Total Interaction Energy Research Articles

On the Permeation of Polychlorinated Dibenzodioxins and Dibenzofurans through Lipid Membranes: Classical MD and Hybrid QM/MM-EDA Analysis.

The permeation of dioxin-like pollutants, namely, chlorinated dibenzodioxins and dibenzofurans, through lipid membranes has been simulated using classic molecular dynamics (CMD) combined with the umbrella sampling approach. The most toxic forms of chlorinated dibenzodioxin and dibenzofuran, 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDF), and a dioleyl-phosphatidylcholine (DOPC) lipid membrane of 50 Å wide have been chosen for our study. The free energy profile shows the penetration process is largely favoured thermodynamically (ΔG ≈ -12 kcal/mol), with a progressively decrease of the free energy until reaching the energy minima at distances of 8 Å and 9.5 Å from the centre of the membrane for, respectively, TCDD and TCDF. At the centre of the membrane, both molecules display subtle local maxima with free energy differences of 0.5 and 1 kcal/mol with respect to the energy minima for TCDD and TCDF, respectively. Furthermore, the intermolecular interactions between the molecules and the lipid membrane have been characterized at the minima and the local maxima using hybrid quantum mechanics/molecular mechanics energy decomposition analysis (QM/MM-EDA). Total interaction energies of -17.5 and -16.5 kcal/mol have been found at the energy minima for TCDD and TCDF, respectively. In both cases, the dispersion forces govern the molecule-membrane interactions, no significant changes have been found at the local maxima, in agreement with the classical free energy profile. The small differences found in the results obtained for TCDD and TCDF point out that the adsorption and diffusion processes through the cell membrane are not related to the different toxicity shown by these pollutants.

Synergistic effect of SPS compound surfactant and diesel oil on low-rank coal slime flotation

ABSTRACT In order to improve the collection performance of diesel oil on low-rank coal slime flotation, SPS (Span80, Polysorbate, sodium dodecyl sulfate) composite emulsifier was mixed with diesel fuel to prepare SPS microemulsion, as a collector for coal slime flotation. When the ratio of SPS emulsifier to diesel oil is 1:2, clean coal yield and the combustible recovery reached the highest, they were 50.16% and 82.65%, respectively. Through Contact angle test, Zeta potential analysis, EDLVO theoretical calculation, the mechanism of SPS microemulsion improving the flotation effect of low-rank coal slime was discussed. The Synergistic adsorption of SPS emulsifier and diesel oil on coal surface increases the coal surface contact Angle by 24.8°, increase the absolute value of Zeta potential on coal surface in solution by 9.14 mV, the energy barrier of the total interaction energy (VT) was increased from 8.4 × 10−18J to 9.8 × 10−18J. The coal particles treated by SPS microemulsion have the best dispersion and contact with collector droplets more fully. SPS microemulsion can be used as a new collector for low-rank coal slime flotation.

Establishing a high-performance anti-fouling PEI-ZIF-PAA membrane with improved Lewis acid-base interactions and hydrophilicity

Membrane fouling and the trade-off between membrane permeability and selectivity restrict the potential applications of membrane filtration for water treatment. ZIF-8 was found having great permeability and antibiofouling performance, but with issue on particle aggregation makes it difficult to achieve high ZIFs loading and fabricate a defect-free molecular sieving membrane in previous research. In this study, we formed a scalable antibiofouling surface with improved permeability and fouling resistance on a PEI-ZIF-PAA membrane using a layer-by-layer assembly technique. The synergistic effects of being sandwiched between two different polyelectrolyte layers with opposite charges endowed the ZIF nanoparticles with improved stability and scalability for membrane modification. The PEI-ZIF-PAA membrane exhibited a satisfactory water flux of 120.78 LMH, which was 46.97% higher than that of the pristine PES membrane. The normalized water flux loss was serious in the absence of ZIF-8, and the flux increased with the ZIF-8 concentration. Antifouling tests suggested that the PEI-ZIF-PAA membrane possessed good antifouling performance due to the much higher surface hydrophilicity and positive Lewis acid-base interactions with foulants. The HA rejection increased with the ZIF-8 concentration and reached a maximum of 92.1% in the presence of 1.00% (w/v) ZIF-8. The membrane regeneration was tested under physical and chemical cleaning with flux recovery rates of about 85% and 95%. XDLVO analysis showed that the total interaction energy between HA and the PEI-ZIF-8-PAA membrane was 26.45 mJ/m2, and the superior antifouling performance was mainly attributed to Lewis acid-base interactions. This study indicates that ZIF-8 nanocrystals are promising materials for fabricating novel membranes for sewage treatment.

Multiscale mechanism exploration and experimental optimization for rosmarinic acid extraction from Rosmarinus officinalis using natural deep eutectic solvents

This study aimed at efficient rosmarinic acid (RA) extraction from rosemary ( Rosmarinus officinalis L.) using natural deep eutectic solvents (NADESs). Quantum chemical calculations and molecular dynamics simulation were used to dissect the microscopic mechanisms. Three choline chloride (ChCl) based NADESs and ethanol (traditional extractant as reference) were investigated. The σ -profiles of RA and each solvent were analyzed. The infinite dilution activity coefficient ( γ ∞ ) of RA in different solvents, the total non-bonded interaction energy and the average H-bond number were calculated, to predict the RA solubility, understand the bond type and strength in each system. Among the solvents studied, ChCl/LA (lactic acid) exhibited wider complementary σ -profile with that of RA, as well as lower ln γ ∞ , higher H-bond number and predominant electrostatic-interaction. Moreover, self-diffusion coefficients revealed that RA had better compatibility with ChCl/LA. Additionally, with different solvents as extractant, ChCl/LA outperformed the others by exhibiting the highest RA extraction yield. Based on results from single factor experiments, Box-Behnken design with response surface methodology was performed for further process optimization. The regressed model obtained was reliable from the ANOVA analysis of variances. RA extraction yield was 6.10% under optimal conditions of 69 °C, 5.6 h, liquid to solid ratio 34 and water content 13.5% (w/w). The combination of simulation and experiment in this study can shed light on the exploration of natural product extraction with high efficiency using green and novel NADESs. • Efficient rosmarinic acid extraction from rosemary with natural deep eutectic solvents. • Solvent evaluation for extractability by molecular simulation and experiment. • Response surface methodology was used for extraction optimization. • Optimal rosmarinic acid yield 6.10% was obtained with Choline chloride/Lactic acid. • Multiscale mechanism exploration inspires solvent screening& design for extraction.

Which Surface Is More Scaling Resistant? A Closer Look at Nucleation Theories for Heterogeneous Gypsum Nucleation in Aqueous Solutions.

Developing engineered surfaces with scaling resistance is an effective means to inhibit surface-mediated mineral scaling in various industries including desalination. However, contrasting results have been reported on the relationship between scaling potential and surface hydrophilicity. In this study, we combine a theoretical analysis with experimental investigation to clarify the effect of surface wetting property on heterogeneous gypsum (CaSO4·2H2O) formation on surfaces immersed in aqueous solutions. Theoretical prediction derived from classical nucleation theory (CNT) indicates that an increase of surface hydrophobicity reduces scaling potential, which contrasts our experimental results that more hydrophilic surfaces are less prone to gypsum scaling. We further consider the possibility of nonclassical pathway of gypsum nucleation, which proceeds by the aggregation of precursor clusters of CaSO4. Accordingly, we investigate the affinity of CaSO4 to substrate surfaces of varied wetting properties via calculating the total free energy of interaction, with the results perfectly predicting experimental observations of surface scaling propensity. This indicates that the interactions between precursor clusters of CaSO4 and substrate surfaces might play an important role in regulating heterogeneous gypsum formation. Our findings provide evidence that CNT might not be applicable to describing gypsum scaling in aqueous solutions. The fundamental insights we reveal on gypsum scaling mechanisms have the potential to guide rational design of scaling-resistant engineered surfaces.

Structure, bonding, and interaction with molecular hydrogen of the β‐D‐glucopyranose―silver+ (1:1) complex

AbstractIn silico studies have been carried out at PBE0‐D3/def2‐TZVP level of theory to disclose the nature of bonding in β‐D‐glucopyranose – silver ion (1:1) complex ([Ag(C6H12O6)]+). Moreover, the interaction of molecular hydrogen with the [Ag(C6H12O6)]+ complex is studied. In doing so, it is found that the metal site can interact with up to three hydrogen molecules with a binding energy of 5.3 kcal/mol per H2. Calculated ∆G298 K values disclose that the interaction of the first H2 with the [Ag(C6H12O6)]+ complex is exergonic, whereas a slightly endergonic nature of interaction is noted for the two and three H2 cases. Electron density and electron density‐based reactivity descriptors help in resolving the nature of interaction between metal ions and C6H12O6 as well as between the metal center and molecular hydrogens. An energy decomposition analysis reveals the orbital interaction as the major contributing factor to the total interaction energy for the one H2 case. Ab initio molecular dynamics study at different temperatures reveals kinetic stability of the hydrogen‐bound silver complex up to 1 ps. Conceptual density functional theory (CDFT) based reactivity descriptors, hardness and electrophilicity, help in assessing the stability trend of the hydrogen‐bound complexes during the simulation.

Surface characterization of indirect restorative materials submitted to different etching protocols

ObjectivesThis in vitro study aimed to evaluate the effect of different times and concentrations of hydrofluoric acid etching on the surface of indirect restorative materials obtained from blocks used in CAD-CAM technology. MethodsSpecimens (4 mm × 4 mm × 0.8 mm) were obtained for each indirect restorative material: zirconia-reinforced lithium monosilicate ceramic (Celtra Duo), nanoceramic resin (Lava Ultimate), and polymer-infiltrated ceramic network material (Vita Enamic). The materials were submitted to etching with 5% or 10% hydrofluoric acid for 20, 40, 60, or 90 s. A control group for each material was evaluated without any surface treatment, totaling nine experimental groups for each material (n = 10). The specimens were evaluated for surface roughness (Ra and Rz), confocal optical microscopy, the contact angle (θ), surface free energy (γs), total free interaction energy (∆G) using a goniometer, and microshear bond strength to resin cement. Specimen images were obtained using scanning electron microscopy, confocal optical microscopy, and atomic force microscopy. Data on the surface roughness, the contact angle, surface free energy, total free interaction energy, and bond strength were subjected to two-way ANOVA and Tukey´s test (α=0.05). ResultsIn general, Celtra Duo showed better results after etching with 10% hydrofluoric acid for 40 or 60 s. Lava Ultimate showed better performance after etching with 10% hydrofluoric acid for 20 or 40 s, whereas Vita Enamic showed better results after etching with 5% hydrofluoric acid for 90 s. ConclusionEach material showed different characteristics after etching with hydrofluoric acid. Knowledge of the proper protocol for each material is essential to ensure improvements in the adhesion process and durability of indirect restorations. In general, Celtra Duo presented mechanical properties superior to those of Lava Ultimate and Vita Enamic. Clinical SignificanceSpecific etching protocols must be recommended for each indirect material because longer exposure to hydrofluoric acid can jeopardize the surface, thus affecting the mechanical and bond strength properties.

Dosage-effectiveness of modified clay flocculating red tide organisms: Mechanical mechanism and mathematical model

Modified clay (MC) technology is the most widely used method to remove red tide organisms from seawater on a large scale. In recent years, frequent outbreaks of red tides worldwide have led to a sharp increase in MC consumption. However, the efficient application of MC for red tides control lacks theoretical basis. The study explored dosage-effectiveness of MC for microalgae (red tide organism) removal. The results showed that the effectiveness of MC for microalgae removal increased first and then decreased with the increase of MC dosage due to the self-flocculation of MC. The self-flocculation of MC was caused by the strong attractive total interaction energy between MCs at close range in seawater. A mathematical model established based on the extended Langmuir model can simulate dosage-effectiveness of MC well, and the quantitative relationship between the dosage of MC and its degree of self-flocculation was deduced based the mathematical model. These studies will help to enrich the flocculation theory of MC application, and provide significant guidance for the efficient application of MC for red tides control.

Biofouling initiation on a microfiltration membrane related to deposition and adhesion of bacteria from the perspective of interface interactions

Inhibition of bacteria deposition and adhesion is an effective strategy to control biofouling in membrane applications. In this study, the deposition and adhesion characteristics of individual bacteria (Pseudomonas aeruginosa) on a polyvinylidene fluoride microfiltration membrane were systematically investigated from the perspective of interface interactions. Preliminary results demonstrated that the deposition process was compatible with and dominated by the intermediate blocking model and cake filtration model However, the adhesion process analyzed by thermodynamic analysis revealed that bacteria should, in theory, not adhere to the virgin membrane, which was inconsistent with the experimental results. Further experiments and theoretical analysis suggested that organic matter in feed water, bovine serum albumin (BSA), or sodium alginate (SA) increased the adhesion capacity of the membrane by changing the properties of the membrane, and the thermodynamic relationship resulted in a reduction in the total interface interaction energy, Lewis acid–base component, and repulsive force. Therefore, biofouling can be controlled by reducing BSA-like or SA-like organics and inhibiting protein secretion by bacteria. This study furthers the understanding of the bacterial attachment process, and provides new insights into the adhesion mechanism from the perspective of interface interactions and thermodynamic analysis.

-Comparison of σ/ π-hole aerogen-bonding interactions based on C2H4···NgOX2 (Ng = Kr, Xe; X = F, Cl, Br) complexes.

The geometric structure, energy properties, and electronic properties of the aerogen-bonding interaction formed by C2H4 and NgOX2 (Ng = Kr, Xe; X = F, Cl, Br) have been studied at the B2PLYP-D3(BJ)/ aug-cc-pVTZ (PP) level. Two kinds of aerogen-bonding interactions were observed among the title systems: the σ-hole and the π-hole complexes. The σ-hole aerogen-bonding complex has a binding energy in the range of - 6.29 ~ - 8.17kcal/mol, which is the most stable. The binding energies of C2H4···NgOX2 increased as X = F < Cl < Br and Ng = KrOX2 < XeOX2 for the σ/π-hole aerogen-bonding complexes. The atoms in molecules (AIM), the non-covalent interaction (NCI) index, and the LMO-EDA energy decomposition analysis were adopted to study the nature of the σ/π-hole aerogen-bonding interaction. The results show that the electrostatic term contributes the most to the total interaction energy for the σ/π-hole aerogen-bonding complexes.

The use of oligodiurethane diisocyanates in cellulose triacetate compositions

We performed the studies on the use of oligodiurethane diisocyanates as an additive to a solution of triacetate cellulose polymer. It was shown that these compounds are effective additives and can be used as structural plasticizers of supramolecular formations of triacetate cellulose (in small quantities of 0.01–0.075 wt.% with respect to the weight of triacetate cellulose), which allows adjusting the viscosity of the solution at the stage of casting films in a wide range. In addition, the investigated compounds are effective modifiers of the properties of the obtained film materials (they approximately double the tensile strength of films, their relative elongation and relative hardness). The improvement in physical-mechanical properties of the test film samples is due to the presence of polar urethane groups in the structure of the additive, which are characterized by higher values of total volumetric energy of cohesive interaction than those of phthalate or adipinate oligomers known as plasticizers.

Involvement of Arsenic Atom of AsF3 in Five Pnicogen Bonds: Differences between X-ray Structure and Theoretical Models.

Bonding within the AsF3 crystal is analyzed via quantum chemical methods so as to identify and quantify the pnicogen bonds that are present. The structure of a finite crystal segment containing nine molecules is compared with that of a fully optimized cluster of the same size. The geometries are qualitatively different, with a much larger binding energy within the optimized nonamer. Although the total interaction energy of a central unit with the remaining peripheral molecules is comparable for the two structures, the binding of the peripherals with one another is far larger in the optimized cluster. This distinction of much stronger total binding within the optimized cluster is not limited to the nonamer but repeats itself for smaller aggregates as well. The average binding energy of the cluster rises quickly with size, asymptotically approaching a value nearly triple that of the dimer.

An Analytical Model for Lithium Storage in Spherical Fullerenes

In this paper, the encapsulation of lithium atoms in spherical fullerenes of varying sizes is investigated. The 6–12 Lennard–Jones potential function and the continuum approximation, in which it is assumed that the atoms can be replaced with a uniform distribution across the surface of the molecules, are exploited to model the interaction energies between lithium atoms and spherical fullerenes. Thus, the total interaction energies can be approximated by applying surface integrations. The results show that for a lithium atom interacting inside a spherical fullerene, the interaction energies are minimized at a position that approaches the fullerene wall as the size of the fullerene increases. However, the results show that an external force would need to be applied to a lithium atom in order to overcome the repulsive energy barrier so that it can be encapsulated in CN fullerenes with a radius of less than 2 Å. The present study indicates that the optimal radius that gives the minimum energy for the storage of Li inside CN fullerenes occurs for a fullerene with a radius of ≈2.4 Å. Overall, this study provides an analytical formulation that may facilitate rapid computational results, and an application of this work is in the design of future high-energy-density batteries that utilize CN fullerenes.

Energetics of carboxylic acid–pyridine heterosynthon revisited: A computational study of intermolecular hydrogen bond domination on phenylacetic acid–nicotinamide cocrystals

Abstract Carboxylic acid–pyridine heterosynthon (CPHS) is one of the most common synthons found in cocrystal packing. Phenylacetic acid (PYC)–nicotinamide (NIC) (PYCNIC) cocrystals were used as a computational model to assess the most important factor in the emergence of the synthon. Geometry optimization was carried out on every possible two molecules of PYC–NIC conformation based on B3LYP-D3BJ/6-311G (d,p). Various energetic parameters, including total energy, interaction energy, and hydrogen bond energy, were used to compare the existing conformation to the putative conformation. The conformation with CPHS has −53.87 kJ mol−1 of single intermolecular hydrogen bond energy (EHB), which is the strongest of all. It turns out that there is no other parameter better than EHB to describe the superiority of CPHS in PYCNIC.

Sandwich, Triple-Decker and Other Sandwich-like Complexes of Cyclopentadienyl Anions with Lithium or Sodium Cations.

Density functional theory, DFT, calculations were carried out on complexes containing cyclopentadienyl anions and lithium or sodium cations; half-sandwich, sandwich and sandwich-like complexes (among them triple-decker ones) are analyzed. Searches performed through the Cambridge Structural Database revealed that crystal structures containing these motifs exist, mostly structures with sodium cations. The DFT calculations performed here include geometry optimization and frequency calculations of the complexes at the ωB97XD/aug-cc-pVTZ level, followed by the partitioning of the energy of interaction via the Energy Decomposition Analysis scheme, EDA, at the BP86-D3/TZ2P level. Additional calculations and analyses were performed using both the Quantum Theory of Atoms in Molecules, QTAIM, and the Natural Bond Orbital analyses, NBO. The results of this work show that the electrostatic interaction energy is the most important attractive contribution to the total interaction energy of each of the complex systems analyzed here, and that complexation itself leads to minor electron charge shifts.

Mechanism study of ternary deep eutectic solvents with protonic acid for lignin fractionation

This paper investigated the fractionation of lignin by ternary DES of different polyols using simulation calculation. ChCl-EG-PTSA showed the highest degradability of lignin with the absolute value of total interaction energy of −8023.80 kJ/mol, and the total number of hydrogen bonds was 91.4. The highest degradability was observed for ChCl:EG: PTSA = 2:4:1. The results show that CL− plays a dominant role in lignin fractionation and readily forms hydrogen bonds with γ-OH. The difference is that the polyol preferred to form hydrogen bonds with α-OH in lignin. The addition of PTSA provided protons to the original system. It formed a new π-π stacking interaction with the lignin benzene ring, which destroyed the π-π stacking interaction between the original lignin. And increased the interaction of DES on lignin from −39.73 kcal/mol to −58.15 kcal/mol based on DFT.

Benzene, an Unexpected Binding Unit in Anion–π Recognition: The Critical Role of CH/π Interactions

We report high-level ab initio calculations (CCSD(T)(full)/CBS//SCS-RI-MP2(full)/aug-cc-pwCVTZ) that demonstrate the importance of cooperativity effects when Anion–π and CH/π interactions are simultaneously established with benzene as the π-system. In fact, most of the complexes exhibit high cooperativity energies that range from 17% to 25.3% of the total interaction energy, which is indicative of the strong influence of the CH/π on the Anion–π interaction and vice versa. Moreover, the symmetry-adapted perturbation theory (SAPT) partition scheme was used to study the different energy contributions to the interaction energies and to investigate the physical nature of the interplay between both interactions. Furthermore, the Atoms in Molecules (AIM) theory and the Non-Covalent Interaction (NCI) approach were used to analyze the two interactions further. Finally, a few examples from the Protein Data Bank (PDB) are shown. All results stress that the concurrent formation of both interactions may play an important role in biological systems due to the ubiquity of CH bonds, phenyl rings, and anions in biomolecules.

Study on the slurrying properties of semi-coke with dispersants and the interaction between semi-coke particles based on E-DLVO theory

It is essential to prepare the semi-coke water slurry (SCWS) using semi-coke powder in China. The preparation of SCWS can not only alleviate the oil demand, but can also provide a new way to utilize semi-coke powder. In this work, the effects of sodium methylene naphthalene sulfonate (NNO), sodium lignosulfonate (SLS), sodium humate (SH), polycarboxylate (PCE), and compound anion-anion (NNO/PCE), on the slurrying performance of semi-coke water slurry (SCWS) were studied. The results showed that the anionic dispersants increased the concentration of SCWSs, and the slurry exhibited the pseudoplastic rheological behavior of shear thinning. Adding PCE and NNO/PCE in the SCWS could improve its stability and fluidity. The absorbed capacity the microstructure of the particles, surface tension, contact angle, as well as Zeta potential of semi-coke powder were impacted by the addition of anionic dispersants in the SCWS. The results showed that anionic dispersants were adsorbed on the surface of semi-coke by a single-molecular layer. The developments of molecular dynamics simulation indicated that all dispersants should be spontaneously adsorbed on the surface of semi-coke, and the addition of anionic dispersants could reduce the contact angle and surface tension of the semi-char particle surface, increase the hydrophilicity and enhance the electronegativity. Finally, extended Derjaguin–Landau–Verwey –Overbeek (E-DLVO) theory calculations showed that adsorption of dispersants could significantly increase the total interaction energy between semi-coke particles. Finally, a model of the interaction mechanism of the anionic dispersants and semi-coke particles was proposed in this paper.

Hydrogen storage capacity of Be2 (NLi)2 cluster with ultra-short beryllium-beryllium distance.

Quantum chemical calculations have been carried out to investigate the hydrogen storage capacity of Be2 (NLi)2 cluster. Calculations reveal that the cluster can take up to eight H2 molecules reaching a maximum gravimetric density of 21.04 wt%. Six H2 molecules bind at the Li atoms and two H2 bind at the Be atoms with moderate binding energy which is required for reversible storage of H2 . Symmetry-adapted perturbation analysis reveals the significant contribution of electrostatic and induction and very minor contribution of dispersion toward the total interaction energy. Molecular dynamics simulations reveal that the H2 molecules are strongly bound at 77 K and get slowly released at elevated temperatures.

Isolation of Elusive Phosphinidene-Chlorotetrylenes: The Heavier Cyanogen Chloride Analogues.

The elusive phosphinidene-chlorotetrylenes, [PGeCl] and [PSiCl] have been stabilized by the hetero-bileptic cyclic alkyl(amino) carbene (cAAC), N-heterocyclic carbene (NHC) ligands, and isolated in the solid state at room temperature as the first neutral monomeric species of this class with the general formulae (L)P-ECl(L') (E=Ge, 3 a-3 c; E=Si, 6; L=cAAC; L'=NHC). Compounds 3 a-3 c have been synthesized by the reaction of cAAC-supported potassium phosphinidenides [cAAC=PK(THF)x ]n (1 a-1 c) with the adduct NHC:→GeCl2 (2). Similarly, compound 6 has been synthesized via reaction of 1 a with NHC:→SiCl2 adduct (4). Compounds 3 a-3 c, and 6 have been structurally characterized by single-crystal X-ray diffraction, NMR spectroscopy and mass spectrometric analysis. DFT calculations revealed that the heteroatom P in 3 bears two lone pairs; the non-bonding pair with 67.8 % of s- and 32 % of p character, whereas the other lone pair is involved in π backdonation to the CcAAC -N π* of cAAC. The Ge atom in 3 contains a lone pair with 80 % of s character, and slightly involved in the π backdonation to CNHC . EDA-NOCV analyses showed that two charged doublet fragments {(cAAC)(NHC)}+ , and {PGeCl}- prefer to form one covalent electron-sharing σ bond, one dative σ bond, one dative π bond, and a charge polarized weak π bond. The covalent electron-sharing σ bond contributes to the major stabilization energy to the total orbital interaction energy of 3, enabling the first successful isolations of this class of compounds (3, 6) in the laboratory.