Preparation Method and Application of a Magnetic Self-Condensing Covalent Organic Framework

Abstract

The present invention relates to the technical fields of the preparation of magnetic covalent organic frameworks and food safety analysis, and discloses a preparation method and application of a magnetic self-condensing covalent organic framework containing sulfonic acid anion groups. The preparation method comprises the following steps: first, preparing amino-functionalized magnetic nanoparticles; then coating a covalent organic framework on the surface of the amino-functionalized magnetic nanoparticles through a self-condensing reaction to obtain a magnetic self-condensing covalent organic framework; and finally, modifying the magnetic self-condensing covalent organic framework through a thiol-alkyne click reaction to obtain a magnetic self-condensing covalent organic framework containing sulfonic acid anion groups. The preparation steps of the present invention are simple and the method is convenient. The magnetic self-condensing covalent organic framework containing sulfonic acid anion groups has good chemical stability, rich adsorption sites and excellent magnetic separation characteristics, can rapidly extract basic orange II from food samples, and in combination with high performance liquid chromatography, can be applied to detecting the content of basic orange II in complex food samples.

Description

Technical Field

[0001] The present invention relates to the technical fields of the preparation of magnetic covalent organic frameworks and food safety analysis, and particularly to a preparation method of a magnetic self-condensing covalent organic framework containing sulfonic acid anion groups and its application in detecting basic orange II in food samples. Background Technique

[0002] Basic orange II is an alkaline azo dye widely used in paper, leather and fabrics. It is toxic and difficult to degrade naturally. Due to its toxic, carcinogenic and mutagenic residues in the human body, it seriously endangers human health. Basic orange II has been listed as a prohibited food additive in many countries. However, because of its excellent dyeing ability for food, in order to obtain more profits, some unscrupulous merchants use basic orange II to dye yellow croaker, chili powder and soy products. Therefore, for food safety and consumer rights, it is very necessary to establish an effective analytical method to extract and detect basic orange II in food.

[0003] Currently, high performance liquid chromatography (HPLC) has become one of the most effective methods for detecting basic orange II in food. Due to the complex matrix in food, the sample must be extracted before HPLC analysis. Magnetic solid phase extraction is a new solid phase extraction technology based on magnetic adsorbents developed in recent years. These magnetic adsorbents have some unique properties, such as excellent superparamagnetism, large surface area, surface modifiability and good biocompatibility. Magnetic solid phase extraction can easily separate the magnetic adsorbent from the sample matrix with the help of an external magnetic field without additional filtration or centrifugation procedures, making sample collection and separation easier and faster. In addition, magnetic solid phase extraction is applicable to directly extract samples containing particles or microorganisms, which are widely present in environmental or biological matrices and may cause blockage of traditional solid phase extraction columns and lead to extraction failure. However, the specific active sites and specific surface area of most magnetic adsorbents are often relatively limited, resulting in low adsorption capacity or low selectivity. Therefore, there is an urgent need to develop magnetic solid phase extraction adsorbents with high adsorption capacity and high selectivity to extract basic orange II in food for these complex samples.

[0004] Covalent organic frameworks are a class of covalent porous crystalline polymers with unique properties such as excellent structural stability, high crystallinity, and high specific surface area. In order to obtain a magnetic solid-phase extraction adsorbent with high adsorption capacity and high selectivity, magnetic nanoparticles can be introduced into covalent organic frameworks to prepare magnetic covalent organic frameworks that can be used as magnetic solid-phase extraction adsorbents. Since there is currently no report on magnetic self-condensing covalent organic frameworks containing sulfonate anionic groups, in the present invention, we propose a method for introducing sulfonate anionic groups onto magnetic self-condensing covalent organic frameworks through thiol-alkyne click chemical reactions. The preparation steps are simple and the method is convenient, and it can be used as a magnetic solid-phase extraction adsorbent to extract basic orange II in food.

[0005] The information disclosed in this background art section is only intended to enhance the overall understanding of the present invention and should not be regarded as an admission or any form of suggestion that this information constitutes prior art already known to those of ordinary skill in the art. Summary of the Invention

[0006] Aiming at the defects or deficiencies existing in the above-mentioned prior art, the purpose of the present invention is to provide a preparation method and application of a magnetic self-condensing covalent organic framework containing sulfonate anionic groups.

[0007] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0008] A preparation method of a magnetic self-condensing covalent organic framework containing sulfonate anionic groups, characterized by comprising the following steps:

[0009] (1) Dissolve FeCl 3 ·6H 2 O in ethylene glycol, then add sodium acetate and 1,6-hexanediamine and stir until dissolved. Then transfer the mixture solution to a polytetrafluoroethylene-lined autoclave, react at 200 °C for 6 hours, and then cool to room temperature. Collect the product with a magnet, wash and dry to obtain amino-functionalized magnetic nanoparticles Fe 3 O 4 -NH 2 ;

[0010] (2) Mix the above Fe 3 O 4 -NH 2 , a self-condensable monomer, tetrahydrofuran, and acetic acid, and carry out a self-condensation reaction at 120 °C for 72 hours. Collect the product with a magnet, wash and dry to obtain a magnetic self-condensing covalent organic framework Fe 3 O 4 @COF;

[0011] (3) React the above magnetic self - condensing covalent organic framework Fe 3 O 4 @COF with sodium 3 - mercapto - 1 - propanesulfonate under the initiation of azobisisobutyronitrile to carry out a thiol - alkyne click reaction. Collect the product with a magnet, and after washing and drying, obtain a magnetic self - condensing covalent organic framework Fe 3 O 4 @COF - SO 3 Na.

[0012] Preferably, in the preparation method, in step (2), the self - condensable monomer is: 1,6 - bis(4 - formylphenyl) - 3,8 - bis((4 - aminophenyl)ethynyl)pyrene.

[0013] Preferably, in the preparation method, in step (3), the reaction conditions are: the reaction is carried out under the protection of an inert gas, the inert gas is argon or nitrogen, the reaction solvent is N,N - dimethylformamide, the reaction temperature is 80 °C, and the reaction time is 8 hours to 24 hours.

[0014] The present invention also discloses the application of a magnetic self - condensing covalent organic framework containing sulfonic acid anionic groups prepared by the preparation method of the present invention in the field of food safety analysis technology. It is characterized in that the food is one or more of yellow croaker, chili powder and dried tofu. The specific method is as follows: Pretreat 2 g of food samples to obtain 20 mL of basic orange II extraction solution, add 8 mg of the magnetic self - condensing covalent organic framework Fe 3 O 4 @COF - SO 3 Na as a magnetic solid - phase extraction adsorbent, vortex for 20 minutes to adsorb and remove the basic orange II extraction solution, separate the magnetic material by using an external magnetic field, add an eluent to the obtained material to recover the analyte basic orange II, and carry out high - performance liquid chromatography analysis.

[0015] Preferably, the pretreatment is: Mix 2 g of the sample and 20 mL of the extractant in a 50 mL centrifuge tube and vortex for 2 minutes, then centrifuge the mixed solution for 10 minutes. Then refrigerate the supernatant for 2 hours to precipitate fat, concentrate the supernatant to 1.0 mL, dilute it to 20 mL with ultrapure water, and adjust its pH to 5 and add NaCl at the same time.

[0016] Preferably, the eluent is a solution with a volume ratio of ammonia water to methanol of 5:95, and the mass concentration of the ammonia water is 25% - 28%.

[0017] Preferably, the extractant is a solution with a volume ratio of acetic acid to methanol of 1:99.

[0018] Compared with the prior art, the advantages of the present invention are as follows:

[0019] (1) In terms of material properties: In the present invention, a self - condensing covalent organic framework containing sulfonic acid anionic groups is coated on the surface of magnetic Fe 3 O 4 , so that the surface of the prepared magnetic solid - phase extraction adsorbent is negatively charged, while the target basic orange II is weakly basic in solution and has a positive charge on its surface. Therefore, the adsorption of the target is better. At the same time, the magnetic self - condensing covalent organic framework containing sulfonic acid anionic groups prepared in the present invention not only has excellent properties such as a large specific surface area and rich adsorption sites of covalent organic frameworks, but also inherits the excellent magnetic separation characteristics of magnetic materials, making this composite material have the characteristics of fast adsorption and separation speed and high adsorption capacity. In addition, the magnetic self - condensing covalent organic framework containing sulfonic acid anionic groups prepared in the present invention has good chemical stability and can be reused more than 6 times.

[0020] (2) In terms of the preparation method: When preparing the magnetic self - condensing covalent organic framework containing sulfonic acid anionic groups in the present invention, the preparation steps are simple and the method is convenient.

[0021] (3) In terms of application: When using the magnetic self - condensing covalent organic framework containing sulfonic acid anionic groups of the present invention to extract basic orange II in food samples, the magnetic solid - phase extraction method is adopted, which does not require the use of a large amount of organic solvents, and the operation is simple and convenient, solving the problems of using a large amount of organic solvents and time - consuming in traditional extraction and separation methods. Description of the Drawings

[0022] Figure 1 SEM images of Fe 3 O 4 -NH 2 and Fe 3 O 4 @COF - SO 3 Na obtained in Example 1; where (a) is Fe 3 O 4 -NH 2 , (b) is Fe 3 O 4 @COF - SO 3 Na;

[0023] Figure 2 X - ray powder diffraction patterns of Fe 3 O 4 -NH 2 , Fe 3 O 4 @COF and Fe 3 O 4 @COF - SO 3 Na obtained in Example 1;

[0024] Figure 3 The Fe obtained in Example 1 3 O 4 -NH 2 、Fe 3 O 4 @COF and Fe 3 O 4 @COF-SO 3 Na infrared spectrogram;

[0025] Figure 4 The Fe obtained in Example 1 3 O 4 -NH 2 、Fe 3 O 4 @COF and Fe 3 O 4 @COF-SO 3 Na hysteresis curve graph;

[0026] Figure 5 For Fe in Example 5 3 O 4 @COF-SO 3 Na reuse rate test graph;

[0027] Figure 6 Typical high performance liquid chromatography graphs obtained after magnetic solid phase extraction (MSPE) of spiked yellow croaker samples in Example 5; where (a) is spiked with 0 μg / L basic orange II, (b) is spiked with 5 μg / L basic orange II, (c) is spiked with 10 μg / L basic orange II, and (d) is spiked with 50 μg / L basic orange II;

[0028] Figure 7 Typical high performance liquid chromatography graphs obtained after magnetic solid phase extraction (MSPE) of spiked dried tofu samples in Example 5; where (a) is spiked with 0 μg / L basic orange II, (b) is spiked with 5 μg / L basic orange II, (c) is spiked with 10 μg / L basic orange II, and (d) is spiked with 50 μg / L basic orange II;

[0029] Figure 8 Typical high performance liquid chromatography graphs obtained after magnetic solid phase extraction (MSPE) of spiked chili powder samples in Example 5; where (a) is spiked with 0 μg / L basic orange II, (b) is spiked with 5 μg / L basic orange II, (c) is spiked with 10 μg / L basic orange II, and (d) is spiked with 50 μg / L basic orange II. Detailed implementation mode

[0030] The present invention will be further described in detail below with reference to the examples given by the inventors.

[0031] Preparation method and application of a magnetic self - condensing covalent organic framework containing sulfonic acid anion groups according to the present invention, the inventors give the following examples.

[0032] Example 1:

[0033] This example discloses the preparation of a magnetic self - condensing covalent organic framework containing sulfonic acid anion groups. The specific steps are as follows:

[0034] Step 1, preparation of amino - functionalized magnetic nanoparticles Fe 3 O 4 -NH 2 : Dissolve 1 g of FeCl 3 ·6H 2 O in 30 mL of ethylene glycol. Then, add 4 g of sodium acetate and 8 mL of 1,6 - hexanediamine and stir until dissolved. Next, transfer the mixture solution to a 100 - mL Teflon - lined autoclave and heat at 200 °C for 6 hours. After cooling to room temperature, collect the product with a magnet and wash it five times with ethanol and deionized water. Finally, dry the product under vacuum at 60 °C for 12 hours to obtain Fe 3 O 4 -NH 2 .

[0035] Step 2, preparation of magnetic self - condensing covalent organic framework Fe 3 O 4 @COF: Add 1,6 - bis(4 - formylphenyl) - 3,8 - bis((4 - aminophenyl)ethynyl)pyrene (64 mg), Fe 3 O 4 -NH 2 (49 mg), and tetrahydrofuran (2 mL) into a 10 - mL Schlenk tube and sonicate for 2 minutes. Then, add 200 μL of 6 mol / L acetic acid and sonicate the mixture for another 2 minutes. Finally, degas the mixture through three freeze - pump - thaw cycles, purge with argon, and heat to 120 °C for 72 hours. After cooling to room temperature, collect the resulting product with a magnet, wash it successively with N,N - dimethylacetamide, tetrahydrofuran, and methanol, and dry it under vacuum at 50 °C overnight to obtain Fe 3 O 4 @COF.

[0036] Step 3, preparation of magnetic self - condensing covalent organic framework Fe 3 O 4 @COF - SO 3 Na: Add anhydrous N,N - dimethylformamide (20 mL), Fe 3 O4 @COF (300 mg), sodium 3-mercapto-1-propanesulfonate (1780 mg), and azobisisobutyronitrile (80 mg) were added to a 50 mL Schlenk tube. After stirring the suspension under argon protection at 80 °C for 12 hours, the resulting black product was collected with a magnet. Finally, the product was washed successively with tetrahydrofuran, ethanol, and acetonitrile and dried in vacuo at 50 °C overnight to obtain Fe 3 O 4 @COF-SO 3 Na.

[0037] Analysis and characterization of Fe 3 O 4 -NH 2 、Fe 3 O 4 @COF, and Fe 3 O 4 @COF-SO 3 Na:

[0038] Figure 1 SEM images of Fe 3 O 4 -NH 2 and Fe 3 O 4 @COF-SO 3 Na obtained in Example 1; where (a) is Fe 3 O 4 -NH 2 , (b) is Fe 3 O 4 @COF-SO 3 Na. In the figure, Fe 3 O 4 -NH 2 exhibits a spherical structure with an average particle size of approximately 160 nm. Compared with Fe 3 O 4 -NH 2 , a film-like structure was observed on the surface of Fe 3 O 4 @COF-SO 3 Na, indicating that the self-condensed covalent organic framework shell was modified on the surface of Fe 3 O 4 -NH 2 and roughened its surface.

[0039] Figure 2 SEM images of Fe 3 O 4 -NH 2 、Fe 3 O 4@COF and Fe 3 O 4 @COF-SO 3 X-ray powder diffraction pattern of Na. Different from Fe 3 O 4 -NH 2 is that Fe 3 O 4 @COF and Fe 3 O 4 @COF-SO 3 Na has diffraction peaks at 2.54°, 4.55°, 5.30°, 6.97° and 9.29°, which respectively represent the diffraction of the self-condensed covalent organic framework shell and the self-condensed covalent organic framework shell containing sulfonic acid anion groups on the (100), (110), (200), (210) and (310) crystal planes.

[0040] Figure 3 Fe 3 O 4 -NH 2 , Fe 3 O 4 @COF and Fe 3 O 4 @COF-SO 3 Na. The infrared spectra of Fe 3 O 4 -NH 2 In the infrared spectrum, the peaks at wavenumbers of 580 cm -1 and 3420 cm -1 can be attributed to Fe-O-Fe and -OH bonds respectively. For Fe 3 O 4 @COF, the peak at 1621 cm -1 is attributed to the -C=N bond, and the peak at 2190 cm -1 is attributed to the -C≡C- bond, proving that Fe 3 O 4 @COF is successfully modified by the self-condensed covalent organic framework shell. Comparing the infrared spectra of Fe 3 O 4 @COF-SO 3 Na and Fe 3 O 4 @COF, it can be seen that the peak corresponding to the -C≡C- bond disappears in the infrared spectrum of Fe 3 O 4 @COF-SO 3 Na. In addition, characteristic peaks of the -S=O bond appear at 1060, 1187 and 1221 cm -1 , proving that Fe 3 O4 @COF-SO 3 Successful addition of sulfonic acid anion groups on Na.

[0041] Figure 4 Fe obtained in Example 1 3 O 4 -NH 2 、Fe 3 O 4 @COF and Fe 3 O 4 @COF-SO 3 Na hysteresis curves. The maximum saturation magnetization values of these three materials are 78.6, 25.8, and 25.0 emu / g in sequence. Although the maximum saturation magnetization value of Fe 3 O 4 @COF-SO 3 Na is lower than that of Fe 3 O 4 -NH 2 , it still has certain magnetism and can be rapidly separated by an external magnetic field within 30 seconds, and can be used for magnetic solid-phase extraction.

[0042] Example 2:

[0043] Other conditions are the same as in Example 1, except that: in step (3), the inert gas is nitrogen, and magnetic self-condensing covalent organic framework Fe containing sulfonic acid anion groups is obtained 3 O 4 @COF-SO 3 Na.

[0044] The SEM image, X-ray powder diffraction pattern, infrared spectrum, and hysteresis curve of Fe 3 O 4 @COF-SO 3 Na prepared in this example are similar to those in Example 1 and will not be given separately here.

[0045] Example 3:

[0046] Other conditions are the same as in Example 1, except that: in step (3), the reaction time is 24 hours, and magnetic self-condensing covalent organic framework Fe containing sulfonic acid anion groups is obtained 3 O 4 @COF-SO 3 Na.

[0047] The SEM image, X-ray powder diffraction pattern, infrared spectrum, and hysteresis curve of Fe 3 O 4 @COF-SO 3 Na prepared in this example are similar to those in Example 1.

[0048] Example 4:

[0049] Others are the same as Example 1, except that in step (3), the reaction time is 8 hours, and magnetic self - polycondensed covalent organic framework Fe 3 O 4 @COF - SO 3 Na is obtained.

[0050] The SEM image, X - ray powder diffraction pattern, infrared spectrum and hysteresis curve of Fe 3 O 4 @COF - SO 3 Na prepared in this example are similar to those in Example 1.

[0051] Example 5:

[0052] Application of magnetic self - polycondensed covalent organic framework containing sulfonic acid anion groups in the field of food safety analysis technology:

[0053] Yellow croaker, chili powder and dried tofu samples were purchased from the market. 2 g of the sample and 20 mL of 1% acetic acid - methanol solution were mixed in a 50 mL centrifuge tube and vortexed for 2 minutes. Then, the mixed solution was centrifuged at 8000 rpm for 10 minutes. Then the supernatant was refrigerated for 2 hours to precipitate fat, concentrated to 1.0 mL using a gentle nitrogen stream, diluted to 20 mL with ultrapure water, adjusted to pH 5, and inorganic salt NaCl was added to a concentration of 40 mmol / L.

[0054] 8 mg of Fe 3 O 4 @COF - SO 3 Na was added to 20 mL of the sample solution and vortexed for 20 minutes. Then, Fe 3 O 4 @COF - SO 3 Na was separated from the solution with a magnet and the supernatant was discarded. Then, 1 mL of 5% ammonia - methanol solution was added and vortexed for 6 minutes. Finally, the eluate was filtered through a 0.22 μm membrane filter. The reusability of Fe 3 O 4 @COF - SO 3 Na was studied. Before each use of Fe 3 O 4 @COF - SO 3 Na, it was washed three times with 5% ammonia - methanol solution and deionized water, and dried in a vacuum oven before the next magnetic solid - phase extraction. The results are as Figure 5 shown, Fe 3 O 4 @COF - SO 3The recovery rate of Na remained above 80% after 6 uses, indicating that Fe 3 O 4 @COF-SO 3 Na had good stability and could be reused at least 6 times.

[0055] High-performance liquid chromatography analysis was carried out on a Shimadzu LC-20AT high-performance liquid chromatograph coupled with an ultraviolet detector SPD-20A. The analytes were separated on an XDB-C18 reversed-phase column (250 mm × 4.6 mm, 5 μm, Agilent, USA) at 30 °C. The mobile phase was a solution of methanol and 0.02 mol / L ammonium acetate with a volume ratio of 35:65, and the flow rate was 1 mL / min. The detection wavelength of basic orange II was 449 nm, and the injection volume was 20 μL.

[0056] Three known concentrations of 5, 10, and 50 μg / L basic orange II mixtures were added to three food samples of yellow croaker, chili peppers, and dried tofu, respectively. After magnetic solid-phase extraction with Fe 3 O 4 @COF-SO 3 Na, high-performance liquid chromatography analysis was performed again. The results are shown in Table 1. Basic orange II was not detected in the unspiked samples of yellow croaker, chili peppers, and dried tofu. The recovery rates of basic orange II in the spiked samples (5, 10, and 50 μg / L) were 90.1 - 98.8%. The typical high-performance liquid chromatography diagrams obtained from the spiked yellow croaker, chili pepper powder, and dried tofu samples after magnetic solid-phase extraction are shown in Figure 6 、 7 and 8 respectively. The matrix interference in the real samples mainly concentrated before 3.5 minutes, and the retention time of basic orange II was 6.4 minutes. These results indicate that the method developed in this study can detect the content of trace basic orange II in complex food samples.

[0057] Table 1 Recovery of basic orange II by Fe 3 O 4 @COF-SO 3 Na in actual food samples

[0058]

[0059] The above-described embodiments are only the preferred embodiments of the present invention, and not an exhaustive list of all feasible embodiments of the present invention. For those skilled in the art, any obvious modifications made without departing from the principles and spirit of the present invention should be considered to be included within the protection scope of the claims of the present invention.

Claims

1. A preparation method of a magnetic self - condensing covalent organic framework containing sulfonic acid anion groups, characterized in that, it includes the following steps: (1) Dissolve FeCl 3 ·6H 2 O in ethylene glycol, then add sodium acetate and 1,6 - hexanediamine and stir until dissolved. Then transfer the mixture solution to a polytetrafluoroethylene - lined autoclave, react at 200 °C for 6 hours, and then cool to room temperature. Collect the product with a magnet, wash and dry to obtain amino - functionalized magnetic nanoparticles Fe 3 O 4 -NH 2 ; (2) Mix the above-mentioned Fe 3 O 4 -NH 2 , self-condensable monomer, tetrahydrofuran and acetic acid, and carry out self-condensation reaction at 120 °C for 72 hours. Collect the product with a magnet, wash and dry it to obtain magnetic self-condensable covalent organic framework Fe 3 O 4 @COF; (3) Carry out thiol-alkyne click reaction on the above-mentioned magnetic self-condensable covalent organic framework Fe 3 O 4 @COF and sodium 3-mercapto-1-propanesulfonate under the initiation of azobisisobutyronitrile. Collect the product with a magnet, wash and dry it to obtain magnetic self-condensable covalent organic framework Fe 3 O 4 @COF-SO 3 Na.

2. The preparation method of a magnetic self - condensing covalent organic framework containing sulfonic acid anion groups according to claim 1, characterized in that, in the step (2), the self - condensable monomer is: 1,6 - bis(4 - formylphenyl)-3,8 - bis((4 - aminophenyl)ethynyl)pyrene.

3. The preparation method of a magnetic self - condensing covalent organic framework containing sulfonic acid anion groups according to claim 1, characterized in that, in the step (3), the reaction conditions are: the reaction is carried out under the protection of an inert gas, the inert gas is argon or nitrogen, the reaction solvent is N,N - dimethylformamide, the reaction temperature is 80 °C, and the reaction time is 8 hours to 24 hours.

4. The application of the magnetic self - condensing covalent organic framework containing sulfonic acid anion groups prepared by the preparation method of a magnetic self - condensing covalent organic framework containing sulfonic acid anion groups according to any one of claims 1 - 3 in the field of food safety analysis technology, characterized in that, The food is one or more of yellow croaker, chili powder and dried tofu. The specific method is as follows: Pretreat 2 g of food sample to obtain 20 mL of basic orange II extraction solution, add 8 mg of the magnetic self-condensing covalent organic framework Fe 3 O 4 @COF-SO 3 Na as the magnetic solid phase extraction adsorbent, vortex for 20 minutes to adsorb and remove the basic orange II extraction solution, separate the magnetic material by using an external magnetic field, add an eluent to the obtained material to recover the analyte basic orange II, and perform high performance liquid chromatography analysis.

5. The application according to claim 4, characterized in that, the pretreatment is: mixing 2 g of the sample and 20 mL of the extractant in a 50 mL centrifuge tube and vortexing for 2 minutes, then centrifuging the mixed solution for 10 minutes, then refrigerating the supernatant for 2 hours to precipitate fat, concentrating the supernatant to 1.0 mL, diluting it to 20 mL with ultrapure water, adjusting its pH to 5 at the same time, and adding NaCl.

6. The application according to claim 4, characterized in that, the eluent is a solution with a volume ratio of ammonia water to methanol of 5∶95, and the mass concentration of the ammonia water is 25% - 28%.

7. The application according to claim 5, characterized in that, the extractant is a solution with a volume ratio of acetic acid to methanol of 1∶99.

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