Assessment of rare earth elements variations in five water systems in Beijing: Distribution, geochemical features, and fractionation patterns.

This study investigates the distribution of rare earth elements (REEs) within the Beijing water system, specifically examining the Yongding, Chaobai, Beiyun, Jiyun, and Daqing rivers. Results indicate that the Beiyun River exhibits the highest REE concentrations, ranging from 35.95 to 59.78 µg/mL, while the Daqing River shows the lowest concentrations, ranging from 15.79 to 17.48 µg/mL. LREEs (La to Nd) predominate with a total concentration of 23.501 µg/mL, leading to a notable LREE/HREE ratio of 7.901. Positive Ce anomalies (0.70-1.11) and strong positive Eu anomalies (1.38-2.49) were observed. The study suggests that the Beijing water system's REEs may originate from geological and anthropogenic sources, such as mining and industrial activities in neighboring regions, including Inner Mongolia. These findings underscore the importance of ongoing monitoring and effective water management strategies to address REE-related environmental concerns.

Research progress on the content and distribution of rare earth elements in rivers and lakes in China.

This study reviewed the content and distribution of rare earth elements (REE) in rivers and lakes in China based on the online literature. The sequence distribution of REE presented the decreasing trends in the order: of Ce > La > Nd > Pr > Sm > Gb > Dy>Er > Yb > Eu > Lu > Ho > Tb > Tm in rivers water. Pearl River and the Jiulong River constitute a significant sediments REE reservoir with an average value mean of 229.6 mg/kg and 266.86 mg/kg, respectively; both have higher concentrations than the global river average (174.8 mg/kg) and higher than the local soil background (Chinese soil background). The Liaohe River is one of China's most polluted rivers, with REE distribution ranging from 106.61 to 174.71 g/L (average 144.59 g/L in water). The total concentrations of dissolved REE in rivers near REE mining areas in China are higher than in other rivers. Increasing anthropogenic inputs to natural systems may permanently alter the natural signatures of REE. The distribution characteristics of REE in Chinese lakes (sediments) varied greatly, and the mean enrichment factor (EF) was sorted as follows: Ce > La > Nd > Pr > Sm > Gd > Dy>Er > Yb > Eu > Ho > Tb > Tm > Lu, where Ce was the most abundant followed by La, Nd, and Pr, and these four elements account for 85.39 % of the total concentration of REE. The REE in the sediments obtained from Poyang Lake and Dongting Lake had an average concentration respectively of 254.0 µg/g and 197.95 µg/g; both are considerably higher than the average upper continental crust (146.4 µg/g) and higher than in other lakes in China and around the world. The distribution and accumulation of LREE in most lake sediments result from the joint action of human activities and natural processes. It concluded that mining tailings were the primary cause of REE pollution in sediments, and industrial and agricultural activities are mainly responsible for water contamination.

Inhibition Effect of Pseudomonas stutzeri on the Corrosion of X70 Pipeline Steel Caused by Sulfate-Reducing Bacteria.

Microbiologically influenced corrosion (MIC) is a common phenomenon in water treatment, shipping, construction, marine and other industries. Sulfate-reducing bacteria (SRB) often lead to MIC. In this paper, a strain of Pseudomonas stutzeri (P. stutzeri) with the ability to inhibit SRB corrosion is isolated from the soil through enrichment culture. P. stutzeri is a short, rod-shaped, white and transparent colony with denitrification ability. Our 16SrDNA sequencing results verify the properties of P. stutzeri strains. The growth conditions of P. stutzeri bacteria and SRB are similar, and the optimal culture conditions are about 30 °C, pH 7, and the stable stage is reached in about seven days. The bacteria can coexist in the same growth environment. Using the weight loss method, electrochemical experiments and composition analysis techniques we found that P. stutzeri can inhibit the corrosion of X70 steel by SRB at 20~40 °C, pH 6~8. Furthermore, long-term tests at 3, 6 and 9 months reveal that P. stutzeri can effectively inhibit the corrosion of X70 steel caused by SRB.

Preparation of pyrrolidinyl diglycolamide bonded silica particles and its rare earth separation properties.

The separation of rare earth elements by solid phase containing diglycolamide-type ligands is a hot topic. In this study, 2-[2-oxo-2-(1-pyrrolidinyl)ethoxy]acetic acid (PYRDGA) was synthesized and attached to the silica. The binding strength of SiO2@PYRDGA for rare earths showed a single increasing trend with the radius of rare earth atoms. IR and XPS spectra demonstrated that carbonyl oxygen and ether bond oxygen are binding sites for rare earth ions. SiO2@PYRDGA was used for the chromatographic separation of REEs, and the primary separation of 16 REEs was achieved at pH = 2.0 using HNO3 solution as the eluent, and La, Ce, Pr, Nd, Sm, and Eu reached the baseline separation level.

Structure, thermostability and magnetic properties of cubic Ce2-x Ti2O7 pyrochlore obtained via sol-gel preparation.

Lanthanum-based titanates have been attracting considerable interest by virtue of their structural operability and hence diverse physical properties. The preparation of lanthanum-based titanates with novel crystal structure is a fascinating task. In this work, we report the preparation of a cubic Ce2-x Ti2O7 pyrochlore using the sol-gel method. The crystal structure, thermostability and magnetism were studied via the temperature dependence of X-ray powder diffraction, X-ray photoelectron spectroscopy and magnetization measurements. It has been revealed that the as-prepared Ce2-x Ti2O7 pyrochlore possesses a cubic symmetry (space group: Fd3̄m), however there is an 18(1)% vacancy of Ce ions in the as-prepared samples. No distinct phase transition and thermal expansion anomaly were observed in the investigated temperature range from 300 K to 700 K. Intriguingly, lattice defects may favor the transformation of Ce valence from +3 to +4 and an unusual weak magnetic ordering state emerged up to 400 K. The persistence of magnetism at such high temperatures is rare and mysterious for cerium titanates. Our findings provide the possibility of adjusting the crystal structure and magnetic properties of cerium titanates, anticipated to the development of lanthanum-based oxides.

Low temperature magnetic behavior and thermal expansion anomaly of cubic CeTiO3.

Lanthanum-based LnBO3 perovskite oxides have demonstrated fascinating magnetic properties and spin-lattice coupling. In this work, we report an unusual thermal expansion anomaly coupled with the magnetic ordering in the cubic CeTiO3 with the vacancy of Ce ions. The magnetic behaviors and lattice thermal expansion at low temperature were systematically investigated using the temperature dependence of the magnetization measurements and low temperature X-ray powder diffraction. It is clearly revealed that there are two magnetic transitions in the cubic CeTiO3 from 5 to 350 K: one is a magnetic ordering-disordering transition at 300 K and the other one might be a change of the magnetic component near 32 K. Both the magnetization and hysteresis change correspondingly upon cooling. Intriguingly, a lattice thermal expansion anomaly is found below the magnetic ordering temperature, which indicates a strong coupling of spin and lattice, i.e., a magnetovolume effect (MVE). Our findings provide the possibility of adjusting thermal expansion behavior and magnetic properties by introducing a vacancy of Ln atoms in lanthanum-based perovskite oxides.

Extraction and Back-Extraction Behaviors of La(III), Ce(III), Pr(III), and Nd(III) Single Rare Earth and Mixed Rare Earth by TODGA.

N,N,N',N'-Tetraoctyl diglycolamide (TODGA), as a new extraction agent, is effective for its excellent performance and low environmental hazard, and it is very welcome for the rare earth separation process. In this paper, by controlling the extraction time, diluent type, acid type and its concentration, rare earth concentration, etc., the optimum extraction and back-extraction effects of TODGA on La(III), Ce(III), Pr(III), and Nd(III) and mixed rare earths were obtained. The experiment showed that 0.10 mol·L-1 TODGA had the best extraction effect on single rare earth under the conditions of using petroleum ether as diluent, 5 mol·L-1 nitric acid, 20 min extraction time, and 0.01 mol·L-1 rare earth. In the mixed rare earth extraction, the percentage concentrations of La(III), Ce(III), Pr(III), and Nd(III) could be achieved from 21.7%, 19.9%, 30.8%, and 22.2% at the initial stage to 90.5%, 37%, 51%, and 62% after extraction, respectively, by controlling the number of back-extraction cycles and the concentrations of hydrochloric acid and nitric acid in the back-extraction system. The TODGA-rare earth carrier system showed the best back-extraction effect when the hydrochloric acid concentration was 1 mol·L-1 and the back-extraction time was 20 min. At the same time, the mixed rare earth liquid system with low initial concentration was selected for extraction and separation of mixed rare earth. The separation effect was better, and the recovery rate was higher than that of mixed rare earth liquid system with a high initial concentration.

Isolation and identification of a 2,3,7,8-Tetrachlorodibenzo-P-dioxin degrading strain and its biochemical degradation pathway.

This study aims to find a high-efficiency degradation strain which can biodegrade the 2,3,7,8-Tetrachlorodibenzo-P-dioxin (2,3,7,8-TCDD). In this paper, a new fungus strain was isolated from activated sludge of Dagu Drainage River in Tianjin which was able to degrade 2,3,7,8-TCDD in the medium. Based on its morphology and phylogenetic analysis of its 18S rDNA sequence, the strain was identified as Penicillium sp. QI-1. Response surface methodology using central composite rotatable design of cultural conditions was successfully employed for optimization resulting in 87.9 % degradation of 2,3,7,8-TCDD (1 µg/mL) within 6 days. The optimum condition for degrading 2,3,7,8-TCDD was at 31℃ and pH 7.4. The biodegradation process was fitted to a first-order kinetic model. The kinetic equation was Ct=0.939e- 0.133t and its half-life was 5.21d. The fungus strain degraded 2,3,7,8-TCDD to form intermediates, they were 4,5-Dichloro-1,2-benzoquinone, 4,5-Dichlorocatechol, 2-Hydrooxy-1,4-benzoquinone, 1,2,4-Trihydroxybenzene and ß-ketoadipic acid. A novel degradation pathway for 2,3,7,8-TCDD was proposed based on analysis of these metabolites. The results suggest that Penicillium sp. QI-1 may be an ideal microorganism for biodegradation of the 2,3,7,8-TCDD-contaminated environments.

Selective extraction and column separation for 16 kinds of rare earth element ions by using N, N-dioctyl diglycolacid grafted silica gel particles as the stationary phase.

The selective extraction and column separation rear earth elements (REEs) were investigated in the present work. Herein, the functional ligand of N, N-dioctyldiglycolic acid (DODGA) was synthesized and chemically grafted on the silica gel (SG) particles to give the novel material SG@DODGA. The obtained SG@DODGA was fully characterized by NMR, BET (Brunauer-Emmett-Teller) N2 physisorption analysis, atom force microscopy (AFM), scanning electronic microscopy (SEM), Fourier transform infrared (FT-IR), X-ray photoelectron spectroscopy (XPS) and thermogravimetric analysis (TGA). After investigating the adsorption capability of the SG@DODGA towards 16 kinds of REEs (La, Ce, Pr, Nd, Sm, Eu, Gd, Td, Dy, Ho, Er, Tm, Yb, Lu, Y and Sc), the results showed that the adsorption kinetic data was better fitted with pseudo-second-order model and Elovich model, the adsorption isotherms data was suitable for Freundlich model. The above result also indicated that the adsorption mechanism between the SG@DODGA and REEs was chemical ion exchange. Moreover, choose SG@DODGA as the column chromatography stationary phase and packed in a glass column for the column studies to obtain breakthroughs profile of each REEs. Furthermore, the column was used to try to separate the mixed 16 kinds of REEs. The first attempt to preliminary separate REEs result showed that this column could be applied for simply separating REEs. The light REEs La, Ce, Pr, Nd exhibited better separation effect than the other REEs.

"Covalent-Assembly"-Based Fluorescent Probe for Detection of a Nerve-Agent Mimic (DCP) via Lossen Rearrangement.

The highly selective and sensitive fluorescence "light-up" probe, 5'-(dimethylamino)-2'-formyl-N-hydroxy-[1,1'-biphenyl]-2-carboxamide(PTS), has been fabricated for the nerve-agent mimic diethyl chlorophosphate (DCP). The probe is designed by combining two novel strategies of "covalent assembly" and Lossen rearrangement. Formation of a phosphoryl intermediate from DCP and a hydroxamic acid group in PTS yields an isocyanate that quickly undergoes Lossen rearrangement to produce an aniline that condenses intramolecularly to a fluorescent phenanthridine system. PTS shows superior properties to probe DCP, such as rapid response (within 100 s), low detection limit (10.4 nM), specificity, and excellent linearity (R2 = 0.9993) in the range from 2 to 16 µM. More importantly, its application of detecting DCP vapor has also been achieved with satisfying results.

Preparation of diglycolamide polymer modified silica and its application as adsorbent for rare earth ions.

Three novel diglycolamide monomers were synthesized and polymerized on silica. The diglycolamide polymer grafted silica were used as adsorbents for rare earth ions. The effects of acid concentration, structure of monomer, initial solution concentration, contact time and coexisting ions on adsorption of rare earth ions were investigated in detail. It was shown that the adsorption capacity increased with increasing acid concentration. Three adsorbents exhibited selectivity for middle and heavy rare earth over light rare earth in different extent. The adsorbent prepared from the monomer having the largest alkyl substituent showed the lowest adsorption capacity but the highest selectivity for different rare earth elements (REEs). Adsorption data were well fitted to the Langmuir isotherm and pseudo-second-order models. The presence of high concentrations (100 fold) of coexisting metal ions, K(I), Cr(II), Cu(II) or Fe(III), does not decrease the adsorption for rare earth ions seriously.

Cell changes and differential proteomic analysis during biodegradation of decabromodiphenyl ether (BDE-209) by Pseudomonas aeruginosa.

Decabromodiphenyl ether (BDE-209) is a brominated flame retardant widely used in the world which, being an emerging persistent organic pollutant, poses a great potential hazard to both human health and the ecological environment. Microbial biodegradation has been considered as an effective and environment-friendly technique to remediate BDE-209. Pseudomonas aeruginosa, a Gram-negative bacterium capable of degrading BDE-209, was isolated from PBDEs-contaminated soil. To promote microbial biodegradation of BDE-209 and gain further insight into its mechanism, cell changes and differential proteomic analysis of P. aeruginosa during biodegradation were studied. The results showed that high cell surface hydrophobicity of P. aeruginosa make the bacteria absorb BDE-209 more easily. The increase in cell membrane permeability was caused by the P. aeruginosa responding to BDE-209 stress. IR spectra showed that hydroxyl, amide and CH2 groups in the P. aeruginosa cell surface were involved in the interactions between BDE-209 with P. aeruginosa. The apoptotic-like cell changes and cell surface morphology changes were observed by flow cytometry (FCM) and field emission scanning electron microscopy (FESEM), respectively. Differentially expressed protein was analysed by two-dimensional electrophoresis (2-DE) and 40 protein spots were identified to be different after 5 days biodegradation.

A novel ratiometric fluorescent probe for the selective determination of HClO based on the ESIPT mechanism and its application in real samples.

Based on the ESIPT fluorescence mechanism, herein, a novel ratiometric fluorescent probe was designed and synthesized for the detection of HClO. The reaction site of diaminomaleonitrile at the ortho-position of the phenolic hydroxyl group made the probe exhibit a ratiometric fluorescence response towards hypochlorous acid (HClO). The specific sensing mechanism was verified via MS, HPLC and 1H NMR spectroscopy. Moreover, the probe showed excellent performance with high sensitivity and good selectivity towards HClO in the presence of other reactive oxygen species. In addition, the probe was successfully applied to detect HClO spiked in tap water, river water and diluted human serum with good recoveries.

Application of a functionalized ionic liquid extractant tributylmethylammonium dibutyldiglycolamate ([A336][BDGA]) in light rare earth extraction and separation.

A type of functionalized ionic liquid extractant, tributylmethylammonium dibutyldiglycolamate ([A336][BDGA]), was synthesized, and its extraction ability of light rare earth elements, namely, La(III), Ce(III), Pr(III) and Nd(III), wascompared with that of other functionalized ionic liquid extractants, including tributylmethylammonium dioctyldiglycolamate and diglycolic amide extractants N,N,N,N-tetrabutyl-3-oxapentane-diamide (TBDGA) and N,N,N,N-tetraoctyl-3-oxapentane-diamide (TODGA). The study of extraction behavior indicated that the ionic liquids exhibited better extraction properties than the amides under lower acidity conditions. The effects of the pH and concentration of the extractant on the extraction behavior of [A336][BDGA] on light rare earths were determined. A separation strategy of mixed light rare earths was investigated by means of extraction chromatography using [A336][BDGA] as the stationary phase. As a result, nearly pure La(III), Ce(III), Pr(III) and Nd(III) were obtained, respectively. A new strategy of separating mixed light rare earths was established by means of extraction chromatography. The stationary phase of [A336][BDGA] was used in this strategy. The four rare earths elements, La(III), Ce(III), Pr(III) and Nd(III), achieved baseline separation.

Removal of MCs by Bi2O2CO3: adsorption and the potential of photocatalytic degradation.

Microcystins (MCs) is a kind of hepatotoxin, which is the secondary metabolite of cyanobacteria. Bi2O2CO3 (BOC) is a kind of cheap and nontoxic semiconductor material. BOC was synthetized by solvothermal method and then microcystin-LR (MC-LR) and microcystin-RR (MC-RR) were removed by BOC, through adsorption and photocatalytic degradation. When the dosage of BOC is 6 g/L, the MC-LR and MC-RR in the natural water sample can be completely adsorbed in 30 min and then after 12 h irradiation, MC-LR and MC-RR were photocatalytically degraded by BOC.

Synthesis and characterization of magnetic mesoporous Fe3O4@mSiO2-DODGA nanoparticles for adsorption of 16 rare earth elements.

In this study, novel magnetic mesoporous Fe3O4@mSiO2-DODGA nanoparticles were prepared for efficiently adsorbing and recycling REEs. Fe3O4@mSiO2-DODGA was characterized by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), vibrating sample magnetometry (VSM), Fourier transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA). The adsorption behavior of Fe3O4@mSiO2-DODGA was investigated by ICP-OES. The results showed that the content of DODGA in the adsorbent was 367 µmol g-1. Fe3O4@mSiO2-DODGA exhibited the highest adsorption rates for 15 REEs, except Tm, in a 2 mol L-1 nitric acid solution. Among these elements, the adsorption rates for Nd, Sm, Eu, Dy, Ho, Yb, Lu, Y and Sc ranged from 85.1% to 100.1%. The desorption rates for all 16 REE ions reached their maximum values when 0.01 mol L-1 EDTA was used as the eluent. The desorption rates for Nd, Ce, Sm, Eu, Ho, Yb, Lu, Y, and Sc were 87.7-99.8%. Fe3O4@mSiO2-DODGA had high stability in 2 mol L-1 HNO3 and could be used five times without significant loss of adsorption capacity. Moreover, these nanoparticles had high selectivity, and their adsorption rate was not affected even in a high-concentration solution of a coexisting ion. Therefore, 8 REE ions (Nd, Sm, Eu, Ho, Yb, Lu, Y, and Sc) were selected for the study of adsorption kinetics and adsorption isotherm experiments. It was demonstrated that the values of Q e (equilibrium adsorption capacity) for Nd, Sm, Eu, Ho, Yb, Lu, Y, and Sc were 14.28-60.80 mg g-1. The adsorption of REEs on Fe3O4@mSiO2-DODGA followed the pseudo-second-order kinetic model, Elovich model and Langmuir isotherm model, which indicated that the adsorption process of Fe3O4@mSiO2-DODGA for REEs comprised single-layer adsorption on a non-uniform surface controlled by chemical adsorption. It was concluded that Fe3O4@mSiO2-DODGA represents a new material for the adsorption of REEs in strongly acidic solutions.

Simultaneous determination of trace rare-earth elements in simulated water samples using ICP-OES with TODGA extraction/back-extraction.

The determination of trace rare-earth elements (REEs) can be used for the assessment of environmental pollution, and is of great significance to the study of toxicity and toxicology in animals and plants. N, N, N', N'-tetraoctyl diglycolamide (TODGA) is an environmental friendly extractant that is highly selective to REEs. In this study, an analytical method was developed for the simultaneous determination of 16 trace REEs in simulated water samples by inductively coupled plasma optical emission spectroscopy (ICP-OES). With this method, TODGA was used as the extractant to perform the liquid-liquid extraction (LLE) sample pretreatment procedure. All 16 REEs were extracted from a 3 M nitric acid medium into an organic phase by a 0.025 M TODGA petroleum ether solution. A 0.03 M ethylenediaminetetraacetic acid disodium salt (EDTA) solution was used for back-extraction to strip the REEs from the organic phase into the aqueous phase. The aqueous phase was concentrated using a vacuum rotary evaporator and the concentration of the 16 REEs was detected by ICP-OES. Under the optimum experimental conditions, the limits of detection (3σ, n = 7) for the REEs ranged from 0.0405 ng mL-1 (Nd) to 0.5038 ng mL-1 (Ho). The relative standard deviations (c = 100 ng mL-1, n = 7) were from 0.5% (Eu) to 4.0% (Tm) with a linear range of 4-1000 ng mL-1 (R2 > 0.999). The recoveries of 16 REEs ranged from 95% to 106%. The LLE-ICP-OES method established in this study has the advantages of simple operation, low detection limits, fast analysis speed and the ability to simultaneously determine 16 REEs, thereby acting as a viable alternative for the simultaneous detection of trace amounts of REEs in water samples.

Effect of copper ion and soil humic acid on biodegradation of decabromodiphenyl ether (BDE-209) by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a good environmental microorganism capable of degrading decabromodiphenyl ether (BDE-209). This paper studied the effect of Cu2+ and humic acid (HA) extracted from e-waste contaminated soils on biodegradation of BDE-209 by P. aeruginosa. The adsorption isotherms of Cu2+ on HA, the crude enzyme activity, cell surface morphology, and biodegradation pathway were also investigated. The results showed that BDE-209 biodegradation by P. aeruginosa was inhibited at Cu2+ concentrations above 5 mg L-1 , but exhibited the best effect at the condition of 40 mg L-1 Cu2+  + 3 g L-1 HA. At the condition of 40 mg L-1 Cu2+  + 3 g L-1 HA, 97.35 ± 2.33% of the initial BDE-209 was degraded after 5 days, debromination efficiency was 72.14 ± 1.89%, crude enzyme activity reached the maximum of 0.519 ± 0.022U g-1 protein, cell surface of P. aeruginosa was smooth with normal short-rod shapes, and biodegradation pathway mainly include debromination, hydroxylation, and cleavage of the diphenyl ether bond. It was suggested that soil HA could eliminate the toxic effect of high Cu2+ concentrations and biodegradation of BDE-209 was improved by synergistic effect of HA and Cu2+ .

Isolation and characterization of a high-efficiency erythromycin A-degrading Ochrobactrum sp. strain.

In this work, Erythromycin A(EA)- degrading bacteria was isolated from the contaminated soil obtained from a pharmaceutical factory in China. The isolate designated as strain WX-J1 was identified as Ochrobactrum sp. by sequence analysis of its 16S rDNA gene. It can grow in a medium containing EA as the sole source of carbon and its optimal growth pH and temperature were 6.5 and 32°C, respectively. Under these conditions, when the initial Erythromycin A concentration was 100mg·L-1, 97% of Erythromycin A has been degraded. HPLC-MS analyses indicated that Erythromycin A degradation produced intermediates contained the following three substances: 3-depyranosyloxy erythromycin A, 7,12-dyhydroxy-6-deoxyerythronolide B, 6-deoxyerythronolide B and propionaldehyde. Since Erythromycin A-degrading Ochrobactrum sp. strain rapidly degraded Erythromycin A, this strain might be useful for bioremediation purposes.

Determination of photoinitiator 4-methylbenzophenone in milk by cloud point extraction.

An efficient and easy sample pretreatment methodology was proposed for the detection of photoinitiator 4-methylbenzophenone from milk before high-performance liquid chromatography. Appropriate conditions for demulsification were studied. The parameters affecting cloud point extraction, such as concentration of Tween-20, electrolyte salt, equilibration temperature, and time, have been investigated. When the spiked level was 200-1000 µg/kg, the average addition standard recovery was 99.14-105.98% with the optimum cloud point extraction conditions (concentration of Tween-20, 138 g/L; mass of anhydrous sodium sulfate, 0.75 g; equilibration temperature, 65°C; equilibration time, 30 min). To decrease the detection limits, further work about the organic solvent, shaking time, and ultrasonic parameters was carried. When the spiked level was 10-100 µg/kg, the average addition standard recovery was 70.40-106.91% with the optimum cloud point extraction and enrichment conditions (optimum cloud point extraction conditions; volume of cyclohexane, 30 mL; shaking time, 20 min; time of ultrasonic, 20 min; temperature of ultrasonic bath, 45°C).