Stopped-flow Apparatus Research Articles

Kinetic study on CO2 absorption by aqueous secondary amine + tertiary amine blends: Kinetic model and effect of the chain length of tertiary amine

To study the reaction kinetic of CO2 absorption in aqueous secondary amine + tertiary amine blends, 1-Diethylamino-2-propanol (DEA2P) and 1-Dimethylamino-2-propanol (DMA2P) were selected to form aqueous amine blended solutions with 2-(Ethylamino) ethanol (EAE), respectively. The reaction kinetics of CO2 absorption in aqueous EAE/DEA2P solutions and EAE/DMA2P solutions were explored using the stopped-flow apparatus, and the reaction mechanism was then explored by Density Functional Theory (DFT) calculations. Based on the zwitterion mechanism and the base-catalyzed hydration mechanism, experimental reaction kinetics models were established. It was found that in secondary amine + tertiary amine blends, tertiary amines played the key role of the proton acceptor in step of zwitterion deprotonation. With the increase of the chain length of tertiary amine, the CO2 absorption rate increased. Through analysis, it was found that the main reason for the increased CO2 absorption rate was the presence of H-bond or the stronger proton affinity of N atom.

A simple method to resolve rate constants when the binding mechanism obeys induced fit or conformational selection

Many interactions involving a ligand and its molecular target are studied by rapid kinetics using a stopped-flow apparatus. Information obtained from these studies is often limited to a single, saturable relaxation that is insufficient to resolve all independent rate constants even for a two-step mechanism of binding obeying induced fit (IF) or conformational selection (CS). We introduce a simple method of general applicability where this limitation is overcome. The method accurately reproduces the rate constants for ligand binding to the serine protease thrombin determined independently from the analysis of multiple relaxations. Application to the inactive zymogen precursor of thrombin, prethrombin-2, resolves all rate constants for a binding mechanism of IF or CS from a single, saturable relaxation. Comparison with thrombin shows that the prethrombin-2 to thrombin conversion enhances ligand binding to the active site not by improving accessibility through the value of kon but by reducing the rate of dissociation koff. The conclusion holds regardless of whether binding is interpreted in terms of IF or CS and has general relevance for the mechanism of zymogen activation of serine proteases. The method also provides a simple test of the validity of IF and CS and indicates when more complex mechanisms of binding should be considered.

Instantaneous extracellular solution exchange for concurrent evaluation of membrane permeability of single cells.

The osmotic stress imposed on microorganisms by hypotonic conditions is perceived to regulate water and solute flux via cell membranes, which are crucial for survival. Some cells that fail to perceive osmotic stress die because this results in the rupture of the cell membrane. The flux through the membrane is characterized by the membrane permeability, which is measured using a stopped-flow apparatus in response to a millisecond-order osmolarity change. However, the obtained data are an ensemble average of each cell response. Additionally, the measurement of permeability, considering cellular viability, contributes to a more accurate evaluation of osmoadaptation. Here, we present a novel on-chip instantaneous extracellular solution exchange method using an air-liquid interface. The presented method provides a concurrent evaluation at the single-cell level in response to a millisecond-order osmotic shock, considering cellular viability by solution exchange. This method utilizes a liquid bridge with a locally formed droplet on the surface of a micropillar fabricated inside a microchannel. We evaluated a solution exchange time of 3.6 ms and applied this method to Synechocystis PCC 6803 under two different osmolarity conditions. The live/dead ratio of 1 M to 0.5 M osmotic down shock condition was 78.8/21.2% while that of 1 M to 0.25 M osmotic down shock condition was 40.0/60.0%. We evaluated the water permeability of two groups: cells that were still live before and after osmotic shock (hereafter named cell type 1), and cells that were live before but were dead 10 minutes after osmotic shock (hereafter named cell type 2). The results indicated that the water permeability of cell type 2 was higher than that of cell type 1. The results obtained using the presented methods confirmed that the effect of osmotic stress can be accurately evaluated using single-cell analysis.

Study on the mechanism and kinetics of amine with steric hindrance absorbing CO2 in non-aqueous/aqueous solution

Several alkanolamines (1A2P, 2A1P, AMP, MAE, EAE, and IPAE) were selected to study the steric hindrance effect on reaction kinetics between amine and CO2 in ethanol and aqueous solutions. Pseudo first-order reaction rate constant k0 was measured by the stopped-flow apparatus. Zwitterion mechanism was applied to describe the reaction and the k0 was well fitted by the corresponding kinetic model with average absolute relative deviation (AARD) in a range of 1.08%-3.89%. Apparent second-order reaction rate constant KA revealed that the reaction rate is simultaneously controlled by steric hindrance and pKa of amines. Additional alkyl groups increase the steric hindrance of amines and accelerate the decomposition of zwitterions. As a result, the reaction rate decreases with stronger steric hindrance effect. The larger pKa is conducive to a better proton receiving of amine. This leads to an acceleration of the deprotonation reaction of zwitterion intermediate. In addition, the relative influence of steric hindrance and pKa on the reaction kinetics is also related to the solvent environment.

Nitrosylation of ferric zebrafish nitrobindin: A spectroscopic, kinetic, and thermodynamic study

Nitrobindins (Nbs) are all-β-barrel heme-proteins present in all the living kingdoms. Nbs inactivate reactive nitrogen species by sequestering NO, converting NO to HNO2, and isomerizing peroxynitrite to NO3− and NO2−. Here, the spectroscopic characterization of ferric Danio rerio Nb (Dr-Nb(III)) and NO scavenging through the reductive nitrosylation of the metal center are reported, both processes being relevant for the regulation of blood flow in fishes through poorly oxygenated tissues, such as retina. Both UV–Vis and resonance Raman spectroscopies indicate that Dr-Nb(III) is a mixture of a six-coordinated aquo- and a five-coordinated species, whose relative abundancies depend on pH. At pH ≤ 7.0, Dr-Nb(III) binds reversibly NO, whereas at pH ≥ 7.8 NO induces the conversion of Dr-Nb(III) to Dr-Nb(II)-NO. The conversion of Dr-Nb(III) to Dr-Nb(II)-NO is a monophasic process, suggesting that the formation of the transient Dr-Nb(III)-NO species is lost in the mixing time of the rapid-mixing stopped-flow apparatus (∼ 1.5 ms). The pseudo-first-order rate constant for the reductive nitrosylation of Dr-Nb(III) is not linearly dependent on the NO concentration but tends to level off. Values of the rate-limiting constant (i.e., klim) increase linearly with the OH− concentration, indicating that the conversion of Dr-Nb(III) to Dr-Nb(II)-NO is limited by the OH−-based catalysis. From the dependence of klim on [OH−], the value of the second-order rate constant kOH− was obtained (5.2 × 103 M−1 s−1). Reductive nitrosylation of Dr-Nb(III) leads to the inactivation of two NO molecules: one being converted to HNO2, and the other being tightly bound to the heme-Fe(II) atom.

Kinetics of CO2 absorption into ethanolamine + water + ethanol system—mechanism, role of water, and kinetic model

The kinetics of CO2 absorption in ethanolamine (MEA) + H2O + ethanol systems was investigated using stopped-flow apparatus. The first-order reaction rate constant (k0) values were tested with varying H2O and MEA concentrations. Transiting from the aqueous to non-aqueous solvents, the k0 decreases and the reaction order for MEA shifts from 1.05 to 1.88. The zwitterion mechanism with the rate-limiting step of deprotonation was used to explore the role of water and its effect. Results showed that water could impact reactions through direct involvement and changing solvation environment. An improved kinetic model based on the zwitterion mechanism was obtained to indicate the quantitative influence of H2O concentration on the reaction kinetics of MEA + H2O + ethanol systems. The predicted values from the model well agreed to the experimental data with the average absolute relative difference of 6.73%. This study highlights the important effects of water on the reaction kinetics of CO2 absorption into water-lean amine solutions.

Time-resolved guanine nitration by peroxynitrite

Purines are nitration targets for Reactive Nitrogen Species such as peroxynitrite (ONOO-), a harmful oxidant produced in vivo by the diffusion-limited combination between nitric oxide and the free radical superoxide. ONOO- is responsible for several types of biological damage, including membrane lipid peroxidation, modification of protein sulfhydryl groups, hydroxylation/nitration of aromatic rings, and DNA damage. We studied the in vitro kinetics of guanine (Gua) nitration by ONOO- as a function of reactant concentrations and temperature (10-40°C), using a SX20 stopped-flow rapid-mixing apparatus with dead-times of ∼1 ms.

A study of kinetics, equilibrium solubility, speciation and thermodynamics of CO2 absorption into benzylamine (BZA) solution

The kinetics between BZA and CO2 were investigated by the stopped-flow apparatus over the BZA concentration range from 0.05 to 0.1 mol/L (0.54–1.07 mass%), and over the temperature range from 293 to 313 K. The results also show that the predicted CO2 absorption rate exhibits good agreement with the experimental data with an absolute average deviation (AAD) of 5.15% and 4.15% with respect to the zwitterion and termolecular models, respectively. Furthermore, the NMR experiments confirmed the formation of carbamates. The VLE plots were made with speciation data obtained from NMR techniques applied to the CO2-BZA-H2O system with CO2 loading of 0–0.67 mol CO2/mol BZA at 298 K. A new VLE model was proposed, and the predicted results exhibit good agreement with the experimentaly measured results.

The Kinetics Investigation of CO2 Absorption into TEA and DEEA Amine Solutions Containing Carbonic Anhydrase

Tertiary amines have been used as alternative absorbents for traditional primary and secondary amines in the process of carbon capture. However, the carbon dioxide (CO2) absorption rates in these kinds of amine are relatively slow, which implies greater investment and construction costs and limits the large-scale application of carbon capture. Carbonic anhydrase (CA) is considered to be an ideal homogeneous catalyst for accelerating the rate of CO2 into aqueous amine solution. In this work, CO2 absorption combining CA with two single aqueous tertiary amines, namely triethanolamine (TEA) and 2-(diethylamino)ethanol (DEEA), was studied by use of the stopped-flow apparatus over temperature ranging from 293 to 313 K. The concentrations of selected aqueous amine solution and CA used in the experiment were ranging among 0.1–0.5 kmol/m3 and 0–50 g/m3 , respectively. Compared to the solution without the addition of CA, the pseudo first-order reaction rate in the presence of CA (k0,withCA) is significantly increased. The values of k0,withCA have been calculated by a new kinetics model. The results of experimental and calculated k0,amine and k0,withCA in CO2-amine-H2O solutions were also investigated,respectively.

A high-flux automated laboratory small-angle X-ray scattering instrument optimized for solution scattering

A commercially available small-angle X-ray scattering (SAXS) NanoSTAR instrument (Bruker AXS) with a liquid-metal-jet source (Excillum) has been optimized for solution scattering and installed at iNANO at Aarhus University. The instrument (named HyperSAXS) employs long high-quality parabolic Montel multilayer optics (Incoatec) and a novel compact scatterless pinhole slit with Ge edges, which was designed and built at Aarhus University. The combination of the powerful source and optimized geometry gives an integrated X-ray intensity close to 109 photons s−1 for a standard range of scattering vector moduli q = 0.0098–0.425 Å−1, where q = (4πsinθ)/λ and λ is the Ga Kα wavelength of 1.34 Å. The high intensity of the instrument makes it possible to measure dilute samples of, for example, protein or surfactant with concentrations of 1 mg ml−1 in a few minutes. A flow-through cell, built at Aarhus University, in combination with an automated sample handler has been installed on the instrument. The sample handler is based on the commercial Gilson GX-271 injection system (Biolab), which also allows samples to be stored under thermostatted conditions. The sample handler inserts and removes samples, and also cleans and dries the sample cell between measurements. The minimum volume of the flow-through capillary is about 20 µl. The high intensity additionally allows time-resolved measurements to be performed with a temporal resolution of seconds. For this purpose a stopped-flow apparatus, (SFM-3000, Bio-Logic) was connected to the flow-through cell by high-performance liquid chromatography tubing. This configuration was chosen as it allows vacuum around the sample cell and thus maintains a low background. The instrument can readily be converted into a low-q setup with a q range of 0.0049–0.34 Å−1 and an X-ray intensity of about 5 × 107 photons s−1.

Dual-Channel Stopped-Flow Apparatus for Simultaneous Fluorescence, Anisotropy, and FRET Kinetic Data Acquisition for Binary and Ternary Biological Complexes.

The Stopped-Flow apparatus (SF) tracks molecular events by mixing the reactants in sub-millisecond regimes. The reaction of intrinsically or extrinsically labeled biomolecules can be monitored by recording the fluorescence, F(t), anisotropy, r(t), polarization, p(t), or FRET, F(t)FRET, traces at nanomolar concentrations. These kinetic measurements are critical to elucidate reaction mechanisms, structural information, and even thermodynamics. In a single detector SF, or L-configuration, the r(t), p(t), and F(t) traces are acquired by switching the orientation of the emission polarizer to collect the IVV and IVH signals however it requires two-shot experiments. In a two-detector SF, or T-configuration, these traces are collected in a single-shot experiment, but it increases the apparatus’ complexity and price. Herein, we present a single-detector dual-channel SF to obtain the F(t) and r(t) traces simultaneously, in which a photo-elastic modulator oscillates by 90° the excitation light plane at a 50 kHz frequency, and the emission signal is processed by a set of electronic filters that split it into the r(t) and F(t) analog signals that are digitized and stored into separated spreadsheets by a custom-tailored instrument control software. We evaluated the association kinetics of binary and ternary biological complexes acquired with our dual-channel SF and the traditional methods; such as a single polarizer at the magic angle to acquire F(t), a set of polarizers to track F(t), and r(t), and by energy transfer quenching, F(t)FRET. Our dual-channel SF economized labeled material and yielded rate constants in excellent agreement with the traditional methods.

Stopped-Flow Dynamics Study on the Escape Behavior of Polyelectrolyte Macromolecules from Microgels: The Influence of the Path Length and Size.

We reported the fabrication of several monodispersed poly(2-vinyl pyridine)-poly(N-isopropylacrylamide) (P2VP-PNIPAM) microgels including the P2VP core (non-cross-linked) and PNIPAM (cross-linked) shell by mature emulsion polymerization. The fast escape behavior (diffusion process) of linear P2VP chains through a porous PNIPAM layer was investigated by a pH jump stopped-flow apparatus. The time-dependent dynamic traces (corresponding to the scattered light intensity) decreased at the initial timescale of several seconds and then reached an apparent equilibrium, confirming the efficient escape of P2VP chains from microgels. Compared with the previously reported literature, such an accelerated escape process resulted from the sharply increased internal charge repulsive force caused by the protonation of P2VP moieties under acidic conditions. The obtained characteristic relaxation times by single exponential fitting of these kinetic traces were dependent on the final pH values, equilibrium temperatures, shell thickness (path length), and cross-linking density (mesh size). We believe that this work can provide an efficient way to investigate hindered diffusion, especially the initial rapid diffusion stage. Not only that, the proposed model can also provide theoretical guidance to some practical applications, such as membrane separation and the exocytosis phenomenon of intracellular proteins or macromolecular substances.

Lipid exchange among polymer-encapsulated nanodiscs by time-resolved Förster resonance energy transfer.

Some amphiphilic copolymers such as diisobutylene/maleic acid (DIBMA) and styrene/maleic acid (SMA) copolymers are able to directly extract cellular membranes into nanosized polymer-bounded lipid-bilayer patches. These polymer-encapsulated nanodiscs offer the possibility to investigate delicate membrane proteins along with their surrounding lipids and, thus, protein/lipid interactions, in a near-native bilayer environment. By dissecting the kinetics of lipid exchange among DIBMA- and SMA-bounded nanodiscs, we have recently shown that the encapsulated lipid bilayer does not represent a static snapshot of the membrane at the time point of solubilisation. Instead, nanoscale lipid-bilayer patches remain in equilibrium with one another through lipid exchange enabled by nanodisc collisions. This finding is important for correctly interpreting any attempts at studying protein/lipid interactions with the aid of polymer-based nanodiscs and will be relevant to characterising the rapidly growing repertoire of new amphiphilic copolymers for membrane extraction. A highly sensitive and efficient technique for measuring the kinetics of lipid transfer among various kinds of nanosized membrane mimics consists in time-resolved Förster resonance energy transfer (TR-FRET) monitored in a stopped-flow apparatus. Here, we provide detailed instructions on how to measure the kinetics and unravel the underlying mechanisms of lipid exchange among lipid-bilayer nanodiscs under various solution conditions.

Comparative kinetics of homogeneous reaction of CO2 and unloaded/loaded amine using stopped-flow technique: A case study of MDEA solution

The stopped-flow technique was applied for the investigation of kinetics of CO2 absorption into loaded MDEA solution over the range of MDEA concentration of 0.4 to 1.0 kmol/m3, CO2 loading of 0.14 to 0.17 mol CO2/mol amine and the temperature of 293 to 308 K. The kinetics of unloaded MDEA was also investigated for the comparison and a huge difference of results which should be equal theoretically was indicated between loaded and unloaded systems. Then, the effect of CO2 loading on the kinetics was studied. As the loading increases, there is a turning point around loading of 0.10, below which the overall reaction constant decreases strongly. It was found that contribution of hydroxyl is a crucial to overall reaction rate constant and cannot be ignored and the influence of reverse reaction of MDEA and CO2 can be ignored. The kinetics of loaded system obtained in this work was modified and interpreted well by the base-catalyzed mechanism with AARD of 6.29% and compares favorably with literature using gas-liquid apparatus. The kinetics of unloaded system in this work is approximately twice as that of literature and loaded system. This work provided literature review and interpretation of MDEA-CO2-H2O and is hopeful to shed light on kinetic study of CO2 absorption in tertiary amine using stopped-flow apparatus.

Observing the Kinetic Pathway of Nanotube Formation from Bolaamphiphiles by Time-Resolved Small-Angle X-ray Scattering.

We investigated the formation kinetics of a single monolayer nanotube from bolaamphiphiles (consisting of a sugar residue, an alkyl chain, and an amino group) in solution. In this bolaamphiphile, a transition from a monomerically dispersed state to the nanotube takes place by changing the solvent condition. This transition was induced by fast mixing with a stopped-flow apparatus. From just after the mixing, this transition process was monitored in situ by time-resolved small-angle X-ray scattering. In this manner, we were able to derive the direct structural information as a function of time during the nanotube formation. The results revealed that disklike aggregates initially formed, which then grew and closed to produce a tubular structure.

The comparative kinetics study of CO2 absorption into non-aqueous DEEA/MEA and DMEA/MEA blended systems solution by using stopped-flow technique

The kinetics of the absorption of CO2 into solutions of (monoethanolamine) MEA-Ethanol, (N, N-diethylethanolamine) DEEA-MEA-Ethanol and (N, N-dimethylethanolamine) DMEA-MEA-Ethanol have been studied by applying a stopped-flow apparatus. In this study, the concentration range of both tertiary amine and MEA were chosen to be 0.2–0.4 kmol/m3 with a temperature range of 293–313 K under non-aqueous conditions. The model based on the termolecular reaction mechanism is proposed to interpret the experimental kinetic data of the reaction occurred in the non-aqueous blended amine systems, and the results indicate that the model is well fitted to the experimental data with an AARD of 5.70% for DEEA-MEA-Ethanol system and 6.53% for DMEA-MEA-Ethanol system. Blended amines show many advantages in kinetics because of the existence of the interaction between the tertiary amine and MEA. The kinetics behaviors of CO2 absorbed in the two non-aqueous blended amine systems are discussed in brief according to the molecular structures of the two tertiary amines. Different from the traditional aqueous systems, this study proved that tertiary amine enhanced the ability of primary amine to absorb CO2 which is due to tertiary amine didn’t react with CO2 directly in a non-aqueous system. The result in the non-aqueous system also shows that DMEA is more easily participate in the termolecular reaction between MEA and CO2 compared with DEEA, which indicate the length of alkyl chain on nitrogen atom also plays an important role in the enhancement effect. Additionally, after comparing the kinetic data of the two blended amine systems under aqueous and non-aqueous conditions, it can be found that tertiary amine can participate in the termolecular reaction between MEA and CO2, and that the protonated MEA can be reconverted to free MEA by the tertiary amine, making it easier for MEA to participate in its own termolecular reactions, so that the overall reaction rate of the blended amine system is accelerated. It can be summarized that there are interactions between the tertiary amine and the primary amine in the non-aqueous blended amine system that positively promote the kinetics.

Kinetics and mechanism of reaction between carbon disulfide and novel aqueous amines solutions

The mechanism and kinetics of carbon disulfide (CS2) capture by aqueous solutions of well known alkanolamines ([monoethanolamine (MEA) and diethanolamine (DEA)] and novel cyclic mono- and polyamines [morpholine (MRPH), piperazine (PZ), n-methyl piperazine (NMPZ) and n-hydroxyethyl piperazine (NHEPZ)] were investigated by stopped-flow apparatus with conductivity detection. The observed reaction rate constants were obtained for a temperature range of 298.15-313.15 K and it was found that cyclic amines react much faster than others. A modified termolecular reaction mechanism was used to analyse the experimental kinetic data. The activation energies for all systems were also obtained by evaluating the Arrhenius equation.

Kinetics and Mechanism of Reaction between Carbon Disulfide and Novel Aqueous Amines Solutions

The mechanism and kinetics of carbon disulfide (CS2) capture by aqueous solutions of well known alkanolamines ([monoethanolamine (MEA) and diethanolamine (DEA)] and novel cyclic mono- and polyamines [morpholine (MRPH), piperazine (PZ), n-methyl piperazine (NMPZ) and n-hydroxyethyl piperazine (NHEPZ)] were investigated by stopped-flow apparatus with conductivity detection. The observed reaction rate constants were obtained for a temperature range of 298.15-313.15 K and it was found that cyclic amines react much faster than others. A modified termolecular reaction mechanism was used to analyse the experimental kinetic data. The activation energies for all systems were also obtained by evaluating the Arrhenius equation.

Study of Equilibrium Solubility, Heat of Absorption, and Speciation of CO2 Absorption into Aqueous 2-Methylpiperazine (2MPZ) Solution

The CO2 reaction rate in aqueous 2-methylpiperazine (2MPZ) solution was investigated in terms of the pseudo-first-order rate constant (k0) by using a stopped-flow apparatus with amine concentration ranges of 0.05–0.125 mol/L and the measuring temperature ranges of 293.15–313.15 K. The results show that the CO2 reaction rate in the 2MPZ solution increased as the temperature and the amine concentration increases. The results also demonstrate that the 2MPZ has a faster reaction rate in terms of k0 than 3-dimethylaminopropylamine, 3-diethylaminopropylamine, and monoethanolamine (MEA) but slower than that of PZ at the same amine concentration. The equilibrium solubility as an important absorption parameter for CO2 capture also were measured at 298.15, 313.15, and 333.15 K with CO2 partial pressures of 5–100 kPa, respectively, in 2 M 2MPZ solution. The results show that the CO2 equilibrium solubility of 2MPZ increased as the CO2 partial pressure increased but decreased as the temperature increased. The heat of ...

Kinetics of Morphological Transition between Cylindrical and Spherical Micelles in a Mixture of Anionic–Neutral and Cationic–Neutral Block Copolymers Studied by Time-Resolved SAXS and USAXS

This study is concerned with the morphological transition kinetics of polyelectrolyte complex micelles formed from an anionic–neutral block copolymer and a cationic–neutral block copolymer in aqueous NaCl solution. The transformation was induced by changing the mixing ratio of the anionic and cationic monomer units in the copolymers. The kinetics of the morphological transition was directly tracked by time-resolved (ultra) small-angle X-ray scattering coupled with rapid mixing of the block copolymer components by a stopped flow apparatus. The transformation from cylindrical to spherical micelles upon changing the mixing ratio of the copolymers was reversible, and the process occurred via the random scission of the cylindrical micelles along their contours. The reverse transition from spherical to cylindrical micelles was found to be a slow process with high activation energy.