CN105572209B - The method that graphene modified determination of electrode Resveratrol content is peeled off using liquid phase - Google Patents

Abstract

The invention discloses a method for measuring the content of resveratrol using a liquid-phase exfoliated graphene modified electrode. In the method, the graphite powder is liquid-phase exfoliated in a liquid medium (i.e., an exfoliation solvent) to obtain a graphene dispersion, and the graphite powder is used to obtain a graphene dispersion. Graphene-modified electrodes were prepared from the ene dispersion, and then the content of resveratrol was determined electrochemically using the graphene-modified electrodes. When the method of the invention is used, under optimal conditions, the resveratrol has good linearity in the range of 0.03-5 μmol/L, and the detection limit is as low as 0.01 μmol/L. The method of the invention is easy to operate, saves time, low cost, and high detection sensitivity, solves the problems of long ultrasonic time and low stripping efficiency of the existing liquid phase stripping method, and is especially suitable for measuring resveratrol in grapes, red wine, knotweed, etc. content and has broad application prospects.

Description

Method for Determination of Resveratrol Content Using Liquid Phase Exfoliated Graphene Modified Electrode

technical field

The invention relates to the technical field of electrochemical analysis of resveratrol, in particular to a method for measuring the content of resveratrol using a liquid-phase exfoliated graphene-modified electrode.

Background technique

Graphene is a carbon nanomaterial with a two-dimensional honeycomb lattice structure densely packed by a single layer of carbon atoms. Since its discovery in 2004, it has set off a wave of research all over the world. Graphene has many peculiar characteristics, has excellent electrical, optical, thermal and mechanical properties, and has caused revolutionary changes in many fields. Compared with fullerenes and carbon nanotubes, graphene has better properties, such as larger specific surface area, higher conductivity, and faster electron transfer rate.

In 2004, Professor Andre K Geim (Andre Geim) and researcher Kostya Novoselov (Kostya Novoselov) from the University of Manchester in the UK used a very simple method - micro-mechanical exfoliation method, in the highly oriented The pyrolytic graphite is repeatedly peeled off with scotch tape to obtain single-layer graphene. In recent years, people have made positive progress in the preparation of graphene. In addition to the micromechanical exfoliation method, various preparation methods such as oxidation-reduction method, crystal epitaxial growth method, chemical vapor deposition method and organic synthesis method have been developed. . With the rapid development of graphene preparation research, the simple liquid-phase exfoliation method is favored by more and more researchers, that is, using graphite intercalation in a specific medium to separate single-layer or multi-layer graphene from graphite by ultrasonic means. This method does not use large-scale precision equipment, only needs ultrasonic equipment, the preparation process is simple and low energy consumption, and has obvious cost advantages; it does not undergo chemical oxidation and reduction processes, does not undergo high-temperature expansion processes, and has few graphene defects. Good quality; but also a green method. However, the usual liquid-phase exfoliation method still has many shortcomings: the concentration of the obtained graphene dispersion is still low, the exfoliation solvent is expensive or has a high boiling point or even high toxicity, and the ultrasonic time is long.

Resveratrol (Resveratrol), also known as stilbene triphenol, chemical name is (E)-5-[2-(4-hydroxyphenyl)-vinyl]-1,3-benzenediol, is a Natural active substances. Studies have found that resveratrol has various pharmacological activities and health functions such as anti-cancer, anti-oxidation, anti-bacterial, anti-inflammatory, anti-hyperlipidemia, anti-atherosclerosis, cardiovascular protection, immune regulation and prevention of osteoporosis. It is more and more widely used in health food. Resveratrol exists in natural plants such as Polygonum cuspidatum, grapes, and mulberry, and the extraction of resveratrol in my country is mainly based on traditional Chinese medicine Polygonum cuspidatum as raw material, and the content of resveratrol is an important indicator of the quality of its raw materials . Therefore, it is of great significance to determine the content of resveratrol in red wine, grapes and other foods, and Chinese medicinal materials such as knotweed.

Currently, methods for the determination of resveratrol mainly include high performance liquid chromatography, gas chromatography/liquid chromatography, and fluorescence spectroscopy. These methods are expensive and require specialized technical training, so it is particularly important to choose a simple, fast, and sensitive assay.

Contents of the invention

The purpose of the present invention is to overcome the defects that the existing resveratrol determination technology is expensive and requires more training time, and provides a method for determining the content of resveratrol using a liquid-phase exfoliated graphene modified electrode. The graphene-modified electrode has the characteristics of high sensitivity and low detection limit for the detection of resveratrol, and the whole method is simple and easy, which can fully meet the content determination of resveratrol.

In order to achieve the above object, the method for measuring the content of resveratrol using a liquid-phase exfoliated graphene modified electrode provided by the present invention has the following steps:

1) Prepare a sample containing resveratrol and dissolve it in an acidic or neutral buffer to obtain a sample solution;

2) using square wave voltammetry, using the graphene-modified electrode as the working electrode, to measure the content of resveratrol in the sample solution;

Wherein, the preparation method of the graphene modified electrode comprises the steps of:

(1) Weighing graphite powder and stripping aid are mixed according to the ratio of 2 to 5:1 by weight, and adding a stripping solvent, and then ultrasonicating for 2 to 3 hours to obtain a graphene dispersion;

The amount of the stripping solvent is mixed with 0.5-4 mg of graphite powder per milliliter of stripping solvent;

Described stripping aid is the mixture that the weight ratio of gallic acid and sodium hydroxide is 5:1;

(2) Polish the glassy carbon electrode to the mirror surface;

(3) Ultrasonic cleaning with nitric acid, ethanol, and double-distilled water for 2 minutes, 2 minutes, and 1 minute respectively, and washing with distilled water before each replacement of the lotion, and drying under an infrared lamp after washing with double-distilled water;

(4) Get the graphene dispersion evenly on the glassy carbon electrode surface of drying, then vacuum dry;

(5) The glassy carbon electrode obtained in step (4) is placed in a buffer solution, and subjected to cyclic voltammetry scanning pretreatment to obtain a graphene-modified electrode for use.

Preferably, the acidic or neutral buffer in step 1) is a phosphate buffer solution with a pH of 5.91.

Preferably, the voltage of the graphene-modified electrode in the step 2) is 0.2-0.8V.

Preferably, the enrichment time is 120s and the enrichment potential is -0.2V when square wave voltammetry is used in the step 2).

Preferably, the amount of stripping solvent used in step (1) of the preparation method of the graphene-modified electrode is 2-3 mg of graphite powder per milliliter of stripping solvent.

Preferably, the stripping solvent in the step (1) of the preparation method of the graphene-modified electrode is N,N-dimethylformamide.

Preferably, the ultrasonic conditions in the step (1) of the preparation method of the graphene-modified electrode are frequency 50kHz and power 220w.

Preferably, neutral alumina powder with a particle size of 0.05 μm is used for polishing the glassy carbon electrode in step (2) of the preparation method of the graphene-modified electrode.

Preferably, in the step (3) of the preparation method of the graphene-modified electrode, the nitric acid is an aqueous nitric acid solution with a volume ratio of 1:1, and the ethanol is absolute ethanol.

Preferably, the step (5) of the preparation method of the graphene-modified electrode is to place the glassy carbon electrode obtained in the step (4) in a Na ₂ HPO ₄ and KH ₂ PO ₄ buffer solution with a pH of 4.91 at a temperature of 0 to 1.0 In the V potential range, cyclic voltammetry scans 60 cycles until the baseline is stable, and then washes to obtain a graphene-modified electrode for use.

In the present invention, the graphite powder is exfoliated in a liquid medium (i.e., a stripping solvent) using a stripping aid to obtain a graphene dispersion, and the graphene dispersion is used to prepare a graphene-modified electrode, and then the graphene-modified electrode is used to electrochemically Determination of resveratrol content, especially in the process of preparing graphene dispersion, using gallic acid as a peeling aid, because it contains benzene rings and can produce π-π interactions with graphene sheets, which is more beneficial Cooperate with solvent molecules for graphite intercalation, so as to improve the efficiency of liquid phase exfoliation. On the other hand, in the process of electrochemical measurement, in addition to the high specific surface of graphene, the gallic acid contained on the surface of graphene electrode is rich in carboxyl and hydroxyl groups, which can The formation of hydrogen bonds with the hydroxyl groups of resveratrol molecules can improve the enrichment efficiency of resveratrol on the surface of graphene electrodes, thereby enhancing the electrochemical response signal of resveratrol.

Beneficial effects of the present invention: the graphene dispersion liquid is prepared in one step by a simple and efficient liquid phase stripping method, the glassy carbon electrode is modified by the drop coating method, and the graphene modified electrode is prepared, and then the graphene modified electrode is used for electrochemical analysis Methods Determination of resveratrol content. When used, under optimal conditions, resveratrol has good linearity in the range of 0.03-5 μmol/L, and the detection limit is as low as 0.01 μmol/L. The method of the invention is easy to operate, saves time, low cost, and high detection sensitivity, solves the problems of long ultrasonic time and low stripping efficiency of the existing liquid phase stripping method, and is especially suitable for measuring resveratrol in grapes, red wine, knotweed, etc. content and has broad application prospects.

Description of drawings

Figure 1 is a transmission electron microscope (TEM) image of a graphene dispersion.

Figure 2 is a scanning electron microscope (SEM) image of a graphene-modified electrode.

Fig. 3 is a graph showing the influence of pH value on the peak current of resveratrol oxidation.

Fig. 4 is a graph showing the influence of enrichment time on the oxidation peak current of resveratrol.

Fig. 5 is a graph showing the influence of enrichment potential on the oxidation peak current of resveratrol.

Figure 6 is a graph of square wave voltammetry curves of different concentrations of resveratrol on graphene electrodes.

Figure 7 is a graph showing the relationship between oxidation peak current and resveratrol concentration.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Example 1

1, the preparation method of graphene modified electrode, comprises the steps:

(1) Weigh 30 mg, 10 mg and 2 mg of graphite powder, gallic acid and sodium hydroxide respectively with an electronic balance. Put the three substances into a 25mL beaker, then take 10mL of DMF (N,N-dimethylformamide) into the beaker with a pipette, put it into an ultrasonic instrument and sonicate for 2-3 hours to obtain a graphene dispersion (see figure 1).

(2) Take a small amount of neutral alumina (0.05 μm) and wet it with water on the polishing cloth, and polish the glassy carbon electrode (Φ=3mm) to a mirror surface on the polishing cloth;

(3) Ultrasonic cleaning with nitric acid, ethanol, and double-distilled water for 2 minutes, 2 minutes, and 1 minute respectively, and washing with distilled water before each replacement of the lotion, and drying under an infrared lamp after washing with double-distilled water;

(4) Take 5 μL of graphene dispersion liquid and drop-coat it on the surface of the dried glassy carbon electrode, and dry it in a vacuum oven at 45°C.

(5) Put the glassy carbon electrode obtained in step (4) in the buffer solution, in the buffer solution (Na ₂ HPO ₄ and KH ₂ PO ₄ ) with pH = 4.91, within the potential range of 0-1.0V, and scan the cyclic voltammetry After 60 cycles until the baseline is stable, wash and obtain a graphene-modified electrode for use. (See Figure 2).

2. Preparation of resveratrol solution:

Weigh 4.6 mg of resveratrol with an electronic balance, dissolve it in 2 mL of water, prepare a 0.01 mol/L resveratrol solution, and dilute it step by step to a concentration of 1.0×10 ^-3 mol/L, 1.0×10 ^-4 mol /L, 1.0× ^{10 -5} mol/L.

3. Determination of resveratrol:

Prepare 0.1mol/L phosphate buffer solutions with pH values of 4.91, 5.39, 5.91, 6.47, 6.98, 7.39, pipette 10mL into a beaker, add the pre-prepared resveratrol solution to make the resveratrol concentration 2.5μmol/L L, using square wave voltammetry, using graphene-modified electrode as the working electrode, investigated the effect of pH value on the current response of resveratrol in the range of 0.2-0.8V, and found that the optimal pH value was 5.91 (see Figure 3).

When the enrichment time is 10s, 40s, 80s, 120s, 160s, and 200s, the current response of 2.5 μmol/L resveratrol is shown in Figure 4, and the optimal enrichment time is 120s.

When the enrichment potential is -0.8V, -0.6V, -0.4V, -0.2V, 0V, 0.2V, the current response of 2.5μmol/L resveratrol is shown in Figure 5, and the best enrichment potential is - 0.2V.

Using graphene-modified electrodes to measure the content of the above-mentioned resveratrol solutions with different concentrations by electrochemical methods, and investigating the relationship between the current response (I) and the concentration of resveratrol (c), it can be seen that the concentration of resveratrol in the range of 0.03 to 5 μmol/L The two have a good linear relationship within the range, and the linear equation is:

I=0.00158+1.04916c,

The correlation coefficient is 0.999 (see Figures 6 and 7), indicating good linearity, and the detection limit is 0.01 μmol/L (S/N=3).

Example 2

This example is basically the same as Example 1, except that the amounts of graphite powder, gallic acid and sodium hydroxide in the preparation method of the graphene-modified electrode are 24 mg, 10 mg and 2 mg, respectively.

The follow-up is still the same as in Example 1. The content of the resveratrol solutions with different concentrations prepared above is determined using a graphene-modified electrode. The results show that it has good linearity and the detection limit is 0.01 μmol/L (S/N=3) .

Example 3

This example is basically the same as Example 1, except that the amounts of graphite powder, gallic acid, and sodium hydroxide in the preparation method of the graphene-modified electrode are 60 mg, 10 mg, and 2 mg, respectively, and the amount of DMF is 15 mL.

The follow-up is still the same as in Example 1. The content of the resveratrol solutions with different concentrations prepared above is determined using a graphene-modified electrode. The results show that it has good linearity and the detection limit is 0.01 μmol/L (S/N=3) .

Test case

According to the method for example 1, the graphene modified electrode prepared in the method of the present invention, N-methylpyrrolidone (NMP) method (stripping condition: stripping solvent is NMP, ultrasonic time: 24h, does not add stripping aid, other conditions and The graphene modified electrode prepared by the same in the present invention and the graphene electrode prepared by Hummers oxidation-reduction method analyze the simulated sample of resveratrol under the same conditions respectively, add different concentrations of resveratrol in the water sample, Measure three times and take the average value.

The measurement results using the graphene-modified electrode of the present invention are shown in the table below, and the results show that the recovery rate is 97.0-102.0%, and the relative deviation range is 2.0-3.0%, indicating that the method for measuring resveratrol using the graphene-modified electrode of the present invention is reproduced Good performance and high sensitivity. However, the other two electrodes did not obtain satisfactory results. Among them, under the condition of 1.5 μmol/L, the current response signals of resveratrol measured by using the graphene modified electrode of the present invention and the graphene electrode prepared by NMP method are 1.5 μA and 0.9 μA respectively, and the Hummers oxidation-reduction The graphene electrode prepared by this method has no obvious response signal to resveratrol.

On this basis, the actual sample is measured, and the measurement object is a commercially available red wine of a certain brand. The wine is previously determined to have a resveratrol content of 3.59 μmol/L by high-performance liquid chromatography, and the graphene-modified electrode of the present invention The average value of the three determination results is 3.68μmol/L, and the relative error is 2.5%, which shows that the method is very reliable and can be applied to the determination of actual samples.

Claims (10)

1. a method utilizing liquid phase exfoliation graphene modified electrode to measure resveratrol content, is characterized in that, the method steps are as follows: 1) Prepare a sample containing resveratrol and dissolve it in an acidic or neutral buffer to obtain a sample solution; 2) using square wave voltammetry, using the graphene-modified electrode as the working electrode, to measure the content of resveratrol in the sample solution; Wherein, the preparation method of the graphene modified electrode comprises the steps of: (1) Weighing graphite powder and stripping aid are mixed according to the ratio of 2 to 5:1 by weight, and adding a stripping solvent, and then ultrasonicating for 2 to 3 hours to obtain a graphene dispersion; The amount of the stripping solvent is mixed with 0.5-4 mg of graphite powder per milliliter of stripping solvent; Described stripping aid is the mixture that the weight ratio of gallic acid and sodium hydroxide is 5:1; (2) Polish the glassy carbon electrode to the mirror surface; (3) Ultrasonic cleaning with nitric acid, ethanol, and double-distilled water for 2 minutes, 2 minutes, and 1 minute respectively, and washing with distilled water before each replacement of the lotion, and drying under an infrared lamp after washing with double-distilled water; (4) Get the graphene dispersion evenly on the glassy carbon electrode surface of drying, then vacuum dry; (5) The glassy carbon electrode obtained in step (4) is placed in a buffer solution, and subjected to cyclic voltammetry scanning pretreatment to obtain a graphene-modified electrode for use. 2. The method for measuring resveratrol content using a liquid-phase exfoliated graphene modified electrode according to claim 1, characterized in that: in the step 1), the acidic or neutral buffer is a phosphate buffered solution of pH5.91. 3. according to claim 1, utilize liquid-phase exfoliated graphene modified electrode to measure the method for resveratrol content, it is characterized in that: in described step 2) when adopting square wave voltammetry to measure, be applied on graphene modified electrode The voltage is 0.2 ~ 0.8V. 4. according to claim 1, utilize liquid-phase exfoliated graphene modified electrode to measure the method for resveratrol content, it is characterized in that: the enrichment time when adopting square wave voltammetry to measure in described step 2) is 120s, The enrichment potential is -0.2V. 5. according to claim 1, utilize liquid phase exfoliation graphene modified electrode to measure the method for resveratrol content, it is characterized in that: in the step (1) of the preparation method of described graphene modified electrode, the consumption of stripping solvent is every Mix 2-3 mg of graphite powder with 1 ml of stripping solvent. 6. according to claim 1, utilize liquid phase exfoliation graphene modified electrode to measure the method for resveratrol content, it is characterized in that: in the step (1) of the preparation method of described graphene modified electrode, stripping solvent is N,N -dimethylformamide. 7. according to claim 1, utilize liquid phase exfoliation graphene modified electrode to measure the method for resveratrol content, it is characterized in that: in the step (1) of the preparation method of described graphene modified electrode, ultrasonic condition is frequency 50kHz, Power 220w. 8. according to claim 1, utilize liquid phase exfoliation graphene modified electrode to measure the method for resveratrol content, it is characterized in that: in the step (2) of the preparation method of described graphene modified electrode, polishing glassy carbon electrode adopts particle size 0.05μm neutral alumina powder. 9. according to claim 1, utilize liquid phase exfoliation graphene modified electrode to measure the method for resveratrol content, it is characterized in that: in the step (3) of the preparation method of described graphene modified electrode, nitric acid is volume ratio 1: 1 nitric acid aqueous solution, ethanol is absolute ethanol. 10. according to claim 1, utilize liquid phase exfoliation graphene modified electrode to measure the method for resveratrol content, it is characterized in that: the step (5) of the preparation method of described graphene modified electrode is to obtain step (4) In Na ₂ HPO ₄ and KH ₂ PO ₄ buffer solutions with a pH of 4.91, in the potential range of 0-1.0V, the glassy carbon electrode was scanned by cyclic voltammetry for 60 cycles until the baseline was stable, and then washed to obtain a graphene-modified electrode spare.