Single Crystal XRD Studies Research Articles

An Insight Into Experimental and Theoretical Investigation of Structure, Photophysical Property by ESIPT Processes and Biological Activities of Schiff Base Copper Complex

ABSTRACTA novel Schiff base (BSSMO) and its copper complex have been synthesized, and their structure was delineated using single crystal XRD studies. Computational techniques were used to design and evaluate BSSMO‐based luminophores, revealing a significant intramolecular hydrogen bond within the molecule. Understanding ESIPT is crucial for optimizing photophysical and luminophore properties of organic molecules, especially for advancing optoelectronic devices. The study also explored the mechanisms of GSIPT and ESIPT for these BSSMO‐based luminophores using transition state theory, charge distribution, molecular orbital analysis, and quantum theory of atoms in molecules. Results advocated that BSSMO‐L2 exhibits higher absorption compared with BSSMO‐L1 and the same trend is observed in emission spectral studies. However, the intensity of enol emissions in BSSMO‐L2 is lower than that of keto (BSSMO‐L3) emissions and the S1 (Keto form) emission of BSSMO‐L3 shows significantly larger values, making it attractive for optoelectronic devices. The findings offer valuable insights for the development of ESIPT emitters with distinct photophysical properties. The in silico antidiabetic study of BSSMO‐L2 explores the interaction with PPAR‐γ protein, revealing a moderate affinity and stable complex, enhancing its bio‐potential for future applications. The in vitro anticancer study of Cu‐BSSMO‐L2 complex shows a potential anticancer effect through mitochondrial and extrinsic death receptor mediated pathways. These insights contribute to the design of novel benzenesulfonamide‐based bioactive molecules.

Hardness, VSM, cyclic voltammetric and DFT studies of Cesium Sulphate-doped TGS crystals

The creation of capacitors, transducers, sensors, and infrared detectors utilise the nonlinear, ferroelectric triglycine sulphate (TGS) crystal. Cesium sulphate is incorporated into the lattice of TGS crystal to change its characteristics. Cesium sulfate-doped triglycine sulphate (CSTGS) single crystals were produced using a solution approach and a slow evaporation process. The title crystal was studied by single crystal XRD, FTIR, hardness, VSM, cyclic voltammetric and density functional theoretical (DFT) studies and the results are discussed in this paper.

Multicolor Emission in o-Carborane Derivatives Induced by Conformation Diversity through C-C Double Bond Substitution.

Rationally designed molecules with versatile conformations are ideal candidates for creating challenging single component-based multicolor emissive materials. Herein, a new strategy is presented by introducing a C-C double bond in an o-carborane derivative. Compared to a linear connection using a C-C triple bond (CbPyY), a C-C double bond connection (CbPyE) exhibited a zigzag structure and unique fluorescence behavior. Yellow, yellowish orange, and red crystals or films of CbPyE can be obtained, while only orange ones of CbPyY were achieved under the same condition. Single crystal XRD and theoretical calculation studies revealed the zigzag structure brings asymmetry to one arm of CbPyE, which causes conformation diversity and leads to the multicolor emission. Interestingly, different emissions showed different responses to acetone vapor; the yellow one showed much higher sensitivity and faster response than the other two. It is believed that the prepared materials and the proposed strategy would contribute to future advances in single-fluorophore-based multicolor emissive materials.

Gold(III) complexes with chloride and cyanopyridines: Facilitated hydrolysis of nitrile ligand to amide and antibacterial activity

A range of novel simple gold(III) compounds has been synthesized in their monocrystalline form, including two previously unknown chloro-complexes of Au3+ with 2-cyanopyridine or 3-cyanopyridine, respectively. Our investigations have revealed the intricate nature of the reaction between 2-cyanopyridine and tetrachloroauric acid, yielding at least three distinct products. The main product, obtained in high yield, is a salt featuring a tetrachloroauric anion and a pyridinium cation stabilized by a hydrogen bond to a further 2-cyanopyridine molecule. Moreover, we observed the in-situ formation of a 2-cyanopyridine-AuCl3 complex, which undergoes hydrolysis of the nitrile bond to yield a picolinamide-Au(III) complex. The complexes were characterized by IR and Raman spectroscopies, NMR spectroscopy, and single-crystal XRD studies. Additional computational studies were conducted to explain unusual spectral features, the observed disparities in the complexation reactions of the three isomeric cyanopyridine ligands and the distinct reactivity of the complex with 2-cyanopyridine. Based on these studies, we propose a mechanism for the catalyzed hydrolysis of the nitrile bond within the Au(III) complex. Finally, we assessed the antimicrobial efficacy of the synthesized gold(III) complexes against a spectrum of bacteria and fungi.

Mixed ligand complexes of Pt(II) Tertiary phosphines and 1-(benzo[d]thiazol-2-yl)-3-phenylthiourea; Crystal structure and DFT of [Pt(C14H9N3S2)(dppe)].CH2Cl2

This paper describes a series of Pt(II) thiourea complexes (1–4). The complexes were synthesized by a reaction between [PtCl2(diphos)] and the ligand 1-(benzo[d]thiazol-2-yl)-3-phenylthioure in the presence of triethylamine, with all reactants present in equimolar amounts. The produced complexes exhibited square planar geometry, which was validated by FT-IR and 1HNMR . Single crystal XRD study revealed that the coordination geometry was distorted square planar in [Pt(Btzt)(dpppe)]. Hirshfeld surface analysis was used to delve deeper into the intermolecular interactions which showed that the combined contribution of H⋯H and H⋯C contacts in the stabilization of the supramolecular assembly was 81.3%. A void analysis was carried out which showed that there was no large cavity in the supramolecular assembly. Theoretical studies were also conducted on [Pt(Btzt)(dpppe)]. The DFT analysis was carried out using the B3LYP Hybrid method in combination with Def2-SVP. The theoretical findings indicated just 3% differences between the theoretical and experimental Figures. Furthermore, the computed HOMO-LUMO gap was 3.4 eV, which was close to the anatase photocatalyst's band gap. DFT predicted that all complexes are non-magnetic with μB=0. There are high electron donation and back-donation between heteroatoms and transition metals in the complexes.

Sporadic binding of perchlorate and dicyanamide ions in trinuclear CuII2NiII complexes with a reduced di-Schiff base ligand: Synthesis, structures and spectral properties

Two new unusual trinuclear linear heterometallic complexes, [(CuLR)2Ni(ClO4)2{CO(CH3)2}2]·0.17H2O (1) and [(CuLR)2Ni(N3C2)2(0.5CH3OH)2] (2) have been synthesized by using [CuLR] as a “metalloligand” (where H2LR=N,N′-bis(2-hydroxybenzyl)-1,3-propanediamine) in presence of anions perchlorate and dicyanamide, respectively. Both the complexes have been diagnosed by elemental analysis, spectroscopic investigations, and single crystal XRD study. In these complexes, the terminal “ligand complex” [CuLR] coordinates to the central nickel atom via double phenoxido oxygen atoms bridge, resulted in a linear trinuclear structures. In both structures, the anions (perchlorate for 1 and dicyanamide for 2) coordinate to the terminal Cu(II) centers of the trinuclear unit (Cu(II)-Ni(II)-Cu(II)) to complete the distorted octahedral geometry around Cu(II); whereas the central Ni(II) metal ion remains in square planar geometry. The binding of perchlorate or dicyanamide to the terminal Cu(II) instead of central Ni(II) ion is rather unusual compared to the complexes of analogous unreduced species.

Synthesis, crystal structure, Hirshfeld, DFT, molecular docking, dynamics studies, and anti-cancer activity of 1-substituted-2-(4-(diethylamino)-2-hydroxyphenyl)- 1H-benzo[d]imidazole-5-ethyl carboxylates

In our present work, a novel series of 1-substituted-2–2-(4-(diethylamino)-2-hydroxyphenyl)- 1H-benzo[d]imidazole-5-ethyl carboxylates were synthesized by an efficient “one-pot” nitro reductive cyclization method using sodium dithionite is reported as potential anticancer agents. IR, 1H & 13C NMR and Mass spectral data confirmed the synthesized compounds' structure. Compound 6a and 6j were confirmed by their single crystal XRD studies. The Hirshfeld surfaces computational method was applied to visualize and analyse the intermolecular interactions within the crystal structure. All the synthesized compounds were assessed for in vitro anticancer activity towards human lung adenocarcinoma A549 cells and breast cancer MCF-7 cell lines. Compound 6j showed a promising activity with an IC50 value 42.7 μg/mL and 89.63 μg/mL against A549 and MCF-7 cell lines respectively. The potent compounds were assessed for cytotoxicity study on human embryonic kidney HEK 293 cell line and found to be non-toxic. Also, apoptosis study indicated compound 6j induced early apoptosis in the A549 lung adenocarcinoma and MCF-7 breast cancer cells. Molecular docking and dynamic simulation studies were carried out on human estrogen receptor protein (PDB: 3ERT).

New mono and dinuclear half-sandwiched organoruthenium(II) complexes: Effect of dinuclearity on the biomolecular interaction and cytotoxicity

The present work describes the syntheses and structural characterization of new half sandwiched mono (complex 1) and dinuclear (complex 2) organoruthenium complexes derived from thiosemicarbazone ligand (L1). The molecular structures of compounds were characterized by analytical and multispectroscopic methods which corroborate well with single crystal XRD studies. DFT and TD-DFT studies of compounds revealed that HOMO electron density in L1 is localised on the thiosemicarbazide moiety while the LUMO electron density is evenly distributed over the ligand. However, in complex 1, the HOMO and LUMO is localised on the arene moiety and partially distributed over metal ion while HOMO and LUMO in complex 2 is equally delocalised throughout the complex. The collaborative preliminary DNA binding studies revealed that complexes 1 and 2 exhibited intercalative mode of interaction. The magnitude of DNA binding strength was determined by calculating Kb, K and Ksv values which demonstrated higher DNA binding propensity of complex 2. Furthermore, HSA binding studies of complexes also revealed better binding efficiency of dinuclear complex in contrast to mononuclear complex. The cytotoxic potential of dinuclear complex 2 was examined against triple negative breast cancer cell lines (MDA-MB-468 and MDA-MB-231) via MTT assay.

Mononuclear and binuclear dioxidomolybdenum(VI) chelates derived from a tridentate ONO donor aroylhydrazone: Spectral, structural, DFT and in silico biological investigations

Four dioxidomolybdenum(VI) complexes, [MoO2(L)]2·H2O (1), [MoO2(L)(py)] (2), [MoO2(L)(3-pic)]·H2O (3) and [MoO2(L)(4-pic)] (4) of a dibasic tridentate ONO donor aroylhydrazone, H2L (where H2L = 3-methoxy-2-hydroxybenzaldehyde-2-furoic acid hydrazone) have been synthesized and well characterized. The stoichiometric reaction of aroylhydrazone with MoO2(acac)2 in methanolic medium yielded phenoxobridged binuclear complex 1 whereas monomeric complexes, 2, 3 and 4 were formed as a result of incorporating different monodentate heterocyclic bases. The tridentate aroylhydrazone coordinates to the MoO22+ core through phenolate oxygen, azomethine nitrogen and iminolate oxygen atoms. Single crystal XRD studies established the coordination geometry of mononuclear dioxidomolybdenum(VI) complexes as distorted octahedron. The crystal structures and various solid state interactions were also investigated here. DFT investigations were conducted to explore the reactivity parameters of the studied aroylhydrazone and complexes. Furthermore, molecular docking analyses unveiled the superior putative binding energy of the investigated compounds in contrast to cisplatin. These findings were particularly pronounced in relation to the selected target proteins, which are indicative of their potential efficacy against lung and breast cancer cell lines.

Crystal structure, spectral characterization, in vitro, molecular docking and DFT studies of pyranopyrazole derivatives

Two Pyranopyrazole derivatives, namely, methyl-11-(2-chlorophenyl)-16-methyl-8-[(4-methylbenzene)sulfonyl], -14-phenyl-12-oxa-8,14,15-triazatetracyclo[8.7.0.02,7.013,17] heptadeca-2(7), 3, 5, 13(17), 15-pentaene-10-carboxylate, C38H37N3S1O5 and butyl‑16-methyl-8-[(4-methylbenzene)sulfonyl]-11,14-diphenyl-12-oxa-8,14,15-triazatetracyclo[8.7.0.02,7.013,17] heptadeca-2(7),3,5, 13(17), 15-pentaene-10-carboxylate, C35 H30 Cl N3 O5 S with phenyl and chlorophenyl substitutions were synthesized successfully, and crystallized by the slow evaporation technique. The pyranopyrazole derivatives were characterized by spectroscopic techniques including UV–visible, FTIR, and mass spectroscopy, and their 3D-structural arrangements were also confirmed by single crystal XRD studies. Both compounds crystallize in the triclinic crystal system with the centrosymmetric space group P-1, which is identified by the X-ray single-crystal structure. These compounds were examined for in vitro and molecular docking studies with the enzymes 1hny, 1pgg, and 4 cox and thus correspond to diabetes and inflammation. The observed results showed better binding energy and inhibition constants for inflammation involving enzymes. The chemical reactivity and electronic arrangement of the compounds have been revealed by DFT studies.

A Crystalline 2D Fullerene‐Based Metal Halide Semiconductor for Efficient and Stable Ideal‐bandgap Perovskite Solar Cells

AbstractDespite advances in mixed tin‐lead (Sn‐Pb) perovskite‐based solar cells, achieving both high‐efficiency and long‐term device stability remains a major challenge. Current device deficiencies stem partly from inefficient carrier transport, originating from defects and improper band energy alignment among the device's interfaces. Developing multifunctional interlayer materials simultaneously addressing the above concerns poses an excellent strategy. Herein, through molecular and crystal engineering, an amine‐functionalized C60 mono‐adduct derivative (C60‐2NH3 = bis(2‐aminoethyl) malonate‐C60) is utilized for the synthesis of the first crystalline fullerene‐based 2D metal halide semiconductor, namely (C60‐2NH3)Pb2I6. Single crystal XRD studies elucidated the structure of the new material, while DFT calculations highlighted the strong contribution of C60‐2NH3 to the electronic density of states of the conduction band of (C60‐2NH3)Pb2I6. Utilization of C60‐2NH3 as an interlayer between a FA0.6MA0.4Pb0.7Sn0.3I3 perovskite and a C60 layer offered superior band energy alignment, reduced nonradiative recombination, and enhanced carrier mobility. The corresponding perovskite solar cell (PSC) device achieved a power conversion efficiency (PCE) value of 21.64%, maintaining 90% of its initial efficiency, after being stored under a N2 atmosphere for 2400 h. This work sets the foundation for developing a new family of functional materials, namely Fullerene Metal Halide Semiconductors, targeting applications from photovoltaics to catalysis, transistors, and supercapacitors.

Synthesis, structural elucidation and characterization of a new organic single crystal Guanidinium adipate monohydrate (GuAM) for nonlinear optical applications

A slow evaporation solution growth process was employed to grow a new organic single crystal Guanidinium adipate monohydrate (GuAM) at room temperature. The structure refinement and cell specifications were elucidated using the X-ray diffraction method. Single crystal XRD study revealed that the grown crystal crystallized in the monoclinic system with unit cell dimensions a = 4.948 (9) Å; b = 16.770 (2) Å; c = 13.554 (2) Å; α and γ = 90.00⁰; β = 95.302⁰ (3), V = 1120.1(3) Å3 and Z = 4. The structure of the crystal was solved by direct method and refined by full matrix least-square method using SHELXL-2018/3 program. The crystalline nature of the sample was evaluated by powder X-ray diffraction (PXRD) study. Fourier transform infrared (FTIR) spectral study was employed to recognize the diverse functional groups present in the synthesized crystal. Optical attributes such as absorbance and emission nature of the GuAM were explored using UV–visible-NIR absorbance study and photoluminescence study respectively. The laser damage threshold value of the formed crystal has been determined by employing an Nd-YAG laser with a wavelength of 1064 nm. The mechanical nature of the GuAM was examined utilizing Vicker's microhardness test and other related parameters were also calculated. TGA-DTA analysis was utilized to estimate the title crystal's thermal stability. The Z-scan technique has been employed to gain insight into the third order nonlinear (NLO) parameters of the GuAM. These findings imply that guanidinium adipate monohydrate (GuAM), a grown single crystal, may be a viable choice for NLO applications.

Synthesis, single crystal XRD, in-vitro, and in-silico studies of polysubstituted tetrahydropyridine as α-amylase inhibitor

The inhibition of α-amylase, an enzyme responsible for the breakdown of starch, has gained opulent attention as a therapeutic target for managing conditions such as diabetes and obesity. In that spirit, the synthesis of densely substituted tetrahydropyridines (PMAAA - PMAAH) as a potential α-amylase inhibitor was herein undertaken. The obtained compounds were thoroughly characterized utilizing 13C-NMR, 1H-NMR, and FT-IR spectroscopic techniques, confirming their structural integrity and purity. Single crystal XRD analysis was also performed to determine the crystal structure of the PMAAA, providing valuable insights into its molecular arrangement and intermolecular interactions. Furthermore, in-silico studies were employed to gain a deeper understanding of the binding interactions between the PMAAA and the active site of α-amylase. The supramolecular assembly was mainly stabilized by H-bonding and explored by Hirshfeld surface analysis. Void analysis predicted the mechanical response of the crystal. α-Amylase inhibition study has shown that PMAAA demonstrated an IC50 value of 40.54±5.0 mg/mL, compared to 8.80±0.21 mg/mL for acarbose as a reference inhibitor. To further explore the interaction of PMAAA with α-amylase, a molecular docking simulation was performed. The docking analysis showed that there is a strong interaction between the PMAAA ligand and α-amylase, with a binding score of −8.4 kcal/mol. These findings pave the way for further research and potential applications in the management of conditions related to α-amylase dysregulation, such as diabetes and obesity.

Fluorescent 2-carbonyl carbazole ketones: Photophysical properties, molecular structure and selective detection of Fe3+ ion

Owing to the diverse implication of carbazole scaffolds in medicinal chemistry, photoelectronic materials and chromophores, the molecules containing the carbazole motif have attracted significant attention over the years. In this work, the vibronic contributions to carbazole structures could facilely change the solvatochromic effect, absorption wavelength, emission wavelength, and Stokes shifts of six 2-carbonyl carbazole ketones fluorophores. Among them, the structure of 1-(1-methyl-9H-carbazol-2-yl)ethanone (3a) were confirmed from single crystal XRD studies. The ratiometric detection of Fe(III) ion of 2-carbonyl carbazole ketone 3a were investigated. Furthermore, it was speculated that chemosensor 3a toward the recognition of Fe3+ cation was dependent on its rigid structure and good coplanarity of the conjugated system, strong intramolecular charge-transfer of 3a.

Fluorescent turn on detection of zinc(II) ion using a hydrazone derived from 4-tert-butyl-2,6-diformylphenol and 3-hydrazineylquinoxalin-2(1H)-one

A polydentate chelating ligand (LH) was synthesized from 4-tertbutyl-2,6-diformylphenol and 3-hydrazineylquinoxalin-2(1H)-one and characterized by mass spectrometry, IR and NMR spectroscopy. LH is weakly fluorescent in EtOH – HEPES buffer (5 mM, pH = 7.4, 8:2, v/v) but exhibited a turn on response with Zn2+ ion having a workable pH range of 6–12 and thus is compatible with biological pH. This sensor is selective for Zn2+ ion, over various metal ions and anions as evident from competition experiments. The probable mechanism of sensing by LH is restriction of ESIPT, PET and C = N isomerization processes and on-set of CHEF process upon coordination to Zn(II) ion. Reversibility was established using EDTA test and applicability in various real samples were evaluated. The calculated limit of detection for Zn2+ ion was 95 nM and Job plot as well as 1H NMR titration suggested a 2:1 binding ratio between Zn2+ ion and LH. From the reaction (in ethanol) of ZnCl2 with LH, in 2:1 ratio, crystalline solids of composition [Zn2(L)Cl3]⋅2H2O 2.5DMF was isolated and its molecular structure was established by single crystal XRD studies. DFT/TDDFT calculation supported the experimental electronic absorption spectrum.

One‐Pot Synthesis of Substituted Benzoxazoles Using Aminophenols and α‐Diazoesters via Annulation of Amide Intermediate Using Co2(CO)8 as a Solid Carbonyl Source

AbstractSynthesis of benzoxazoles via amide formation from amino phenols and α‐diazoesters using dicobalt octacarbonyl as a solid CO source is reported. Our methodology provides a strategy for synthesizing substituted benzoxazoles with methylene linker between the benzoxazole and aromatic ring systems. Substrates with different substituents were employed to produce corresponding benzoxazoles. The synthesized compounds were characterized by using standard spectroscopic and analytical techniques including single crystal XRD studies.

Investigation of corticosteroid-NSAID prodrugs for TNF-α and IL6 receptors, their characterization, single crystal XRD and DFT studies: A combined experimental and theoretical approach

In continuation of our research towards the synthesis of steroidal derivatives, we have synthesized corticosteroids-NSAIDs derivatives 3i-3p as potent anti-inflammatory agents. The products obtained have been characterized using elemental analysis, spectroscopic techniques (FT-IR, 1H NMR, 13C NMR, UV–Vis and mass spectrometry) and product 3k has also been characterized with the help of single crystal X-ray crystallography. The inhibition of pro-inflammatory cytokines is a key approach to abate severe inflammation. In this work a series of corticosteroids-NSAIDs derivatives are reported whose activity were performed on RAW264.7 cell line and have been identified as potential TNF alpha and IL6 inhibitors. Our work demonstrates that these corticosteroid derivatives may be useful for the development of anti-inflammatory agents. Among these derivatives, 11,17-dihydroxy-3,20-diketo-pregn-1,4-diene-21-yl-2-(1,8‑diethyl-1,3,4,9-tetrahydropyrano[3,4-β]indol-1-yl)acetate (3j) was identified as the most potent anti-inflammatory agent, which showed significant anti-inflammatory activity by inhibiting the LPS-induced pro-inflammatory mediator TNF-α and IL6 in a dose-dependent manner without any cytotoxicity.

Pyrene aroylhydrazone-based Pd(II) complexes for DNA/protein binding, cellular imaging and in vitro anticancer activity via ROS production

A novel class of Pd(II) pyrene aroylhydrazone complexes (Pd1–Pd4) of general formula [Pd(L)(PPh3)Cl] (L = pyrene aroylhydrazones) was synthesized and their solid-state structures were determined by single-crystal XRD study. In vitro cytotoxicity of Pd1–Pd4 were examined against two cancer cell lines, HeLa and MCF-7 and two non-cancerous cell line, HEK 293T and 3T3-L1 to assess their selectivity. Among these, Pd2 and Pd3 had the most significant cytotoxic effects in comparison to cisplatin, a traditional anticancer medication featuring undesirable side effects, in tested cancer cell lines. Moreover, they showed noticeably less toxicity towards non-cancerous cell lines. Their interactions with serum albumin were confirmed experimentally as well as with molecular docking. Pd3 was also found to effectively interact with DNA as revealed by DNA interactions studies. Pd3-treated HeLa cells exhibited nuclear apoptotic features such as chromatin condensation and nuclear fragmentation and was also found to induce ROS which further led to decrease in mitochondrial membrane potential in cancer cells. Further, Pd3 was efficiently internalized by HeLa cells as evident from cellular imaging using confocal microscopy. Taken together, we report a novel class of Pd(II) pyrene aroylhydrazone complexes displaying strong protein/DNA binding and anti-cancer activity via ROS generation under in vitro circumstances.

Synthesis, crystal structure, Hirshfeld surface analysis and DFT calculations of 1,2-bis(1-phenylethylidene)hydrazine

The compound 1,2-bis(1-phenylethylidene)hydrazine was synthesized by refluxing acetophenone and hydrazine hydrate in the presence of ethanol. The 1,2-bis(1-phenylethylidene)hydrazine was characterized by various spectroscopic techniques and the 3D molecular structure was confirmed by single crystal X-ray diffraction experiment. The single crystal XRD studies show that the molecule crystallizes in the monoclinc crystal system with the space group P21/n. The intermolecular interactions in the crystal were visualized using Hirshfeld surface analysis. The interaction energies, 3D energy frameworks were computed to visualize the packing modes of the molecules. The density functional theory calculations were performed to optimize the structure using B3LYP/6–31 G(d,p) functional. The energy levels of the frontier molecular orbitals were studied to estimate the kinetic stability, and chemical reactivity of the molecule. MEP surface was generated to investigate the charge distribution over the molecule and to find possible chemical reactive sites in the molecule.

Single crystal XRD and FTIR studies of 4H,4H′-trimethylenedipiperidine-diium hydrogen sulfate hydrate and investigation its catalytic activity in Fischer esterification using the in-situ continuous liquid–liquid extraction technique

Single crystal XRD and FTIR studies of 4H,4H′-trimethylenedipiperidine-diium hydrogen sulfate hydrate and investigation its catalytic activity in Fischer esterification using the in-situ continuous liquid–liquid extraction technique