Solvents Of Different Polarity Research Articles

On-line identification of the chemical constituents of Polygoni Multiflori Radix by UHPLC-Q-ToF MS/MS.

Introduction: Polygoni Multiflori Radix (PMR) is a type of Chinese herbal medicine with rich chemical composition and pharmacological activity used widely in medicine and food. However, in recent years, there have been increasing numbers of negative reports about its hepatotoxicity. Identification of its chemical constituents for quality control and safe use is very important. Methods: Three solvents of different polarities (water, 70% ethanol, and 95% ethanol solution) were used to extract the compounds from PMR. Extracts were analyzed and characterized by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-ToF MS/MS) in the negative-ion mode. Results: 152 compounds were detected and identified: 50 anthraquinones, 33 stilbene derivatives, 21 flavonoids, seven naphthalene compounds, and 41 other compounds. Eight other compounds were reported for the first time in the PMR-related literature, and eight other compounds were potentially new compounds. Discussion: This study lays a solid foundation for the screening of toxicity and quality-control indicators of PMR.

Polarization-dependent reconfigurable light field manipulation by liquid-immersion metasurface.

Traditional grating lenses can accumulate phase for adjusting wavefronts, and plasmonic resonances can be excited in metasurfaces with discrete structures for optical field modulation. Diffractive and plasma optics have been developing in parallel, with easy processing, small size, and dynamic control advantages. Due to theoretical hybridization, structural design can combine advantages and show great potential value. Changing the shape and size of the flat metasurface can easily produce light field reflections, but changes in height are rarely cross-explored. We propose a graded metasurface with a single-structure periodic arrangement, which can mix the effects of plasmonic resonance and grating diffraction. As for solvents of different polarities, strong polarization-dependent beam reflections are produced, enabling versatile beam convergence and deflection. Dielectric/metal nanostructures with selective hydrophobic/hydrophilic properties can be arranged by the structural material specification to selectively settle the location of the solution in a liquid environment. Furthermore, the wetted metasurface is actively triggered to achieve spectral control and initiate polarization-dependent beam steering in the broadband visible light region. Actively reconfigurable polarization-dependent beam steering has potential applications in tunable optical displays, directional emission, beam manipulation and processing, and sensing technologies.

Halogenated Schiff base complexes: A new type of molecular probe for specific detection of o-Nitrophenol

o-Nitrophenol (2-NP) is common nitroaromatic (NACs) explosion-prone chemical and environmental pollutant. Due to the similarities in structure and composition of nitroaromatic compounds, it is difficult to distinguish 2-NP from other NACs by conventional probes. Hence, there is an urgent need to develop a simple and fast method for 2-NP trace detection. Herein, we report a new molecular fluorescent sensor constructed from the halogenated Schiff base complex [Zn(3Br5Cl-salpn)(H2O)2] (Zn-χ-L). Identifiability analysis revealed that Zn-χ-L can be used as a highly selective and sensitive molecular sensor for 2-NP, and the fluorescence signal can rapidly identify 2-NP and distinguish 2-NP from other nitroaromatics at a detection limit of (LOD) of 2.20 μM with a response speed of less than 15 s. Unexpectedly, compared with conventional NACs probes, the fluorescence signal of Zn-χ-L is not affected by solvents, which intensity remained essentially constant in the solvents of different polarities. Based on the experimental data, the potential roles of the halogenated ligands, as well as the coordination water molecules in improving the sensitivity and specificity are discussed in detail.

Nature of Linear Spectral Properties and Fast Relaxations in the Excited States and Two-Photon Absorption Efficiency of 3-Thiazolyl and 3-Phenyltiazolyl Coumarin Derivatives.

Coumarin-based fluorescent agents play an important role in the manifold fundamental scientific and technological areas and need to be carefully studied. In this research, linear photophysics, photochemistry, fast vibronic relaxations, and two-photon absorption (2PA) of the coumarin derivatives, methyl 4-[2-(7-methoxy-2-oxo-chromen-3-yl)thiazol-4-yl]butanoate (1) and methyl 4-[4-[2-(7-methoxy-2-oxo-chromen-3-yl)thiazol-4-yl]phenoxy]butanoate (2), were comprehensively analyzed using stationary and time-resolved spectroscopic techniques, along with quantum-chemical calculations. The steady-state one-photon absorption, fluorescence emission, and excitation anisotropy spectra, as well as 3D fluorescence maps of 3-hetarylcoumarins 1 and 2 were obtained at room temperature in solvents of different polarities. The nature of relatively large Stokes shifts (∼4000-6000 cm-1), specific solvatochromic behavior, weak electronic π → π* transitions, and adherence to Kasha's rule were revealed. The photochemical stability of 1 and 2 was explored quantitatively, and values of photodecomposition quantum yields, on the order of ∼10-4, were determined. A femtosecond transient absorption pump-probe technique was used for the investigation of fast vibronic relaxation and excited-state absorption processes in 1 and 2, while the possibility of efficient optical gain was shown for 1 in acetonitrile. The degenerate 2PA spectra of 1 and 2 were measured by an open aperture z-scan method, and the maximum 2PA cross-sections of ∼300 GM were obtained. The electronic nature of the hetaryl coumarins was analyzed by quantum-chemical calculations using DFT/TD-DFT level of theory and was found to be in good agreement with experimental data.

PHYTOCHEMICAL SCREENING AND EVALUATION OF FREE RADICAL SCAVENGING ACTIVITY OF EXTRACT PREPARED FROM LEAVES OF DIGITALIS PURPUREA

Objective: The present study deals with the phytochemical screening and free radical scavenging activity of leaf extract of Digitalis purpurea. Methods: The phytochemicals from the leaf extract of D. purpurea were extracted qualitatively in methanol and confirmed by performing various chemical tests. The free radical scavenging activity of leaves extract was performed using DPPH method with solvents of different polarity, namely, petroleum ether, ethyl acetate, and methanol. The IC50 (50 % inhibition of DPPH) value of free radical scavenging activity of leaf extract was calculated from the graph for each solvent. Results: The leaves extract showed presence of various biologically important phytochemicals such as cardiac glycosides, flavonoids, saponins, anthraquinone, carbohydrates, steroids, phenolic compounds, and aminoacids. The IC50 value of DPPH free radical scavenging activity in petroleum ether, ethyl acetate, and methanolic extract of leaves was found 0.982±0.009 mg/mL, 0.538±0.006 mg/mL and 0.480±0.01 mg/mL respectively. Conclusion: The present study reveals that D. purpurea is medicinally important plant and it can be well exploited for making a diverse range of pharmaceutical drugs. The present investigations also give an insight to scientists and researchers to come up with innovative ideas for effective eradication of different diseases.

Blue‐Emitting 2‐Fluoroaryl‐1,2,3‐Triazole Fluorophores: Synthesis, Theoretical Calculations, and Optical Properties

AbstractA convenient multistep synthetic methodology to afford prospective push‐pull fluorophores based on the N(2)‐fluoroaryl‐1,2,3‐triazole scaffold as valuable functional blocks in the design of blue‐emitting organic materials is reported. The strategy includes five synthetic steps with yields of 60–99% at each stage. The obtained photoactive molecules, 2‐fluoroaryl‐5‐aryl‐4‐methyl‐1,2,3‐triazoles, were found to possess a blue‐violet emission in the range of λ=330–440 nm and the absolute quantum yield of >99% in solvents of different polarities. The electron transfer mechanistic features in the excited states for the developed N(2)‐fluoroaryl‐1,2,3‐triazoles compared to the 1,2,3‐triazoles‐1‐oxide analogues were also studied by using DFT and CIS calculations.

Old Meets New: Mass Spectrometry-Based Untargeted Metabolomics Reveals Unusual Larvicidal Nitropropanoyl Glycosides from the Leaves of Heteropterys umbellata.

The Aedes aegypti (Diptera: Culicidae) mosquito is the vector of several arboviruses in tropical and subtropical areas of the globe, and synthetic pesticides remain the most widely used combat strategy. This study describes the investigation of secondary metabolites with larvicidal activity from the Malpighiaceae taxon using a metabolomic and bioactivity-based approach. The workflow initially consisted of a larvicidal screening of 394 extracts from the leaves of 197 Malpighiaceae samples, which were extracted using solvents of different polarity, leading to the selection of Heteropterys umbellata for the identification of active compounds. By employing untargeted mass spectrometry-based metabolomics and multivariate analyses (PCA and PLS-DA), it was possible to determine that the metabolic profiles of different plant organs and collection sites differed significantly. A bioguided approach led to the isolation of isochlorogenic acid A (1) and the nitropropanoyl glucosides karakin (2) and 1,2,3,6-tetrakis-O-[3-nitropropanoyl]-beta-glucopyranose (3). These nitro compounds exhibited larvicidal activity, possibly potentialized by synergistic effects of their isomers in chromatographic fractions. Additionally, targeted quantification of the isolated compounds in different extracts corroborated the untargeted results from the statistical analyses. These results support a metabolomic-guided approach in combination with classical phytochemical techniques to search for natural larvicidal compounds for arboviral vector control.

Mitigation of Paeoniae Radix Alba extracts on H2O2-induced oxidative damage in HepG2 cells and hyperglycemia in zebrafish, and identification of phytochemical constituents.

Paeoniae Radix Alba (PRA), as a Traditional Chinese Medicine, is widely used in Chinese cuisine due to high health-benefits and nutrition, but the effect of different polarity of solvents on the extraction of antioxidant and hypoglycemic constituents, as well as the major active compounds remain unclear. In this research, 40, 70, and 95% ethanol were firstly applied to extract the polyphenols from PRA, the extraction yields, total phenolics, and total flavonoids content, free radical scavenging ability, α-glucosidase inhibition ability, and anti-glycation ability of extracts were evaluated spectroscopically. The oxidative damage protection, hypoglycemic activity, and alleviation on peripheral nerve damage were evaluated by H2O2-induced HepG2 cells and hyperglycemic zebrafish models. UPLC-QTOF-MS/MS was used to identify the major chemical constituents. The results showed that 40, 70, and 95% ethanol exhibited insignificant difference on the extraction of phenolics and flavonoids from PRA. All extracts showed promising DPPH⋅ and ABTS⋅+ scavenging ability, α-glucosidase inhibition and anti-glycation ability. In addition, PRA extracts could restore the survival rate of HepG2 cells induced by H2O2, and alleviate the oxidative stress by reducing the content of MDA and increasing the levels of SOD, CAT, and GSH-Px. The 70% ethanol extract could also mitigate the blood glucose level and peripheral motor nerve damage of hyperglycemic zebrafish. Thirty-five compounds were identified from 70% ethanol extract, gallotannins, gallic acid and its derivatives, and paeoniflorin and its derivatives were the dominant bioactive compounds. Above results could provide important information for the value-added application of PRA in functional food and medicinal industry.

3-Aryl-5-aminobiphenyl Substituted [1,2,4]triazolo[4,3-c]quinazolines: Synthesis and Photophysical Properties.

Amino-[1,1']-biphenyl-containing 3-aryl-[1,2,4]triazolo[4,3-c]quinazoline derivatives with fluorescent properties have been designed and synthesized. The type of annelation of the triazole ring to the pyrimidine one has been unambiguously confirmed by means of an X-ray diffraction (XRD) method; the molecules are non-planar, and the aryl substituents form the pincer-like conformation. The UV/Vis and photoluminescent properties of target compounds were investigated in two solvents of different polarities and in a solid state. The samples emit a broad range of wavelengths and display fluorescent quantum yields of up to 94% in toluene solutions. 5-(4'-Diphenylamino-[1,1']-biphenyl-4-yl)-3-(4-(trifluoromethyl)phenyl)-[1,2,4]triazolo[4,3-c]quinazoline exhibits the strongest emission in toluene and a solid state. Additionally, the solvatochromic properties were studied for the substituted [1,2,4]triazolo[4,3-c]quinazolines. Moreover, the changes in absorption and emission spectra have been demonstrated upon the addition of water to MeCN solutions, which confirms aggregate formation, and some samples were found to exhibit aggregation-induced emission enhancement. Further, the ability of triazoloquinazolines to detect trifluoroacetic acid has been analyzed; the presence of TFA induces changes in both absorption and emission spectra, and acidochromic behavvior was observed for some triazoloquinazoline compounds. Finally, electronic-structure calculations with the use of quantum-chemistry methods were performed for synthesized compounds.

Compositing Nanostructured Polyaniline with Single-Walled Carbon Nanotubes for High Thermoelectric Performance

In the face of the growing energy crisis and demand on environmental protection, organic thermoelectric (TE) materials have been coming under the spotlight from global researchers as a potential countermeasure. However, there have been few studies on the relationship between the morphology of conductive polymers (CPs) and their TE performance. In this work, nanostructured polyaniline (PANI) was synthesized by interfacial polymerization with the addition of organic solvents of different polarities, including cyclohexane (CYH), carbon tetrachloride (CCl4), toluene (MB), dichloromethane (DCM), and dimethyl sulfoxide (DMSO). Physical mixing PANI with single-walled carbon nanotubes (SWCNTs) affords PANI/SWCNT composites. The composite of PANI nanobelts and 10 wt% SWCNTs achieves a power factor (PF) of 5.13 ± 0.56 μW m-1 K-2 at room temperature (RT). Spherical PANI doped with 50 wt% SWCNTs followed by pressing affords maximal room temperature PF value of 178.10 ± 3.77 μW m-1 K-2. A prototype TE generator (TEG) constructed with seven pairs of legs of as-prepared PANI/SWCNT composites and their n-type counterparts generates voltage of 10.27 ± 0.15 mV and output power of 36.65 ± 1.18 nW at temperature difference of about 50°C. This work illustrates that well-regulated PANI nanostructure could significantly affect the TE properties of PANI/SWCNT composites, which are promising in future preparation of high-performance polymer-based composites regarding the fabrication of TE devices suitable for harnessing low-grade waste heat.

Effect of the sol-gel condition on photostability and optical properties of alkoxy-substituted zinc phthalocyanine in the hybrid glass matrix

The series of sol-gel synthesis was prepared using different organic and inorganic acids and bases, and with different polarity of solvents, and with various concentration of the luminophore to immobilize zinc phthalocyanine to the matrix. Immobilization was achieved by coordination of zinc phthalocyanine in the axial position with isonicotinamide substituted by alkoxysilane. By using siloxane precursors, matrices of variable hydrophilicity were obtained by the sol-gel method in order to select the optimal environment for phthalocyanine immobilization. The optical properties of phthalocyanine (fluorescence and thermoluminescence) of hybrid materials were investigated. Finally, tests of material photostability under the influence of high-energy radiation were carried out with emission measurement in the ultraviolet and visible range. The aim of this study was to optimize the conditions for obtaining hybrid material containing phthalocyanine for potential application in optoelectronic systems, such as, photodiodes.

Synthesis and Optical Properties of a Series of Push-Pull Dyes Based on Pyrene as the Electron Donor.

Fifteen push-pull dyes comprising the tetracyclic polyaromatic pyrene have been designed and synthesized. The optical properties of the fifteen dyes have been examined in twenty-two solvents of different polarities. Surprisingly, contrarily to what is classically observed for push-pull dyes of D-π-A structures, a negative solvatochromism could be found for numerous dyes. The photoluminescence and thermal properties of the dyes were also examined. Theoretical calculations were carried out to support the experimental results.

Diversity of Citrullus colocynthis (L.) Schrad Seeds Extracts: Detailed Chemical Profiling and Evaluation of Their Medicinal Properties.

Seeds and fruits of Citrullus colocynthis have been reported to possess huge potential for the development of phytopharmaceuticals with a wide range of biological activities. Thus, in the current study, we are reporting the potential antimicrobial and anticancer properties of C. colocynthis seeds extracted with solvents of different polarities, including methanol (M.E.), hexane (H.E.), and chloroform (C.E.). Antimicrobial properties of C. colocynthis seeds extracts were evaluated on Gram-positive and Gram-negative bacteria, whereas, anticancer properties were tested on four different cell lines, including HepG2, DU145, Hela, and A549. All the extracts have demonstrated noteworthy antimicrobial activities with a minimum inhibitory concentration (MIC) ranging from 0.9-62.5 µg/mL against Klebsiella planticola and Staphylococcus aureus; meanwhile, they were found to be moderately active (MIC 62.5-250 µg/mL) against Escherichia coli and Micrococcus luteus strains. Hexane extracts have demonstrated the highest antimicrobial activity against K. planticola with an MIC value of 0.9 µg/mL, equivalent to that of the standard drug ciprofloxacin used as positive control in this study. For anticancer activity, all the extracts of C. colocynthis seeds were found to be active against all the tested cell lines (IC50 48.49-197.96 µg/mL) except for the chloroform extracts, which were found to be inactive against the HepG2 cell line. The hexane extract was found to possess the most prominent anticancer activity when compared to other extracts and has demonstrated the highest anticancer activity against the DU145 cell line with an IC50 value of 48.49 µg/mL. Furthermore, a detailed phytoconstituents analysis of all the extracts of C. colocynthis seeds were performed using GC-MS and GC-FID techniques. Altogether, 43 phytoconstituents were identified from the extracts of C. colocynthis seeds, among which 21, 12, and 16 components were identified from the H.E., C.E., and M.E. extracts, respectively. Monoterpenes (40.4%) and oxygenated monoterpenes (41.1%) were the most dominating chemical class of compounds from the hexane and chloroform extracts, respectively; whereas, in the methanolic extract, oxygenated aliphatic hydrocarbons (77.2%) were found to be the most dominating chemical class of compounds. To the best of our knowledge, all the phytoconstituents identified in this study are being reported for the first time from the C. colocynthis.

Successive Solvent Extraction, Characterization and Antioxidant Activities of Cardoon Waste (Leaves and Stems) Extracts: Comparative Study.

The main interest in the valorization of vegetable wastes is due to the peculiarity of their chemical composition in substances that present important properties. Among these substances, antioxidants could replace those industrially manufactured. In the present study, three solvents of different polarities (hexane, ethanol, and water) were applied for the extraction of phenolic compounds from Cynara cardunculus L. waste using two extraction methods: Soxhlet Extraction (SE) and Ultrasonic-Assisted Extraction (UAE). The obtained extracts were then characterized by Fourier-Transform Infrared (FTIR) spectroscopy and spectrophotometric determination of Total Phenolics (TPC), Total Flavonoids (TFC), and Condensed Tannins (CT). Total Antioxidant Capacity (TAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity of ethanol and water extracts of leaves and stems were also evaluated. High extraction yields were obtained by UAE. Water extracts had high yield regardless of the technique used for leaves and stems, and these extracts showed high TAC of 534.72 ± 3.83 mg AAE/g FM for leaves and 215.70 ± 8.87 mg AAE/g FM (mg of ascorbic acid equivalent per g of FM) for stems, and IC50 of 2077.491 μg/mL for leaves and 1248.185 μg/mL for stems. We explain the latter by the high total phenolic contents (TPCs), which reach 579.375 ± 3.662 mg GAE/g FM (mg of gallic acid equivalents per g of fresh matter) for leaves and 264.906 ± 3.500 mg GAE/g FM for stems. These results confirmed that the leaves and stems of the studied cardoon waste were, indeed, interesting sources of natural antioxidants.

A General Approach to Stabilize Nanocrystal Superlattices by Covalently Bonded Ligands

Self-assembled inorganic nanocrystal (NC) superlattices are powerful material platforms with diverse structures and emergent functionalities. However, their applications suffer from the low structural stability against solvents and other stimuli, due to the weak interparticle interactions. Existing strategies to stabilize NC superlattices typically require the design and incorporation of special ligands prior to self-assembly and are only applicable to superlattices of certain NCs, ligands, and structures. Here we report a general method to stabilize superlattices of various NC compositions and structures via strong, covalently bonded ligands. The core is the use of light-triggered, nitrene-based cross-linkers that do not interfere the self-assembly process while nonspecifically and effectively bonding the native ligands of NCs. The stabilized 2D and 3D superlattices of metal, semiconductor, and magnetic NCs retain their structures when being exposed to solvents of different polarities (from toluene to water) and show high thermal stability and mechanical rigidity. This can further stabilize binary NC superlattices, beyond those achievable in previous methods. Stabilized superlattices show robust and reproducible functionalities, for instance, when serving as reusable substrates for surface enhanced Raman spectroscopy. These results create more possibilities in exploiting the impressive library of NC superlattices in a broad scope of applications.

Yellow-Green and Blue Fluorescent 1,8-Naphthalimide-Based Chemosensors for Metal Cations

Two new 1,8-naphthalimides (NI), emitting yellow-green or blue fluorescence depending on the type of substituents at the C-4 position, have been synthesized and characterized. Their basic photophysical characteristics have been investigated in organic solvents of different polarities. Their ability to detect metal ions (Ag+, Cu2+, Zn2+ Ca2+, Mg2+, Ni2+, and Fe3+) has been studied in DMF solution. The amino (NI1) and alkoxy (NI2) functionalized 1,8-naphthalimides exhibit different optical and metal ion sensing properties attributable to the nature of the C-4 substituents. In addition, theoretical calculations based on the affordable but effective density functional theory (DFT) and time-dependent DFT (TDDFT) methods were performed in order to investigate the geometric and electronic structure of the title NI compounds.

Construction of carbon dots having aggregation-induced emission and intramolecular charge transfer properties

Aggregation-induced emission–fluorescent carbon dots (AIE-CDs) have applications in both academia and industry. In this study, triphenylamine-based CDs (T-CDs) prepared in acetic acid showing both intramolecular charge transfer (ICT) and aggregation-induced emission (AIE) were designed and synthesized. Intriguingly, the T-CDs fluoresced with different colors in solvents of different polarities: for example, in acetic acid, they showed blue fluorescence. In contrast, in the presence of water, aggregation and precipitation occurred because of the hydrophobic nature of the compound. With time, the increasing aggregation resulted in a loss in intramolecular rotational degrees of freedom, resulting in bright red emissions as a result of ICT and AIE. Interestingly, the T-CDs could be used for intracellular imaging and the lysosome-specific targeting of live cells.

Conformational Screening of Arbidol Solvates: Investigation via 2D NOESY.

Understanding of the nucleation process's fundamental principles in saturated solutions is an urgent task. To do this task, it is necessary to control the formation of polymorphic forms of biologically active compounds. In certain cases, a compound can exist in a single polymorphic form, but have several solvates which can appear in different crystal forms, depending on the medium and conditions of formation, and show different pharmaceutical activity. In the present paper, we report on the analysis of Arbidol conformational preferences in two solvents of different polarities-deuterated chloroform and dimethyl sulfoxide-at 25 °C, using the 2D NOESY method. The Arbidol molecule has various solvate forms depending on the molecular conformation. The method based on the nuclear Overhauser effect spectroscopy was shown to be efficient in the analysis of complex heterocyclic compounds possessing conformation-dependent pseudo-polymorphism. It is one of the types of polymorphism observed in compounds forming crystal solvates. Combined use of NMR methods and X-ray data allowed determining of conformer populations of Arbidol in CDCl3 and DMSO-d6 which were found to be 8/92% and 37/63%, respectively. The preferred conformation in solution is the same that appears in stable crystal solvates of Arbidol.

Physico-chemical characterization of betel leaf and its effect on oxidative stability and shelf-life of coconut oil

Plant-based bioactive components have shown immense potential and have recently gained considerable attention. Nevertheless, studies pertaining to betel leaves are scarce. The current study investigated the physio-chemical, antioxidant, and antimicrobial properties of two cultivars of Piper betel named Nadan and Koottakkodinadan. The study also evaluated the effects of ethanolic extract of Koottakkodinadan on the storage stability of the coconut oil at room temperature. Both cultivars contained high amounts of sodium, potassium, calcium, and iron. The bioactive components of betel leaf were extracted using solvents of different polarities. The acetone extract of Koottakkodinadan had the highest phenolic content, (154.3 ± 0.21 mg GAE/g), while the DMSO and dichloromethane extract of Nadan had the highest tannin content (100 ± 0 mg TAE/g) and flavonoid content (37.5 ± 0.2 mg RE/g). Nevertheless, the methanol extract of the Nadan cultivar showed high total antioxidant activity by DPPH and ABTS assay. It also showed significant antimicrobial properties against Staphylococcus aureus. The acetone extract of Koottakkodinadan showed potent antimicrobial effects against E. coli, which could be attributed to phenolic content. The coconut oil storage stability showed promising results when treated with ethanolic extract, emphasizing the significance of betel leaf extracts in the food and pharmaceutical industry.

Negative solvatochromism and sign inversion of circularly polarized luminescence in chiral exciplexes as a function of solvent polarity

The properties of chiral donor–acceptor systems displaying CPL sign inversion are investigated in solvents of different polarity. The solvent enables control of their deexcitation pathways favoring either locally excited or charge-transfer states.