Mass-overload Conditions Research Articles

Retention properties of hydrophobically end-capped poly(ethylene glycol)s on a β-cyclodextrin support

High-performance liquid chromatography (HPLC) was used to examine the retention behavior of monomethoxypoly(ethylene glycol)s bearing one hydrophobic naphthyl end group (Nap-MPEG) on β-cyclodextrin polymer (poly-β-CD) immobilized on a silica support, under isocratic elution conditions and using water as mobile phase. Studies of retentions and theoretical plate heights H were conducted at infinite dilution by comparing the behavior of Nap-MPEGs having different molecular weight (750, 1000 and 5000 g/mol). The larger is its molecular size, the lower is the retention of the polymer. The linear increase of H with mobile phase velocity reveals slow mass-transfer kinetics arising from the restricted diffusion into the pores of the support. The complexation constants between the Nap-MPEGs and β-CD in solution (around 500 M −1) were determined from the decrease of retention observed by adding increasing concentrations of hydroxypropyl β-CD into the eluent. The peak profiles in mass-overload conditions were studied by fitting a model based upon bi-Langmuir kinetics which assumes a non-uniform support having two types of binding sites and apparent adsorption rate constants are used to describe mass-transfer kinetics. A three-parameter adsorption equilibrium isotherm was sufficient to account for the modifications of peak shapes observed when increasing amounts of polymer were injected. This result indicates an interaction with a heterogeneous poly-β-CD support mainly composed of low affinity groups, non-saturable in the range of polymer concentration studied. An upper limit was estimated for the equilibrium constant (<1000 M −1) characterizing the affinity of Nap-MPEG for the non-saturable sites of the poly-β-CD support. Large affinity constants (8–9 × 10 4 M −1) were found for the interaction of Nap-MPEGs with a small percentage of active sites.

Study of the retention properties of warfarin enantiomers on a β-cyclodextrin polymeric support

High-performance liquid chromatography was used to study the retention properties of ( R)- and ( S)-warfarins on a silica support coated with a β-cyclodextrin polymer. The influence of the methanol content of the acetate buffer eluent was investigated at pH 4. The measure of the variations of retention time with temperature enables one to determine the enthalpy and the entropy of adsorption. The plot of the two thermodynamic functions shows a minimum around 30% (v/v) methanol. At low methanol contents, the decrease of the hydrophobic interactions with increasing methanol content explains the decrease of the enthalpic and entropic terms. Above 40% (v/v) methanol, the decrease of the adsorption enthalpy absolute value is due to the solvation by the organic component. From the analysis of peak shape in mass-overload conditions, the column capacity toward each enantiomer was determined. A lower capacity was found toward ( S)-warfarin, the more retained enantiomer. Peak shape analysis in mass-overload conditions was used to determine the adsorption isotherm. A Langmuir-type adsorption isotherm accounts well for the experimental data.

Study of affinity supports based on reactive polymers immobilized on silica: affinity constant determination from isocratic zonal elution

Polymers bearing benzamidine moieties have been prepared from reactive copolymer containing chloroformate functions and deposited on porous silica matrices. These high-performance affinity chromatography supports were characterized by quantitative methods, which analyse the zonal elution behaviour of trypsin in the presence of a soluble competitor ( l-arginine). The column loading capacity for trypsin was measured by the zonal elution method in mass overload conditions. On the basis of a Langmuir isotherm, the influence of the protein capacity and the concentration of the soluble ligand on the elution volume was studied for the determination of the binding constants. The plate heights determined for silica supports of various porosities and particle diameters show that the strong affinity interactions between trypsin and p-aminobenzamidine are mainly responsible for the low efficiencies observed.

Multivalent ion-exchange model of biopolymer chromatography for mass overload conditions

The simple model of multivalent ion-exchange biopolymer chromatography is analyzed on the basis of classical quasi-chemical temperature. The rigorous isotherm equation deduced from the stoichiometric displacement model (SDM) was used to stimulate the migration of a solute through the chromatographic column in the isocratic and gradient elution modes. The peak profiles generated for various sample sizes were compared with those obtained on the basis of a Langmuir isotherm. Peak tailing increases with the value of the exponent Z, defined as the ratio of the protein valency to the displacing counter-ion valency. For large Z the asymmetries due to the non-linearity of the isotherm are still present for small sample sizes, the ion-exchange model was applied to analyse the zonal elution behaviour of bovine serum albumin on a polymeric anion-exchange stationary phase deposited on silica. The effective charge of the protein ( m = 8) was determined at infinite dilution with mono- and a divalent counter-ions. This value was introduced into the SDM isotherm to predict the elution behaviour in mass-overload conditions and the maximum loading capacity of the protein was determined. Good agreement between theory and experiment was obtained: for about the same capacity factor at infinite dilution, a larger peak asymmetry due to non-linear effects is found with a monovalent counter ion.

Application of the split-peak effect to study the adsorption kinetics of human serum albumin on a reversed-phase support

The adsorption step of human serum albumin on a reversed-phase support was analyzed by studying the “split-peak” effect in mass-overload conditions. This behavior is characterized by the occurrence of a first non-retained fraction and is described by an analytical expression in the case of a Langmuirian adsorption isotherm. The method was applied to determine the column loading capacity, the number of mass-transfer units and the apparent adsorption rate constant measured at a given flow-rate. The nature of the organic modifier influences the split-peak effect: it increases with the eluotropic strength of the organic solvent added to the buffer. Compared to the results with pure buffer, it is the association of two effects, the decrease of the column loading capacity and that of the apparent adsorption rate constant, which increases the split-peak effects observed when methanol and 2-propanol are added to the eluent. These results allow us to gain a better understanding of the role of the organic solvent in the elution behavior of proteins in reversed-phase high-performance liquid chromatography.

Preparative high-performance liquid chromatography under gradient conditions : I. Band broadening in gradient elution as a function of sample size

Previous studies of gradient elution under mass-overload conditions are continued. Use of the Craig distribution model shows that there is a simple relationship between bandwidth, sample size and gradient conditions for single-solute samples. this allows the use of two or three experimental measurements to determine the saturation capacity of a column by a given sample. Several (reversed-phase) experimental studies with small-molecule samples provide quantitative confirmation of this model. For the case of protein samples the model works equally well, but it appears that only 20–44% of the column capacity (as measured by saturation uptake) is available under separation conditions. the column capacity for lysozyme was measured as a function of pore diameter and the accessibility of the surface under separation conditions was found to increase with increasing pore size.

Analysis of the information in a preparative chromatogram for further optimization of the operating conditions

It was demonstrated how the most salient theoretical aspects of linear and non-linear elution liquid chromatography can be used, without the aid of sophisticated optimization software, to analyse the information in a preparative chromatogram. It is important. First, to be able to recognize whether a given preparative chromatogram was obtained under volume- or mass-overload conditions or both. This permits further optimization of the injection conditions (size, volume and concentration of the sample), depending on the preparative objectives, and improvement of the stationary phase particle size and of the mobile phase flow-rate. This approach can also provide insight into the performance characteristics of the injection device, the efficiency of the column packing and the stationary phase capacity. Some examples taken from the recent literature are discussed.

Preparative separation of peptide and protein samples by high-performance liquid chromatography with gradient elution : I. The craig model as a basis for computer simulations

The Craig model (assuming a Langmuir isotherm) has been used by us previously to successfully simulate isocratic high-performance liquid chromatographic (HPLC) separation in a mass-overload mode. Here we have extended this approach to the case of gradient elution for large samples. These simulations support our earlier conclusion that so-called “corresponding” isocratic and gradient separations provide similar sample resolution when the sample size is the same. “Corresponding” separations refer to the case where isocratic retention k′ is equal to average gradient retention k̄, and where other conditions (column, flow-rate, etc.) are the same. Craig simulations reported here also provide further insight into the factors that affect preparative HPLC separations under mass-overload conditions.

Experimental Characterization of Strongly Nonlinear Elution Peaks in Liquid Chromatography

Abstract The shape of nonlinear elution peaks was studied experimentally in various chromatographic modes and a wide sample size range encompassing the case where the distribution isotherm curvature is the prevailing factor of band broadening. It is shown that as sample size is varied, the tailing part of all peaks adjoins a common curve that can be fitted with an exponential function whose parameters are independent of mobile phase linear velocity and stationary phase particle size. The effect of solute capacity factor on these parameters was also investigated. These findings provide valuable help for the optimization of linear velocity, particle size, as well as sample size in preparative liquid chromatography under mass overload conditions.

Simplified description of high-performance liquid chromatographic separation under overload conditions, based on the Craig distribution model : IV. Comparison of the model with experimental data for samples containing two or more solutes

The preceding paper presented a quantitative description of high-performance liquid chromatography (HPLC) separation under mass-overload conditions for samples that contain two or more solutes. Specifically it was suggested that such separations differ from corresponding single-solute separations in terms of a so-called “blockage” effect. The present paper describes the experimental verification of this model for samples containing two or more solutes. Examples of blockage are shown, and these are predicted quantitatively by means of our previous model. Mixtures of β-hydroxyethyl- and 7-β-hydroxypropyltheophylline were separated by reversed-phase HPLC on a 15 x 0.46 cm C 8 column. Total sample mass w was varied over the range 0.002 ⩽ w ⩽ 27 mg, leading to pronounced changes in separation. Similar studies were carried out with mixtures of methyl, ethyl, propyl and butyl parabens, varying total sample mass over the range 0.004 ⩽ w ⩽ 20 mg. Resulting separations were generally in good agreement with predictions (“model simulations”).

Some Sampling Effects in Liquid Chromatography

Abstract The relationship between column packing particle size and column efficiency under mass overload conditions was examined with three silica-based packing materials of moderate surface area (SBET range 200–500 m2 g−1). An equivalent relation between reduced plate height, H/dp, and relative sample load, sample mass/mass packing, was shown for all materials examined, both in the adsorption and reversed phase modes of chromatography over a narrow range of reduced velocity, udp/Dm. The data were used in a simple calculation to establish the minimum dimensions of columns packed with dp=5μm and 20μm particles for a typical semi-preparative separation of a mixture of 20 mg each of two substances. A short study on the effect of variation of reduced velocity on reduced plate height at ‘analytical’ and semipreparative sample loadings showed that performance of LC columns in the preparative mode could be described reasonably well as a three dimensional surface in which the Y axis is reduced plate height, the X axis is reduced load, and the Z axis is reduced velocity. Some aspects of the performance of a ‘curtain-flow’ preparative precolumn/column system are described along with an outline of the principles of design of this apparatus.