Supercritical carbon dioxide (SC-CO2) extraction was employed to extract oil from Nigella glandulifera Freyn seed in this study. Response surface methodology (RSM) was applied to evaluate the effects of the proces...Supercritical carbon dioxide (SC-CO2) extraction was employed to extract oil from Nigella glandulifera Freyn seed in this study. Response surface methodology (RSM) was applied to evaluate the effects of the process parameters (pressure, temperature, and CO2 flow rate) on oil yield of N. glandulifera seed. A Box-Behnken design was used to optimize the extraction parameters. The analysis of variance indicated that the linear coefficients of pressure and CO2 flow rate, the quadratic term coefficients of pressure and temperature and the interactions between pressure and temperature, as well as temperature and CO2 flow rate, had significant effects on the oil yield (P〈0.05). The optimal conditions to obtain the maximum oil yield from N. glandulifera seed were pressure 30.84 MPa, temperature 40.57°C, and CO2 flow rate 22.00 L h-1. Under these optimal conditions, the yield of oil was predicted to be 38.19%. The validation experiment results agreed with the predicted values. The fatty acid composition of N. glandulifera seed oil extracted using SC-CO2 was compared with that of oil obtained by Soxhlet method. The results showed that the fatty acid compositions of oil extracted by the two methods were similar. Identification of oil compounds with gas chromatography-mass spectrometry (GC-MS) showed that the contents of unsaturated fatty acids linoleic acid (48.30%), oleic acid (22.28%) and saturated fatty acids palmitic acid (16.65%), stearic acid (4.17%) were the most abundant fatty acids in seed oil from N. glandulifera.展开更多
Water soluble organic carbon (WSOC) is considered the most mobile and reactive soil carbon source and its characterization is an important issue for soil ecology study. A biodegradability test was set up to study WS...Water soluble organic carbon (WSOC) is considered the most mobile and reactive soil carbon source and its characterization is an important issue for soil ecology study. A biodegradability test was set up to study WSOC extracted from 7 soils differently managed. WSOC was extracted from soil with water (soil/water ratio of 1:2, W/V) for 30 min, and then tested for biodegradability by a liquid state respirometric test. Result obtained confirmed the finding that WSOC biodegradability depended on the both land use and management practice. These results suggested the biodegradability test as suitable method to characterize WSOC, and provided useful information to soil fertility.展开更多
The aim of this study was to determine the extraction technique of supercritical fluid carbon dioxide(SF-CO 2) for the essential oil from Inula britannica flowers and its antifungal activities against plant pathogen...The aim of this study was to determine the extraction technique of supercritical fluid carbon dioxide(SF-CO 2) for the essential oil from Inula britannica flowers and its antifungal activities against plant pathogenic fungi for its potential application as botanical fungicide.The effects of factors,including extraction temperature,extraction pressure,SF-CO 2 flow rate,flower powder size,and time on the essential oil yield were studied using the single factor experiment.An orthogonal experiment was conducted to determine the best operating conditions for the maximum extraction oil yield.Adopting the optimum conditions,the maximum yield reached 10.01% at 40°C temperature,30 MPa pressure,60 mesh flower powder size,20 L h-1SF-CO 2 flow rate,and 90 min extraction time.The antifungal activities of I.britannica essential oil using the SF-CO 2 against the most important plant pathogenic fungi were also examined through in vitro and in vivo tests.Sixteen plant pathogenic fungi were inhibited to varying degrees at 1 mg mL-1concentration of the essential oil.The mycelial growth of Gaeumannomyces graminis var.tritici was completely inhibited.The radial growths of Phytophthora capsici and Fusarium monilifome were also inhibited by 83.76 and 64.69%,respectively.In addition,the essential oil can inhibit the spore germination of Fusarium oxysporum f.sp.vasinfectum,Phytophthora capsici,Colletotrichum orbiculare,and Pyricularia grisea,and the corresponding inhibition rates were 98.26,96.54,87.89,and 87.35% respectively.The present study has demonstrated that the essential oil of I.britannica flowers extracted through the SF-CO 2 technique is one potential and promising antifungal agent that can be used as botanical fungicide to protect crops.展开更多
In this work, a metal-organic framework derived nanoporous carbon (MOF-5-C) was fabricated and modified with Fe3O4 magnetic nanoparticles. The resulting magnetic MOF-5-derived porous carbon (Fe304@MOF-5-C) was the...In this work, a metal-organic framework derived nanoporous carbon (MOF-5-C) was fabricated and modified with Fe3O4 magnetic nanoparticles. The resulting magnetic MOF-5-derived porous carbon (Fe304@MOF-5-C) was then used for the magnetic solid-phase extraction of chlorophenols (CPs) from mushroom samples prior to high performance liquid chromatography-ultraviolet detection. Scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and N2 adsorption were used to characterize the adsorbent. After experimental optimization, the amount of the adsorbent was chosen as 8.0 mg, extraction time as 10 min, sample volume as 50 mL, desorption solvent as 0.4 mL (0.2 mL × 2) of alkaline methanol, and sample pH as 6. Under the above optimized conditions, good linearity for the analytes was obtained in the range of 0.8-100.0 ng g 1 with the correlation coefficients between 0.9923 and 0.9963. The limits of detection (SIN= 3) were in the range of 0.25-0.30 ng g-1, and the relative standard deviations were below 6.8%. The result showed that the Fe304@MOF-5-C has an excellent adsorption capacity for the analytes.展开更多
In this work, a new sample pretreatment method prior to HPLC separations was developed for the determination of auxins in plant samples, Owing to its large surface area and high adsorption capacity, multi-walled carbo...In this work, a new sample pretreatment method prior to HPLC separations was developed for the determination of auxins in plant samples, Owing to its large surface area and high adsorption capacity, multi-walled carbon nanotube (MWCNT) was chosen as the adsorbent for the extraction of auxins from plant samples. In this study, two important auxins were selected as model analytes, namely indole-3- butyric acid (IBA) and 1-naphthylacetic acid (NAA). They could be extracted and concentrated due to their π-π stacking interactions with MWCNT. Then HPLC-UV was introduced to detect IBA and NAA after sample pretreatment. Factors that may affect the enrichment efficiency were investigated and optimized. Comparative studies showed that MWCNT was superior to CI 8 for the extraction of the two analytes. Validation experiments showed that the optimized method had good linearity (0.9998 and 0.9960), high recovery (81.4%-85.4%), and low detection limits (0.0030 mg/L and 0.0012 mg/L). The results indicated that the novel method had advantages of convenience, good sensitivity, high efficiency, and it was feasible for the determination of auxins in plant samples.展开更多
The humification degree of fresh litter directly controls the accumulation of soil humus derived from plant litter,but very little information on this process is available.Planted forests are well known to restrict so...The humification degree of fresh litter directly controls the accumulation of soil humus derived from plant litter,but very little information on this process is available.Planted forests are well known to restrict soil fertility,which is often indicated by the soil humus level.In this study,fresh litter was collected during different plant phenological stages during 2016 and 2017 in a mixed plantation in Southwest China.The values of hue coefficientΔlogK(absorbance ratio of 400 nm and 600 nm on a logarithmic scale),optical density E4/E6(absorbance ratio of 400 nm and 600 nm)and A600/C(absorbance at 600 mm per mg of carbon per ml of extraction)and the concentration of extractable humus carbon(HC)were determined in four litter components(foliar,twigs,reproductive organs and miscellaneous)of the dominant species(Pterocarya stenoptera,Quercus acutissima,Cunninghamia lanceolata and Toxicodendron vernicifluum).All of the litter components exhibited obvious humification characteristics,and showed the highest concentration of extractable HC during the leaf maturation period.The miscellaneous and foliar litters showed greater humification than the other litter types.The components of Pterocarya stenoptera litter exhibited greater degrees of humification than those of the other species,with lowerΔlogK and E4/E6 values and higher A600/C values.The litter from coniferous and evergreen species showed lower humification than that from broad-leaf and deciduous species regardless of the litter component examined.The present results provide new insights into the management of plantations and theoretical data to accurately improve the quality of plantations and maintain soil fertility under a global change.展开更多
A sensitive and selective preconcentration method using solid-phase extraction (SPE) disk made from oxidized multiwalled carbon nanotubes (OMWCNTs), has been developed for the determination of aniline derivatives,...A sensitive and selective preconcentration method using solid-phase extraction (SPE) disk made from oxidized multiwalled carbon nanotubes (OMWCNTs), has been developed for the determination of aniline derivatives, such as 2-nitroaniline (2-NA), 4- nitroaniline (4-NA), and 2,4-dichloroaniline (2,4-DCA) in water samples. Anilines were extracted onto OMWCNT disk and then determined by high performance liquid chromatography (HPLC) with UV detector. Several parameters on the recovery of the analytes were investigated. The experimental results showed that it was possible to obtain quantitative analysis when the solution pH was 8 using 200 mL of validation solution containing 2 ug of anilines and 10 mL of acetonitrile/ethanol (8/2, v/v) as an eluent. Relative standard deviations for five determinations were 7.5% (2-NA), 6.5% (4-NA) and 3.8% (2,4-DCA) under optimum conditions. The linear range of calibration curves were 0.5 ng.mL 1 to 15 ng.mL-1 for each analyte with good correlation coefficients. The detection limits (3S/N) of 2-NA, 4-NA and 31 pg.mL -1 and 26 pg.mL-1 was successfully applied to 2,4-DCA were 30pg.mL -1, respectively. Our method the determination of aniline compounds in fiver water sample with high precision and accuracy.展开更多
Ordered mesoporous carbon(CMK-3) was synthesized and functionalized with 2-amino-5-mercapto-1,3,4-thiadiazole groups(AMT-OCMK-3) for Hg(Ⅱ) removal from aqueous solution. The modified CMK-3 was characterized by ...Ordered mesoporous carbon(CMK-3) was synthesized and functionalized with 2-amino-5-mercapto-1,3,4-thiadiazole groups(AMT-OCMK-3) for Hg(Ⅱ) removal from aqueous solution. The modified CMK-3 was characterized by X-ray diffraction, N2adsorption-desorption isotherm, scanning electron microscopy and Fourier transform infrared spectroscopy. The effects of solution pH, contact time, initial Hg(Ⅱ) concentration and matrix effect were studied. The adsorption data were successfully fitted with the Langmuir model, exhibiting high adsorption capacity of 450.45 mg/g of AMT-OCMK-3. In the solid-phase extraction system a series of experimental parameters such as sample flow rate, sample volume,eluent volume and concentration of the eluent solution have been investigated and established for preconcentration of Hg(Ⅱ) in aqueous solution. The results showed that the enrichment factor for Hg(Ⅱ) was 250, the precision(relative standard deviation(RSD), %) for six replicate measurements was 2.05% and the limit of detection for Hg(Ⅱ) was achieved at0.17 μg/L.展开更多
In this research, supercritical carbon dioxide extraction (SFE) showed better extraction etlect when compared with Solid- liquid extraction (SLE), Soxhlet extraction (SEt and Ultrasonic extraction (UE), not onl...In this research, supercritical carbon dioxide extraction (SFE) showed better extraction etlect when compared with Solid- liquid extraction (SLE), Soxhlet extraction (SEt and Ultrasonic extraction (UE), not only in the rate but also the time. The comparison among these three extraction modifiers, including acetone, ethanol and methanol demonstrated that ethanol was preferred to SFE due to its high extraction effect and low toxicology. In addition, parameter of SFE, influence of temperature and pressure were investigated, and the best extraction effect was achieved at the optima conditions, temperature of 40℃ and the pressure of 35 MPa. Thus, SFE is a highly effective method for flavonols extraction, requiring minimum energy and producing non-toxic byproduct. SFE-GC system is applied for the evaluation on flavonols that plays a key role in plant resistance to heavy metal, with its content and synthetase gene expression significantly increasing in plant when threatened by heavy metal. Besides, results indicated that flavonols can improve plant resistance to oxidative stress by quenching the redundant ROS in matrix.展开更多
To give a further research on strengthening mechanism of binary Fe-Cu alloys dur- ing the aging process, the compositions, size distributions and volume fractions of precipitates were investigated by carbon extraction...To give a further research on strengthening mechanism of binary Fe-Cu alloys dur- ing the aging process, the compositions, size distributions and volume fractions of precipitates were investigated by carbon extraction replicas. Both the size and the volume fraction of precipitates increase with the aging time prolonged. The precip- itate sizes are corresponding with Weibull distribution. The stability of the sample aged for 35 rain is worse than that aged for 1800 min and 18000 min. The precipi- tate is Cu-rich phase in the early aging process, and Cu content increases with the prolonging aging time. Before aging peak, the precipitation of Cu-rich phase plays a dominant role in strengthening. The growth and the change in crystal structure of the precipitate particles are the main factors leading to the decrease in strengthening after the aging peak.展开更多
基金supported by the Public Welfare Industry (Agriculture) Research Program,China (200903018)
文摘Supercritical carbon dioxide (SC-CO2) extraction was employed to extract oil from Nigella glandulifera Freyn seed in this study. Response surface methodology (RSM) was applied to evaluate the effects of the process parameters (pressure, temperature, and CO2 flow rate) on oil yield of N. glandulifera seed. A Box-Behnken design was used to optimize the extraction parameters. The analysis of variance indicated that the linear coefficients of pressure and CO2 flow rate, the quadratic term coefficients of pressure and temperature and the interactions between pressure and temperature, as well as temperature and CO2 flow rate, had significant effects on the oil yield (P〈0.05). The optimal conditions to obtain the maximum oil yield from N. glandulifera seed were pressure 30.84 MPa, temperature 40.57°C, and CO2 flow rate 22.00 L h-1. Under these optimal conditions, the yield of oil was predicted to be 38.19%. The validation experiment results agreed with the predicted values. The fatty acid composition of N. glandulifera seed oil extracted using SC-CO2 was compared with that of oil obtained by Soxhlet method. The results showed that the fatty acid compositions of oil extracted by the two methods were similar. Identification of oil compounds with gas chromatography-mass spectrometry (GC-MS) showed that the contents of unsaturated fatty acids linoleic acid (48.30%), oleic acid (22.28%) and saturated fatty acids palmitic acid (16.65%), stearic acid (4.17%) were the most abundant fatty acids in seed oil from N. glandulifera.
文摘Water soluble organic carbon (WSOC) is considered the most mobile and reactive soil carbon source and its characterization is an important issue for soil ecology study. A biodegradability test was set up to study WSOC extracted from 7 soils differently managed. WSOC was extracted from soil with water (soil/water ratio of 1:2, W/V) for 30 min, and then tested for biodegradability by a liquid state respirometric test. Result obtained confirmed the finding that WSOC biodegradability depended on the both land use and management practice. These results suggested the biodegradability test as suitable method to characterize WSOC, and provided useful information to soil fertility.
基金supported by the Scientific and Technological Key Project of Henan Province, China (082102350006 and 102102310242)the College Young Teachers Projects of Henan Province, China (2010GGJS046)
文摘The aim of this study was to determine the extraction technique of supercritical fluid carbon dioxide(SF-CO 2) for the essential oil from Inula britannica flowers and its antifungal activities against plant pathogenic fungi for its potential application as botanical fungicide.The effects of factors,including extraction temperature,extraction pressure,SF-CO 2 flow rate,flower powder size,and time on the essential oil yield were studied using the single factor experiment.An orthogonal experiment was conducted to determine the best operating conditions for the maximum extraction oil yield.Adopting the optimum conditions,the maximum yield reached 10.01% at 40°C temperature,30 MPa pressure,60 mesh flower powder size,20 L h-1SF-CO 2 flow rate,and 90 min extraction time.The antifungal activities of I.britannica essential oil using the SF-CO 2 against the most important plant pathogenic fungi were also examined through in vitro and in vivo tests.Sixteen plant pathogenic fungi were inhibited to varying degrees at 1 mg mL-1concentration of the essential oil.The mycelial growth of Gaeumannomyces graminis var.tritici was completely inhibited.The radial growths of Phytophthora capsici and Fusarium monilifome were also inhibited by 83.76 and 64.69%,respectively.In addition,the essential oil can inhibit the spore germination of Fusarium oxysporum f.sp.vasinfectum,Phytophthora capsici,Colletotrichum orbiculare,and Pyricularia grisea,and the corresponding inhibition rates were 98.26,96.54,87.89,and 87.35% respectively.The present study has demonstrated that the essential oil of I.britannica flowers extracted through the SF-CO 2 technique is one potential and promising antifungal agent that can be used as botanical fungicide to protect crops.
基金Financial support from the National Natural Science Foundation of China (Nos. 31471643, 31571925)the Innovation Research Program of the Department of Education of Hebei for Hebei Provincial Universities (No. LJRC009)
文摘In this work, a metal-organic framework derived nanoporous carbon (MOF-5-C) was fabricated and modified with Fe3O4 magnetic nanoparticles. The resulting magnetic MOF-5-derived porous carbon (Fe304@MOF-5-C) was then used for the magnetic solid-phase extraction of chlorophenols (CPs) from mushroom samples prior to high performance liquid chromatography-ultraviolet detection. Scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and N2 adsorption were used to characterize the adsorbent. After experimental optimization, the amount of the adsorbent was chosen as 8.0 mg, extraction time as 10 min, sample volume as 50 mL, desorption solvent as 0.4 mL (0.2 mL × 2) of alkaline methanol, and sample pH as 6. Under the above optimized conditions, good linearity for the analytes was obtained in the range of 0.8-100.0 ng g 1 with the correlation coefficients between 0.9923 and 0.9963. The limits of detection (SIN= 3) were in the range of 0.25-0.30 ng g-1, and the relative standard deviations were below 6.8%. The result showed that the Fe304@MOF-5-C has an excellent adsorption capacity for the analytes.
基金supported by National Natural Science Foundation of China(Nos.20975056,21275082 and 81102411)Natural Science Foundation of Shandong(Nos. ZR2011BZ004,ZR2011BQ005)+2 种基金JSPS and NSFC under the Japan-China Scientific Cooperation Program(No.21111140014)State Key Laboratory of Analytical Chemistry for Life Science(No. SKLACLS1110)the National Key Basic Research Development Program of China(973 Special Preliminary Study Plan,No. 2012CB722705)
文摘In this work, a new sample pretreatment method prior to HPLC separations was developed for the determination of auxins in plant samples, Owing to its large surface area and high adsorption capacity, multi-walled carbon nanotube (MWCNT) was chosen as the adsorbent for the extraction of auxins from plant samples. In this study, two important auxins were selected as model analytes, namely indole-3- butyric acid (IBA) and 1-naphthylacetic acid (NAA). They could be extracted and concentrated due to their π-π stacking interactions with MWCNT. Then HPLC-UV was introduced to detect IBA and NAA after sample pretreatment. Factors that may affect the enrichment efficiency were investigated and optimized. Comparative studies showed that MWCNT was superior to CI 8 for the extraction of the two analytes. Validation experiments showed that the optimized method had good linearity (0.9998 and 0.9960), high recovery (81.4%-85.4%), and low detection limits (0.0030 mg/L and 0.0012 mg/L). The results indicated that the novel method had advantages of convenience, good sensitivity, high efficiency, and it was feasible for the determination of auxins in plant samples.
基金This study was supported by the National Natural Science Foundation of China(31470636)Specialized Fund for the Post-Disaster Reconstruction and Heritage Protection in Sichuan Province(No.5132202019000128)the Open Fund for Ecological Restoration and Conservation for Forest and Wetland Key Laboratory of Sichuan Province(No.2020KFKT01).
文摘The humification degree of fresh litter directly controls the accumulation of soil humus derived from plant litter,but very little information on this process is available.Planted forests are well known to restrict soil fertility,which is often indicated by the soil humus level.In this study,fresh litter was collected during different plant phenological stages during 2016 and 2017 in a mixed plantation in Southwest China.The values of hue coefficientΔlogK(absorbance ratio of 400 nm and 600 nm on a logarithmic scale),optical density E4/E6(absorbance ratio of 400 nm and 600 nm)and A600/C(absorbance at 600 mm per mg of carbon per ml of extraction)and the concentration of extractable humus carbon(HC)were determined in four litter components(foliar,twigs,reproductive organs and miscellaneous)of the dominant species(Pterocarya stenoptera,Quercus acutissima,Cunninghamia lanceolata and Toxicodendron vernicifluum).All of the litter components exhibited obvious humification characteristics,and showed the highest concentration of extractable HC during the leaf maturation period.The miscellaneous and foliar litters showed greater humification than the other litter types.The components of Pterocarya stenoptera litter exhibited greater degrees of humification than those of the other species,with lowerΔlogK and E4/E6 values and higher A600/C values.The litter from coniferous and evergreen species showed lower humification than that from broad-leaf and deciduous species regardless of the litter component examined.The present results provide new insights into the management of plantations and theoretical data to accurately improve the quality of plantations and maintain soil fertility under a global change.
文摘A sensitive and selective preconcentration method using solid-phase extraction (SPE) disk made from oxidized multiwalled carbon nanotubes (OMWCNTs), has been developed for the determination of aniline derivatives, such as 2-nitroaniline (2-NA), 4- nitroaniline (4-NA), and 2,4-dichloroaniline (2,4-DCA) in water samples. Anilines were extracted onto OMWCNT disk and then determined by high performance liquid chromatography (HPLC) with UV detector. Several parameters on the recovery of the analytes were investigated. The experimental results showed that it was possible to obtain quantitative analysis when the solution pH was 8 using 200 mL of validation solution containing 2 ug of anilines and 10 mL of acetonitrile/ethanol (8/2, v/v) as an eluent. Relative standard deviations for five determinations were 7.5% (2-NA), 6.5% (4-NA) and 3.8% (2,4-DCA) under optimum conditions. The linear range of calibration curves were 0.5 ng.mL 1 to 15 ng.mL-1 for each analyte with good correlation coefficients. The detection limits (3S/N) of 2-NA, 4-NA and 31 pg.mL -1 and 26 pg.mL-1 was successfully applied to 2,4-DCA were 30pg.mL -1, respectively. Our method the determination of aniline compounds in fiver water sample with high precision and accuracy.
基金the Iran National Science Foundation for the financial support to this project
文摘Ordered mesoporous carbon(CMK-3) was synthesized and functionalized with 2-amino-5-mercapto-1,3,4-thiadiazole groups(AMT-OCMK-3) for Hg(Ⅱ) removal from aqueous solution. The modified CMK-3 was characterized by X-ray diffraction, N2adsorption-desorption isotherm, scanning electron microscopy and Fourier transform infrared spectroscopy. The effects of solution pH, contact time, initial Hg(Ⅱ) concentration and matrix effect were studied. The adsorption data were successfully fitted with the Langmuir model, exhibiting high adsorption capacity of 450.45 mg/g of AMT-OCMK-3. In the solid-phase extraction system a series of experimental parameters such as sample flow rate, sample volume,eluent volume and concentration of the eluent solution have been investigated and established for preconcentration of Hg(Ⅱ) in aqueous solution. The results showed that the enrichment factor for Hg(Ⅱ) was 250, the precision(relative standard deviation(RSD), %) for six replicate measurements was 2.05% and the limit of detection for Hg(Ⅱ) was achieved at0.17 μg/L.
文摘In this research, supercritical carbon dioxide extraction (SFE) showed better extraction etlect when compared with Solid- liquid extraction (SLE), Soxhlet extraction (SEt and Ultrasonic extraction (UE), not only in the rate but also the time. The comparison among these three extraction modifiers, including acetone, ethanol and methanol demonstrated that ethanol was preferred to SFE due to its high extraction effect and low toxicology. In addition, parameter of SFE, influence of temperature and pressure were investigated, and the best extraction effect was achieved at the optima conditions, temperature of 40℃ and the pressure of 35 MPa. Thus, SFE is a highly effective method for flavonols extraction, requiring minimum energy and producing non-toxic byproduct. SFE-GC system is applied for the evaluation on flavonols that plays a key role in plant resistance to heavy metal, with its content and synthetase gene expression significantly increasing in plant when threatened by heavy metal. Besides, results indicated that flavonols can improve plant resistance to oxidative stress by quenching the redundant ROS in matrix.
基金supported by the National Natural Science Foundation of China(No. 50671012)
文摘To give a further research on strengthening mechanism of binary Fe-Cu alloys dur- ing the aging process, the compositions, size distributions and volume fractions of precipitates were investigated by carbon extraction replicas. Both the size and the volume fraction of precipitates increase with the aging time prolonged. The precip- itate sizes are corresponding with Weibull distribution. The stability of the sample aged for 35 rain is worse than that aged for 1800 min and 18000 min. The precipi- tate is Cu-rich phase in the early aging process, and Cu content increases with the prolonging aging time. Before aging peak, the precipitation of Cu-rich phase plays a dominant role in strengthening. The growth and the change in crystal structure of the precipitate particles are the main factors leading to the decrease in strengthening after the aging peak.