Multidimensional Separation Research Articles

Self-cleaning micro/nano graded porous groove structure fiber membranes by coaxial spinning for purification of dye wastewater.

Adjusting the structure of the membrane and improving its performance proved to be an effective technique for accomplishing efficient dye wastewater purification. Water erosion of polyvinylpyrrolidone (PVP) core in polyacrylonitrile (PAN) nanofiber membrane modified with UiO-66-NH2 was successfully achieved, in this study, using coaxial electrospinning, and ZIF-8 with excellent performance was further epitaxy-grown in situ. Two differently shaped and positively charged MOFs confer strong adsorption capacity (adsorption capacity >2042mg/g) on cationic dyes. In addition, the multi-dimensional separation pores brought by the micro/nano graded porous groove structure and MOFs not only make the membrane have excellent static adsorption performance, but also have excellent dynamic separation performance under the influence of toxic heavy ions (separation efficiency >99 %; Flux >1666Lm-2 h-1 bar-1). More importantly, this special structure of the membrane has an excellent photocatalytic activity for the dye, so the membrane can be used for a long time in a green and environmentally friendly way. Together, membranes show a significant deal of potential for the treatment of wastewater containing dyes due to the combination of these outstanding characteristics.

Toward Multi-Dimensional Separation of Nanoparticles in Tubular Centrifuges

The processing and preparation of particulate products is an important process in modern industry and science. The enormous potential for innovation in research and development is due to the complex interactions of solids with their environment. The aim of advanced particle production is to achieve high yields of narrowly distributed particle sizes, shapes or material compositions that provide advantageous product specifications. The integration of solid–liquid separation into these processes expands the process engineering scope in terms of product quality and efficiency. Designing these processes to accommodate a wide range of separation characteristics at small-particle-size scales is a major challenge. Taking these aspects into account, the present work aims to improve a dynamic simulation tool for tubular centrifuges that models the time- and space-dependent mass transport and thus, for the first time, can predict separation outcomes when processing both single- and multi-component systems. Utilizing an optical measurement technique, nanosuspension properties can be measured in real time during separation to support model validation. The simulation results align closely with experimental findings and offer plausible insights when addressing multi-dimensional property distributions of non-spherical particles. This study contributes to advanced modeling of separation experiments in tubular centrifuges in real time, taking into account multiple particle properties such as material density and particle form.

Characterization of Sugammadex-Related Isomeric Cyclodextrin Impurities Using Cyclic Ion Mobility High-Resolution Mass Spectrometry.

Cyclic ion mobility-mass spectrometry (cIM-MS) is a powerful technique for separating and identifying isomeric mixtures of compounds. When coupled with chromatography, cIM-MS creates a multidimensional separation system, with high resolving power and peak capacity. In this study, we report the cyclic ion mobility separation and high-resolution mass spectrometry identification of four regioisomers of a Sugammadex-related impurity, abbreviated as Di-OH-SGM. Separation using multipass cyclic ion mobility was achieved by selecting the [M + 2Na]2+ ion, while other adducts, such as [M + Na]+, [M + 2H]2+, [M + H + Na]2+, and [M - 2H]2- did not yield isomer separation. Two methods were developed for ion mobility separation of the isomers: a conventional multipass method and a slicing method. Isomer assignment was based on the characteristic fragment ions. The collision cross section values (cTWCCSN2) of the resolved cyclodextrin isomers were also determined. Ion mobility separation of structurally different fragment ions was demonstrated. Additionally, by coupling cIM-MS with reversed-phase liquid chromatography (HPLC-cIM-MS), two-dimensional separation of the isomers was achieved. The isomers, separated using HPLC-cIM-MS, were identified with the same approach as with cIM-MS alone, and their elution order provided insights into their relative hydrophobicity.

PEPPI-MS: gel-based sample pre-fractionation for deep top-down and middle-down proteomics.

Top-down analysis of intact proteins and middle-down analysis of proteins subjected to limited digestion require efficient detection of traces of proteoforms in samples, necessitating the reduction of sample complexity by thorough pre-fractionation of the proteome components in the sample. SDS-PAGE is a simple and inexpensive high-resolution protein-separation technique widely used in biochemical and molecular biology experiments. Although its effectiveness for sample preparation in bottom-up proteomics has been proven, establishing a method for highly efficient recovery of intact proteins from the gel matrix has long been a challenge for its implementation in top-down and middle-down proteomics. As a much-awaited solution to this problem, we present an experimental protocol for efficient proteoform fractionation from complex biological samples using passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry (PEPPI-MS), a rapid method for extraction of intact proteins separated by SDS-PAGE. PEPPI-MS allows recovery of proteins below 100 kDa separated by SDS-PAGE in solution with a median efficiency of 68% within 10 min and, unlike conventional electroelution methods, requires no special equipment, contributing to a remarkably economical implementation. The entire protocol from electrophoresis to protein purification can be performed in <5 h. By combining the resulting PEPPI fraction with other protein-separation techniques, such as reversed-phase liquid chromatography and ion mobility techniques, multidimensional proteome separations for in-depth proteoform analysis can be easily achieved.

Multidimensional Particle Separation by Tilted-Angle Standing Surface Acoustic Waves—Physics, Control, and Design

Lab-on-a-Chip devices based on tilted-angle standing surface acoustic waves (tasSAWs) emerged as a promising technology for multidimensional particle separation, highly selective in particle size and acoustic contrast factor. For this active separation method, a tailored acoustic field is used to focus and separate particles on stationary pressure nodes by means of the acoustic radiation force. However, additional non-linear acoustofluidic phenomena, such as the acoustically induced fluid flow or dielectrophoretic effects, are superimposed on the separation process. To obtain a particle separation of high quality, control parameters that can be adjusted during the separation process as well as design parameters are available. The latter are specified prior to the separation and span a high-dimensional parameter space, ranging from the acoustic wavelength to the dimensions and materials used for the microchannel. In this paper, the physical mechanisms to control and design tasSAW-based separation devices are reviewed. By combining experimental, semi-analytical, and numerical findings, a critical channel height and width are derived to suppress the influence of the acoustically induced fluid flow. Dealing with the three-dimensional nature of the separation process, particles are focused at different height levels of equal force balance by implementing a channel cover of high acoustic impedance while achieving an approx. three-times higher acoustic pressure. Using this improved channel design, the particle shape is identified as an additional separation criterion, rendering the continuous acoustofluidic particle separation as a multidimensional technology capable of selectively separating microparticles below 10 μm with regard to size, acoustic contrast, and shape.

Identification and Structural Elucidation of Acylsugars in Tomato Leaves Using Liquid Chromatography-Ion Mobility-Tandem Mass Spectrometry (LC-IM-MS/MS).

Leaves of tomato plants contain various glandular trichomes that produce a wide range of metabolic products including acylsugars, which may serve as a defense mechanism against various insect pests. Acylsugars exhibit significant structural diversity, differing in their sugar cores, acylated positions, and type of acyl chains. This work demonstrated a comprehensive approach using multidimensional separation techniques, specifically liquid chromatography-ion mobility-tandem mass spectrometry (LC-IM-MS/MS), for structural characterization, and the discrimination of different tomato plants (one cultivar and five accessions) was demonstrated using tomato leaf extracts; six genotypes from five species of Solanum were represented. As a result, we identified 16 acylsugars through their molecular formulas and annotations using LC and MS analyses. The incorporation of ion mobility (IM) analysis revealed an additional 9 isomeric forms, resulting in a comprehensive total of 25 isomeric acylsugars identified. Furthermore, the experimental collision cross section (CCSexp) values agreed reasonably well with the corresponding predicted values (CCSpred), with an overall estimated error of less than 2%. These findings pave the way for research into how the different structural isomers of acylsugars might influence the self-defense mechanism in plants. Moreover, this work demonstrated that the investigated cultivar and accessions of tomatoes can be distinguished from each other based on their metabolite profile, e.g., acylsugars, with principal component analysis (PCA) and linear discriminant analysis (LDA) statistical models, yielding a prediction rate of 98.3%.

Coffee: Lighting Its Complex Ground Truth and Percolating Its Molecular Brew

Coffee is one of the most widely traded commodities worldwide and its popularity is only increasing. The International Coffee Organisation (ICO) reported a 6% increase in global production in 2020 to 10.5 million tonnes. Coffee production is quite involved (from sowing to harvesting, processing, packaging, and storage); consequently, the industry faces major challenges in terms of the assessment of its quality, flavour, and the components which contribute to coffee’s characterisation, as well as the sustainability of coffee production and global trade. This has prompted multiple studies on the nature of the aroma and taste of the many varieties of coffee around the world, which has resulted in the identification of approximately 1000 volatile compounds and the development and implementation of upwards of 100 lexicons to describe the specific sensory characteristics of coffee. The complex nature of coffee has necessitated the development and incorporation of new analytical methodologies, such as multidimensional separation technologies and spectroscopy coupled with multivariant analysis, to qualify the essential characteristics of coffee’s flavour. This work aims to review the research on coffee’s flavour, covering the roasting process of coffee beans, the volatile and non-volatile components generated by this process, and the chemical reactions responsible for their formation, as well as coffee’s sustainability, the coffee value chain, and various forms of regulation, particularly the current emphasis on ‘fair trade’.

Proteomic and Lipidomic Plasma Evaluations Reveal Biomarkers for Domoic Acid Toxicosis in California Sea Lions.

Domoic acid is a neurotoxin secreted by the marine diatom genus Pseudo-nitzschia during toxic algal bloom events. California sea lions (Zalophus californianus) are exposed to domoic acid through the ingestion of fish that feed on toxic diatoms, resulting in domoic acid toxicosis (DAT), which can vary from mild to fatal. Sea lions with mild disease can be treated if toxicosis is detected early after exposure. Therefore, rapid diagnosis of DAT is essential but also challenging. In this work, we performed multiomics analyses, specifically proteomic and lipidomic, on blood samples from 31 California sea lions. Fourteen sea lions were diagnosed with DAT based on clinical signs and post-mortem histological examination of brain tissue, and 17 had no evidence of DAT. Proteomic analyses revealed 31 statistically significant proteins in the DAT individuals compared to the non-DAT individuals (adjusted p < 0.05). Of these proteins, 19 were decreased in the DAT group of which three were apolipoproteins that are known to transport lipids in the blood, prompting lipidomic analyses. In the lipidomic analyses, 331 lipid species were detected with high confidence and multidimensional separations, and 29 were found to be statistically significant (adjusted p < 0.05 and log2(FC) < -1 or >1) in the DAT versus non-DAT comparison. Of these, 28 were lower in the DAT individuals, while only 1 was higher. Furthermore, 15 of the 28 lower concentration lipids were triglycerides, illustrating their putative connection with the perturbed apolipoproteins and potential use in rapid DAT diagnoses.

The rCOS framework for multi-dimensional separation of concerns in model-driven engineering

The software industry increasingly turns to Model-Driven Engineering (MDE) to mitigate complexity by automating model creation and transformation. Many organisations are pursuing Integrated Development Platforms (IDPs) to enhance automation in their software development processes within MDE. However, the adoption of MDE and engagement with IDPs remain limited due to concerns over their efficacy. We address these challenges in this review paper by introducing a framework for the formal refinement of component and object systems (rCOS). It provides: (1) a formal theory that consists of a modelling language (named OPL) with a calculus of refinement for object-oriented models and component models; (2) a suite of analysis and design techniques that facilitate abstractions and decompositions, leading to a multidimensional separation of concerns; and (3) an IDP (named rCOS Modeller) that supports modelling, design and verification from requirements elicitation through to coding. By advocating for an rCOS-enabled multidimensional approach to separating concerns, this paper offers a comprehensive solution to the challenges facing MDE and IDPs, paving the way for their successful implementation in practice. By delineating the emerging challenges and prospects associated with integrating formal methods for modelling and designing human-cyber–physical systems (HCPS), we show the potential of extending rCOS for MDE in HCPS.

Super-resolution electromagnetic imaging with an optical frequency comb sampling via multi-dimensional signal separation

Electromagnetic sources show wide distribution, broad frequency coverage, and numerous quantities, posing challenges for traditional sensing techniques to achieve ultra-wideband, large-scale detection and localization. The “electromagnetic eye” imaging technique, inspired by the human eye, utilizes a Luneberg lens and a wideband optoelectronic sensing array as the electromagnetic “lens” and “retina,” respectively. This technique utilizes femtosecond optical pulse sampling reception to down-convert wideband signals, facilitating rapid, large range, and wideband sensing of multiple targets in complex electromagnetic environments. However, the limited aperture of the Luneberg lens results in diffraction-limited blurring, and optical down-conversion may lead to spectral aliasing, causing time-frequency-space overlap and reduced system resolution. In this paper, the frequency variation of the point spread function (PSF) in the wideband degraded images is analyzed, and a multi-dimensional joint super-resolution algorithm is proposed, which involves joint time-frequency-space diagonalization of eigenmatrices based on convolutional mixing array model. The concept is demonstrated through a four-sources imaging simulation achieving 2° resolution, breaking the Rayleigh limit 7.25 times. Furthermore, experimental results show 4-10 GHz imaging breaks the Rayleigh limit 4.5 times.

Multidimensional Characterization and Separation of Ultrafine Particles: Insights and Advances by Means of Froth Flotation

Particle systems and their efficient and precise separation are becoming increasingly complex. Therefore, instead of focusing on a single separation feature, a multidimensional approach is needed where more than one particle property is considered. This, however, requires the precise characterization of the particle system, which is especially challenging for fine particles with sizes below 10 µm. This paper discusses the benefits and limitations of different characterization techniques, including optical contour analysis, inverse gas chromatography, flow cytometry, and SEM-based image analysis. The separation of ultrafine particles was investigated for a binary system using froth flotation, where a novel developed flotation apparatus is used. A special focus was placed on the multidimensional evaluation of the separation according to the particle properties of size, shape, and wettability, which was addressed via multivariate Tromp and entropy functions. The results emphasize the intricacy of the flotation process and the complex interaction of the individual particle properties and process parameters. The investigations contribute to the understanding of the characterization of particulate properties as well as the separation behavior of ultrafine particles via froth flotation, especially in the case of a multidimensional approach.

A water-resistant, ultrathin, conformable organic photodetector for vital sign monitoring.

Ultrathin flexible photodetectors can be conformably integrated with the human body, offering promising advancements for emerging skin-interfaced sensors. However, the susceptibility to degradation in ambient and particularly in aqueous environments hinders their practical application. Here, we report a 3.2-micrometer-thick water-resistant organic photodetector capable of reliably monitoring vital sign while submerged underwater. Embedding the organic photoactive layer in an adhesive elastomer matrix induces multidimensional hybrid phase separation, enabling high adhesiveness of the photoactive layer on both the top and bottom surfaces with maintained charge transport. This improves the water-immersion stability of the photoactive layer and ensures the robust sealing of interfaces within the device, notably suppressing fluid ingression in aqueous environments. Consequently, our fabricated ultrathin organic photodetector demonstrates stability in deionized water or cell nutrient media over extended periods, high detectivity, and resilience to cyclic mechanical deformation. We also showcase its potential for vital sign monitoring while submerged underwater.

Chromatographic parameters: Transport efficiency—A meaningful alternative to the plate number parameter

The plate number parameter officially defined in GC and LC as N = (tR/σ)2 (tR and σ are peak retention time and standard deviation, respectively, under no-programming conditions) can be substantially smaller than the actual number of plates. Therefore, N does not always represent what its name implies it does, and its interpretation is not clear. This is even more so for the extensions of N to multidimensional separations. N has other shortcomings. Metrics of separation performance of chromatographic analyses (peak resolution, Rs, peak capacity, nc, and others) are not proportional to N.A column transport efficiency, Q = tR/σ, is free of the shortcomings of N. Metrics Rs and nc are proportional to Q. Q also has a transparent interpretation – it is the number of σ-wide segments in the time-intervals of certain significance in static and programmable GC and LC analyses. Q also has meaningful extensions to multi-dimensional separations.

Hyphenation of supercritical fluid chromatography and trapped ion mobility-mass spectrometry for quantitative lipidomics

BackgroundLipidomics studies require rapid separations with accurate and reliable quantification results to further elucidate the role of lipids in biological processes and their biological functions. Supercritical fluid chromatography (SFC), in particular, can provide this rapid and high-resolution separation. The combination with trapped ion mobility spectrometry (TIMS) has not yet been applied, although the post-ionization separation method in combination with liquid chromatography or imaging techniques has already proven itself in resolving isomeric and isobaric lipids and preventing false identifications. However, a multidimensional separation method should not only allow confident identification but also provide quantitative results to substantiate studies with absolute concentrations. ResultsA SFC method was developed and the hyphenation of SFC and TIMS was further explored towards the separation of different isobaric overlaps. Furthermore, lipid identification was performed using mass spectrometry (MS) and parallel accumulation serial fragmentation (PASEF) MS/MS experiments in addition to retention time and collision cross section (CCS). Quantification was further investigated with short TIMS ramps and performed based on the ion mobility signal of lipids, since TIMS increases the sensitivity by noise filtering. The final method was, as an exemplary study, applied to investigate the function of different ceramide synthases (CerS) in the nematode and model organism Caenorhabditis elegans (C. elegans). Loss of three known CerS hyl-1, hyl-2 and lagr-1 demonstrated different influences on and alterations in the sphingolipidome. SignificanceThis method describes for the first time the combination of SFC and TIMS-MS/MS, which enables a fast and sensitive quantification of lipids. The results of the application to C. elegans samples prove the functionality of the method and support research on the metabolism of sphingolipids in nematodes.

Multidimensional Separation by Magnetic Seeded Filtration: Theoretical Study

Magnetic seeded filtration (MSF) is a multidimensional solid–liquid separation process capable of fractionating a multimaterial suspension based on particle size and surface properties. It relies on the selective hetero-agglomeration between nonmagnetic target and magnetic seed particles followed by a magnetic separation. Experimental investigations of multimaterial suspensions are challenging and limited. Therefore, a Monte Carlo model for the simulation of hetero-agglomeration processes is developed, validated, and compared to a discrete population balance model. The numerical investigation of both charge-based and hydrophobicity-based separation in an 11-material system, using synthetic agglomeration kernels based on real-world observations, yields results consistent with prior experimental studies and expectations: Although a multidimensional separation is indeed possible, unwanted hetero-agglomeration between target particles results in a reduced selectivity. This effect is more pronounced when separation is based on a dissimilarity rather than a similarity in the separation criterion and emphasizes the advantages of hydrophobicity-based systems. For the first time, 2D grade efficiency functions T(φ,d) are presented for MSF. However, it is shown that these functions strongly depend on the initial state of the suspension, which casts doubt on their general definition for agglomeration-based processes and underlines the importance of a simulation tool like the developed MC model.

Human arm redundancy: a new approach for the inverse kinematics problem.

The inverse kinematics (IK) problem addresses how both humans and robotic systems coordinate movement to resolve redundancy, as in the case of arm reaching where more degrees of freedom are available at the joint versus hand level. This work focuses on which coordinate frames best represent human movements, enabling the motor system to solve the IK problem in the presence of kinematic redundancies. We used a multi-dimensional sparse source separation method to derive sets of basis (or source) functions for both the task and joint spaces, with joint space represented by either absolute or anatomical joint angles. We assessed the similarities between joint and task sources in each of these joint representations, finding that the time-dependent profiles of the absolute reference frame's sources show greater similarity to corresponding sources in the task space. This result was found to be statistically significant. Our analysis suggests that the nervous system represents multi-joint arm movements using a limited number of basis functions, allowing for simple transformations between task and joint spaces. Additionally, joint space seems to be represented in an absolute reference frame to simplify the IK transformations, given redundancies. Further studies will assess this finding's generalizability and implications for neural control of movement.

Capillary zone electrophoresis-high field asymmetric ion mobility spectrometry-tandem mass spectrometry for top-down characterization of histone proteoforms.

Characterization of histone proteoforms with various post-translational modifications (PTMs) is critical for a better understanding of functions of histone proteoforms in epigenetic control of gene expression. Mass spectrometry (MS)-based top-down proteomics (TDP) is a valuable approach for delineating histone proteoforms because it can provide us with a bird's-eye view of histone proteoforms carrying diverse combinations of PTMs. Here, we present the first example of coupling capillary zone electrophoresis (CZE), ion mobility spectrometry (IMS), and MS for online multi-dimensional separations of histone proteoforms. Our CZE-high-field asymmetric waveform IMS (FAIMS)-MS/MS platform identified 366 (ProSight PD) and 602 (TopPIC) histone proteoforms from a commercial calf histone sample using a low microgram amount of histone sample as the starting material. CZE-FAIMS-MS/MS improved the number of histone proteoform identifications by about 3 folds compared to CZE-MS/MS alone (without FAIMS). The results indicate that CZE-FAIMS-MS/MS could be a useful tool for comprehensive characterization of histone proteoforms with high sensitivity.

Using Multidimensional Separations to Distinguish Isomeric Amino Acid-Bile Acid Conjugates and Assess Their Presence and Perturbations in Model Systems.

Bile acids play key roles in nutrient uptake, inflammation, signaling, and microbiome composition. While previous bile acid analyses have primarily focused on profiling 5 canonical primary and secondary bile acids and their glycine and taurine amino acid-bile acid (AA-BA) conjugates, recent studies suggest that many other microbial conjugated bile acids (or MCBAs) exist. MCBAs are produced by the gut microbiota and serve as biomarkers, providing information about early disease onset and gut health. Here we analyzed 8 core bile acids synthetically conjugated with 22 proteinogenic and nonproteogenic amino acids totaling 176 MCBAs. Since many of the conjugates were isomeric and only 42 different m/z values resulted from the 176 MCBAs, a platform coupling liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) was used for their separation. Their molecular characteristics were then used to create an in-house extended bile acid library for a combined total of 182 unique compounds. Additionally, ∼250 rare bile acid extracts were also assessed to provide additional resources for bile acid profiling and identification. This library was then applied to healthy mice dosed with antibiotics and humans having fecal microbiota transplantation (FMT) to assess the MCBA presence and changes in the gut before and after each perturbation.

GeLC-FAIMS-MS workflow for in-depth middle-down proteomics.

Middle-down proteomics (MDP) is an analytical approach in which protein samples are digested with proteases such as Glu-C to generate large peptides (>3kDa) that are analyzed by mass spectrometry (MS). This method is useful for characterizing high-molecular-weight proteins that are difficult to detect by top-down proteomics (TDP), in which intact proteins are analyzed by MS. In this study, we applied GeLC-FAIMS-MS, a multidimensional separation workflow that combines gel-based prefractionation with LC-FAIMS MS, for deep MDP. Middle-down peptides generated by optimized limited Glu-C digestion conditions were first size-fractionated by polyacrylamide gel electrophoresis, followed by C4 reversed-phase liquid chromatography separation and additional ion mobility fractionation, resulting in a significant increase in peptide length detectable by MS. In addition to global analysis, the GeLC-FAIMS-MS concept can also be applied to targeted MDP, where only proteins in the desired molecular weight range are gel-fractionated and their Glu-C digestion products are analyzed, as demonstrated by targeted analysis of integrins in exosomes. In-depth MDP achieved by global and targeted GeLC-FAIMS-MS supports the exploration of proteoform information not covered by conventional TDP by increasing the number of detectable protein groups or post-translational modifications (PTMs) and improving the sequence coverage.

Right in two: capabilities of ion mobility spectrometry for untargeted metabolomics.

This mini review focuses on the opportunities provided by current and emerging separation techniques for mass spectrometry metabolomics. The purpose of separation technologies in metabolomics is primarily to reduce complexity of the heterogeneous systems studied, and to provide concentration enrichment by increasing sensitivity towards the quantification of low abundance metabolites. For this reason, a wide variety of separation systems, from column chemistries to solvent compositions and multidimensional separations, have been applied in the field. Multidimensional separations are a common method in both proteomics applications and gas chromatography mass spectrometry, allowing orthogonal separations to further reduce analytical complexity and expand peak capacity. These applications contribute to exponential increases in run times concomitant with first dimension fractionation followed by second dimension separations. Multidimensional liquid chromatography to increase peak capacity in metabolomics, when compared to the potential of running additional samples or replicates and increasing statistical confidence, mean that uptake of these methods has been minimal. In contrast, in the last 15years there have been significant advances in the resolution and sensitivity of ion mobility spectrometry, to the point where high-resolution separation of analytes based on their collision cross section approaches chromatographic separation, with minimal loss in sensitivity. Additionally, ion mobility separations can be performed on a chromatographic timescale with little reduction in instrument duty cycle. In this review, we compare ion mobility separation to liquid chromatographic separation, highlight the history of the use of ion mobility separations in metabolomics, outline the current state-of-the-art in the field, and discuss the future outlook of the technology. "Where there is one, you're bound to divide it. Right in two", James Maynard Keenan.