chromatography column is a cross-linked resin hydrogen ion chromatography column; more preferably a cross-linked sulfonated styrene-divinylbenzene hydrogen ion chromatography column; most preferably a Rezex ROA-Organic Acid H + Chromatographic column.

[0009] Preferably, the chromatographic conditions include a column temperature of 55 to 75°C, an automatic sampler temperature of 10°C, a flow rate of 0.3 to 0.6 mL / min, a run time of 20 to 40 min, an injection volume of 10 to 40 μL, a mobile phase of 2.5 mM sulfuric acid solution, and a cleaning solution of deionized water filtered through a 0.22 μm filter membrane.

[0010] Preferably, in the high performance liquid chromatography method, the phenol standard solution is diluted stepwise with a 2.5 mM sulfuric acid solution to form 5 to 6 standard solutions of different concentrations, which are then placed in a liquid chromatography automatic injection rack for injection and detection according to the chromatographic conditions of the method, and the peak area corresponding to each concentration is plotted to obtain a concentration-peak area standard curve of phenol.

[0011] Preferably, the high performance liquid chromatography sample detection step comprises:

[0012] (1) Sample processing: After the liquid sample is thoroughly mixed, place it in a centrifuge tube, dilute it with 2.5 mM sulfuric acid solution, mix it and react for 5-10 minutes, centrifuge it at high speed, and collect the supernatant to obtain the liquid sample to be tested;

[0013] (2) Sample detection: 1 mL of the liquid sample to be tested was drawn into a liquid sample bottle using a needle filter and detected using a high performance liquid chromatograph. The retention time was used for qualitative analysis, and the peak area was used to correspond to the standard curve to obtain the phenol concentration in the sample.

[0014] The beneficial effects of the present invention are:

[0015] (1) The method of the present invention adopts high performance liquid chromatography. By selecting a suitable chromatographic column, mobile phase and controlling suitable chromatographic conditions, the phenol content in the bio-based fermentation broth can be accurately detected. It has the advantages of good reproducibility, method linearity, and high spike recovery rate (90% to 106%).

[0016] (2) The present invention requires less sample, has a simple operation process, and can quickly achieve separation and detection.

[0017] (3) The present invention uses aqueous sulfuric acid solution as the mobile phase, and does not contain toxic liquids such as methanol and acetonitrile. The amount of solvent used in the entire detection process is small, which avoids the use of a large amount of chemical reagents, reduces environmental pollution, is green and environmentally friendly, and the experimental waste liquid is also easy to handle, making it suitable for popularization and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a phenol standard curve diagram of Example 1 of the present invention.

[0019] Figure 2 This is a liquid chromatogram of a phenol standard sample from Example 1 of the present invention.

[0020] Figure 3 This is the liquid chromatogram of the fermentation broth sample of Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0021] In order to make the technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0022] The equipment and standards used in the present invention are:

[0023] High performance liquid chromatograph: Shimadzu pump LC-20AD; automatic sampler: SIL-20AC; column box: CTO-20A; detector: RID-20A; controller: CBM-20A;

[0024] Centrifuge: Eppendorf Centrifuge 5424R;

[0025] Phenol solution standard substance: phenol solution in water standard substance purchased from Beijing North Weiye Metrology Technology Research Institute with a concentration of 1000 μg / mL.

[0026] Example 1

[0027] This embodiment provides a method for detecting phenol content in bagasse fermentation broth by high performance liquid chromatography, comprising:

[0028] Testing equipment and conditions:

[0029] Shimadzu high-performance liquid chromatograph; autosampler: SIL-20AC; column oven: CTO-20A; detector: RID-20A; column: Phenomenex Rezex ROA-Organic Acid H+ (8%) New Column 00F-0138-KO, 150 x 7.8 mm (max. pressure 67 bar); guard column: Phenomenex Carbo-H, 4 x 3.0 mm AJ0-4490 (VwK: 1112 / 852235) and SecurityGuard Guard Cartridge Kit KJ0-4282 (VwK: 1112 / 852235); column temperature: 65°C; autosampler temperature: 10°C; flow rate: 0.45 mL / min; injection volume: 40 μL; run time: 35 min; mobile phase: 2.5 mM sulfuric acid solution; cleaning solution: deionized water filtered through a 0.22 μm filter membrane.

[0030] Test steps for detection:

[0031] Prepare standard samples for the standard curve: dilute the purchased phenol solution in water (1000 μg / mL) with the mobile phase, using the mobile phase as the zero point to obtain six standard solutions of different concentrations (0.3 g / L, 0.15 g / L, 0.075 g / L, 0.0375 g / L, 0.01875 g / L, and 0 g / L).

[0032] Sample preparation: After thoroughly mixing the sugarcane bagasse fermentation broth, take 1 mL and place it in a 2 mL centrifuge tube. Dilute it with 1 mL of 2.5 mM sulfuric acid solution. After thoroughly mixing, centrifuge at 14,000 rpm for 10 min. Take the supernatant to obtain the sample to be tested. Repeat the same procedure to obtain a total of 6 parallel samples.

[0033] Standard sample addition: Take 1 mL of fermentation broth and add 1 mL of 0.06 g / L, 0.03 g / L, and 0.015 g / L phenol standard solutions in step (1), respectively. Mix thoroughly and centrifuge at 14,000 rpm for 10 min. Take the supernatant to obtain the liquid sample to be tested. Follow the same steps to obtain a total of 6 parallel standard samples.

[0034] Blank spiked sample: Take 1 mL of the above mobile phase and add 1 mL of the 0.04 g / L phenol standard solution in step (1) to obtain a blank spiked sample. Follow the same steps to obtain a total of 6 parallel blank spiked samples;

[0035] The above-mentioned standard samples, test samples, spiked samples, and blank spiked samples were all injected into liquid sample bottles using syringe filters, placed in the sample rack of the high performance liquid chromatograph, and the content was determined according to the above-mentioned chromatographic conditions. Figure 1 This is the phenol standard curve diagram of this embodiment, Figure 2 This is the liquid chromatogram of the phenol standard sample in this embodiment, Figure 3 1 is a liquid chromatogram of the fermentation broth sample of this embodiment.

[0036] The linear correlation coefficient of the phenol standard sample was analyzed to be r=0.9999, indicating good linearity. The phenol content test results are shown in Table 1:

[0037] Table 1 Analysis of phenol detection accuracy

[0038]

[0039]

[0040] Example 2

[0041] This embodiment provides a method for detecting phenol content in wheat straw fermentation broth by high performance liquid chromatography, comprising:

[0042] Testing equipment and conditions:

[0043] Chromatographic conditions of Shimadzu HPLC: chromatographic column: Phenomenex Rezex ROA-Organic Acid H+ (8%) New Column 00F-0138-KO.150 x 7.8 mm (max.pressure 67 bar); guard column: Phenomenex Carbo-H 4 x 3.0 mm AJ0-4490 (VwK: 1112 / 852235) and SecurityGuard GuardCartridge Kit KJ0-4282 (VwK: 1112 / 852235); column temperature: 70°C; autosampler temperature: 10°C; flow rate: 0.5 mL / min; injection volume: 20 μL; run time: 30 min; mobile phase: 2.5 mM sulfuric acid solution; cleaning solution: deionized water filtered through a 0.22 μm filter membrane.

[0044] Test steps for detection:

[0045] (1) Preparation of standard samples for the standard curve: Take the purchased phenol solution standard substance in water (1000 μg / mL) and dilute it stepwise with the mobile phase by 2 times, 4 times, 8 times, and 16 times. Use the mobile phase as the zero point to obtain 5 standard solutions with different concentrations (0.5 g / L, 0.25 g / L, 0.125 g / L, 0.0625 g / L, and 0 g / L);

[0046] (2) Sample preparation: After thoroughly mixing the wheat straw fermentation broth, 1 mL was taken and placed in a 2 mL centrifuge tube. 1 mL of 2.5 mM sulfuric acid solution was added for dilution. After thorough mixing, the broth was centrifuged at 14,000 rpm for 10 min. The supernatant was collected to obtain the sample to be tested. Six parallel samples were obtained by the same procedure.

[0047] (3) Standard sample addition: Take 1 mL of fermentation broth and add 1 mL of 0.5 g / L, 0.25 g / L, and 0.125 g / L phenol standard solutions in step (1), respectively. Mix thoroughly and centrifuge at 14,000 rpm for 10 min. Take the supernatant to obtain the liquid sample to be tested. Follow the same steps to obtain a total of 6 parallel standard samples.

[0048] (4) Blank spiked sample: Take 1 mL of the above mobile phase and add 1 mL of the 0.125 g / L phenol standard solution in step (1) to obtain a blank spiked sample. Follow the same steps to obtain a total of 6 parallel blank spiked samples.

[0049] The above-mentioned standard samples, test samples, spiked samples, and blank spiked samples were all injected into liquid sample bottles using syringe filters, placed in the sample rack of the high performance liquid chromatograph, and the content was determined according to the above-mentioned chromatographic conditions. Figure 1The linear correlation coefficient of the phenol standard sample was r = 0.9999, which showed good linearity. The phenol content test results are shown in Table 2:

[0050] Table 2 Analysis of phenol detection accuracy

[0051]

[0052] Example 3

[0053] This embodiment provides a method for detecting phenol content in corncob fermentation broth by high performance liquid chromatography, comprising:

[0054] Testing equipment and conditions:

[0055] Shimadzu high-performance liquid chromatograph; autosampler: SIL-20AC; column oven: CTO-20A; detector: RID-20A; column: Phenomenex Rezex ROA-Organic Acid H+ (8%) New Column 00F-0138-KO, 150 x 7.8 mm (max. pressure 67 bar); guard column: Phenomenex Carbo-H, 4 x 3.0 mm AJ0-4490 (VwK: 1112 / 852235) and SecurityGuard Guard Cartridge Kit KJ0-4282 (VwK: 1112 / 852235); column temperature: 55°C; autosampler temperature: 10°C; flow rate: 0.6 mL / min; injection volume: 40 μL; run time: 40 min; mobile phase: 2.5 mM sulfuric acid solution; cleaning solution: deionized water filtered through a 0.22 μm filter membrane.

[0056] Test steps for detection:

[0057] Prepare standard samples for the standard curve: dilute the purchased phenol solution in water (1000 μg / mL) with the mobile phase, using the mobile phase as the zero point to obtain five standard solutions of different concentrations (0.14 g / L, 0.07 g / L, 0.035 g / L, 0.0175 g / L, and 0 g / L).

[0058] Sample preparation: After thoroughly mixing the corncob fermentation broth, take 1 mL and place it in a 2 mL centrifuge tube. Dilute with 1 mL of 2.5 mM sulfuric acid solution. After thorough mixing, centrifuge at 14,000 rpm for 10 min. Collect the supernatant to obtain the sample to be tested. Repeat the same procedure to obtain a total of 6 parallel samples.

[0059] Standard sample addition: Take 1 mL of fermentation broth and add 1 mL of 0.14 g / L, 0.07 g / L, and 0.035 g / L phenol standard solutions in step (1), respectively. Mix thoroughly and centrifuge at 14,000 rpm for 10 min. Take the supernatant to obtain the liquid sample to be tested. Follow the same steps to obtain a total of 6 parallel standard samples.

[0060] Blank spiked sample: Take 1 mL of the above mobile phase and add 1 mL of the 0.07 g / L phenol standard solution in step (1) to obtain a blank spiked sample. Follow the same steps to obtain a total of 6 parallel blank spiked samples;

[0061] The above standard samples, test samples, spiked samples, and blank spiked samples were all injected into liquid sample bottles using syringe filters and placed in the sample rack of a high performance liquid chromatograph. The content was determined according to the above chromatographic conditions. The linear correlation coefficient of the phenol standard sample was r = 0.9999, indicating good linearity. The phenol content test results are shown in Table 3:

[0062] Table 3 Analysis of phenol detection accuracy

[0063]

[0064] The embodiment described above is only a preferred solution of the present invention and does not limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solution described in the claims.

Claims

1. A method for detecting phenol content in a bio-based fermentation broth, characterized in that: The method is a high performance liquid chromatography method, and the chromatographic conditions include: using an ion exclusion chromatography column, using a sulfuric acid aqueous solution as the mobile phase, a detector RID-20A, a column temperature of 55 to 75° C., and a flow rate of 0.3 to 0.6 mL / min; The ion exclusion chromatography column is a cross-linked sulfonated styrene-divinylbenzene hydrogen ion chromatography column; the concentration of the sulfuric acid aqueous solution is 2.5mM; The bio-based fermentation liquid is one or more of the fermentation liquids of crop straw, wheat bran, corn cobs, sugarcane bagasse, and wood fiber processing liquid.

2. The method for detecting phenol content in a bio-based fermentation broth according to claim 1, wherein: The ion exclusion chromatography column is Rezex ROA-Organic Acid H + Chromatographic column.

3. The method for detecting phenol content in a bio-based fermentation broth according to claim 2, wherein: The chromatographic conditions are as follows: the autosampler temperature is 10° C., the running time is 20 to 40 min, the injection volume is 10 to 40 μL, the mobile phase is 2.5 mM sulfuric acid solution, and the cleaning solution is deionized water filtered through a 0.22 μm filter membrane.

4. The method for detecting phenol content in a bio-based fermentation broth according to claim 3, wherein: In the high performance liquid chromatography method, the phenol standard solution is diluted stepwise with a 2.5 mM sulfuric acid solution to form 5 to 6 standard solutions of different concentrations, which are then placed in a liquid chromatography automatic injection rack for injection and detection according to the chromatographic conditions of the method, and the peak area corresponding to each concentration is plotted to obtain a phenol concentration-peak area standard curve.

5. The method for detecting phenol content in a bio-based fermentation broth according to claim 4, wherein: The high performance liquid chromatography sample detection step comprises: (1) Sample processing: After the liquid sample is thoroughly mixed, place it in a centrifuge tube, dilute it with 2.5 mM sulfuric acid solution, mix it and react for 5-10 minutes, centrifuge it at high speed, and collect the supernatant to obtain the liquid sample to be tested; (2) Sample detection: 1 mL of the liquid sample to be tested was drawn into a liquid sample bottle using a needle filter and detected using a high performance liquid chromatograph. The retention time was used for qualitative analysis, and the peak area was used to correspond to the standard curve to obtain the phenol concentration in the sample.

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