Stationary Phase Concentration Research Articles

Development and characterisation of whey protein micro-beads as potential matrices for probiotic protection

This study evaluated the efficacy of whey protein isolate (WPI) as an encapsulation matrix for the maintenance of Lactobacillus rhamnosus GG viability during simulated gastro-intestinal studies. Micro-bead characteristics were investigated using microscopy, chromatography, laser diffractometry and zeta potential analysis. Heat-treated WPI (11%, w/v) blended with stationary phase cultures demonstrated an instant gelation impetus in acetate buffer (0.5 M), tempered to 35 °C in the presence of Tween-20 (0.04%). Atomic force microscopy (AFM) demonstrated that micro-bead extrusion at pH 4.6 fuelled strong cohesive interactions within protein-probiotic amalgams; an electrostatic alliance further highlighted by zeta potential analysis. Optimization of encapsulation conditions generated self-supporting structures (200 ± 1.2 μm) with high micro-bead strength and individual loading capacity of 2.7 × 10 4 cfu/micro-bead. Plate enumeration demonstrated that micro-bead extrusion had no detrimental effect on cell viability due to the perpetuation of stationary phase concentrations (10 9 cfu/mL). This finding was further validated by LIVE/DEAD microscopy staining, which visualized the homogenous distribution of live probiotics throughout micro-bead matrices. Following 3 h in vitro stomach incubation (pH 1.8; 37 °C), micro-beads laden with 10 10 cfu demonstrated acid-stability and peptic-resistance, characteristics required for optimum probiotic refuge. However, enzyme-activated intestinal conditions catalysed a synergistic response engaging rapid matrix disintegration and controlled probiotic release. Matrix digestion was monitored by chromatography, which witnessed the sequential release of peptides <2 kDa after 30 min. In conclusion, this study led to the development and design of a protein encapsulation polymer based on congruent matrix interactions for reinforced probiotic protection during challenging situations for their targeted delivery to intestinal absorption sites.

Validation of the Tracer-Pulse Method for Multicomponent Liquid Chromatography, A Classical Paradox Revisited

The tracer-pulse method was extended and validated for the determination of multicomponent adsorption isotherms in liquid chromatography. Competitive adsorption isotherms can be determined for any number of solutes, up to the column resolution limit. The basic principle is to equilibrate the column with an eluent containing a mixture of the solutes and then measure the migration velocity of each of them through the column. It is easy to calculate the stationary phase concentrations from these velocities, given the eluent composition. As in frontal analysis, real competitive isotherm data are measured using this method, unlike other methods, which only produce parametric estimates. The method was used to measure the binary isotherms of beta-blockers on a Kromasil C8 column. The data were fitted to competitive bi-Langmuir adsorption isotherm functions and was found to agree well with the results of frontal analysis and the perturbation method. Computer simulations based on the isotherm parameters were performed and displayed very good agreement with the experimental chromatograms. An intriguing and seemingly paradoxical property is visualized and discussed: the fact that the injected molecules are not found in the detected peaks.

The Fractional Charge Approach in Ion‐Interaction Chromatography of Zwitterions: Influence of the Stationary Phase Concentration of the Ion Interaction Reagent and pH

The fractional charge approach to Ion‐interaction chromatography (IIC) of zwitterions was used to quantitatively explain their retention behavior as a function of the Ion Interaction Reagent (H) concentration in the stationary phase and also to account for the influence of the zwitterion ionization degree, according to the mobile phase pH. The theory was validated using two Hs and two reversed phase columns. The good predictive abilities of the retention equations lend strong support to the hypotheses made by the fractional charge approach in the IIC of zwitterions. For the investigated homologous series the increase in retention upon H addition can be quantitatively related to the estimated fractional charge. The estimates of the operative charges and bonded phase coverages are very reasonable since they make sense physically. This way, they offer a cross validation of the fractional charge approach, since they compare well with those obtained from the fitting of parallel retention data, obtained as a function of the mobile phase concentration of H. This confirms that retention modelling may instruct the chromatographer through the optimization procedure in the parameter space.

Extended Thermodynamic Approach to Ion Interaction Chromatography: Effect of the Electrical Charge of the Solute Ion

The chromatographic behavior of multiply charged analytes in ion interaction chromatography (IIC) was theoretically investigated. Practical equations that describe the relationship between the retention factor and the concentration of the ion interaction reagent (IIR) were developed. They can be used to model analyte retention as a function of both the mobile and stationary phase concentrations of the IIR. A comparison between the retention behaviour of singly and doubly charged analytes is given.

THE DIPOLE APPROACH IN ION INTERACTION CHROMATOGRAPHY OF ZWITTERIONS. USE OF THE LINEARIZED POTENTIAL EXPRESSION FOR LOW SURFACE POTENTIAL

The chromatographic behaviour of zwitterionic analytes in Ion Interaction Chromatography (IIC) was theoretically investigated for low surface potential. For the first time, simplified retention equations were obtained via the linearized potential expression. They can be used to model zwitterions retention as a function of both the mobile and stationary phase concentration of the Ion-Interaction Reagent (IIR), if the surface potential is below 25 mV.

Separation of zirconium and hafnium using hollow fibres: Part II. Membrane chromatography

A novel extraction chromatography system using hollow fibre membranes as supports is reported for separation of zirconium and hafnium. The effects of flow rate and flow mode, stationary phase concentration loaded on the hollow fibre membranes and fibre length on the separation were investigated. The separation performance of hollow fibre membrane chromatography can be analysed from existing theories of conventional extraction chromatography using particle supports.

Extended thermodynamic approach to ion-interaction chromatography for high surface potential: Use of potential approximation for simplified retention equations

The chromatographic behaviour of charged analytes in ion interaction chromatography (IIC) has been investigated theoretically. A potential approximation for high surface potential was used to obtain simplified retention equations that are able to model analyte retention as a function of both the mobile and stationary phase concentrations of the ion-interaction reagent (IIR). The main advantage of using this potential approximation is that it allows calculation of the surface potential, without needing detailed information on physical and chemical properties of the mobile phase.

EXTENDED THERMODYNAMIC APPROACH TO ION INTERACTION CHROMATOGRAPHY FOR LOW SURFACE POTENTIAL. USE OF THE LINEARIZED POTENTIAL EXPRESSION

The chromatographic behaviour of charged analytes in Ion Interaction Chromatography (IIC) was theoretically investigated. Simplified retention equations were obtained via the linearized potential expression. They can be used to model analyte retention as a function of both the mobile and stationary phase concentration of the Ion-Interaction reagent (IIR), if the surface potential is below 25 mV. Simplified retention equations were compared to those, which can be obtained from two of the most important retention models in IIC. They reduce to stoichiometric or electrostatic retention model equations if the surface potential or pairing equilibria are respectively neglected.

A correction to the calculation of the Gibbs free energy of adsorption for biomolecules in ion-exchange systems

We wish to propose a correction to the methodology introduced by Gerstner et al. [J.A. Gerstner, J.A. Bell, S.M. Cramer, Biophys. Chem. 52 (1994) 97–106] for the calculation of Gibbs free energies of adsorption of biomolecules to ion-exchange systems. Our approach is based on the requirement that the mobile phase and stationary phase concentrations be expressed in exactly the same units and the equilibrium constant be strictly dimensionless. The Gibbs free energies of ion-exchange calculated based on this correction appear to be more negative than those originally calculated by Gertner et al.

Chiral discrimination using a quartz crystal microbalance and comparison with gas chromatographic retention data

Quartz crystal microbalances (QCMB) have been constructed using 10 MHz AT cut quartz crystals coated with heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, heptakis(6-O-methyl-2,3-di-O-pentyl)-β-cyclodextrin, and octakis(6-O-methyl-2,3-di-O-pentyl)-γ-cyclodextrin as 50% and 20% (w/w) solutions in OV1701. The reduction in frequency seen on exposure of each coated QCMB to pure enantiomeric forms of α- and β-pinene and cis- and trans-pinane show that statistically significant (P = 0.05, n = 7) differences are observed between the enantiomeric pairs. The apparent preferential binding shown by the QCMB for enanciomers of α- and β-pinene and cis- and trans-pinane have been compared with the elution order observed on the corresponding gas chromatographic stationary phase. The magnitude of the observed separation factor (calculated as the ratio of the OV1701 normalised frequency shift) is seen to be dependent upon the chiral stationary phase concentration. These results indicate that on-line determination of enantiomeric excess and concentration of certain monoterpenes is possible at room temperature using QCMB in conjunction with chiral gas chromatographic stationary phases. Chirality 9:225–232, 1997. © 1997 Wiley-Liss, Inc.

Adsorption isotherms in protein chromatography combined influence of protein and salt concentration on adsorption isotherm

The distribution coefficient ( K) of a protein in ion-exchange chromatography is dependent on the protein and salt concentration in the mobile phase. Conventional isotherms only describe the relationship between the mobile phase and stationary phase concentrations of the protein, the influence of salt being neglected. In this paper, the dependence of K on salt is described by transition functions [ ϕ( I)] such as logistic dose response and hyperbolic tangent functions. The relationship between K and protein concentration is described by conventional isotherms; the K value is considered as the first derivative of the isotherm, or as the ratio between concentrations in the mobile and stationary phases. The stationary phase concentration can be expressed as a function of the mobile phase concentration. Combinations of these functions with transition functions can be used to approximate the binding characteristics of a protein dependent on protein and salt concentration.

Thermodynamics of the adsorption of Tröger's base enantiomers from ethanol on cellulose triacetate

The adsorption isotherms of the (−)- and (+)-enantiomers of Tröger's base on microcrystalline cellulose triacetate with ethanol as solvent were measured at 30, 40, 50 and 60°C using frontal analysis. The isotherms of the first eluted (−)-enantiomer can be described satisfactorily with the Langmuir equation at all temperatures. By contrast, the isotherms of the longer retained (+)-enantiomer exhibit a pronounced inflection point in the lower temperature range. This point nearly vanishes at 60°C where the isotherm is almost linear in the concentration range investigated. All these isotherms can be satisfactorily described by a quadratic isotherm equation. The standard thermodynamic functions were derived from these equilibrium data. The enthalpies and the isosteric heats of adsorption are not constant in the temperature region studied. The adsorption enthalpies of both enantiomers increase with increasing temperature. At constant temperature, the isosteric heat of adsorption of the (−)-enantiomer depends only slightly on the stationary phase concentration whereas that of the (+)-enantiomer increases strongly with increasing concentration.

Determination of competitive adsorption isotherms for modeling large-scale separations in liquid chromatography

Preparative and process-scale high-performance liquid chromatography has assumed an increasingly important role in the production of highly purified substances, such as proteins expressed by recombinant DNA technology for use as human pharmaceuticals. The theory for modeling chromatographic separations is well-developed, but requires data on the competitive adsorption behavior of all mixture components for accurate predictions and process design. This paper describes two new methods for determining competitive adsorption isotherms from multi-component frontal chromatography experiments. The first new method reported here complements a method described previously that employed the theory of chromatography to estimate Langmuir isotherm parameters from the breakthrough volumes in frontal chromatography. The new method estimates Langmuir parameters from the experimentally determined compositions of the breakthrough zones, rather than from retention volumes, and so provides a check on the magnitudes of these parameters, but also yields values that may more accurately predict the concentrations of zones in a chromatogram than the Langmuir parameters estimated by other methods. The second method is based on both the mass balance and composition velocity approaches to analysis of the profile obtained in frontal chromatography. The method does not require measurement of concentrations in the profile, instead, concentrations are estimated according to the theory of chromatography from the breakthrough volumes of zones of different composition. The resulting method enables calculation of data points for mobile and stationary phase concentrations at equilibrium, yet it eliminates the need for a tandem rapid analytical high-performance liquid chromatographic unit to monitor effluent compositions.

Adsorption isotherms on silica for methanol and 1-hexanol modifiers from supercritical carbon dioxide

Adsorption isotherms were determined for methanol and 1-hexanol on silica from supercritical carbon dioxide at four temperatures and three mobile phase densities. Maximum stationary phase concentrations were extrapolated from linear least squares fits of the data to the Langmuir equation for monolayer adsorption. From these results, maximum surface area coverages were calculated using the mean molecular area of each modifier. The maximum, molar stationary phase concentration of methanol was found to exceed that of 1-hexanol under all experimental conditions; however, in each case the surface area coverage by hexanol was calculated to be larger. The capacity factors of several substituted and unsubstituted aromatic hydrocarbons were determined in 0–1% (w/v) methanol modifier carbon dioxide. From the linearity of capacity factor versus modifier concentration plots, the ability of the solutes to compete with methanol for active column sites was determined. Unsubstituted aromatic hydrocarbons do not appear to compete with the modifier for direct adsorption onto modifier-sorbing active sites.

Cation Exchange in Chemical Flooding: Part 2--The Effect of Dispersion, Cation Exchange, and Polymer/Surfactant Adsorption on Chemical Flood Environment

Abstract In this paper, we extend some of the results of Part 1 by discussing aspects of the effects of Part 1 by discussing aspects of the effects of dispersion, two-cation exchange, and adsorption on a chemical flood:we show that when chemical slug sizes are small and dispersion is moderate, the inclusion of dispersion in the calculation can modify results that are calculated by methods which assume no dispersion;we describe a new phenomenon in which dispersion itself can initiate cation exchange; andfinally, we describe an asymptotic concentration profile toward which dispersive, self-sharpening fronts tend. Introduction One of the most important factors affecting the interfacial activity, phase behavior, and mobility control of a chemical flood is the ionic environment in which the chemical slug passes through the reservoir. Factors affecting this environment are the in-place and injected-fluid compositions and dispersive mixing. Equally important, but perhaps not so obvious, is the role that cation exchange can play in determining the chemical slug's environment. The importance of surfactant and polymer adsorption to the practicality and economics of the chemical flood process long has been recognized. This is because adsorption directly affects the quantity and rate at which surfactant and polymer can be propagated through the reservoir. The interfacial activity and, to a lesser extent, the mobility of the surfactant and polymer are as strongly affected by their ionic environment, particularly the divalent ion concentration, as by particularly the divalent ion concentration, as by their own concentration levels for some ranges. Concern about this sensitivity has led to formulations that include cosurfactants to increase salinity tolerance in the chemical slug, and to preflooding schemes that lower reservoir salinity to acceptable levels. Since cation exchange profoundly affects the flowing phase ionic environment, it must play a central role in the design and implementation of a chemical flood. Toward this goal, this paper tries to illustrate some consequences of cation exchange and polymer/surfactant adsorption in porous media, polymer/surfactant adsorption in porous media, including the effect of fluid-dynamic dispersion. This paper is a companion to Part 1, dealing with cation exchange in nondispersing systems. We, therefore, use some conventions and definitions from that paper. ASSUMPTIONS The assumptions used here are similar to those in Part 1, except thatfluid-dynamic dispersion is not neglected,all material-balance relations are restricted to single-phase flow (there is no oil in the system), andthe system can have no more than five components: calcium (C1 - meq/ml) - a divalent cationic component; sodium (C2 - meq/ml) - a monovalent cationic component; chloride or total salinity (C3 - meq/ml) - a nonadsorbing anionic component; surfactant (C4 - meq/mi) - an adsorbing component; and polymer (C5 - ppm) - a nonionic adsorbing component. Note that all concentrations for C1 through C4 (including the stationary phase concentrations) are in milliequivalents per milliliters of pore volume. The foregoing assumptions restrict somewhat the usefulness of the following results when predicting the behavior of real systems. Nevertheless, these results effectively illustrate the combined influence of dispersion, cation exchange, and polymer/ surfactant adsorption on a chemical flood's environment. SPEJ P. 435

Evaluation of glass capillary columns with different pre-coating layers

An evaluation is made of glass capillary columns pre-coated with dispersions of various materials (Silanox, Carbopack, Chromosorb, perlite and Porapak) and then coated with polyethylene glycol (PEG 20M) at various concentrations as stationary phase by analysing alkanes and alcohols in terms of the peak-asymmetry factor, separation factor and kinetic characteristics. Pre-coating with Silanox, Carbopack and Chromosorb yielded similar results, but with different amounts of stationary phase. Use of Carbopack for pre-coating offers the advantage that, according to the concentration of stationary phase, separation can be achieved by gas-liquid or gas-liquid-solid chromatography.

The phosphoglyceride composition of gram-negative bacteria and the changes in composition during growth

1. 1. With one exception, Agrobacterium tumefaciens in stationary growth phase, phosphatidylethanolamine was the major phospholipid present at all stages of growth in 8 species of Gram-negative bacteria studied. Phosphatidylglycerol and diphosphatidylglycerol were also always present. 2. 2. The species studied could otherwise be divided into two groups on the basis of whether N- methyl derivatives of phosphatidylethanolamine were also present. This classification and the discrimination of two additional groups on the basis of published reports are discussed. 3. 3. In addition to the major lipids indicated above, phosphatidic acid was also generally detected and phosphatidylglycerol phosphate was found in measurable amounts in some strains. 4. 4. With a few exceptions, phosphatidylethanolamine, phosphatidylglycerol, phosphatidyl- N- monomethylethanolamine and phosphatidylcholine rise to a maximum concentration in late log or early stationary phase and then decrease in concentration. Diphosphatidylglycerol rises during the transition between log and stationary phase and then increases to a maximum concentration in stationary phase.

Separation and Identification of Barbiturates and Some Related Compounds by Means of Gas-Liquid Chromatography

Barbiturates and certain related hypnotics have been separated and identified by direct gas chromatography on a number of different stationary phases. Depending on the type and concentration of stationary phase, column temperatures ranging from about 140 to 200° were used. No evidence of decomposition of the substances was observed. The method is rapid and extremely sensitive.

The separation of C 6F 14 isomers by gas chromatography and the effect of stationary phase concentration

The separation of the isomers of perfluorohexane by gas-liquid chromatography is difficult for two reasons: (1) the isomers have similar vapor pressures in that they all boil within a range of one or two degrees, and (2) they all show essentially identical thermodynamic behavior in any given solvent. The latter behavior precludes the existence of selective solvent effects, so useful in hydrocarbon systems, to aid in the separation. Satisfactory stationary phases for this isomer separation are hydrocarbons at concentrations of 0.3 c.c. per c.c. of column or chlorofluorocarbons at 0.05 c.c. per c.c. of column volume. Approximate retention volumes for n-C 6F 14 are 8 c.c. per c.c. of n-hexadecane at 28°, and 77 c.c. per c.c. of Cl(CF 2CFCl) 3CF 2COOC 2H 5 at 24°. The relative behaviors of the partitioning media are explained on the basis of the G olay theory. The resolution of the hydrocarbon hexanes is poor on perfluorocarbon substrates because of large differences in the thermodynamics among the hydrocarbon isomers in these solvents.