Supernatant Phase Research Articles

Evaluation of the use of volatile electrolyte system produced by ammonia and carbon dioxide in water for the salting-out of proteins: Precipitation of porcine trypsin

In this work, the precipitation of porcine trypsin in solutions of volatile electrolytes formed by ammonia and carbon dioxide was investigated. The solubility curve of trypsin at 4 °C was determined by measuring the compositions of the precipitate and supernatant phases. Analysis showed that ammonium carbamate is able to induce trypsin salting-out. The verified salting-out property of ammonium carbamate, the easy removal of the salt out of the protein precipitate and mother-solution and the high activity recovery demonstrate the feasibility of using ammonium carbamate as precipitant agent.

Evaluation of electrolysis for oxidative deodorization of hog manure

Electrolysis was investigated as a method for the amelioration of odor from the supernatant phase of centrifuged liquid hog manure. Methods examined included the use of dual anodes to provide both oxidizing intermediates from water electrolysis and a sacrificial input of iron into solution to remove organic constituents of hog manure by coagulation. The most promising approaches used a “dimensionally stable anode” composed of tin dioxide coated on a base of titanium, and the newly developed material boron-doped diamond, which has an exceptional stability to both oxidizing and reducing electrolytic conditions. These anodes promote the formation of hydroxyl radicals which initiate oxidation of organic constituents of the manure.

Kinetics of phase separation in systems exhibiting simple coacervation

The kinetics of phase separation of a homogeneous polyelectrolytic solution into a dense polymer-rich coacervate and the dilute supernatant phase is discussed through statistical thermodynamics. It has been shown that the coacervate phase is associated with higher internal pressure, consequently giving rise to syneresis. Physical conditions for phase separations has been deduced explicitly which reveals that sigma(2)/qrt[I] > or = constant (where sigma is polyelectrolyte charge density and I is solution ionic strength), consistent with experimental observations. In the lattice model, r is the number of sites occupied by the polymer having a volume critical fraction psi(2c), it was found that phase separation would ensue when sigma(3)r > or = (64/9 alpha(2)) [psi(2c)/(1 - omega(2c))(2)], which reduces to (sigma(3)r/psi(2c)) > or = (64/9 alpha(2)) approximately 0.45 at 20 degrees C for psi(2c) < 1. The separation kinetics mimics a spinodal decomposition process. Rate of release of supernatant due to syneresis was found to be independent of the initial coacervate mass. Syneresis results are discussed in the context of temporal evolution of self-organization in polymer melts through Avrami model.

Evaluation of different primary recovery methods forE. coli-derived recombinant human growth hormone and compatibility with further down-stream purification

Due to advances in fermentation technology, it is now possible to obtain fermentation broth with over 30% solids. The high solid content makes the clarification step difficult, especially at large scale. The primary protein recovery step is challenging due to the heterogeneous solution of soluble and insoluble material. In this study, we compare different primary recovery routes and the compatibility with the initial capture chromatography step. The primary recovery routes studied are standard clarification by centrifugation and extraction in aqueous two-phase systems. The compatibility of the feed streams from the different primary recovery steps with the first chromatography step is addressed. An anion-exchange column was used as the first capture column in the purification process. The aqueous two-phase system was composed of a random copolymer of ethylene oxide and propylene oxide (EOPO) in combination with a waxy starch. The target protein in this study was human growth hormone (hGH) produced in recombinant Escherichia coli. The purity of hGH in the top phase after aqueous two-phase extraction was found to be significantly higher than in clarified homogenate supernatant and increased as the EOPO polymer concentration in the aqueous two-phase system increased. Stability of the supernatant and EOPO top phases and hGH were determined by turbidity measurements and LC-MS assay. All of the feed-streams from the primary recovery steps were compatible with the anion-exchange chromatography step; however, the capacity of the resin was strongly dependent on the purity of the load. Different process aspects, e.g., resin capacity, viscosity, purification, and yield of hGH and scalability are compared.

Clusters of Optimum Size Formed by Hydrophobically Associating Polyelectrolyte in Homogeneous Solutions and in Supernatant Phase in Equilibrium with Macroscopic Physical Gel

AbstractSummary: The formation of finite‐sized microgel particles was revealed by photon correlation spectroscopy in aqueous solutions of hydrophobically associative polyelectrolyte polystyrene‐block‐poly(sodium methacrylate)‐block‐polystyrene. Starting from the lowest polymer concentration (1 × 10−6 g · L−1) up to 1 g · L−1 a single relaxation process dominates the spectrum of relaxation times of the polymer solutions. This dominant mode was attributed to the diffusive motion of supramolecular microgel particles with a size of about 100 nm. Above a polyelectrolyte concentration of 1.0 g · L−1, the size of the microgel grows up to ca. 300 nm. At a polymer concentration 20 g · L−1, macroscopic phase separation occurs: the liquid phase of the microgel‐cluster solution coexists with the phase of the macroscopic gel.Schematic representation of the formation of supramolecular structures in aqueous solutions of hydrophobically associative polyelectrolyte.magnified imageSchematic representation of the formation of supramolecular structures in aqueous solutions of hydrophobically associative polyelectrolyte.

Influence of Humic Acid on Adsorption and Desorption of Atrazine, Hydroxyatrazine, Deethylatrazine, and Deisopropylatrazine onto a Clay-Rich Soil Sample

Adsorption and desorption properties of atrazine and some of its metabolites, hydroxyatrazine (AT-OH), deethylatrazine (DEA), and deisopropylatrazine (DIA), were studied with a clay-rich soil sample (clay content of 53%). A part of this soil was treated with humic acid (Soil-HA) to assess the influence of this important component of natural organic matter on adsorption and desorption processes. This study was performed using the batch approach with 1.0 g of soil, or Soil-HA, in 5.0 mL of 0.010 mol L(-)(1) CaCl(2) solution containing the herbicide and the metabolites in a concentration range between 0.010 and 5.0 mg L(-)(1). After 24 h of contact time, the suspensions were centrifuged and the four compounds were quantified in the supernatant phases by high-performance liquid chromatography. The adsorption and desorption data of both Soil and Soil-HA were properly fitted by the linearized Freundlich equation. For the untreated soil, the adsorption affinity order evaluated as a function of the K(f) values was AT-OH > AT > DIA > DEA, while desorption followed the order DEA > DIA approximately AT > AT-OH. The presence of humic acid increased significantly the adsorption of all compounds, following the same affinity order observed for the untreated soil. Increase in adsorption was especially high for AT-OH and AT. On the other hand, the dealkylated metabolites, DEA and DIA, were more easily desorbed from the Soil-HA sample, suggesting that natural organic matter facilitates the leaching of these compounds. Desorption order in the presence of humic acid was DEA > DIA > AT > AT-OH.

Structural and Rheological Properties of Hydrophobically Modified Polysaccharide Associative Networks

The phase behavior of hydrophobically modified chitosans (HMCs) in aqueous solution has been investigated using scattering and rheology experiments. We observed four regions on the phase diagram of the associative polymer: (i) a supernatant phase (unimers phase) at low polymer concentration; (ii) a dilute solution of intermolecularly bridged flowerlike micelles at intermediate concentration; (iii) an associative gel phase at high polymer content; and (iv) a phase separation. In the present paper, we discuss the structural and dynamical properties of the HMC associative networks (c > c*) at a fixed hydrophobic degree of substitution of 2% and fixed alkyl side chains (stickers) length C8 (domains iii and iv of the phase diagram). As the polymer concentration is increasing, a connecting network is formed from the percolation of bridges between micellar aggregates. In this regime, small-angle neutron scattering and light scattering measurements show that -50-nm flower aggregates are acting like junction points in the network. The effect of the concentration, the stress, and the shear on the structure of the network is discussed. In particular, we observe bridge-to-loop transitions and then the formation of microgels or a low-connected network under shear. Therefore, our results are compared to recent theoretical models and to the results reported for telechelic systems.

Preparation and adsorption of refined polyelectrolyte complex nanoparticles

We report on bulk and surface properties of centrifuged nonstoichiometric polyelectrolyte complex (PEC) dispersions. PECs were prepared by mixing poly(diallyldimethylammonium chloride) (PDADMAC) and sodium poly(maleic acid-co- α-methylstyrene) (PMA-MS) at the monomolar mixing ratio of 0.6 and polymer concentration ⩾1 mmol/l. Centrifugation of initial PEC dispersions revealed three phases: supernatant (SUP), coacervate (COAC), and an insoluble precipitate. Mass, turbidity, particle hydrodynamic radii ( R h ), and the titratable charge amount were determined for those phases. The turbid COAC phase consisted of 200-nm nanoparticles and carried 60% of the polymer mass and 20% of the titratable charge amount of the initial PEC dispersion. The SUP phase showed no turbidity and no such nanoparticles, but carried 80% of the initial titratable charge amount, presumably caused by excess polycations. Furthermore, linear dependences of turbidity and R h on COAC concentration was observed. COAC adsorption was studied at polyelectrolyte multilayer (PEM) modified silicon surfaces in dependence on both adsorption time and concentration using attenuated total-reflection–Fourier transform infrared (ATR–FTIR) spectroscopy. The adsorption data were fitted by the simple Langmuir model. Comparison of COAC particles and polystyrene latices revealed similar adsorption features. SEM and AFM measurements resulted in hemispherically shaped adsorbed COAC particles with coverages ⩾25%, whose calculated volumes correlated well with those in dispersion obtained by PCS.

Selenium chemistry in amorphous iron (hydr)oxide-applied soil as influenced by redox potential (Eh) and pH

A laboratory study was carried out that focused on the stability of immobilized Se in amorphous iron (hydr)oxide-applied soils in relation to the redox potential (Eh) and pH. Applied soils were incubated at 28°C (± 0.1) under submerged conditions. Without the addition of a microbial nutrient source, no significant changes in the incubated soil Eh and, hence, in the amount of soluble Se were observed. On the other hand, the Eh changed within a few days of incubation with the addition of 1% glucose to the samples. Under oxidative conditions (Eh > 200 mV), the amount of soluble Se in the incubated samples ranged from 2 to 22 μg L−1. Under mildly oxidative conditions (200 to 0 mV), the Eh decreased and pH increased simultaneously, resulting in a subsequent increase in the amount of soluble Se. After 28 d of incubation, the Eh drastically decreased to a strongly reductive state in both soil and supernatant phase, and the amount of soluble Se increased to a maximum value followed by a decrease to a low level. Reductive dissolution of applied amorphous iron (hydr)oxide occurred during this transition period that led to the subsequent desorption of immobilized Se into the solution phase. The amount of desorbed Se further reduced to insoluble Se forms in a strongly reductive condition. Se species in total soluble Se in soils with mixed [Se(IV) + Se(VI)] contamination were determined under different Eh-pH conditions. The amount of soluble Se(VI) inversely followed the changes in the Eh values in an oxidative to mildly reductive condition and that closely followed the changes in the Eh values in a reductive to strongly reductive condition in this experiment. The amount of soluble Se(VI) increased followed by a decrease in the amount of total soluble Se; however, Se(VI) in soluble Se was the major species throughout the incubation period. The increase in the amount of soluble Se(IV) and organic Se species was inversely related to the decrease in the Eh values throughout the incubation period. These results suggest that immobilized Se in amorphous iron (hydr)oxide-applied soil is stable either under oxidative or strongly reductive environment.

Effect of light on birnessite catalysis of the Maillard reaction and its implication in humification

The Maillard reaction between carbohydrates and nitrogenous compounds originally investigated in 1912 has subsequently been proposed as a possible pathway for the formation of humic substances in natural environments. However, the role of mineral catalysis of the Maillard reaction is little understood and the promoting effect of light on such catalysis is not known. Birnessite (δ-MnO2), which is commonly present in soil environments, was investigated for its activity in promoting the Maillard reaction between glucose and glycine at a light intensity of 168 µE s–1 m–2 or in the dark. The presence of substantial quantities of Mn(II) was detected in both the supernatant and solid phase of the glucose-glycine-birnessite systems. The spectroscopic evidence indicates that birnessite, in the presence of light, is a very effective catalyst in abiotic browning of solutions of glucose and glycine. Furthermore, birnessite significantly promoted the reaction even in the absence of light. Therefore, the abiotic heterogeneous catalytic role of soil minerals such as birnessite in polycondensation of simple sugars and amino acids merits close attention in the formation of humic substances in natural environments. Key words: Maillard reaction, heterogeneous catalysis, light, birnessite, humic substance formation, XANES

Phase Behavior of Associating Polyelectrolyte Polysaccharides. 1. Aggregation Process in Dilute Solution

We have examined the structure of hydrophobically modified chitosans in a selective aqueous solution at T = 25 °C, i.e., a nonsolvent for the alkyl grafted chains and a good solvent for the polyelectrolyte chitosan backbone. The behavior of the hydrophobically modified chitosan was investigated using viscosity and static and dynamic light scattering. We observed three regions on the phase diagram of the associative polymer in aqueous solution: (i) a supernatant phase (unimers phase) at low polymer concentration; (ii) solutions of intermolecularly bridged “flowerlike” micelles at intermediate polymer concentration; (iii) an associative gel phase at high polymer content. The aggregation process in dilute regime was investigated using combined static and dynamic light scattering. In particular, the concentration dependence of the aggregation number, of the micellar aggregates size, of the micelle concentration, and of the unimer concentration is discussed.

Poind Soil pH Measurement

Soil pH often is measured in samples from the bottoms of aquaculture ponds. Several different techniques for soil pH are used. This study considered the differences in pH obtained by the different methods and determined which methods appeared most useful. Dual electrodes (indicating and reference) and a single-probe combination electrode gave similar pH values when inserted into 1:1 mixtures of dry soil and distilled water. There were slight differences in pH between readings with dual and combination electrodes when the dual electrodes were arranged with the indicating electrode in the sediment phase and the reference electrode in the supernatant phase of the mixture. The two-phase method with the dual electrode does not appear warranted because of greater difficulty in making measurements. Dry soil: distilled water ratios of 1:2.5, 1:5, and 1:10 had progressively greater pH readings than obtained at a 1:1 ratio. Measurements made in 0.01 M CaCl2 and 1.0 M KCl had much different values than those made in distilled water. Higher pH resulted when pH was measured without stirring or in filtrates of soil-water mixtures. A 20-min period of intermittent stirring before making measurements was necessary for a stable pH value. Particle size did not influence pH in aliquots passing 0.053 to 2.36-mm sieves. Drying temperature had a strong influence on pH, and measurements made on samples dried at 40 to 60 C are probably most reliable. Measurements of in situ pH in wet soil with standard pH electrode or a portable acidity tester differed greatly from those made in 1:1 dry soil to distilled water mixtures. Pond bottom soil pH measurement should be standardized. Based on findings of this study, the following method is suggested: dry soil at 60 C in a forced-draft oven; pulverize soil to pass a 2-mm sieve; mix soil and distilled water in a 1:1 ratio (weight: volume); stir intermittently with glass rod for 30 min; insert dual electrodes or a combination electrode into the mixture; measure pH while stirring.

High-performance liquid chromatographic determination of bradykinin in saliva: a critical review and a new method

Because of difficulties or dubious results with previously published methodologies, a new semi-automated HPLC method with UV absorbance detection was developed and applied to the determination of bradykinin (BK) in human saliva. The new method consisted of an uncomplicated sample preparation involving the addition to saliva of an equal volume of 0.1 M orthophosphoric acid to stabilize BK, vortex-mixing, centrifugation, and separation, followed by chromatography of the supernatant phase on a C 8, 150×3.9-mm (I.D.) stainless steel column. The mobile phase was composed of 19% acetonitrile/0.1% trifluoroacetic acid at flow-rate of 0.4 ml/min. Using UV detection at 220 nm, the detection limit was 1 ng/ml for the BK standard, and 7 ng/ml for the assay of endogenous salivary BK. The orthophosphoric acid initially added to the saliva allowed BK to be stabilized from enzymic degradation at 20°C for 5 days and at 4°C for 60 days. Assignment made to the peak with the chromatographic properties of salivary BK was confirmed by HPLC–MS with an electrospray interface. This paper presents a new method that is reproducible, reliable and allows kinetic studies of salivary BK to be performed for clinical investigations.

Swelling and Collapse of Physical Gels Formed by Associating Telechelic Polyelectrolytes

A theory is proposed for the swelling and collapse of physical gels formed by associating telechelic polyelectrolytes. It is shown that increasing the of degree of ionization makes the formation of an associative gel less favorable and increases the density of the supernatant phase. This leads to the contraction of the associative gel, in comparison with an analogous chemically cross-linked gel. The results of calculation show that the destruction of the associative gel happens monotonically in good solvent, while in poor solvent it can proceed in a jump-like fashion accompanied by the first-order phase decollapse transition of the associative gel. The addition of a low molecular weight salt to the solution facilitates the association of telechelic molecules which produces the associative gel and a further increase of salt concentration leads to the contraction of the associative gel.

Expression of human PAI-2 in the baculovirus expression system

Using pSXIVVI + X3 as an expressing vector, an occluded recombinant Trichoplusia ni nuclear polyhedrosis virus carrying the cDNA encoding plasminogen activators inhibitor-2 (PAI-2) under the control of the Syn and XIV promoters, has been constructed. SDS-PAGE and immunoblot analysis revealed that the virus-mediated PAI-2, with a molecular weight of ∼45 kDa, was synthesized in the Sf cells at a level of ∼16% of total intracellular protein and in the supernatant phase at a level of ∼64% of total extracellular protein secreted into the hemolymph of infected larvae. The expressed protein was similar to its authentic counterpart in terms of immunoreactivity and bioactivity.

Some molecular and colloidal aspects of tea cream formation

The formation of tea cream for a range of black tea materials was studied by turbidity measurements, time-resolved light scattering, gravimetric measurements and High Performance Liquid Chromatography (HPLC). These techniques demonstrate that, in the absence of caffeine and/or gallate esters the solubility of tea solids in black tea infusions is enhanced compared with the ‘standard’ black tea material. All the tea system show an increase in cream-point, amount of tea cream and particle size with increasing tea solids concentration. Decaffeination has a larger impact on enhancing the solubility of tea colloids than degallation. HPLC studies indicate that theaflavins have a preference for the cream phase, whereas ie. catechin C is preferentially partitioned into the aqueous supernatant phase. Caffeine is preferentially partitioned into the cream phase, albeit less favourably than theaflavins and other relatively high-molecular mass polyphenols. Comparison between ‘standard’ and decaffeinated black tea shows that in the absence of caffeine, epigallocatechin gallate (EGCG) is preferentially partitioned into the cream phase.

Initial protein concentration effects on precipitation by salt

The influence of initial protein concentration on the performance of salting-out precipitation is examined. In the precipitation of bovine albumin by ammonium sulfate, a peak occurs in the plot of protein solubility versus initial protein concentration; that is, the solubility first increases and then falls with increasing initial protein concentration. In addition, the dependence of solubility on the initial protein concentration is less significant if using higher salt concentrations. The solubility behavior of bovine albumin may be representative, because it covers all possible alternatives; namely, the solubility is independent of, increases with, decreases with, or first increases and then decreases with the initial protein concentration. The appearance of a solubility peak can be explained based on the occurrence of a primary particle during the precipitation process. However, inclusion of the influence of initial protein concentration into the Cohn equation is not feasible with the use of a logarithmic scale, which does not sensitively reflect the change in protein solubility. Increasing initial protein concentration favors protein recovery because it reduces the resultant volume of the supernatant phase. (c) l997 John Wiley & Sons, Inc.

Single-step purification of recombinant Bm86 protein produced in Pichiapastoris by salting-out and by acid precipitation of contaminants

Two single-step purification methods were used to isolate the recombinant protein, rBm86, produced in Pichia pastoris. Salting-out results in a compromise between final purity and recovery of rBm86. At 15, 25 and 35% of ammonium sulphate saturation (pH 7), rBm86 concentration in the supernatant phase was proportional to the initial amount of protein. Acid precipitation of contaminants resulted in 98% purity and 98% recovery of rBm86. High aggregation of rBm86, forming particles of 28 nm, changed the isoelectric point of monomers (5.5), considering only the aminoacid sequence, to 4.5 for particles.

Determination of contact phase activation by the measurement of the activity of supernatant and membrane surface-adsorbed factor XII (FXII): Its relevance as a useful parameter for thein vitro assessment of haemodialysis membranes

We investigated hemodialysis membrane biocompatibility with respect to contact phase activation by determination of FXII-like activity (FXIIA) on the membrane surface and in the supernatant phase, during plasma contact with various hemodialysis membranes using an in vitro incubation test cell. The results were compared to the influence of these membranes on the activation of purified FXII. A time course for the generation of activated FXII using purified FXII solution at physiologic concentrations on two similar negatively charged polymers was performed. The membranes assessed were regenerated cellulose (Cuprophan; Akzo Faser AG, Germany), modified cellulosic (Hemophan; Akzo Faser AG), acrylonitrile-sodium methallyl copolymer-based membrane AN69S (Hospal, France), and SPAN, a new polyacrylonitrile-based copolymer (akzo Nobel AG). The plasma FXIIA at the membranes surface was significantly different between the membranes, while the supernatant phase FXIIA exhibited no significant differences. In contrast, activation of purified FXII in a plasma-free system with respect to supernatant activity indicated significant differences between the materials. A similar finding for the membrane-bound factor XIIA was also observed when purified factor XII was used. The membrane-bound FXIIA values observed in the plasma system containing heparin were significantly greater than in citrated plasma. This demonstrated the strong influence of heparin and the interaction of other plasma components to the membrane surface on the activation of contact phase of coagulation.

Phase Separation of Lactan Gum from Fermentation Broths by Addition of Water-Miscible Organic Solvents

Abstract A microbial polysaccharide (lactan gum) produced by bacterium ATCC 55046 was precipitated from fermentation broths by the addition of ethanol, acetone, isopropanol, or tert-butanol. Compositions of the precipitate and supernatant phases were determined as a function of organic solvent concentration and used to construct binodal solubility curves. Lactan did not precipitate at bulk-mixture organic solvent concentrations below 35% (wt) ethanol, 35% acetone, 33% isopropanol, or 25% tert-butanol. At organic solvent concentrations just exceeding the solubility transition point, the precipitates were soft, moist, and sponge-like in texture, with low lactan concentrations. At higher organic solvent concentrations the precipitates were compact and dense. The maximum lactan concentration in the precipitate was 25–37%, depending on the organic solvent type and concentration. Increasing the organic solvent concentration beyond 50% for ethanol, or 70% for acetone, decreased the lactan concentration in the precipitate. No such decrease occurred for isopropanol and tert-butanol. Thus, organic solvent usage, from greatest to least, was in the order ethanol, acetone, isopropanol, and tert-butanol, but the maximum lactan concentration in the precipitate, from greatest to least, was in the order acetone, isopropanol, ethanol, and tert-butanol.