A direct nitrogen-15 NMR study of neodymium(III)-nitrate complex formation in aqueous solvent mixtures

A study of contact ion-pair formation between the neodymium (III) and nitrate ions in aqueous solvent mixtures has been carried out by a direct, low temperature, nitrogen-15 (¹⁵N) nuclear magnetic resonance (NMR) technique. At low temperatures, –90 to –120°C ligand exchange is slow enough to permit the observation of¹⁵N NMR signals for uncomplexed nitrate ion, and this anion in the primary solvation shell of Nd(III). In aqueous mixtures with inert acetone and Freon-12, resonance signals for Nd(NO₃)²⁺, Nd(NO₃) ₂ ¹⁺ , and two higher complexes are observed. Signal areas indicate these additional species are possibly a combination of the tetra-, penta-, and hexanitrato complexes, but not the trinitrato. In water-methanol, a medium of higher dielectric constant, complexation is much less and signals only for the mono-and dinitrato complexes are observed. The effect of solvent on complexation is demonstrated more clearly by a series of measurements in water-methanol-acetone mixtures.     

A direct nitrogen-15 NMR study of cerium(III)-nitrate complexes in aqueous solvent mixtures

A study of the complex formation which occurs between cerium(III) and nitrate ions in aqueous solvent mixtures has been carried out by a direct, low-temperature, nitrogen-15 (¹⁵N) NMR technique. At temperatures in the range of –95 to –110°C, ligand exchange is slow enough to permit the observation of separate¹⁵N NMR signals for bulk nitrate, and this anion in the cerium(III) principal coordination shell. In water-acetone-Freon-12 mixtures, the spectra reveal the nitrato complexes do not form consecutively. Rather, signals are observed for Ce(NO₃)²⁺, Ce(NO₃) ₂ ¹⁺ , and only two other higher order complexes, even at very high NO ₃ ^– to Ce(III) mole ratios. Signal area evaluations were used to identify the possible higher order complexes. At comparable salt concentrations in aqueous-methanol mixtures, only Ce(NO₃)²⁺ and Ce(NO₃) ₂ ¹⁺ are formed, reflecting a decreased tendency for complexation in media of higher dielectric constant.     

A direct carbon-13 and nitrogen-15 NMR study of samarium(III) complexation with nitrate and isothiocyanate in aqueous solvent mixtures

The extent of inner-shell, contact ion-pairing between samarium(III)-nitrate and in a preliminary manner, samarium(III)-isothiocyanate, has been determined by a direct, low-temperature, multinuclear magnetic resonance technique. In water-acetone mixtures containing Freon-12 or Freon-22, the slow exchange condition is achieved at –110 to –120°C, permitting the observation of¹⁵N NMR resonance signals for bulk and coordinated nitrate. In these mixtures, signals are observed for Sm(NO₃)²⁺, Sm(NO₃) ₂ ⁺ , and two higher complexes, possibly the tetranitrato with either the penta-or hexanitrato.¹H NMR signals for bound water molecules in these mixtures were observed, but accurate hydration numbers can not yed be determined. In anhydrous or aqueous methanol mixtures,¹⁵N NMR signals for only three complexes are observed, with the dinitrato clearly dominating. Using¹⁵N and³⁵Cl NMR chemical shift and linewidth measurements, the superior complexing ability of nitrate compared to perchlorate and chloride, was demonstrated. Successful preliminary¹³C and¹⁵N NMR measurements of Sm³⁺-NCS^– interactions in water-acetone-Freon-22 mixtures also have been made. The¹³C NMR spectra reveal signals for five complexes, presumably Sm(NCS)²⁺ through Sm(NCS) ₅ ^2– . In the¹⁵N NMR spectra, signals for only three complexes are observed (the result of insufficient spectral resolution.) displaced about +240 ppm from bulk anion.     

A direct nitrogen-15 NMR study of praseodymium(III)-nitrate complex formation in aqueous solvent mixtures

A direct, low-temperature nitrogen-15(¹⁵N) NMR technique has been applied to the study of inner-shell complex formation between praseodymium(III) and nitrate ion in aqueous solvent mixtures. In water-acetone mixtures at –95°C, ligand exchange is slow enough to permit the observation of¹⁵N NMR signals for uncomplexed and coordinated nitrate ion, but satisfactory resolution is obtained only by the addition of Freon-12 to these systems for study at –110 to –115°C. Four coordinated nitrate signals are generally observed and a very small signal for an additional complex, or an isomer of one of the others, appears at the highest nitrate concentrations. Signals for the mono-and dinitrato complexes are unambiguously identified, but with the exception of the trinitrato complex, several possibilities exist for the remaining peaks. To overcome excessive viscosity signal broadening, measurements in methanol and ethanol are possible only with praseodymium trifluoromethanesulfonate (triflate). Coordinated nitrate signals in aqueous and anhydrous methanol are observed only for the mono-and dinitrato species, and signal areas indicate a maximum of two moles of nitrate per Pr(III) are complexed. A third signal is evident in the ethanol solution spectra, and the presence of this higher complex was confirmed by area measurement of the fraction of bound nitrate. The extent of complex formation in these solvent systems is attributed to differences in the dielectric constant. A comparison of the complexing tendencies of Pr(III) to other ions studied by this NMR method suggests the possibility of a coordination number change across the lanthanide series. Preliminary¹⁵N NMR results for metal-ion complexes with the isothiocyanate ion are presented.     

A Direct Carbon-13 and Nitrogen-15 NMR Study of Praseodymium(III)- and Neodymium(III)-Isothiocyanate Complex Formation in Aqueous Solvent Mixtures

Multinuclear magnetic resonance spectroscopic studies of the trivalent lanthanide complexes with isothiocyanate have been completed for the praseodymium(III) and neodymium(III) ions. In water–acetone–Freon mixtures, at temperatures low enough to slow ligand exchange, usually –85 to –125°C for isothiocyanate, separate carbon-13 and nitrogen-15 NMR signals can be observed for free anion and NCS^- in each metal–ion complex. For both metal ions, ¹⁵N NMR signals are observed for four complexes, displaced about +1500 ppm downfield from free NCS^- for Pr³⁺ and about +2000 ppm for Nd³⁺. In the ¹³C NMR spectra, only three peaks are observed for the complexes of both metal anions, with signal overlap obscuring the resonance for the fourth complex. However, the metal ion coordination numbers, obtained by integration of the resonance signals, are comparable in the ¹⁵N and ¹³C spectra, approaching a maximum value of about 3. These spectral data indicate the formation of Ln(NCS)²⁺ through Ln(NCS) ₄ ^1- occurs for both lanthanides in these solvent systems, a result also observed previously for Ce³⁺, Sm³⁺, and Eu³⁺ in our laboratory. Attempts to study these complexes in water–methanol were unsuccessful, due to the inability to achieve low enough temperatures to slow ligand exchange sufficiently. Results for NCS^- and Cl^- competitive-binding studies by ³⁵Cl NMR for both metal ions will also be described.     

A direct carbon-13 and nitrogen-15 NMR study of europium(III) complexation-nitrate and europium(III)-isothiocyanate complexation in aqueous solvent mixtures

A direct, low-temperature nuclear magnetic resonance spectroscopic study of europium(III)-nitrate contact ion-pairing has been completed, and preliminary results for europium(III)-isothiocyanate have been obtained. In water-acetone-Freon mixtures, at –110°C to –120°C, four¹⁵N NMR signals are observed for coordinated nitrate ion. Area evaluations of the signals and their concentration dependence indicate the formation of Eu(NO₃)²⁺, Eu(NO₃) ₂ ¹⁺ , and two higher complexes, possibly the tetra-, with either the penta-or hexanitrato. This correlates well with similar¹⁵N NMR results obtained for Ce(III), Pr(III), Nd(III), and Sm(III). As a result of a higher dielectric constant, complex formation is significantly less in water-methanol mixtures, wheein only three complexes form with Eu(NO₃) ₂ ¹⁺ dominating at the highest anion concentrations. Competitive complexing experiments in water-methanol also were made by³⁵Cl NMR chemical shift and linewidth measurements, as well as¹⁵N NMR. Initial experiments with the Eu³⁺-NCS^– system show four coordinated anion signals, displaced from the bulk anion peak by about –250 ppm and –2,500 ppm in the¹³C and¹⁵N NMR spectra, respectively. Area evaluations are consistent with the presence of Eu(NCS)²⁺ through Eu(NCS) ₄ ^1- in these solutions. A consideration of the chemical shifts identified the nitrogen atom as the site of binding in the NCS^–. A discussion of these preliminary results, as well as those for several other metal-ions, will be presented.     

A direct carbon-13 and nitrogen-15 nmr study of europium(iii)-isothiocyanate complex formation in aqueous solvent mixtures

A continuation of the contact ion-pairing studies of the trivalent lanthanides by direct, low-temperature, multinuclear magnetic resonance techniques has been completed for the europium(III)-isothiocyanate system. In water-acetone-Freon-22 solvent mixtures, ligand exchange is sufficiently slow at — 100°C to - 125°C to permit the observation of¹³C and¹⁵N NMR signals for Eu³⁺-NCS^- contact ion-pair complexes. With each nuclide, signals for four complexes are observed, displaced approximately 250 ppm upfield from free anion in the^13C spectra, and 2,500 ppm upfield from bulk NCS^- in the¹⁵N spectra. The concentration dependence of the signal areas is consistent with the formation of Eu(NCS)²⁺ through Eu(NCS) ₄ ^1- , with water molecules completing the solvation shell. In the¹⁵N NMR spectra, the large chemical shifts identified the nitrogen atom as the NCS^- binding site. Also, the observation of two¹⁵N NMR signals for isomers of Eu(NCS) ₂ ¹⁺ was possible in several spectra. In methanol, a medium of higher dielectric constant, complex formation was diminished, with signal area integrations confirming the dominance of Eu(NCS) ₁ ²⁺ . A comparative binding study of Cl^- and NCS^- also was made by³⁵C1 NMR chemical shift and linewidth measurements in water-methanol mixtures. The much stronger coordinating ability of NCS^- was evident in these experiments, but there is a strong possibility of Eu³⁺-Cl^- ion-pairing in the absence of this anion.     

Direct 1H, 13C, and 15N NMR Study of Lanthanum(III), Thulium(III), and Ytterbium(III) Complex Formation with Nitrate and Isothiocyanate

An ¹H, ¹³C, and ¹⁵N NMR study has been completed for the complexes of La(III), Tm(III), and Yb(III) with nitrate and isothiocyanate in aqueous solvent mixtures. Signals for four complexes are observed for both the Tm³⁺–NO₃ ^– and Yb³⁺–NO₃ ^– solutions, with the species identified as the mono-, di-, tetra-, and either the penta - or hexanitrato. These results are consistent with those determined for the nitrate complexes of the Ce(III)–Eu(III) metal ions. The chemical shifts for the Tm(III) and Yb(III) nitrate complexes indicate a pseudocontact binding mechanism prevails. The complexes of diamagnetic La(III) with NO₃ ^– produce three signals in the ¹⁵NO₃ ^– spectra, with assignments paralleling those observed with the paramagnetic lanthanides. Three complexes are formed in the La³⁺–NCS^– solutions, with signals assigned to the mono-, di-, and triisothiocyanato species.     

A direct hydrogen-1, carbon-13, and nitrogen-15 NMR study of lutetium(III)-isothiocyanate complexation in aqueous solvent mixtures

A direct, low-temperature hydrogen-1, carbon-13, and nitrogen-15 nuclear magnetic resonance study of lutetium(III)-isothiocyanate complex formation in aqueous solvent mixtures has been completed. At –100°C to –120°C in water-acetone-Freon mixtures, ligand exchange is slowed sufficiently to permit the observation of separate¹H,¹³C, and¹⁵N NMR signals for coordinated and free water and isothiocyanate ions. In the¹³C and¹⁵N spectra of NCS^–, resonance signals for five complexes are observed over the range of concentrations studied. The¹³C chemical shifts of complexed NCS^– varied from –0.5 ppm to –3 ppm from that of free anion. For the same complexes, the¹⁵N chemical shifts from free anion were about –11 ppm to –15 ppm. The magnitude and sign of the¹⁵N chemical shifts identified the nitrogen atom as the binding site in NCS^–. The concentration dependence of the¹³C and¹⁵N signal areas, and estimates of the fraction of anion bound at each NCS^–:Lu³⁺ mole ratio, were consistent with the formation of [(H₂O)₅Lu(NCS)]²⁺ through [(H₂O)Lu(NCS)₅]^2–. Although proton and/or ligand exchange and the resulting bulk-coordinated signal overlap prevented accurate hydration number measurements, a good qualitative correlation of the water¹H NMR spectral results with those of¹³C and¹⁵N was possible.     

H NMR study of interparticle interactions in the system rare-earth element-acetic acid-diamagnetic salt

Complexation of acetic acid in 5.0 M NaNO₃ at 298 K with yttrium-group rare-earth ions (Dy³⁺, Er³⁺, Tm³⁺, Yb³⁺) in the absence and in the presence of Mg²⁺ ions was studied by ¹H NMR spectroscopy in combination with mathematical simulation of complex equilibria. In the presence of Mg²⁺, the stability constants of monoacetate complexes of rare-earth ions decrease relative to the Mg-free systems.     

A hydrogen-1, nitrogen-15, and chlorine-35 NMR coordination study of Lu(ClO4)3 and Lu(NO3)3 in aqueous solvent mixtures

A coordination study of Lu(III) has been carried out for the nitrate and perchlorate salts in aqueous mixtures of acetone-d₆ and Freon-12 by¹H,¹⁵N and³⁵Cl NMR spectroscopy. At temperatures lower than –90°C, proton and ligand exchange are slow enough to permit the direct observation of¹H resonance signals for coordinated and free water molecules, leading to an accurate measure of the Lu(III) hydration number. In perchlorate solution, in the absence of inner-shell ion-pairing, Lu(III) exhibits a maximum coordination number of six over the allowable concentration range of study, contrasting markedly with the report of values of six to nine or greater as determined by a similar NMR method. The absence of contact ion-pairing was confirmed by³⁵Cl NMR chemical shift and linewidth measurements. Extensive ion-pairing was observed in the nitrate solutions as reflected by the lower Lu(III) hydration numbers of two to three in these systems, the observation of two coordinated water signals, and¹⁵N NMR signals for two complexes. The¹H and¹⁵N NMR spectra and the hydration number could be accounted for by the presence of (H₂O)₄Lu(NO₃)²⁺ and (H₂O)₂Lu(NO₃) ₂ ¹⁺ .     

Direct hydrogen-1, carbon-13, and nitrogen-15 NMR study of magnesium(II)-isothiocyanate complexing

A hydrogen-1, carbon-13, and nitrogen-15 NMR study of magnesium(II)-isothiocyanate complexation in aqueous mixtures has been completed. At temperatures low enough to slow proton and ligand exchange, separate¹H,¹³C, and¹⁵N NMR signals are observed for coordinated and bulk water molecules and anions. The¹H NMR spectra reveal signals for the hexahydrate and the mono-through triisothiocyanato complexes, as well as two small signals attributed to [Mg(H₂O)₅(OH)]¹⁺ and [Mg(H₂O)₄(OH)(NCS)]. Accurate hydration numbers were obtained from signal area integrations at each NCS^– concentration. In the¹⁵N NMR spectra, signals also were observed for the mono-through triisothiocyanato complexes, and a small signal believed to be due to [Mg(H₂O)₄(OH)(NCS)]. Coordination number contributions for NCS^– were measured from these spectra and when combined with the hydration numbers they totalled essentially six at each anion concentration. Signals for [Mg(H₂O)₅(NCS)]¹⁺ through [Mg(H₂O)₃(NCS)₃]^1– also were observed in the¹³C NMR spectra and the area evaluations were comparable to the¹⁵N NMR results. An analysis of the magnitude and sign of the coordinated NCS^– chemical shifts identified the nitrogen atom as the anion binding site. All spectra indicated [Mg(H₂O)₅(NCS)]¹⁺ and [Mg(H₂O)₄(NCS)₂] were the dominat isothiocyanato complexes over the entire range of anion concentrations. The inability to detect evidence for complexes higher than the triisothiocyanato reflects the competitive binding ability of water molecules and perhaps the decreased electrostatic interaction between NCS^– and negatively charged higher complexes.     

Thermodynamics of reactions of complex formation for Ce<Superscript>3+</Superscript> and Er<Superscript>3+</Superscript> ions with glycine in an aqueous solution

Enthalpies of complex formation for glycine (HL^±) with Ce³⁺ and Er³⁺ ions at 298.15 K and the value of the ionic strength of 0.5 (KNO₃) are determined by calorimetric means using two independent procedures. Thermodynamic characteristics of the reactions of formation for complexes of glycine with Ce³⁺ and Er³⁺ ions at various [metal]: [ligand] molar ratios are calculated.     

Exceptionally Inert Lanthanide(III) PARACEST MRI Contrast Agents Based on an 18‐Membered Macrocyclic Platform

We report a macrocyclic ligand based on a 3,6,10,13‐tetraaza‐1,8(2,6)‐dipyridinacyclotetradecaphane platform containing four hydroxyethyl pendant arms (L¹) that forms extraordinary inert complexes with Ln³⁺ ions. The [EuL¹]³⁺ complex does not undergo dissociation in 1 M HCl over a period of months at room temperature. Furthermore, high concentrations of phosphate and Zn²⁺ ions at room temperature do not provoke metal‐complex dissociation. The X‐ray crystal structures of six Ln³⁺ complexes reveal ten coordination of the ligand to the metal ions through the six nitrogen atoms of the macrocycle and the four oxygen atoms of the hydroxyethyl pendant arms. The analysis of the Yb³⁺‐ and Pr³⁺‐induced paramagnetic ¹H NMR shifts show that the solid‐state structures are retained in aqueous solution. The intensity of the ¹H NMR signal of bulk water can be modulated by saturation of the signals of the hydroxy protons of Pr³⁺, Eu³⁺, and Yb³⁺ complexes following chemical‐exchange saturation transfer (CEST). The ability of these complexes to provide large CEST effects at 25 and 37 °C and pH 7.4 was confirmed by using CEST magnetic resonance imaging experiments.     

A Direct Carbon-13 and Nitrogen-15 NMR Study of Samarium(III)–Isothiocyanate Complexation in Aqueous Solvent Mixtures

Multinuclear magnetic resonance studies of trivalent lanthanide inner-shell ion-pairing with nitrate and isothiocyanate are continuing. For NCS^– solutions in water–acetone–Freon mixtures at low temperature, generally –100 to –125°C, ligand exchange is slow enough to permit the observation of ¹³C and ¹⁵N NMR signals for coordinated and free anions. For samariuni(III) solutions, four coordinated NCS^–signals, displaced about +35 ppm and +250 ppm from free anion, are observed in the ¹³C and ¹⁵N NMR spectra, respectively. The ¹³C and ¹⁵N NMR data are complementary, showing a signal area concentration dependence and measured coordination numbers consistent with the formation of Sm(NCS)²⁺ through Sm(NCS) ₄ ¹ . The coordination numbers reach a maximum of about three moles of NCS^– per mole of Sm(III) with both nuclides, a result confirmed by spectral appearance showing the dominance of Sm(NCS)₃ at the highest concentration studied. An analysis of the chemical shifts indicates that binding occurs at the nitrogen atom of NCS^–. In water–methanol, due to the higher dielectric constant of such mixtures, coordination was less extensive. A competitive binding study with Ci^– by ³⁵Ci NMR demonstrated conclusively the superior coordinating ability of NCS^–.     

A High Performance Theory for Thermodynamic Study on the Binding of Human Serum Albumin with Erbium Chloride

A thermodynamic study of the interaction between erbium(III) chloride (Er3＋) and human serum albumin (HSA) was studied at pH＝7.0, 27 and 37 ℃ in phosphate buffer by isothermal titration calorimetry (ITC). The present study reports the thermodynamic parameters that govern HSA-Er3＋ interactions. The extended solvation theory was used to reproduce the enthalpies of HSA-Er3＋ interactions over the whole range of Er3＋ concentrations. The binding parameters recovered from the new model were attributed to the structural change of HSA and its biological activity. The results obtained indicate that there is a set of two identical binding sites for Er3＋ ions with negative cooperativity. The enhancement of complex formation by Er3＋ and concomitant increase in ∆S suggest that the metal ion plays a role in increasing the number of hydrophobic contacts. The binding parameters discovered from the extended solvation model indicate that the stability of HSA molecule is increased as a result of its interaction with Er3＋ ions.     

DNA Intercalating Near-Infrared Luminescent Lanthanide Complexes Containing Dipyrido[3,2-a:2′,3′-c]phenazine (dppz) Ligands: Synthesis,Crystal Structures,Stability, Luminescence Properties and CT-DNA Interaction

In order to create near-infrared (NIR) luminescent lanthanide complexes suitable for DNA-interaction, novel lanthanide dppz complexes with general formula [Ln(NO₃)₃(dppz)₂] (Ln = Nd³⁺, Er³⁺ and Yb³⁺; dppz = dipyrido[3,2-a:2′,3′-c]phenazine) were synthesized, characterized and their luminescence properties were investigated. In addition, analogous compounds with other lanthanide ions (Ln = Ce³⁺, Pr³⁺, Sm³⁺, Eu³⁺, Tb³⁺, Dy³⁺, Ho³⁺, Tm³⁺, Lu³⁺) were prepared. All complexes were characterized by IR spectroscopy and elemental analysis. Single-crystal X-ray diffraction analysis of the complexes (Ln = La³⁺, Ce³⁺, Pr³⁺, Nd³⁺, Eu³⁺, Er³⁺, Yb³⁺, Lu³⁺) showed that the lanthanide’s first coordination sphere can be described as a bicapped dodecahedron, made up of two bidentate dppz ligands and three bidentate-coordinating nitrate anions. Efficient energy transfer was observed from the dppz ligand to the lanthanide ion (Nd³⁺, Er³⁺ and Yb³⁺), while relatively high luminescence lifetimes were detected for these complexes. In their excitation spectra, the maximum of the strong broad band is located at around 385 nm and this wavelength was further used for excitation of the chosen complexes. In their emission spectra, the following characteristic NIR emission peaks were observed: for a) Nd³⁺: ⁴F_3/2 → ⁴I_9/2 (870.8 nm), ⁴F_3/2 → ⁴I_11/2 (1052.7 nm) and ⁴F_3/2 → ⁴I_13/2 (1334.5 nm); b) Er³⁺: ⁴I_13/2 → ⁴I_15/2 (1529.0 nm) c) Yb³⁺: ²F_5/2 → ²F_7/2 (977.6 nm). While its low triplet energy level is ideally suited for efficient sensitization of Nd³⁺ and Er³⁺, the dppz ligand is considered not favorable as a sensitizer for most of the visible emitting lanthanide ions, due to its low-lying triplet level, which is too low for the accepting levels of most visible emitting lanthanides. Furthermore, the DNA intercalation ability of the [Nd(NO₃)₃(dppz)₂] complex with calf thymus DNA (CT-DNA) was confirmed using fluorescence spectroscopy.     

Synthesis and properties of potassium salts of per-<Emphasis Type="Italic">O</Emphasis>-carboxymethyl-calix[4]pyrogallols and their complexes with Cu<Superscript>2+</Superscript>, Fe<Superscript>3+</Superscript>, and La<Superscript>3+</Superscript>

Synthesis of water-soluble potassium salts of carboxymethyl derivatives of calix[4]pyrogallols and dodeca(carboxylatomethyl)tetramethylcalix[4]pyrogallol (L) complexes with transition metal ions (Cu²⁺, Fe³⁺, La³⁺) is described. Their structures in the solid state and in solution were characterized by NMR spectroscopy, ESR, and IR spectroscopy. Calix[4]pyrogallol dodecacarboxylates exist in the rccc-configuration. Calix[4]pyrogallol with methyl substituents at the lower rim in a wide range concentrations exists in water predominantly in the dimeric form. The obtained polynuclear transition metal complexes possess less symmetric structure than potassium salt of calix[4]pyrogallol (K₁₂L). All studied complexes contain water molecules bound by rather strong hydrogen bonds. At room temperature the Fe₄L complex is characterized by the environment of the Fe³⁺ ions close to octahedral. The absence of signals in the ESR spectrum of the Cu₆L complex indicates the strong antiferromagnetic interaction Cu²⁺-Cu²⁺.     

Al3+ solvation in solutions of aluminum nitrate in a protic ionic liquid: Nuclear magnetic resonance verification of the solvation shell composition

The closest environment of Al³⁺ cations was analyzed in detail in solutions of aluminum nitrate in the prototypical protic ionic liquid ethyl ammonium nitrate (EAN) using ¹H and ¹⁴N nuclear magnetic resonance (NMR) spectra. For Al (NO₃)₃–EAN mixtures with different water content, a quantitative analysis of the integral intensities of the ¹H and ¹⁴N signals was carried out and the composition of the first solvation shell of the aluminum cation was refined.