Immiscible Solvent Phases Research Articles

Polyelectrolyte based technique for sequestration of protein from an aqueous phase to an organic solvent

Abstract Separation of proteins from a complex multi-component suspension of broken down cells is technically challenging. In this regard, a technique that is capable of distinguishing proteins from other major cellular components (such as carbohydrates, lipids, and other cellular debris) could immensely simplify downstream processing. In this work, we report the ability to use the inherent zwitterionic properties of proteins to latch amphiphilic cationic electrolytes on to protein surfaces so that proteins can be extracted from an aqueous suspension into a water-immiscible organic solvent. Here we show the separation of egg albumin (a model protein) from an aqueous suspension to adjacent hexane phase using poly-(diallyl dimethyl ammonium) chloride (PolyDADMAC). Results show that, under optimum pH and polyelectrolyte concentrations, proteins can be successfully migrated from water to the adjacent hexane phase. The amount and molecular weight of electrolyte, and solution pH played vital roles in determining the amount of protein that could be migrated. Low molecular weight polyelectrolyte under neutral pH conditions, low equilibration time and 1:2 hexane: water ratio favored highest protein separation (∼85% dry weight). FTIR studies indicated that the secondary structure of the protein was preserved after migration of the protein from the aqueous phase to solvent under the protection of the polyelectrolyte coating. The results suggest that ionic electrolytes with hydrophobic moieties could be used as molecular transport vehicles to safely separate proteins from a bulk aqueous phase to an immiscible solvent phase.

Improved partition efficiency with threaded cylindrical column in vortex counter-current chromatography

Type-I coil planet centrifuge produces a uniformly circulating centrifugal force field to produce vortex motion of two immiscible solvent phases in a cylindrical cavity of the separation column to perform efficient countercurrent chromatography. The partition efficiency obtained from the original vortex column was substantially improved by threading the cylindrical cavity to increase the area of mass transfer between the two phases. Partition efficiency of the threaded column was evaluated by three different two-phase solvent systems with a broad range of hydrophobicity each with a set of suitable test samples. Overall results of the present studies indicated that the threaded cylindrical column substantially improves the partition efficiency in terms of theoretical plate number, peak resolution, and height equivalent of one theoretical plate. The results also indicated that higher peak resolution is produced by eluting either the upper phase in the head to tail direction or the lower phase in the reversed direction. When there is a choice in the mobile phase, a better separation is achieved by using the less viscous phase as the mobile phase. Since the present system gives extremely low column pressure, it may be a potential alternative to the conventional type-J HSCCC system for a large-scale preparative separation.

Mathematical model of computer-programmed intermittent dual countercurrent chromatography applied to hydrostatic and hydrodynamic equilibrium systems

Dual high-speed countercurrent chromatography (dual CCC) literally permits countercurrent flow of two immiscible solvent phases continuously through the coiled column for separation of solutes according to their partition coefficients. Application of this technique has been successfully demonstrated by separation of analytes by gas–liquid and liquid–liquid two-phase systems. However, the method cannot be directly applied to the system with a set of coiled columns connected in series, since the countercurrent process is interrupted at the junction between the columns. However, this problem can be solved by intermittent dual CCC by eluting each phase alternately through the opposite ends of the separation column. This mode of application has an advantage over the conventional dual CCC in that the method can be applied to all types of CCC systems including hydrostatic equilibrium systems such as toroidal coil CCC and centrifugal partition chromatography. Recently, the application of this method to high-speed CCC (hydrodynamic system) has been demonstrated for separation of natural products by Hewitson et al. using a set of conventional multilayer coil separation columns connected in series. Here, we have developed a mathematical model for this intermittent dual CCC system to predict retention time of the analytes, and using a simplified model system the validity of the model is justified by a series of basic studies on both hydrodynamic and hydrostatic CCC systems with a computer-programmed single sliding valve. The present method has been successfully applied to spiral tube assembly high-speed CCC (hydrodynamic system) and toroidal coil CCC (hydrostatic system) for separation of DNP-amino acid samples with two biphasic solvent systems composed of hexane–ethyl acetate–methanol–0.1 M hydrochloric acid (1:1:1:1 and 4:5:4:5, v/v).

Separation with Multi-Layer Coil Counter-Current Chromatography (MLCCC)

Counter-current chromatography (CCC) is a low-pressure, all-liquid chromatographic technique which requires no solid support. [ 1 ] Commonly it utilizes two immiscible solvent phases plus an auxiliary solvent which is miscible in both phases. The mobile phase is pumped through the stationary phase, causing the compounds to partition between two immiscible solvents, based on the partition coefficients of the solutes. MLCCC is an advanced form of CCC and employs teflon tubing wound onto a bobbin, which is centrifugally rotated in a planetary fashion [ 2 ] (Fig. 1). The planetary motion sets up alternating zones of mixing and settling which travel along the length of the coil and result in the partitioning of the components. The main advantages of CCC over conventional chromatography techniques are the absence of a solid support, the use of inexpensive solvents, and complete recovery of highly functional and reactive compounds. In contrast to other forms of CCC, MLCCC can be used on both an analytical and preparative scale. Due to high centrifugal forces, an increase in the number oftheoretical plates is achieved, thereby allowing up to 50000 mixing zones to occur per hour. This results in efficient partitioning and separation of the components in a complex mixture. Hence MLCCC is beginning to be more widely accepted as a method of choice for the separation and isolation of highly polar natural products.

Modelling of mass transfer from falling droplets

The prediction of mass transfer rates to and from moving drops has traditionally used the Whitman two-film theory approach in which the resistances to mass transfer on each side of the interface are described by film mass transfer coefficients. These are correlated in terms of the hydrodynamic conditions, the physical properties of the fluids, and the geometry of the system. The performance of liquid–liquid contactors in which mass transfer occurs between swarms of moving droplets of one phase and the other liquid phase as the continuous phase, has similarly been correlated in terms of mass transfer coefficients. These lump together the combined effects of interfacial mass transfer resistances and those associated with the bulk phases, the effects of interfacial disturbances, coalescence and break-up phenomena, specific surface area, and the effects of axial mixing. In this paper we build upon earlier work and other published research which uses finite element methods to quantitatively calculate flow field data and trajectory predictions for single particles and drops in a two phase system. The differential equations were discretised using the finite element approach employing a procedure based on the Lagrangian framework developed earlier. Here we extend the approach to calculate mass transfer rates between single aqueous drops and a continuous immiscible solvent phase. The calculated values of drop velocity and mass transfer rates are compared with experimental values determined for single drops of ethanol/water mixtures extracting into a continuous phase of n-decanol. Good agreement between the experimental and predicted values was obtained, thus demonstrating that in this case, interfacial mass transfer in liquid–liquid systems can be predicted from the fundamental transport equations. The results of the work indicate the potential of further development of this approach for swarming drops and hence quantitative prediction of the behaviour of liquid–liquid contactors.

Fluctuations and instabilities of model amphiphile interfaces

We study the stability of planar, cylindrical and spherical interfaces with respect to shape and width fluctuations for a model amphiphile solution described by a free energy density functional with square-gradient and square-Laplacian terms. That is, we determine the stability matrix when the stationary state consists of an interface with given geometry that separates two immiscible solvent phases. From the spectrum and the related eigenfunctions of this matrix we establish where lamellar and micellar domain-structured phases occur, and contrast our results with those for a simple square-gradient fluid model for which these phases are always unstable. We also characterize some instability properties such as the buckling of lamella, the undulation of cylindrical structures and the nucleation of micelles.

TOROIDAL COIL COUNTERCURRENT CHROMATOGRAPHY SEPARATION AND ANALYSIS OF STAPHYLOCOCCAL ENTEROTOXIN A (SEA) IN MILK

Countercurrent Chromatography (CCC) utilizes continuous partitioning of solute between two immiscible solvent phases without a solid support. The absence of solid support makes CCC a suitable method for food analysis because it permits analysis of crude and complex materials that are not amenable to conventional solid-supported chromatography. CCC was evaluated for its ability to separate Staphylococcal enterotoxin A (SEA), a common cause of food poisoning, from milk. Although many foods can be analyzed for SEA directly by Western blot analysis, milk samples generally require some purification because the high concentration of milk proteins distorts SDS-PAGE mobility. Milk samples containing SEA were separated by toroidal coil CCC and the fractions were analyzed by Western immunoblotting. Fractions containing SEA were pooled, concentrated by ultrafiltration, and rechecked by Western immunoblotting. Concentrating the fractions increased the sensitivity of Western immunoblotting by approximately an order of magnitude.

Resolution studies on counter-current chromatography using supercritical fluid carbon dioxide

Counter-current chromatography (CCC) is a form of liquid–liquid partition chromatography. It requires two immiscible solvent phases; the stationary phase is retained in the separation column, generally by centrifugal force, while the mobile phase is eluted. We recently replaced the mobile phase with supercritical fluid carbon dioxide (SF CO 2). Since the solvent strength of SF CO 2 can be varied by changing the temperature and pressure of the system, separation adjustments are thus more versatile. We investigated the pressure and temperature effects on resolution using water and low-carbon alcohol mixtures as the stationary phases. It was demonstrated that these special properties of SF CO 2 were indeed beneficial to the optimization of separations. In addition, the phase retention ratio was examined in terms of separation resolution. The results appeared very similar to those obtained from conventional traditional CCC. This study should be helpful for the future development of SF applications in CCC.

Speculation on the Mechanism of Unilateral Hydrodynamic Distribution of Two Immiscible Solvent Phases in the Rotating Coil

Abstract The proposed hypothesis is based on the interplay between two force components acting on the fluid in the rotating coil. The tangential force component generates the Archimedean screw effect to move two solvent phases toward the head of the coil whereas the radial force component acts against the Archimedean force to establish a hydrostatic distribution of the two phases throughout the coil. The unilateral hydrodynamic distribution of the two phases is governed by the degree of asymmetry in the radial force field on the coil in both simple rotation and synchronous planetary motion. The present hypothesis successfully explains all the observed hydrodynamic phenomena reported in the past.

Strategies for reducing solvent toxicity in extractive fermentations

The toxicity of an Alamine 336/oleyl-alcohol extraction system on Lactobacillus delbrueckii was investigated. It was shown that the solvent affected the cells through the water-soluble portion and the immiscible portion of the solvent. While immobilization significantly protected the cells from the immiscible solvent phase, the water-soluble part of the solvent still caused toxicity to the microorganisms due to diffusion of the solvent into the matrix. Adding soybean oil to the kappa-carrageenan matrix could trap the diffusing solvent molecules, and therefore reduce the toxic effect from the water soluble portion of the solvent. The protective ability of soybean oil was quantified through mathematical modeling and experimentation.

Studies on Hydrodynamic Distribution of Two Immiscible Solvent Phases in Rotating Coils

Abstract Hydrodynamic distribution of two immiscible solvent phases in rotating coils was studied with a simple rotary device. Experiments were performed with 1–2 cm I.D. glass coils, either nontreated or silicone coated, ranging in helical diameter from 2.5 to 20 cm. In general two solvent phases display characteristic four-stage distribution according to the applied rpm governed by interplay between the Archimedean screw force and radial centrifugal force. Under slow rotation the two solvent phases are evenly distributed on the head side of the coil by the Archimedean screw effect (Stage I). As the rotational speed is increased, one phase, usually the heavier phase, tends to dominate on the head side of the coil and at a critical rpm the two solvent phases are completely separated in the coil, one phase entirely occupying the head side and the other phase the tail side (Stage II). At this stage, high interfacial tension binary systems such as chloroform/water disclosed a typical effect of solvent-wall i...

Experimental observations of the hydrodynamic behavior of solvent systems in high-speed counter-current chromatography

Extensive studies have been performed on hydrodynamic distribution of two immiscible solvent phases in helical and spiral columns by means of coil planet centrifugation. Under the Scheme IV [see Fig. 1 in Y. Ito, J. Chromatogr., 301 (1984) 377] planetary motion, the two phases display unilateral distribution in the coil, i,e., one phase travels toward the head and the other phase toward the tail. This hydrodynamic trend is sensitively affected by various factors such as physical properties of the solvent system, helical diameter of the column, revolutional radius, etc. Phase distribution diagrams obtained from a set of conventional two-phase solvent systems under various experimental conditions provide extremely useful information for both basic hydrodynamic studies and practical application in high-speed counter-current chromatography.

A general model to account for the liquid/liquid kinetics of extraction of metals by organic acids

A model based on two-film theory is developed for the case of the rate of extraction of a divalent metal from an aqueous acid phase with an organic acid HR held in a second immiscible solvent phase. The model involves a reaction zone of variable thickness so that the case of reaction at an interface of molecular dimensions can be accommodated as well as the case of reaction extending into the diffusion film on the aqueous side of the interface. Four parameters are used, one involving a partition coefficient for the acid, one involving diffusivities in the films together with the two-film mass-transfer coefficients. Rate data from three techniques used in laboratory liquid/liquid contacting are fitted by the model.

New continous extraction method with a coil planet centrifuge

A compact table-top model of the coil planet centrifuge simultaneously enables both preliminary purification and enrichment of samples from crude extracts or biological fluids. The method uses hydrodynamic behavior of two immiscible solvent phases in a rotating coiled tube to retain the stationary phase against a high flow-rate of mobile phase. Consequently, a small quantity of the sample present in a large volume of mobile phase is efficiently extracted into a small volume of the stationary phase within a short period of time and at a high recovery rate. The capability of the present method was demonstrated in the extraction of dinitrophenylamino acids (used as a comparative performace standard) with a set of two-phase solvent systms composed of ethyl acetate and 0.5 M NaH 2PO 4.