Free Amine Research Articles

Simple model for water wash performance in amine scrubbing for CO2 capture

In amine-based capture systems, a water wash is necessary to remove gaseous amine to minimize atmospheric emissions and maximize amine recovery. Water wash simulation requires rigorous thermodynamic and kinetic models like those for absorption and stripping systems. An offline water wash model has been developed based on simplified thermodynamic and mass transfer assumptions to simulate a single-stage water wash for flue gas from gas-fired turbines with little or no aerosol, and to expedite simulations without needing to utilize process modeling software. Calculation of activity coefficients using both Pitzer–Debye–Hückel and Debye–Hückel theory suggest that the liquid phase in a typical water wash will be ideal due to the dilute concentration of molecular and ionic species.Vapor phase piperazine (PZ) is predicted with a driving force based on the volatility of the free amine that has not reacted with other species or dissociated into its ionic form, and the predicted outlet concentration is nearly identical to that predicted by rigorous Aspen PlusⓇ simulations. The predicted UT FEED outlet concentration is 220 ppb compared to 260 ppb predicted by Aspen PlusⓇ.Single-stage water washes are predicted to have single ppm concentration of MEA leaving the wash outlet flue gas. Amino-methyl-propanol (AMP) is predicted to be reduced to 10–30 ppm by a single-stage wash, and is predicted to be a difficult-to-capture amine without further washing steps. This prediction correlates with published TCM data from an ALIGN-CCUS campaign. Amine compounds at a similar or lower volatility to that of PZ are predicted to be reduced to ppb levels via a single-stage wash.

The NMR signature of maltose-based glycation in full-length proteins

Reducing sugars can spontaneously react with free amines in protein side chains leading to posttranslational modifications (PTMs) called glycation. In contrast to glycosylation, glycation is a non-enzymatic modification with consequences on the overall charge, solubility, aggregation susceptibility and functionality of a protein. Glycation is a critical quality attribute of therapeutic monoclonal antibodies. In addition to glucose, also disaccharides like maltose can form glycation products. We present here a detailed NMR analysis of the Amadori product formed between proteins and maltose. For better comparison, data collection was done under denaturing conditions using 7 M urea-d4 in D2O. The here presented correlation patterns serve as a signature and can be used to identify maltose-based glycation in any protein that can be denatured. In addition to the model protein BSA, which can be readily glycated, we present data of the biotherapeutic abatacept containing maltose in its formulation buffer. With this contribution, we demonstrate that NMR spectroscopy is an independent method for detecting maltose-based glycation, that is suited for cross-validation with other methods.

Comparative Studies of Wear and Corrosion Behaviors of Conventional and Nano Filler Based Solvent Free Tarfree Epoxy-amine Coatings

Tar-free epoxy coatings aid in protecting intact of Mild Steel (MS) substrate immersion condition of atmosphere. From lot of study revised and assorted that tar free (light colour) epoxy amine coating developed which give better performance for protection in long term. By ASTM method, wear behaviors as well as mechanical properties of quartz (micro silica) and organosiliane (nano silica) based tar-fee epoxy coatings were studied. DETA cured epoxy composites coatings were characterized by FTIR spectroscopic technique, powder X-Ray Diffraction (XRD), SEM and FESEM analysis. Transmission Electron Microscope (TEM) confirmed the size of nano particles. The data of wear index of nano fillers based epoxy coating was observed 8.3mg/1000cycles which indicate such an improvement of of abrasion behavior. Corrosion behaviors of the coated MSspecimens were evaluated by Cathodic Disbondment (CD) test immersion in 3.5% NaCl solution. Water absorption and chemical resistance also studied of composite coatings. All over tar-free epoxy-amine nano composite coating shows good result.

Pan msr gene deleted strain of Salmonella Typhimurium suffers oxidative stress, depicts macromolecular damage and attenuated virulence

Salmonella encounters but survives host inflammatory response. To defend host-generated oxidants, Salmonella encodes primary antioxidants and protein repair enzymes. Methionine (Met) residues are highly prone to oxidation and convert into methionine sulfoxide (Met-SO) which compromises protein functions and subsequently cellular survival. However, by reducing Met-SO to Met, methionine sulfoxide reductases (Msrs) enhance cellular survival under stress conditions. Salmonella encodes five Msrs which are specific for particular Met-SO (free/protein bound), and ‘R’/‘S’ types. Earlier studies assessed the effect of deletions of one or two msrs on the stress physiology of S. Typhimurium. We generated a pan msr gene deletion (Δ5msr) strain in S. Typhimurium. The Δ5msr mutant strain shows an initial lag in in vitro growth. However, the Δ5msr mutant strain depicts very high sensitivity (p < 0.0001) to hypochlorous acid (HOCl), chloramine T (ChT) and superoxide-generating oxidant paraquat. Further, the Δ5msr mutant strain shows high levels of malondialdehyde (MDA), protein carbonyls, and protein aggregation. On the other side, the Δ5msr mutant strain exhibits lower levels of free amines. Further, the Δ5msr mutant strain is highly susceptible to neutrophils and shows defective fitness in the spleen and liver of mice. The results of the current study suggest that the deletions of all msrs render S. Typhimurium highly prone to oxidative stress and attenuate its virulence.

Amine-functionalized single-layered 2D nanosheets of α-zirconium phosphate for highly efficient removal of Pb2+ ions and Congo red dye

This paper reports a facile route for preparing single-layered and crystalline 2D nanosheets of α-zirconium phosphate (α-ZrP), which were further functionalized through covalent attachment of amino organosilane on its exfoliated surfaces (AF-ZrP) to enhance their sorption capacity for Pb2+ and Congo red removal. Two possible surface arrangements of NH2 groups were observed: one as free amine groups without bonding and the other as protonated or hydrogen-bonded amine groups. AF-ZrP exhibits extraordinary adsorption capacities of 1640 and 1213 mg g−1 for removing Pb2+ ions and CR dye. Further, linear and non-linear regression methods have been studied and compared for fitting four adsorption isotherm models and two kinetics models. Non-linear Freundlich isotherm and non-linear pseudo-second order kinetic models prove the most suitable. The high adsorption capacity, easy regeneration, and reusability rendered AF-ZrP a suitable adsorbent for removing Pb2+ ions and Congo red (CR) dye at industrial scale.

Regioselective Organocatalyzed Monochlorination of Arenes with Electrophilic Chlorosulfoniums

AbstractThis manuscript presents a regioselective chlorination of arenes, yielding almost exclusively the monochlorinated product. It tolerates free hydroxy groups, 1°, 2° and 3° amines, and carboxylic acids, among other polar and nonpolar functional groups, as well as mono‐ and polysubstituted aromatic rings. Additionally, the utilization of chlorosulfonium salts as the chlorinating agent is demonstrated. The method emerges as an alternative to other processes where organic byproducts must be separated from the reaction mixture. In addition, the main byproducts are gaseous materials such as CO and CO2. This publication introduces the application of sulfoxides, oxalyl chloride, and aqueous hydrogen peroxide as the chlorination mixture.

Total and Free Hydrogen Cyanide Content and Profile of Bioactive Amines in Commercial Tucupi, a Traditionally Derived Cassava Product Widely Consumed in Northern Brazil.

Tucupi is a broth derived from cassava roots which is produced after the spontaneous fermentation of manipueira (the liquid portion obtained by pressing cassava roots), followed by cooking. This product is widely consumed along with traditional dishes in the Brazilian Amazonia and is already used in different places worldwide. In this study, tucupi obtained from the markets of Belém (Pará, Brazil) and produced using agroindustrial (11 samples) and non-agroindustrial (11 samples) units were investigated to determine their physicochemical characteristics, total and free HCN contents, and free bioactive amine profiles. Most of the samples showed significant variations (p ≤ 0.05) in pH (2.82-4.67), total acidity (0.14-1.36 g lactic acid/100 mL), reducing sugars (up to 2.33 g/100 mL), and total sugars (up to 4.35 g/100 mL). Regarding the amines, four biogenic amines (0.5-4.2 mg/L tyramine, 1.0-23.1 mg/L putrescine, 0.5-66.8 mg/L histamine, and 0.6-2.9 mg/L tryptamine) and one polyamine (0.4-1.7 mg/L spermidine) were identified in the tucupi samples. Even in the tucupi produced using the agroindustrial units, which had quality seals provided by the local regulatory agency, high levels of biogenic amines (4.4-78.2 mg/L) were observed, as well as high dosages of total (8.87-114.66 mg/L) and free (0.80-38.38 mg/L) HCN. These facts highlight the need for better knowledge regarding the product manufacturing process to establish standardization and high-quality conditions for tucupi processing since high contents of biogenic amines and HCN are commonly associated with adverse health effects.

Complexes of N, N- and N, N, N- Sulfonamide Ligands as Therapeutic and Diagnostic Agents

Ethylenediamine, diethylenetriamine and dipicolylamine have been used as the carrier ligands to synthesize bidentate (N,N) or tridentate (N,N,N) chelate systems that coordinate with metal centers. The terminal amine groups of ethylenediamine and diethylenetriamine and the central amine group of dipicolylamine can be easily substituted to give sulfonamide ligands having bulky aromatic fragments. In the formation of metal sulfonamides, the sulfonamide nitrogens of primary or secondary sulfonamidesdeprotonate and coordinate with metal centers forming M-N bonds while the free amines coordinate to metal centers through lone pairs. The reported synthetic approaches and the properties of these complexes and ligands are discussed in this review. The bulky sulfonamide moieties bring about unique biological features to the ligand system while enhancing the fluorescent properties of the ligand. The metal center itself incorporates interesting biological features to the complex. These properties of the metal center andthe ligand show a synergistic effect in the complexes that may come in handy when designing therapeutic or diagnostic agents. The increased lipophilicity of the ligands and thereby the complexes will ensure better uptake by target cells making them ideal candidates for biological applications.&#x0D; Keywords: Sulfonamide, ethylenediamine, diethylenetriamine, dipicolylamine

Bio-based polyester-polyurethane foams: synthesis and degradability by Aspergillus niger and Aspergillus clavatus.

In this article, the degradability by Aspergillus niger and Aspergillus clavatus of three bio-based polyurethane (PU) foams is compared to previous degradability studies involving a Pseudomonas sp. bacterium and similar initial materials (Spontón et al. in Int. Biodet. Biodeg. 85:85-94, 2013, https://doi.org/10.1016/j.ibiod.2013.05.019 ). First, three new polyester-polyurethane foams were prepared from mixtures of castor oil (CO), maleated castor oil (MACO), toluene diisocyanate (TDI), and water. Then, their degradation tests were carried out in an aqueous medium, and employing the two mentioned fungi, after their isolation from the environment. From the degradation tests, the following was observed: (a) the insoluble (and slightly collapsed) foams exhibited free hydroxyl, carboxyl, and amine moieties; and (b) the water soluble (and low molar mass) compounds contained amines, carboxylic acids, and glycerol. The most degraded foam contained the highest amount of MACO, and therefore the highest concentration of hydrolytic bonds. A basic biodegradation mechanism was proposed that involves hydrolysis and oxidation reactions.

Ligand-Enabled γ-C(sp3)-H Hydroxylation of Free Amines with Aqueous Hydrogen Peroxide.

Selective oxidation of the γ-C-H bonds from abundant amine feedstocks via palladium catalysis is a valuable transformation in synthesis and medicinal chemistry. Despite advances on this topic in the past decade, there remain two significant limitations: C-H activation of aliphatic amines requires an exogenous directing group except for sterically hindered α-tertiary amines, and a practical catalytic system for C(sp3)-H hydroxylation using a green oxidant, such as oxygen or aqueous hydrogen peroxide, has not been developed to date. Herein, we report a ligand-enabled selective γ-C(sp3)-H hydroxylation using sustainable aqueous hydrogen peroxide (7.5-10%, w/w). Enabled by a CarboxPyridone ligand, a series of primary amines (1°), piperidines, and morpholines (2°) were hydroxylated at the γ-position with excellent monoselectivity. This method provides an avenue for the synthesis of a wide range of amines, including γ-amino alcohols, β-amino acids, and azetidines. The retention of chirality in the reaction allows rapid access to chiral amines starting from the abundant chiral amine pool.

Synthesis, Characterization, and Impact of Water on the Stability of Postmodified Schiff Base Containing Metal–Organic Frameworks

In this study, the synthesis of a Schiff base containing metal–organic frameworks (MOFs) of the UiO-67 family has been investigated. MOFs featuring free amine groups were successfully synthesized under mild solvothermal conditions using 2-amino-[1,1′-biphenyl]-4,4′-dicarboxylic acid and 2,2′-diamino-[1,1′-biphenyl]-4,4′-dicarboxylic acid as bridging ligands, resulting in MOFs with amine groups covalently linked to the bridging ligands. Both types of functionalized MOFs were post-synthetically modified with 4-formylbenzonitrile that resulted in imine formation. All the obtained compounds were characterized by PXRD, TGA, DTA, BET, NMR, and FTIR spectroscopy, while stability in water was monitored with SEM, EDS, and UV–VIS spectroscopy.

Anonna crassiflora suppresses colonic carcinogenesis through its antioxidant effects, bioactive amines, and phenol content in rats

Marolo (Annona crassiflora) is an underutilized Brazilian Cerrado fruit with few reports in the literature about its bioactive compounds and functional properties. In this context, the chemoprevention against the carcinogen 1,2-dimethylhydrazine (DMH)-induced pre-neoplastic lesions in Wistar rat colon was investigated and correlated with marolo’s antioxidant activity and the contents of phenolic compounds and bioactive amines. Total phenolic compounds (TPC) and total flavonoids compounds (TFC) were determined in the marolo pulp extract by spectrophotometric and Ultra-Performance Liquid Chromatography and diode array detection (UPLC-DAD) analysis. Free bioactive amines were determined by High Performance Liquid Chromatography and fluorescence detection (HPLC-FLD) after post column derivatization with o-phthalaldehyde. In addition, the in vitro antioxidant activity was determined by DPPH, and ABTS. Wistar rats were treated orally with marolo pulp at 0.7, 1.4 and 2.8 g/kg body weight (bw)/day added to a standard ration. Four subcutaneous injections of DMH (40 mg/kg bw) were used to induce a pre-neoplastic lesion that was assessed by the aberrant crypt foci (ACF) assay. The marolo pulp (fresh weigh) showed high content of total phenolic compounds (9.16 mg GAE/g), with predominance of chlorogenic acid (1.86 µg/g) and epicatechin (0.99 µg/g), and total flavonoids (7.26 mg CE/g), ∼85 % of the TPC. The marolo pulp had significant contents of tyramine (31.97 mg/kg), putrescine (20.65 mg/kg), and spermidine (6.32 mg/kg). The marolo pulp inhibited (p < 0.05) pre-neoplastic lesions induced by DMH administration at the all concentrations tested. These findings indicate that marolo pulp has a colon carcinogenesis chemopreventive effect, which could be due to, at least in parts, its antioxidant action associated with its phenolics and flavonoids content as well of spermidine.

A novel graphene oxide-1,3,4-oxadiazole-2,5-diamine nanomaterial with enhanced bactericide activity

BackgroundThe development of effective nanomaterials is becoming important to safeguard the human health from the bacteria-induced infectious diseases. Among the popular nanomaterials, graphene oxide (GO) owns functional groups and exhibits noticeable benefits which are helpful for biological applications. For raising the antibacterial activity with the reinforcement of the active organic molecules like oxadiazoles, a novel GO-1,3,4-oxadiazole-2,5-diamine (ODA) nanomaterial was prepared and characterized for the first time in our study. MethodsGO-diamine nanomaterials were synthesized by simply reacting GO with ODA or aliphatic diamine in a water bath at room temperature. In an in vitro antimicrobial activity investigation, 5 mg of the nanomaterial was immersed in 20 mL of the Escherichia coli culture in agar media at 37 °C, and the growth of bacteria was evaluated after 24 h. Significant FindingsBy XRD analysis, it was found that the increase in d-spacing after the intercalation of aliphatic diamine into GO layers was close to the molecular length of aliphatic diamine, indicating a perpendicular bridging configuration. Conversely, the aromatic ODA reacted with the oxygenated functionalities on the outer surface of GO layer and a free amine was left behind in the terminal. In the bactericide activity test, a near complete E. coli inhibition was achieved only with GO-ODA, much better than pure ODA (30–60 %) and pristine GO (0–20 %). The pure aliphatic diamines showed some extent of antibacterial activity (20–60 %) due to their natural toxicity, but their GO-aliphatic diamine derivatives demonstrated negligible bactericide activity (0–10 %). Conclusively, this study provides a greener and effective antibacterial material without using any catalyst or organic solvent.

Optimum relative humidity enhances CO2 uptake in diamine-appended M2(dobpdc)

Diamine-appended M2(dobpdc) metal–organic frameworks (MOFs) are a promising class of CO2 adsorbents, but the impact of relative humidity (RH) on their adsorption performance has yet to be carefully studied. We evaluated the CO2 uptake of (2-ampd)2M2(dobpdc) at varying relative humidities and associated water loadings using gravimetric and breakthrough adsorption methods. A significant enhancement in uptake is observed between 20 and 40 % RH, with an optimum of 30 % RH, at which a maximum CO2 uptake of 5.7 ± 0.2 mmol/g is achieved at 0.044 atm of CO2. These uptakes – which are above 1 CO2:diamine – suggest that humidity promotes an additional sorption mechanism beyond the well-known cooperative insertion between the metal and diamine. Temperature-programmed desorption and IR spectroscopy also indicated a second adsorption site accounting for the additional uptake, hypothesized to be carbamic acid pairs on the free amine within the MOF channel. Further increasing the humidity to 50 % RH resulted in a dramatic loss of capacity due to water-filled pores at these high humidity conditions. These results reveal the potential for a mixed mechanism in (2-ampd)2M2(dobpdc) promoted by humidity and highlight an optimum humidity range that will maximize the MOF’s utilization in CO2 capture applications.

Maillard reaction in different food products: Effect on product quality, human health and mitigation strategies

Maillard reaction is a non-enzymatic reaction takes place between reducing sugar and free amine in food products. This reaction alters the organoleptic properties and possess negative health consequences due to production of mutagenic compounds as well as makes lysine unavailable. Numerous attempts have been made for controlling Maillard reaction in the past decades. In present article, the Maillard reaction occurring in different products like cereals, fruits and vegetables, milk, meat, etc., has been discussed. In addition, the recent advances in strategies for mitigating Maillard reaction in food system has been reviewed critically. The underlying mechanism, strength and weakness of each strategies including use of polyphenols, non-thermal techniques, enzymes, food additives that influence the food quality and safety have been discussed. The trapping of Maillard targets, altering the initial or intermediate Maillard products prevents the food product from Maillard browning during processing and storage.

Measurement of degree of hydrolysis and molecular weight distribution of protein hydrolysates by liquid chromatography-mass spectrometry

Enzymatic hydrolysis of milk protein is an effective way to improve protein digestibility, to reduce their allergenicity and to produce peptides with better functionalities. Among the process, the degree of hydrolysis (DH) and molecular weight distribution (MWD) of protein hydrolysates are two important parameters that need to be monitored. In this work, a new method based on liquid chromatography-mass spectrometry (LC-MS) was developed for the first time to accurately detect the DH and the MWD of proteolytic peptides. With LC-MS, the content of free amine groups released during hydrolysis was acquired by direct analysis of free trinitrobenzene sulfonic acid (TNBS) for DH assay, overcoming the disadvantage of TNBS-based spectral method. Based on this method, the DH% values of five protein hydrolysis samples were determined and consistent with file specification values. Compared to the size-exclusion high-performance liquid chromatography (SE-HPLC) method, LC-MS was capable of measuring MWD (similar results with file specification values) while additionally providing precise molecular weight and amino acid sequence information for each proteolytic peptide. This method was characterized by its simplicity, accuracy, and reproducibility, making it a valuable technology for monitoring proteolysis and producing peptides with better functionality.

Dual Tunability for Uncatalyzed N-Alkylation of Primary Amines Enabled by Plasma-Microdroplet Fusion.

The fusion of non-thermal plasma with charged microdroplets facilitates catalyst-free N-alkylation for a variety of primary amines, without halide salt biproduct generation. Significant reaction enhancement (up to >200×) is observed over microdroplet reactions generated from electrospray. This enhancement for the plasma-microdroplet system is attributed to the combined effects of energetic collisions and the presence of reactive oxygen species (ROS). The ROS (e.g., O2•-) act as a proton sink to increase abundance of free neutral amines in the charged microdroplet environment. The effect of ROS on N-alkylation is confirmed through three unique experiments: (i) utilization of radical scavenging reagent, (ii) characterization of internal energy distribution, and (iii) controls performed without plasma, which lacked reaction acceleration. Establishing plasma discharge in the wake of charged microdroplets as a green synthetic methodology overcomes two major challenges within conventional gas-phase plasma chemistry, including the lack of selectivity and product scale-up. Both limitations are overcome here, where dual tunability is achieved by controlling reagent concentration and residence time in the microdroplet environment, affording single or double N-alkylated products. Products are readily collected yielding milligram quantities in eight hours. These results showcase a novel synthetic strategy that represents a straightforward and sustainable C-N bond-forming process.

Dual Tunability for Uncatalyzed N‐Alkylation of Primary Amines Enabled by Plasma‐Microdroplet Fusion

AbstractThe fusion of non‐thermal plasma with charged microdroplets facilitates catalyst‐free N‐alkylation for a variety of primary amines, without halide salt biproduct generation. Significant reaction enhancement (up to &gt;200×) is observed over microdroplet reactions generated from electrospray. This enhancement for the plasma‐microdroplet system is attributed to the combined effects of energetic collisions and the presence of reactive oxygen species (ROS). The ROS (e.g., O2⋅−) act as a proton sink to increase abundance of free neutral amines in the charged microdroplet environment. The effect of ROS on N‐alkylation is confirmed through three unique experiments: (i) utilization of radical scavenging reagent, (ii) characterization of internal energy distribution, and (iii) controls performed without plasma, which lacked reaction acceleration. Establishing plasma discharge in the wake of charged microdroplets as a green synthetic methodology overcomes two major challenges within conventional gas‐phase plasma chemistry, including the lack of selectivity and product scale‐up. Both limitations are overcome here, where dual tunability is achieved by controlling reagent concentration and residence time in the microdroplet environment, affording single or double N‐alkylated products. Products are readily collected yielding milligram quantities in eight hours. These results showcase a novel synthetic strategy that represents a straightforward and sustainable C−N bond‐forming process.

Rational Design of Porous Ionic Liquids for Coupling Natural Gas Purification with Waste Gas Conversion

AbstractRemoval of trace impurities for natural gas purification coupled with waste gas conversion is highly desired in industry. We here report a type of porous ionic liquids (PILs) that can realize the continuous flow separation of CH4/CO2/H2S and the conversion of the captured H2S to useful products. The PILs are synthesized through a step‐by‐step surface modification of ionic liquids (ILs) onto UiO‐66‐OH nanocrystals. The introduction of free tertiary amine groups on the nanocrystal surface endows these PILs with an exceptional ability to enrich H2S from CO2 and CH4 with impressive selectivity, while the permanent pores of UiO‐66‐OH act as containers to store an exceptionally higher amount of the selectively captured H2S than the corresponding nonporous ILs. Simultaneously, the tertiary amines as dual functional moieties offer effective catalytic sites for the conversion of the H2S stored in PILs into 3‐mercaptoisobutyric acid, a key intermediate required for the synthesis of Captopril (an antihypertensive drug). Molecular dynamics, density functional theory calculations and Grand Canonical Monte Carlo simulations help understand both the mechanisms of separation and catalysis performance, confirming that the tertiary amines as well as the permanent pores in UiO‐66‐OH play vital roles in the whole procedure.

Rational Design of Porous Ionic Liquids for Coupling Natural Gas Purification with Waste Gas Conversion.

Removal of trace impurities for natural gas purification coupled with waste gas conversion is highly desired in industry. We here report a type of porous ionic liquids (PILs) that can realize the continuous flow separation of CH4 /CO2 /H2 S and the conversion of the captured H2 S to useful products. The PILs are synthesized through a step-by-step surface modification of ionic liquids (ILs) onto UiO-66-OH nanocrystals. The introduction of free tertiary amine groups on the nanocrystal surface endows these PILs with an exceptional ability to enrich H2 S from CO2 and CH4 with impressive selectivity, while the permanent pores of UiO-66-OH act as containers to store an exceptionally higher amount of the selectively captured H2 S than the corresponding nonporous ILs. Simultaneously, the tertiary amines as dual functional moieties offer effective catalytic sites for the conversion of the H2 S stored in PILs into 3-mercaptoisobutyric acid, a key intermediate required for the synthesis of Captopril (an antihypertensive drug). Molecular dynamics, density functional theory calculations and Grand Canonical Monte Carlo simulations help understand both the mechanisms of separation and catalysis performance, confirming that the tertiary amines as well as the permanent pores in UiO-66-OH play vital roles in the whole procedure.