Effect of the flow rate on the measurement of adsorption data by dynamic frontal analysis

The adsorption data of propyl benzoate were acquired by frontal analysis (FA) on a Symmetry-C18 column, using a mixture of methanol (65%, v/v) and water as the mobile phase, at three different flow rates, 0.5, 1.0 and 2.0 mL/min. The exact flow rates Fv were measured by collecting the mobile phase in volumetric glasses (deltaFv / Fv < or = 0.2%). The extra-column volumes and the column hold-up volume were accurately measured at each flow rate by tracer injections. The detailed effect of the flow rate on the value of the amount adsorbed was investigated. The best isotherm model accounting for the adsorption data was the same BET isotherm model at all three flow rates. Only slight differences (always less than 5%) were found between the three different sets of isotherm parameters (saturation capacity, q(s), equilibrium constant on the adsorbent, b(s) and equilibrium constant on successive layers of propyl benzoate, bL). The reproducibility of the same isotherm parameters measured by the inverse method (IM) is less satisfactory, leading to R.S.D.s of up to 10%. A flow rate increase is systematically accompanied by a slight increase of the amount adsorbed. This phenomenon is consistent with the influence of the pressure on the equilibrium constant of adsorption due to the difference between the partial molar volumes of the solute and the adsorbate. The larger average pressure along the column that is required to achieve a larger flow rate causes a larger amount of solute to be adsorbed on the column at equilibrium. This result comforts the high sensitivity and versatility of the FA method for isotherm determination under any kind of situation.     

Study of adsorption from solutions by frontal chromatography

Summary The automatic frontal chromatography installation was described. By this chromatographic method the adsorption isotherm of benzene from n-heptane solutions on hydroxylated surface of silica with various porosity has been determined. This investigation was performed at different flow rates of eluent and in a wide range of concentrations. The isotherm of adsorption obtained by this chromatographic method has been compared with the results of the static measurements. The coincidence of adsorption isotherms measured at the various flow rates are shown to be a criterion of proximity the chromatographic process to the equilibrium.     

Adsorption from binary solvent mixtures onto silica gel by HPLC frontal analysis

Summary The chromatographic technique of frontal analysis is applied to measuring adsorption from binary liquid mixtures by silica gel. The complete adsorption isotherm of a solvent mixture is obtained by measuring the break-through curves for a series of small concentration steps of the mobile phase. This method offers a direct way to determine the composition of the stationary phase in liquid-solid chromatography with mixed mobile phases. The surface excess isotherms of all binary systems formed by benzene, cyclohexane, and 1,2-dichloroethane, at the solution-silica gel interface at 25 °C are presented. The data of the three systems are shown to be thermodynamically mutually consistent.     

Application of the chromatographic step profile method for determination of water film pressure and surface free energy of quartz

Summary Investigation of water adsorption by the step profile method (Glueckauf method) was carried out with the help of a modified gas chromatography equipped with thermal-conductivity detector. On the basis of the adsorption isotherm obtained, the water film pressure and the polar component of the surface free energy of quartz were calculated. The calculated value of the polar component of the surface free energy of quartz agrees with analogous values obtained by other methods.     

Theoretical study of the accuracy of the pulse method,frontal analysis,and frontal analysis by characteristic points for the determination of single component adsorption isotherms

The adsorption isotherms of selected compounds are our main source of information on the mechanisms of adsorption processes. Thus, the selection of the methods used to determine adsorption isotherm data and to evaluate the errors made is critical. Three chromatographic methods were evaluated, frontal analysis (FA), frontal analysis by characteristic point (FACP), and the pulse or perturbation method (PM), and their accuracies were compared. Using the equilibrium-dispersive (ED) model of chromatography, breakthrough curves of single components were generated corresponding to three different adsorption isotherm models: the Langmuir, the bi-Langmuir, and the Moreau isotherms. For each breakthrough curve, the best conventional procedures of each method (FA, FACP, PM) were used to calculate the corresponding data point, using typical values of the parameters of each isotherm model, for four different values of the column efficiency (N=500$N=500$, 1000, 2000, and 10,000). Then, the data points were fitted to each isotherm model and the corresponding isotherm parameters were compared to those of the initial isotherm model. When isotherm data are derived with a chromatographic method, they may suffer from two types of errors: (1) the errors made in deriving the experimental data points from the chromatographic records; (2) the errors made in selecting an incorrect isotherm model and fitting to it the experimental data. Both errors decrease significantly with increasing column efficiency with FA and FACP, but not with PM.     

Determination of the adsorption isotherm of fluoxetine by adjustable factor h-root method (AF-HRM)

Summary Conventional methods to determine competitive adsorption isotherm are tedious, and require large quantities of pure isomers. In some cases pure enantiomers are not available, thereby making the full experiments to determine the competitive adsorption isotherm of enantiomers impossible. A new method, adjustable factor h-root method (AF-HRM), had been presented for the measurement of langmuir equilibrium coefficients. In the new approach, the retention factors and the frontal retention factors are required to determine the parameters of isotherms. As the ultimate goal is to separate enantiomers of fluoxetine on a preparative scale, a convenient method to determine the adsorption isotherm is essential. In this paper, we have demonstrated that AF-HRM can be applied to determine the parameters in the extended Langmuir model. Experimental results agree well with the prediction.     

The direct inverse method: A novel approach to estimate adsorption isotherm parameters

A novel method to estimate adsorption isotherm parameters is presented and its applicability is studied through synthetic as well as experimental data. This approach assumes a linear dependency of the UV absorption intensity on the solute concentration in the fluid phase, at least in certain ranges of the UV spectra. It was demonstrated that by fitting the absorption profiles, i.e. the new direct inverse method, and by fitting the concentration profiles, i.e. the classical inverse method, very similar adsorption isotherm parameters can be obtained. The findings presented in this work have as important implication the elimination of the requirement of converting a measured absorption intensity into a concentration value, i.e. the elimination of the calibration of the UV signal.     

Expanding the elution by characteristic point method for determination of various types of adsorption isotherms

Important improvements have recently been made on the elution by characteristic point (ECP) method to increase the accuracy of the determined adsorption isotherms. However, the method has so far been limited/used for only type I adsorption isotherms (e.g. Langmuir, Tóth, bi-Langmuir). In this study, general strategies are developed to expand the ECP method for the determination of more complex adsorption isotherms including such containing inflection points. We will exemplify the methodology with type II, type III and type V isotherms. Guidelines are given for how to determine such isotherms using the ECP method and for the experimental considerations that must be taken into account or that may be eliminated in the particular case.     

A chromatographic analysis of hindered diffusion of saccharides in silica gels

Summary The chromatographic technique as a means for experimental studies of diffusion in porous solids has been demonstrated. In this paper we report the effective diffusion coefficients which we have chromatographically obtained for two dextran samples and three low molecular weight solutes. The chromatographic method of measuring diffusional resistances in porous solids has the advantages of rapidity and simplicity. This method also provides information on the axial dispersion and distribution of a solute between the moving phase and the solvent held stationary in the pore. The elution curves of a packed column in pulse response experiments were analyzed by the method of moments to give the parameters of interest. The results indicate that pore diffusion of the solutes was appreciably restricted in comparison with diffusion in bulk solution. The degree of hindrance depended on the size of the diffusing substances.     

Application of a novel affinity adsorbent for the capture and purification of recombinant Factor VIII compounds

Recombinant Factor VIII (FVIII) therapies have been created to provide treatment for Hemophilia A, an inherited bleeding disorder caused by mutation in the FVIII gene. A major challenge in the purification of recombinant FVIII molecules is the development of an affinity chromatography step. Such a step must be highly specific and selective for the FVIII molecule, but also must be designed appropriately to ensure the FVIII molecule can be effectively recovered without resorting to harsh elution conditions which may be harmful to the product. Additionally, it is desirable to have affinity adsorbents designed to be reusable over a large number of column cycles while maintaining consistent purification performance. In this work, we describe the use of VIIISelect, a commercially available affinity adsorbent designed specifically for the purification of FVIII compounds. The VIIISelect adsorbent consists of a 13 kDa recombinant protein ligand attached to a cross-linked agarose base matrix. The structure of the recombinant ligand is based upon Camelid-derived single domain antibody fragments. The VIIISelect adsorbent is produced using a process free of animal-derived raw materials, which is a highly desirable attribute for adsorbents used in the purification processes of recombinant protein therapeutics. The VIIISelect adsorbent was used as the initial capture column to purify a FVIII compound directly from clarified cell culture fluid prior to further downstream purification. The purification performance of the VIIISelect was evaluated, which included measurement of the static binding capacity, dynamic binding capacity, product recovery, impurity clearance, and adsorbent reuse. Following laboratory-scale process development, the VIIISelect adsorbent was scaled up and used in the large scale manufacturing of a FVIII compound.     

Recombinant protein purification using gradient-assisted simulated moving bed hydrophobic interaction chromatography. Part I: selection of chromatographic system and estimation of adsorption isotherms

The design of gradient simulated moving bed (SMB) chromatographic processes requires an appropriate selection of the chromatographic system followed by the determination of adsorption isotherm parameters in the relevant range of mobile phase conditions. The determination of these parameters can be quite difficult for recombinant target proteins present in complex protein mixtures. The first part of this work includes the estimation of adsorption isotherm parameters for streptokinase and a lumped impurity fraction present in an Escherichia coli cell lysate for a hydrophobic interaction chromatography (HIC) matrix. Perturbation experiments were carried out using a Butyl Sepharose matrix with purified recombinant protein on buffer equilibrated columns as well as with crude cell lysate saturated columns. The Henry constants estimated for streptokinase were found to exhibit in a wide range a linear dependence on the salt concentration in the mobile phase. These parameters were applied in subsequent investigations to design a simulated moving bed (SMB) process capable to purify in a continuous manner recombinant streptokinase from the E. coli cell lysate.     

Gas chromatographic method for the analysis of nitrochlorobenzene isomers

Summary An efficient, reproducible and rapid gas chromatographic method for the analysis of nitrochlorobenzenes has been described in this paper. The method uses FFAP as stationary phase and 3,4-dichloronitrobenzene as internal standard. The total analysis time of the method is less than 15 minutes with a coefficient of variation ranging from 0.41% to 1.19% for different levels of three isomers.     

A rapid gas chromatographic method for the analysis of acidic fermentation products

Summary A rapid and simple method for the identification of acidic fermentation products is described. Methyl esters of volatile and non-volatile acids are separated on a single chromatographic column. Retention time data for a number of acids are tabulated.     

A novel technique for the sampling and gas chromatographic analysis of vapours

Summary A conventional pyrolysis gas chromatography system has been adapted to the analysis of vapours. A Curie point pyrolysis wire is used as a holder for granular active carbon on which the sample can be adsorbed. Desorption is achieved in the pyrolysis unit, but no pyrolysis occurs. The present study reports on the completeness of adsorption by the sampler and on the degree of release of adsorbate from it.     

A routine gas chromatographic method for the analysis of chlorofluorocarbons in aerosol cans

Summary A gas chromatographic (GC) method for the routine analysis of fully halogenated chlorofluorocarbons (CFCs) in aerosol cans is described. The identification of CFCs by GC was found to be in full agreement with those by GC-mass-spectrometery. The method has been applied to the analysis of CFCs in 448 aerosol products. The most commonly used fully halogenated CFC propellants in aerosol cans were found to be CFC11, CFC12 and CFC114.     

New clean-up method for gas chromatographic analysis of pyrethroid residues

Summary A fast and efficient method has been developed for clean-up of fenpropathrin and fluvalinate for gas chromatographic determination of their residues in crops, grain and soil. Charcoal (Darco-G-60) was used as adsorbent and the pyrethroids recovered by washing the adsorbent with hexane-acetone (9∶1). The method provides a clear filtrate with no interfering peaks in the chromatogram. Recoveries ranged 91.00–100%.     

Enhanced chromatographic NMR with polyethyleneglycol. A novel resolving agent for diffusion ordered spectroscopy

NMR analysis of complex mixtures can be significantly simplified using polyethyleneglycol (PEG) as resolving additive in DOSY NMR technique, which allows the extraction of individual spectra of mixture components with differing polarity. Resolving power of PEG‐assisted DOSY was demonstrated with natural product mixtures. Copyright © 2010 John Wiley & Sons, Ltd.     

A chromatographic method for hydrophobicity investigations of sorbents

Summary The energy of interaction (₀) between the methylene group and the surface of a sorbent is proposed for use in the quantitative evaluation of the hydrophobicity of a sorbent. A method was developed to determine the ₀ parameter from chromatographic measurements using the investigated sorbent as the stationary phase and linear methylene-containing homologous series, e.g., alkanols, amino-alkanes, etc., as the test compounds. The effectiveness of this method is demonstrated by using, as examples, a series of bonded stationary phases and a series of cross-linked copolymeric sorbents differing in the concentration of the hydrophobic groups. The proposed method is compared with the Martin rule used extensively in the chromatography of oligomers.     

Comparison of the adsorption mechanisms of pyridine in hydrophilic interaction chromatography and in reversed-phase aqueous liquid chromatography

The adsorption isotherms of pyridine were measured by frontal analysis (FA) on a column packed with shell particles of neat porous silica (Halo), using water–acetonitrile mixtures as the mobile phase at 295 K. The isotherm data were measured for pyridine concentrations covering a dynamic range of four millions. The degree of heterogeneity of the surface was characterized by the adsorption energy distribution (AED) function calculated from the raw adsorption data, using the expectation-maximization (EM) procedure. The results showed that two different retention mechanisms dominate in Per aqueous liquid chromatography (PALC) at low acetonitrile concentrations and in hydrophilic interaction chromatography (HILIC) at high acetonitrile concentrations. In the PALC mode, the adsorption mechanism of pyridine on the silica surface is controlled by hydrophobic interactions that take place on very few and ultra-active adsorption sites, which might be pores on the irregular and rugose surface of the porous silica particles. The surface is seriously heterogeneous, with up to five distinct adsorption sites and five different energy peaks on the AED of the packing material. In contrast, in the HILIC mode, the adsorption behavior is quasi-homogeneous and pyridine retention is governed by its adsorption onto free silanol groups. For intermediate mobile phase compositions, the siloxane and the silanol groups are both significantly saturated with acetonitrile and water, respectively, causing a minimum of the retention factor of pyridine on the Halo column.