Optimization of desorption parameters using response surface methodology for enhanced recovery of arsenic from spent reclaimable activated carbon: Eco-friendly and sorbent sustainability approach.

Desorption and adsorbent regeneration are imperative factors that are required to be taken into account when designing the adsorption system. From the environmental, economic, and practical points of view, regeneration is necessary for evaluating the efficiency and sustainability of synthesized adsorbents. However, no study has investigated the optimization of arsenic species desorption from spent adsorbents and their regeneration ability for reuse as well as safe disposal. This study aims to investigate the desorption ability of arsenic ions adsorbed on hybrid granular activated carbon and the optimization of the independent factors influencing the efficient recovery of arsenic species from the spent activated carbon using central composite design of the response surface methodology. The activated carbon before the sorption process and after the adsorption-desorption of arsenic ions have been characterized using SEM-EDX, FTIR, and TEM. The study found that all the investigated independent desorption variables greatly influence the retrievability of arsenic ions from the spent activated carbon. Using the desirability function for the optimization of the independent factors as a function of desorption efficiency, the optimum experimental conditions were solution pH of 2.00, eluent concentration of 0.10 M, and temperature of 26.63 ℃, which gave maximum arsenic ions recovery efficiency of 91 %. The validation of the quadratic model using laboratory confirmatory experiments gave an optimum arsenic ions desorption efficiency of 97 %. Therefore, the study reveals that the application of the central composite design of the response surface methodology led to the development of an accurate and valid quadratic model, which was utilized in the enhanced optimization of arsenic ions recovery from the spent reclaimable activated carbon. More so, the desorption isotherm and kinetic data of arsenic were well correlated with the Langmuir and the pseudo-second-order models, while the thermodynamics studies indicated that arsenic ions desorption process was feasible, endothermic, and spontaneous.

Rapid determination of chromium species in environmental waters using a diol-bonded polymer-stationary column under water-rich conditions coupled with ICPMS.

Chromium speciation analysis in environmental water is of great significance for the monitoring of water pollution and assessing its influences on human health. This study proposes a rapid analytical approach for the simultaneous determination of Cr(VI) and Cr(III) in environmental waters by hydrophilic interaction chromatography (HILIC) coupled with ICPMS under a water-rich condition. 2,6-Pyridinedicarboxylic acid (PDCA) was used to unify Cr(III) species in various chemical forms into a stable Cr(III)-PDCA anion complex and then separated from Cr(VI) oxyanion on a diol-bonded polymer-based HILIC column. An aqueous mobile phase containing 50 mmol L-1 ammonium acetate (pH 7.0), 2 mmol L-1 PDCA, and 4% acetonitrile successfully separates chromium species as well as chloride ions. In addition, our method elutes Cr(VI) preferentially over Cr(III)-PDCA, enabling rapid determination of Cr(VI), and both chromium species were analyzed within 6.2 min. The detection limits of 0.19 µg L-1 for Cr(VI) and 0.35 µg L-1 for Cr(III) at m/z 52 under He collision mode are comparable to or better than the conventional ion exchange chromatography-ICPMS methods, and quantitative recovery was obtained from spike-recovery tests on river water samples containing various levels of matrix. Optimization experiments of the HPLC conditions indicate that the retentions of Cr(VI) and Cr(III)-PDCA are characterized by electrostatic and nonpolar interactions, respectively. The retention behavior of inorganic anions and cations in water-rich conditions observed in this study will provide new insights into the separation mechanism in polymer-based HILIC columns, which has been poorly understood.

Chromium Recovery from Chromium-Loaded Cupressus lusitanica Bark in Two-Stage Desorption Processes.

Hexavalent chromium (Cr(VI)) contamination poses serious health and environmental risks. Chromium biosorption has been employed as an effective means of eradicating Cr(VI) contamination. However, research on chromium desorption from chromium-loaded biosorbents is scarce despite its importance in facilitating industrial-scale chromium biosorption. In this study, single- and two-stage chromium desorption from chromium-loaded Cupressus lusitanica bark (CLB) was conducted. Thirty eluent solutions were evaluated first; the highest single-stage chromium desorption efficiencies were achieved when eluent solutions of 0.5 M NaOH, 0.5 M H2SO4, and 0.5 M H2C2O4 were used. Subsequently, two-stage kinetic studies of chromium desorption were performed. The results revealed that using 0.5 M NaOH solution in the first stage and 0.5 M H2C2O4 in the second stage enabled the recovery of almost all the chromium initially bound to CLB (desorption efficiency = 95.9-96.1%) within long (168 h) and short (3 h) desorption periods at each stage. This study clearly demonstrated that the oxidation state of the recovered chromium depends on the chemical nature and concentration of the eluent solution. The results suggest the possible regeneration of chromium-loaded CLB for its subsequent use in other biosorption/desorption cycles.

Phenanthrene removal from soil washing eluent by Bacillus subtilis embedded in alginate-carboxymethyl cellulose-diatomite beads.

The processes of surfactant-enhanced soil washing have been widely applied to the remediation of polycyclic aromatic hydrocarbon (PAH)-contaminated soil, accompanied by the production of soil washing eluent. In this study, novel composite materials of beads containing alginate, carboxymethyl cellulose and diatomite (SCD) were used to encapsulate Bacillus subtilis to remove phenanthrene (PHE) from simulated soil washing eluent with rhamnolipid. The effects of dosage, pH and temperature on the PHE removal performance were explored, and the optimal PHE removal conditions [SCD bead dose 16.2% (w:v), pH 7.1 and 30.6°C] were determined using response surface methodology. After incubation in simulated soil washing eluent for 7 d, SCD beads removed 84.92% of PHE, which was 49.18% higher than by free bacteria. In addition, SCD beads mainly removed PHE through biodegradation processes, and the degradation rate (1.38 mg L-1 d-1) was higher than that of free bacteria (0.64 mg L-1 d-1). Characterization results revealed that the immobilized substrate provided the micro-environment for bacteria and reduced the intense effect of high rhamnolipid concentration. Reusability results showed that SCD beads could be recycled four times to remove 80.05% of PHE. Collectively, SCD beads have great prospects for the decontamination of soil-washing eluent containing complex components.

Exploitation of the enantioselectivity space of coated amylose tris(3,5-dimethylphenylcarbamate) in mixtures of 2-propanol and acetonitrile.

Chiral stationary phases (CSPs) with coated amylose tris(3,5-dimethylphenylcarbamate) (ADMPC) selector have long been recognized for their excellent chiral recognition ability in liquid chromatography. The conformational versatility behind this feature is the source of their known hysteretic behavior, which has been previously observed in polar organic (PO) mode eluents containing 2-propanol (IPA). Mixtures of IPA and acetonitrile (MeCN), a typical PO mode eluent system, have not been examined in this aspect yet, even though hysteresis is promising for finding unique unexplored enantioselectivities. Not only was the hysteresis detectable on ADMPC using mixtures of IPA and MeCN, but it was the typical behavior in a diverse set of test compounds. The difference in the retention time of the same analyte under conditions which only differed in the eluent history on the column can go up to 20-fold. The assumed hindered conformational changes of the selector were reflected in retention drift at certain eluent compositions. On the two sides of the transitions, distinct, useful states of the selector were detected. A series of IPA - MeCN compositions with defined pretreatment was selected and recommended as an extension of the preliminary, first choice method screening set that used only alcohols. The incorporation of a solvent possessing substantially different characteristics enhances the potential in practical applications, while keeping the technical simplicity. Stability and robustness of the additional states of the CSP were characterized. The examined columns of different brands shared the observed behavior. Kinetic stability of a column state is adequate for successful application. The evaluated states of ADMPC provide multiple enantiorecognition potential by using mixtures of IPA and MeCN also considering the pretreatment of the column. Unprecedented double and triple elution order reversals along the composition range supported the versatility of the available states. Our findings further enhance the usefulness of ADMPC-containing CSPs. We provide instructions for the application of the widespread chiral selector in common eluent mixtures to avoid pitfalls regarding reproducibility and robustness.

Photoreversible DNA nanoswitch-based eluent-free strategy for the direct and effective isolation of highly-active thrombin from whole blood.

This study proposes an eluent-free isolation strategy for the direct isolation of thrombin from whole blood via tandem temperature/pH dual-responsive polyether sulfone monolith and photoreversible DNA nanoswitch-functionalized metal-organic framework (MOF) aerogel. Temperature/pH dual-responsive microgel immobilized on polyether sulfone monolith was adopted to remove the matrix complexity of blood sample via size/charge screening effect. Photoreversible DNA nanoswitches, comprising thrombin aptamer, aptamer complementary ssDNA (cDNA) and the azobenzene-modified ssDNA (control DNA), were functionalized on MOF aerogel to offer efficient capturing of thrombin under irradiation of ultraviolet light (365 nm), driven by electrostatic and hydrogen bond interactions. The release of captured thrombin was easily achieved by changing the complementary behaviors of DNA strands via blue light (450 nm) irradiation. Thrombin with purity higher than 95 % can be directly obtained from whole blood using this tandem isolation procedure. Fibrin production and substrate chromogenic tests showed that the released thrombin possessed high biological activity. The photoreversible thrombin capturing-release strategy is merited with eluent-free, avoiding the loss of activity of thrombin in chemical circumstances and undesired dilution, providing a robust guarantee for subsequent application.

Evidence for the Hydration of Some Organic Compounds during Reverse-Phase HPLC Analysis.

Some polar analytes (X) can reversibly form hydrates in water-containing eluents under the conditions of reversed-phase HPLC analysis, X + H2O ⇄ X × H2O. One of the methods to detect their formation is the recurrent approximation of the net retention times of such analytes, tR(C + ΔC) = atR(C) + b, where ΔC = const is the constant step in the variation of the organic modifier content of an eluent. These dependencies are linear if hydrates are not formed, but in the case of hydrate formation, they deviate from linearity under high water content. It has been shown that UV spectroscopic parameters, namely, relative optical densities: Arel = A(λ1)/A(λ2), depend on eluent composition for some organic compounds, but their variations cannot be used as indicators for hydrate formation. The coefficients that characterize the dependence of the analyte retention indices on the organic component concentration of an eluent, dRI/dC, appeared to be the most informative additional criterion for hydration. The values of these coefficients for most polar analytes are largely negative (dRI/dC < 0), whereas, for nonpolar compounds, they are largely positive (dRI/dC > 0).

A review for recent advances on soil washing remediation technologies.

Contaminated soils have caused serious harm to human health and the ecological environment due to the high toxicity of organic and inorganic pollutants, which has attracted extensive attention in recent years. Because of its low cost, simple operation and high efficiency, soil washing technology is widely used to permanently remove various pollutants in contaminated soils and is considered to be the most promising remediation technology. This review summarized the recent developments in the field of soil washing technology and discusses the application of conventional washing agents, advanced emerging washing agents, the recycling of washing effluents and the combination of soil washing and other remediation technologies. Overall, the findings provide a comprehensive understanding of soil washing technology and suggest some potential improvements from a scientific and practical point of view.

A protocol to replace dedication to either normal phase or polar organic mode for chiral stationary phases containing amylose tris(3,5-dimethylphenylcarbamate).

Alteration of the enantiorecognition ability of polysaccharide-based chiral columns in the shipping normal phase (NP) eluent after exposition to polar organic (PO) mode eluents can be conceived as an incomplete hysteresis cycle. Non-standard solvents provide a solution to overcome this issue with immobilized stationary phases, but a procedure was missing so far to regenerate coated stationary phases from the altered state. Recent results with alcohol mixtures within the PO mode showed that an appropriate order of standard solvents may also be suitable to complete hysteresis. Using an analogous approach, a simple protocol was established to regenerate the original NP retentions on various stationary phases containing amylose tris(3,5-dimethylphenylcarbamate) (ADMPC) chiral selector after the change induced by flushing with 2-propanol or ethanol. The members of a chemically diverse compound set indicated that alterations in retentions and selectivities using different brands and types of ADMPC-based stationary phases can be quite different, but the recovery of the original state was very good for all of them. The proposed protocol eliminates the need of the costly dedication of a chiral column with ADMPC selector to either NP or PO mode. Furthermore, the limits of the alcohol content in the mobile phase compositions where the system is free of hysteresis were determined.

Sequential washing and eluent regeneration with agricultural waste extracts and residues for facile remediation of meta-contaminated agricultural soils.

Washing with organic acids and dissolved organic carbon (DOC) is a promising technique for effective removal of potentially toxic metals from agricultural soils and the two key factors are the screening of inexpensive, high-efficiency, and environmentally friendly washing agents and the safe treatment of waste eluent. We used extracts from agro-forestry wastes (pineapple peel, lemon peel, grapefruit peel and gardening crabapple fruit) to develop a facile two-stage sequential washing method (extracts and/or citric acid (CA) and coupled with extracts) and regenerated waste eluent. The washing efficiencies of Cd and Cu were significantly increased by pineapple peel (PP) using two-stage sequential washing with the sequence of PP + CA-PP > CA-PP > PP-PP. The potential pollution risk from soil Cd was lowered by 33.0% from moderate to low risk, and soil nutrient contents increased. 80.9% of Cd and 81.3% of Cu in waste eluent were efficiently removed by the PP residues. The removal mechanisms of metals in soils and eluents by PP washing agents and residues can be attributed to acid activation, cation exchange and complexation between metal ions and carboxyl groups. Therefore, the PP extracts and residues are potentially suitable for the removal of Cd and Cu from polluted agricultural soils and washing waste eluents.

Investigation of the effects of solvent-mismatch and immiscibility in normal-phase × aqueous reversed-phase liquid chromatography.

Comprehensive two-dimensional liquid chromatography (LC × LC) is an attractive separation technique that allows achieving high peak capacities and information on chemical correlations. Unfortunately, its application in industrial practice is still not widespread due to limiting factors such as complex method development, tedious method optimization and solvent-incompatibility (such as solvent-strength mismatch or immiscibility experienced during fraction transfer). A severe case of solvent-incompatibility is encountered in the comprehensive coupling of normal-phase LC and reversed-phase LC (NPLC × RPLC). NPLC × RPLC is considered a desirable LC × LC system, especially for the characterization of synthetic polymers, due to the high orthogonality of the two retention mechanisms. However, its experimental realization often suffers from solvent-injection effects in the RPLC dimension, such as peak-deformation, peak-splitting, or even unretained elution ("breakthrough") of sample components. Such a decrease in performance or loss of retention is highly dependent on the types of solvents used. To explore the boundaries of solvent compatibility, we applied large-volume injections (LVI) of reference analytes (e.g. alkyl benzenes; ethoxylate and propoxylate polymers) dissolved in water-immiscible sample solvents, such as dichloromethane, n-hexane, and isooctane in fast water-based gradient RPLC separations (using methanol or acetonitrile as eluent). It was found that, when using highly aqueous initial gradient conditions, hydrophobic sample diluents were retained and eluted during the applied gradient. Depending on the relative retention of the retained diluent and the sample analytes, good chromatograms for LVI of immiscible solvents were obtained, comparable with injections under ideal conditions. The conclusions from injection experiments in aqueous RPLC were verified by coupling an NPLC system with a gradient from isooctane to tetrahydrofuran and an RPLC system with a gradient from water to acetonitrile in an online comprehensive NPLC × RPLC separation of a mixture of propoxylate polymers. The separation provided separation of the polymers based on their number of hydroxyl end-groups (NPLC) and oligomer chain-length (RPLC), without suffering from significant band-broadening effects due to solvent-mismatch upon injection in the second-dimension RPLC system.

Kinetics and thermodynamics of organo-sulfur-compound desorption from saturated neutral activated alumina.

Desulfurization of liquid fuels mitigates the amount of noxious sulfur oxides and particulates released during fuel combustion. Existing literature on oxidative-adsorptive desulfurization technologies focus on sulfur-in-fuel removal by various materials, but very little information is presented about their desorption kinetics and thermodynamics. Herein, we report for the first time, the mechanism of sulfur desorption from neutral activated alumina saturated with dibenzothiophene sulfone. Batch experiments were conducted to examine the effects of agitation rate, desorption temperature, sulfur content, and eluent type on sulfur desorption efficiencies. Results show enhanced desorption capacities at higher agitation rate, desorption temperature, and initial sulfur content. Desorption efficiency and capacity of acetone were found to be remarkably superior to ethanol, acetone:ethanol (1:1), and acetone:isopropanol (1:1). Desorption kinetics reveal excellent fit of the nonlinear pseudo-second-order equation on desorption data, indicating chemisorption as the rate-determining step. Results of the thermodynamics study show the spontaneous (ΔG° ≤ -2.08 kJ mol-1) and endothermic (ΔH° = 32.35 kJ mol-1) nature of sulfur desorption using acetone as eluent. Maximum regeneration efficiency was attained at 93% after washing the spent adsorbent with acetone followed by oven-drying. Scanning electron microscopy, Fourier transform infrared, and X-ray diffraction spectroscopy analyses reveal the intact and undamaged structure of neutral activated alumina even after adsorbent regeneration. Overall, the present work demonstrates the viability of neutral activated alumina as an efficient and reusable adsorbent for the removal of sulfur compounds from liquid fossil fuels.

Water, electricity and ion exchange; how Hamish Small sustained the evolution of ion chromatography.

Since the development of synthetic ion exchange resins in 1935, the electrochemical properties of these unique materials have intrigued researchers. In 1951, Hamish Small was introduced to synthetic ion exchange resin when he studied the electromigration of cations in these new materials. In 1955, he began working in the ion exchange division of the Physical Research Laboratory at Dow Chemical. In 1975, Small and co-workers described a new analytical tool, ion chromatography, which would revolutionize ion analysis. Twenty years after the commercial introduction of ion chromatography, Small demonstrated how an electrically polarized ion exchange resin bed could be used to produce an acid or base eluent using water as the pumped phase. Today, Small's inventions remain the core technology in state-of-the-art ion chromatography systems for electrolytic eluent production, purification, and suppression. This is a personal account of my collaboration with Hamish and how he sustained the evolution of ion chromatography for more than 50 years.

[Advances of electrodialytic technologies used in ion chromatography].

An electrodialysis device is used to transport salt ions from one solution to another through an ion-exchange membrane under the influence of an applied electric potential. They can produce hydronium or hydroxide ions via electrodialysis water, which can be used for online preparation of eluents or suppression or detection in ion chromatography (IC). As opposed to the manual preparation of eluents or regenerant solutions, electrodialysis devices have the advantages such as green, efficient and high purity. Thus, IC systems equipped with electrodialysis devices dominate the majority uses of IC system, and more importantly, find widespread application. The present thesis briefly summarizes the latest developments in this area.

Separation and preconcentration of perrhenate from ionic solutions by ion exchange chromatography.

Technetium poses an environmental hazard because of its radioactivity and long half-life. It exists in the form of pertechnetate in the environment and can be modeled by the nonradioactive ion perrhenate, since pertechnetate and perrhenate have the same geometry and similar chemical properties. In this research, a new zinc cyclen resorcinarene cavitand (ZCR) column was used in ion chromatography (IC) to efficiently separate perrhenate. Ion chromatography has the advantage of requiring almost no sample preparation for water samples. The ZCR column demonstrated the ability to separate anions: fluoride, chloride, nitrate, sulfate, phosphate, perchlorate, and perrhenate by gradient 2-60 mM NaOH. Unlike other columns, the new column material was selective in retaining perrhenate. The ZCR column also gave a linear range from 2.0 to 1000 mg L-1 for perrhenate with R2 > 0.997. There was a logarithmic relationship between the concentration of perrhenate and its retention time. Excellent perrhenate recovery was achieved on the ZCR column when river water was spiked with perrhenate and perrhenate was preconcentrated. The efficient separations of perrhenate by the ZCR column will potentially assist in pertechnetate separations.

A two-membrane electrodialytic carbonate eluent generator for ion chromatography.

A two-membrane electrodialytic carbonate eluent generator (EDG) for ion chromatography (IC) is described. It is in a sandwich-like configuration, in which the central eluent channel is spatially isolated from two outer regenerant channels by stacked cation exchange membranes (CEMs) and anion exchange membranes (AEMs). A platinum screen electrode is placed in each of two outer regenerant channels. The electrode at the CEMs side is set as an anode with respect to the electrode at the AEMs side being cathode. Potassium carbonate and/or bicarbonate solution is pumped into the regenerant channel as feed solution. The electromigration of carbonate and/or bicarbonate and potassium from feed solution respectively through AEMs and CEMs will form a gas-free eluent. With this configuration, ion transport behavior through AEMs was explored. The device demonstrated good reproducibility, as indicated by the relative standard deviation of retention time of less than 0.08%.

Recycling of Chemical Eluent and Soil Improvement After Leaching.

Ethylenediaminetetraacetic acid disodium salt (EDTA) was selected among various eluents due to its highest removal efficiency for lead (Pb) (43.7%) and zinc (Zn) (57.1%) leaching from Pb-Zn contaminated soil by soil column experiment. Compared with newly prepared EDTA eluent, using recycled EDTA eluent can still leaching down 71.1% of Pb and 63.2% of Zn respectively, which showed the reusable benefits of recycled EDTA eluent. After soils were leached by EDTA, soil quality decline, such as reducing of urease, catalase, invertase activities and microorganism numbers. However, adding 5% nutrition soil or earthworm fertilizer can significantly improve the quality of EDTA leached soil, and promote growth of peas and ryegrass compared with EDTA treatments. Overall, the improvement of EDTA leached soil by adding nutrition soil or earthworm fertilizer is important, and recycled EDTA eluent can recycle and re-use for Pb-Zn contaminated soil remediation.

Retention of acidic and basic analytes in reversed phase column using fluorinated and novel eluent additives for liquid chromatography-tandem mass spectrometry.

This research focuses on retention mechanisms in a LC column with C18 stationary phase when novel eluent additives (HFIP, HFTB and TFE as well as NFTB and perfluoropinacol) are used. The retention factors between novel eluent additives and conventional ones like ammonium acetate and ammonium bicarbonate at different eluent pH values were compared. A simple set of drug-like molecules, widely spread over different logP values, containing protonated and deprotonated acids and bases was selected for this investigation. HFIP, HFTB, NFTB and PP demonstrated strong influence on basic polar analytes in basic medium. These additives drastically increased retention. A decrease in retention was observed for acidic analytes when novel eluent additives were used. Additionally, for the first time, the absolute pH (pHabs) scale was used for expressing the mobile phase pH.

Mismatch between sample diluent and eluent: Maintaining integrity of gradient peaks using in silico approaches.

The mismatch of elution strength between the sample diluent and the eluent causes undesirable peak deformations for large sample volumes in gradient liquid chromatography. The solution to that problem consists in diluting the sample solution in a weak solvent. But the minimum dilution factor has to be determined by the user given some performance objectives. In silico approaches are applied in this work to find such adequate dilution factors. Two calculations methods are proposed for the prediction of peak distortions. The first comprehensive method is based on solving numerically the mass balance equations for all the analytes and the strong solvent. An excellent agreement between the experimental and the calculated gradient chromatograms is observed (sample diluent: acetonitrile/water, 50/50, v/v; injection volume: 15 µL; linear gradient: 5%-95% acetonitrile during 3 min) for five compounds (acetanilide, coumarin, benzoin, bi-naphthol, and dibutylphthalate) injected into a 2.1 × 50 mm column packed with 1.7 µm XBridge-C18 particles. This first method happens to be highly time-consuming and impractical for common users. Experimental work and calculation times are then minimized by applying a second method based on the basics of retention and dispersion of injected pulses. Despite being less accurate than the first method, the agreement between the experimental and calculated peak width remains physically meaningful allowing the experimenter to rapidly guess the required sample dilution factor for any combination of injected volume and strong solvent concentration in the sample solution.

Green analytical chromatographic assay method for quantitation of cyclobenzaprine in tablets, spiked human urine and in-vitro dissolution test.

Cyclobenzaprine hydrochloride, a skeletal muscle relaxant has been determined using an ecofriendly micellar HPLC method in its pure form and tablets. The chromatographic determination was performed using C8 monolithic column (100mm×4.6mm i.d., 5µm particle size) and micellar eluent which was composed of sodium dodecyl sulfate (0.15M), n-propanol (15%), 0.02M orthophosphoric acid (pH 4.5) and 0.3% triethylamine using UV detection of effluent was set at 225nm. The calibration plot showed good linearity over concentration range from 2-40µg/mL. The assay results were statistically validated for linearity, accuracy, precision and specificity according to ICH guidelines. Additionally, regarding USP guidelines, the uniformity of tablets content and in-vitro dissolution test of the tablets was tested using the proposed method. Simple and rapid applicability of the developed method allowed determination of the drug in its pure and tablet dosage forms. Moreover, the major advantage of micellar HPLC technique is to determine the drug in biological fluids without prior extraction steps. Depending on this, the estimation of cyclobenzaprine in spiked human urine was so simple without traditional tedious procedures. The proposed method offers the advantages of sensitivity and simplicity in addition to short analysis time which didn't exceed 6 minutes.