Study of ion chromatography with ion-exchange fibers as the stationary phase

The application of ion-exchange fibers as the stationary phase in ion chromatography for the separation of inorganic anions has been studied. Results indicate that a separator column packed with VS-2 anion-exchange fibers and a suppressor column packed with VS-1 cation-exchange fibers have a similar separation efficiency to small-particle resin columns, but that the column pressure drop (ΔP) in fiber columns in only one-tenth of that in resin columns, at the same flow-rate. This allows the separation to be performed efficiently at a higher flow-rate and with lower presure, as shown for the separation of an anion mixture.     

Binary adsorption of globular proteins on ion-exchange media

Adsorption equilibrium of binary pairs of lysozyme (LYS), cytochrome c (CYC) and ribonuclease A (RNase) has been measured on different cation-exchange media at various solution conditions. Adsorption patterns largely follow the intrinsic protein–surface interactions, but can differ significantly for different pairs or even for one pair at different solution conditions. LYS/CYC adsorption shows similar behavior on all the adsorbents examined, with competitive adsorption dominated by LYS and the presence of LYS reducing the adsorption of CYC significantly. Simultaneous and sequential measurements for LYS/CYC show that the order of adsorption does not have a significant effect on the adsorption equilibrium. For LYS/RNase, LYS is consistently more strongly adsorbed. For CYC/RNase, both proteins can display significant adsorption, depending on the pH and salt concentration. A model based on colloidal energetics is developed to calculate the binary adsorption isotherms using parameter values obtained from single-component isotherms. The calculated adsorption is in good agreement with experimental results, with significantly better representation than for other commonly used binary isotherms.     

Effect of hindered diffusion on the adsorption of proteins in agarose gel using a pore model

The hindered diffusion and binding of proteins of different sizes (lysozyme, BSA and IgG) in an agarose gel is described using adsorption kinetic and diffusional data together with an experimentally determined pore size distribution in the gel. The validity of the pore model, including variable diffusion coefficients and porosities is tested against experimental confocal microscopy data. No fitting parameters were used in the present model. The importance of knowing the gel structure is demonstrated especially for large proteins such as IgG. Experimental confocal microscopy data can be explained by the present model.     

Relative retention of the fibroblast growth factors FGF-1 and FGF-2 on strong cation-exchange sorbents

The isocratic retention of two heparin-binding fibroblast growth factors, FGF-1 (acidic FGF) and FGF-2 (basic FGF), was compared on a set of six preparative strong cation-exchange adsorbents. The FGFs comprise a solute pair that are structurally equivalent, yet differ in protein parameters of potential importance in cation-exchange chromatography, such as isoelectric point, net charge, and the number and distribution of basic amino acids. The cation-exchange adsorbents comprise a diverse set of materials in common use for protein purification, with physical and chemical properties that have been characterized and described previously. Isocratic k' values for the two proteins obtained on each adsorbent at several different [NaCl] are compared with one another and with corresponding data for hen egg lysozyme, which is also strongly retained on cation-exchangers. Of the six adsorbents examined, three showed strong retention of both FGFs, with equivalent k' values for FGF-1 and FGF-2. Three others, which showed weaker overall retention for the FGF pair, showed much larger retention differences between FGF-1 and FGF-2. The trends in retention order among the stationary phases are very similar to those seen previously with other unrelated proteins. However, retention differences between the two FGFs, and between the FGFs and lysozyme, do not correlate well with simple charge properties such as net charge, indicating, as in some previous studies, the importance of local regions on the protein surface in determining retention. These observations are interpreted in terms of the structural features of the proteins and the physicochemical properties of the adsorbents.     

Modeling of the dynamic adsorption of an anionic dye through ion-exchange membrane adsorber

The present study examines the dynamic adsorption through ion-exchange membrane adsorbers. The model used in the study includes convection, axial dispersion with simultaneous adsorption and desorption of the solute in the membrane. Adsorption and desorption processes give the Langmuir isotherm for the equilibrium. The mathematical model makes use of dimensionless parameters in terms of characteristic times for the different mechanisms that take place during the process (convection, dispersion, adsorption and desorption characteristic times). The model has five independent dimensionless parameters. Three of these parameters are related to the equilibrium isotherm and the other two are related to the dynamic process. Equilibrium and dynamic experiments were carried out in order to fit their respective parameters. In order to examine the suitability of the model to describe real processes, the adsorption of an anionic dye (Orange-G) through the ion-exchange membrane adsorber was investigated as a function of dye and KCl concentration, obtaining strong correlation between fitted and experimental breakthrough curves. The results show the relative importance of axial dispersion, adsorption and desorption as a function of operational variables.     

Comparison of chromatographic ion-exchange resins IV. Strong and weak cation-exchange resins and heparin resins

A comparative study was performed on heparin resins and strong and weak cation exchangers to investigate the pH dependence, efficiency, binding strength, particle size distribution, static and dynamic capacity, and scanning electron microscopy pictures of chromatographic resins. The resins tested include: Heparin Sepharose FF, SP Sepharose FF, CM Sepharose FF, Heparin Toyopearl 650 m, SP Toyopearl 650 m, CM Toyopearl 650 m, Ceramic Heparin HyperD M, Ceramic S HyperD 20, and Ceramic CM HyperD F. Testing was performed with four different proteins: anti-FVII Mab (IgG), aprotinin, lysozyme, and myoglobin. Dependence of pH on retention was generally very low for proteins with high isoelectric point (pI), though some decrease of retention with increasing pH was observed for CM Ceramic HyperD F and S Ceramic HyperD 20. Binding of anti-FVII Mab with pI < 7.5 was observed on several resins at pH 7.5. Efficiency results show the expected trend of increasing dependence of the plate height with increasing flow rate of Ceramic HyperD resins followed by Toyopearl 650 m resins and the highest flow dependence of the Sepharose FF resins corresponding to their pressure resistance. Determination of particle size distribution by two independent methods, coulter counting and SEM, was in good agreement. Binding strength of cation-exchange resins as a function of ionic strength varies depending on the protein. Binding and elution at high salt concentration may be performed with Ceramic HyperD resins, while binding and elution at low salt concentration may be performed with model proteins on heparin resins. Employing proteins with specific affinity for heparin, a much stronger binding is observed, however, some cation exchangers may still be good substitutions for heparin resins. Dynamic capacity at 10% breakthrough compared to static capacity measurements and dynamic capacity displays that approximately 40-80% of the total available capacity is utilized during chromatographic operation depending on flow rate. A general good agreement was obtained between results of this study and data obtained by others. Results of this study may be used in the selection of resins for testing during protein purification process development.     

Adsorption of inulinases in ion-exchange columns

Theuse of adsorption columns packed with ion-exchange resins for recovering, concentrating and purifying proteins is now widespread. The present work consists of a study on the dyamic behavior ofadsorption columns that uses two kinds of adsorbents: a cationic and an anionic resin. A frontal analysis of the columns was performed with experimental data obtained from Fructozyme, a mixture of inulinase en zymes. The parameters of a Langmuir type of isotherm and adsorption kinetics were obtained from experimental tests in a batch system. A numerical technique based on orthogonal colocation and a fourth-order Runge-Kutta method was coupled with a nonlinear optimization method to predict the coefficients of the rate equations, which are fundamental for scale-up purposes.     

Comprehensive study of the macropore and mesopore size distributions in polymer monoliths using complementary physical characterization techniques and liquid chromatography

Poly(styrene‐co‐divinylbenzene) monolithic stationary phases with two different domain sizes were synthesized by a thermally initiated free‐radical copolymerization in capillary columns. The morphology was investigated at the meso‐ and macroscopic level using complementary physical characterization techniques aiming at better understanding the effect of column structure on separation performance. Varying the porogenic solvent ratio yielded materials with a mode pore size of 200 nm and 1.5 μm, respectively. Subsequently, nano‐liquid chromatography experiments were performed on 200 μm id × 200 mm columns using unretained markers, linking structure inhomogeneity to eddy dispersion. Although small‐domain‐size monoliths feature a relatively narrow macropore‐size distribution, their homogeneity is compromised by the presence of a small number of large macropores, which induces a significant eddy‐dispersion contribution to band broadening. The small‐domain size monolith also has a relatively steep mass‐transfer term, compared to a monolith containing larger globules and macropores. Structural inhomogeneity was also studied at the mesoscopic level using gas‐adsorption techniques combined with the non‐local‐density‐function‐theory. This model allows to accurately determine the mesopore properties in the dry state. The styrene‐based monolith with small domain size has a distinctive trimodal mesopore distribution with pores of 5, 15, and 25 nm, whereas the monolith with larger feature sizes only contains mesopores around 5 nm in size.     

Thin-layer adsorption chromatography with mixed mobile phases. 4. Extension of Ościk's equation to heterogeneous adsorbents

In this paper the equation for thin-layer adsorption chromatography with multicomponent mobile phases, proposed by O?cik in 1965 is extended to energetically heterogeneous solid surfaces. Other forms of this equation, more convenient in practical applications, are presented. Model calculations are made for TLC with binary mobile phases according to the modified form of O?cik's equation. Finally, this equation is compared with that of Snyder.     

Practical aspects of fast reversed-phase high-performance liquid chromatography using 3 microm particle packed columns and monolithic columns in pharmaceutical development and production working under current good manufacturing practice

The potential and limitations of fast reversed-phase high-performance liquid chromatographic separations for assay and purity of drug substances and drug products were investigated in the pharmaceutical industry working under current good manufacturing practice using particle packed columns and monolithic columns. On particle packed columns, the pressure limitation of commercially available HPLC systems was found to be the limiting factor for fast separations. On 3 microm particle packed columns, HPLC run times (run to run) for assay and purity of pharmaceutical products of 20 min could be achieved. As an interesting alternative, monolithic columns were investigated. Monolithic columns can be operated at much higher flow rates, thus allowing for much shorter run times compared to particle packed columns. Compared to particle packed columns, the analysis time could be reduced by a factor up to 6. However, some compounds investigated showed a dramatic loss of efficiency at higher flow rates. This phenomenon was observed for some larger molecules supporting the theory that mass transfer is critical for applications on monolithic columns. At flow rates above 3 ml/min some HPLC instruments showed a dramatic increase in noise, making quantifications at low levels impossible. For very fast separations on monolithic columns, the maximum data acquisition rate of the detector is the limiting factor.     

Protein adsorption and transport in dextran-modified ion-exchange media. III. Effects of resin charge density and dextran content on adsorption and intraparticle uptake

Custom-synthesized variants of the commercial Capto S resin were used to examine the effects of resin charge density and dextran content on protein adsorption and intraparticle uptake. For the small protein lysozyme, resin charge density had the greatest effect on equilibrium capacity, consistent with calculations suggesting that lysozyme capacity should be limited by the available charge on the resin. Isocratic retention data and confocal microscopy imaging for this protein revealed a consistent ordering of the resins linking stronger protein-resin interactions with higher static capacities but slower intraparticle uptake rates over the range of properties studied. For the larger protein lactoferrin, it was found that increasing dextran content led to increased protein exclusion from the dextran layer, but that increasing resin charge density helped overcome the exclusion, presumably due to the increased electrostatic attraction between the resin and protein. Collectively examining the lysozyme and lactoferrin data along with information from previous studies suggests that a trade-off in maximizing dynamic capacities should exist between static capacities that increase to a finite extent with increased resin charge density and uptake rates that decrease with increased charge density. Column breakthrough data for lysozyme and lactoferrin appear to support the hypothesis, though it appears that whether a resin charge density is low or high must be considered in relation to the protein charge density. Using these trends, this work could be useful in guiding resin selection or design.     

Investigation of particle-based and monolithic columns for cation exchange protein displacement chromatography using poly(diallyl-dimethylammonium chloride) as displacer

The overall topic of the investigation was the separation of basic proteins by cation exchange displacement chromatography. For this purpose two principal column morphologies were compared for the separation of ribonuclease A and alpha-chymotrypsinogen, two proteins found in the bovine pancreas. These were a column packed with porous particles (Macro-Prep S, 10 microm, 1000 A) and a monolithic column (UNO S1). Both columns are strong cation exchangers, carrying -SO3(-)-groups linked to a hydrophilic polymer support. Poly(diallyl-dimethylammonium chloride) (PDADMAC), a linear cationic polyelectrolyte composed of 100-200 quaternary pyrrolidinium rings, was used as displacer. The steric mass action (SMA) model and, in particular, the operating regime and dynamic affinity plots were used to aid method development. To date the SMA model has been applied primarily to simulate non-linear displacement chromatography of proteins using low molar mass displacers. Here, the model is applied to polyelectrolytes with a molar mass below 20000 g mol(-1), which corresponds to a degree of polymerization below 125 and an average contour length of less than 60 nm. The columns were characterized in terms of the adsorption isotherms (affinity, capacity) of the investigated proteins and the displacer.     

Two-resistance mass transfer model for the adsorption of the pesticide deltamethrin using acid treated oil shale ash

Adsorption characteristics of the pesticides Deltamethrin were studied in aqueous solutions using acid treated Oil Shale Ash (ATOSA) in a series of batch adsorption experiments. The maximum loading capacity of the adsorbent and the rate of adsorption were found to increase with increasing the pesticide initial concentration, mixing speed and were found to decrease with temperature and particle size. Langmuir as well as Freundlich isotherm models fit the adsorption data with R ²>0.97 in all cases. The maximum adsorption capacity for Deltamethrin was 11.4 mg/g. The two-resistance mass transfer model based on the film resistance and homogeneous solid phase diffusion was used to fit the experimental data. A computer program has been developed to estimate the theoretical concentration-time dependent curves and to compare them with the experimental curves by means of the best-fit approach. The model predicts that the external mass transfer coefficient K was affected by varying the initial pesticide concentration, the agitation speed and temperature whereas the diffusion coefficient D was affected by the initial pesticide concentration, and temperature.     

Band splitting in overloaded isocratic elution chromatography II. New competitive adsorption isotherms

The equations of two new binary competitive isotherms models are derived. The first of these models assumes that the isotherms of the two pure, single compounds have distinct monolayer capacities. Its derivation is based on kinetic arguments. The ideal adsorbed solution (IAS) framework was applied to derive the second model that is a thermodynamically consistent competitive isotherm. This second model predicts the competitive adsorption isotherm behavior of a mixture of two compounds that have single-component adsorption behavior following a BET and/or a Langmuir isotherms. Both models apply well to the binary adsorption of ethylbenzoate and 4-tert.-butylphenol on a Kromasil-C18 column (with methanol-water, 62:38, v/v, as the mobile phase). The best single-solute adsorption isotherms of these two compounds are the liquid-solid extended multilayer BET and the Langmuir isotherms, respectively. The kinetic and thermodynamic new competitive models were compared, regarding the accuracy of their prediction of the elution band profiles of mixtures of these two compounds. A better agreement between experimental and calculated profiles was observed with the kinetic model. The IAS model failed because the behavior of the ethylbenzoate/4-tert.-butylphenol adsorbed phase mixture is probably non-ideal. The most striking result is the qualitative prediction by these models of the peak splitting of 4-tert.-butylphenol during its elution in presence of ethylbenzoate.     

Studies of the mixed adsorption layer on a mercury electrode in the systemp-toluidine-polyethyleneglycol-1M NaClO4

Summary The properties of the mixed adsorption layer on a mercury electrode in the system 1M NaClO₄-p-toluidine-polyethyleneglycol (average molecular masses of polyethyleneglycols: 400 or 10000) are discussed. The parameters of theFrumkin and virial isotherms were determined in the range of strong adsorption potentials. In the range of more negative potentials, a mixed adsorption layer was found by investigating the kinetics of the reduction of Zn(II) ion as a pilot ion. Depending on the concentration ratio of the studied organic substances, inhibition, acceleration, or compensation of both with respect to Zn(II) electroreduction was observed. In the presence of polyethyleneglycol, the efficiency of the Zn(II) ion electroreduction increases due to a greater adsorption lability ofp-toluidine molecules on the mercury surface.

---

Untersuchung der gemischten Adsorptionsschicht an einer Quecksilberelektrode im Systemp-Toluidin-Polyethyleneglycol-1M NaClO₄

Zusammenfassung Die Eigenschaften der gemischten Adsorptionsschicht an einer Quecksilberelektrode trode im System 1M NaClO₄-p-Toluidin-Polyethyleneglycol(durchschnittliche Molmassen der Polyethylenglycole: 400 bzw. 1000) werden diskutiert. Die Parameter derFrumkin- und Virialisotherme wurden im Bereich hoher Adsorptionspotentiale bestimmt. Im Bereich negativerer Potentiale wurde durch Untersuchung der Kinetik der Elektroreduktion von Zn(II)-Ionen als Pilotionen eine gemischte Adsorptionsschicht gefunden. In Abhängigkeit von der Konzentration der organischen Verbindungen beobachtet man bezüglich der Elektroreduktion von Zn(II) Hemmung, Beschleunigung oder eine gegenseitige Kompensation beider Effekte. In Gegenwart von Polyethyleneglycol nimmt die Effizienz der Elektroreduktion von Zn(II) aufgrund einer höheren Adsorptionslabilität derp-Toluidin-Moleküle an der Quecksilberoberfläche zu.

     

Adsorption isotherms and retention behavior of 1,1'-bis(2-naphthol) on CHIRIS AD1 and CHIRIS AD2 columns

The separation of the atropoisomers of 1,1'-bis(2-naphthol) was studied on CHIRIS AD1 and CHIRIS AD2, two Pirkle-type chiral stationary phases. Satisfactory selectivity was found only on CHIRIS AD2. The ternary mobile phases comprised hexane, dichloromethane and methanol. The effects of their composition and of the temperature on the retention under analytical conditions and on the single-component and competitive isotherms were investigated. The retention of the R- and S-isomers on CHIRIS AD1 and CHIRIS AD2 is controlled by the enthalpic contribution to adsorption, but the effect of the mobile phase on the retention should be attributed mainly to the entropic contribution. The adsorption of the less retained R-isomer is controlled by the achiral interactions, which are the same as for the S-isomer. The single-component and competitive isotherms of the R- and S-isomers are adequately described by the sum of a Langmuir term for the achiral contribution to adsorption and a linear-term characterising the selective or chiral adsorption of the S-isomer in the concentration range experimentally available, i.e. within the solubility limit of 1,1'-bis(2-naphthol).     

Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode

The adsorption isotherms of four model proteins (lysozyme, α-lactalbumin, ovalbumin, and BSA) on eight commercial phenyl hydrophobic interaction chromatography media were measured. The isotherms were softer than those usually seen in ion-exchange chromatography of proteins, and the static capacities of the media were lower, ranging from 30 to 110 mg/mL, depending on the ammonium sulfate concentration and the protein and adsorbent types. The protein-accessible surface area appears to be the main factor determining the binding capacity, and little correlation was seen with the protein affinities of the adsorbents. Breakthrough experiments showed that the dynamic capacities of the adsorbents at 10% breakthrough were 20-80% of the static capacities, depending on adsorbent type. Protein diffusivities in the adsorbents were estimated from batch uptake experiments using the pore diffusion and homogeneous diffusion models. Protein transport was affected by the adsorbent pore structures. Apparent diffusivities were higher at lower salt concentrations and column loadings, suggesting that adsorbed proteins may retard intraparticle protein transport. The diffusivities estimated from the batch uptake experiments were used to predict column breakthrough behavior. Analytical solutions developed for ion-exchange systems were able to provide accurate predictions for lysozyme breakthrough but not for ovalbumin. Impurities in the ovalbumin solutions used for the breakthrough experiments may have affected the ovalbumin uptake and led to the discrepancies between the predictions and the experimental results.     

Hydrophobic and cation exchange mechanisms in the retention of basic compounds in a polymeric column

A cation exchange retention mechanism concomitant with the well-known hydrophobic partition mechanism in a polymeric column has been observed and investigated. This exchange process is attributed to ionization of some acidic sites present in the polymer column at basic mobile phase pH values. Several drugs of different basicity have been chromatographed on a polymeric PLRP-S column with methanol-water and acetonitrile-water mobile phases. The cation exchange between the protonated basic drug and the buffer cations (Na+, K+ and BuNH4+) is observed at the pH range where the protonated drug and the ionized sites of the column coexist. This process produces a shift of the retention versus pH plot of the base to pH values lower than those expected from the pKa of the base as well as a maximum in the plot at basic pH values. These effects are more pronounced for acetonitrile-water mobile phases.     

Capillary electrochromatographic separation of bovine milk proteins using a G-quartet DNA stationary phase

DNA oligonucleotides that form G-quartet structures were used as stationary phase reagents for separation of bovine milk proteins, including alpha-casein, beta-casein, kappa-casein, alpha-lactalbumin and beta-lactoglobulin. Both artificial protein mixtures and a skim milk sample were analyzed. The separations were performed using open-tubular capillary electrochromatography, in which the oligonucleotides were covalently attached to the inner surface of a fused-silica capillary. Better resolution was achieved using the G-quartet-coated capillaries than was achieved using either a bare capillary or a capillary coated with an oligonucleotide that does not form a G-quartet structure. A 4-plane G-quartet-forming stationary phase was able to resolve three peaks for alpha-casein and to detect thermal denaturation of the proteins in the milk sample. The results suggest that G-quartet stationary phases could be used to separate very similar protein structures, such as those arising from genetic variations or post-translational modifications.     

Use of lipophilic ion adsorption isotherms to determine the surface area and the monolayer capacity of a chromatographic packing, as well as the thermodynamic equilibrium constant for its adsorption

A method that champions the approaches of two independent research groups, to quantitate the chromatographic stationary phase surface available for lipophilic ion adsorption, is presented. For the first time the non-approximated expression of the electrostatically modified Langmuir adsorption isotherm was used. The non approximated Gouy-Chapman (G-C) theory equation was used to give the rigorous surface potential. The method helps model makers, interested in ionic interactions, determine whether the potential modified Langmuir isotherm can be linearized, and, accordingly, whether simplified retention equations can be properly used. The theory cultivated here allows the estimates not only of the chromatographically accessible surface area, but also of the thermodynamic equilibrium constant for the adsorption of the amphiphile, the standard free energy of its adsorption, and the monolayer capacity of the packing. In addition, it establishes the limit between a theoretical and an empirical use of the Freundlich isotherm to determine the surface area. Estimates of the parameters characterising the chromatographic system are reliable from the physical point of view, and this greatly validates the present comprehensive approach.