Overloaded gradient elution chromatography on heterogeneous adsorbents in reversed-phase liquid chromatography

Overloaded band profiles of phenol were measured on a C18-Kromasil column in gradient elution conditions. The mobile phase used was a mixture of methanol and water. The volume fraction of methanol was allowed to vary between 0 and 0.5. A general adsorption model, which expresses the amount of phenol adsorbed q* as a function of both its concentration C and the composition phi of the organic modifier (methanol) in the mobile phase, was empirically derived from previous independent adsorption experiments based on frontal analysis (FA) and frontal analysis by the characteristic point (FACP). Accordingly, the general model was an extension of the simplest heterogeneous model, the Bilangmuir model, to non-isocratic conditions. The low-energy sites followed the classical linear solvent strength model (LSSM), but not the high-energy sites whose saturation capacity linearly decreased with phi. The general model was validated by comparing the experimental and simulated band profiles in gradient elution conditions, in linear and non-linear conditions, as well. The band profiles were calculated by means of the equilibrium-dispersive model of chromatography with a finite difference algorithm. A very good agreement was observed using steps gradient (delta phi) from 0 to 50% methanol and gradient times t(g) of 20, 25, 30, 40, 60, 80 and 100 min. The agreement was still excellent for steps gradient from 5 to 45% (t(g) = 25 min), 5 to 35% (t(g) = 50 min), 5 to 25% (t(g) = 50 min) and 5 to 15% (t(g) = 50 min). Significative differences appeared between experience and simulation when the slope of the gradient (delta phi/t(g)) became too strong beyond 3.3% methanol per minute. This threshold value probably mirrored the kinetic of arrangement of the G18-bonded chains when the methanol content increased in the mobile phase. It suggested that the chromatographic system was not in a full thermodynamic equilibrium state when very steep mobile phase gradients were applied.     

High speed gradient elution reversed-phase liquid chromatography

A major disadvantage of gradient elution in terms of speed results from the need to adequately re-equilibrate the column. This work distinguishes two states of re-equilibration: (1) run-to-run repeatability and (2) full equilibration. We find that excellent repeatability (+/-0.002 min in retention time) is achieved with at most 2 column volumes of re-equilibration whereas full equilibration can require considerably more than 20 column volumes. We have investigated the effects of adding ancillary solvents (e.g. n-propanol, n-butanol) to the eluent and changing the particle pore size, initial eluent composition and type, column temperature and flow rate on the speed of full equilibration. Full equilibration seems to be more thermodynamically limited than kinetically controlled. Also, we show that the main limitation to reducing the full equilibration time is related to instrument design issues; a novel approach to overcome these instrumental issues is described.     

Limits of multi-linear gradient optimisation in reversed-phase liquid chromatography

The concept of limiting peak purity was applied to quantify the degree of completion of the separation capability of a chromatographic system using multi-linear gradients. The objective was to check whether the complexity of a gradient program deserves be increased to enhance resolution by inserting more linear segments, or on the contrary, no significant improvements can be expected under more complex gradients. A set of 19 isoindole derivatives of primary amino acids was selected to test the performance of isocratic, single linear and multi-linear gradients. Accurate simulated chromatograms were obtained via numerical integration of the general equation of gradient elution, using pre-established start and end conditions of the gradient program. The overall peak purity was selected as objective function. Good--although not baseline--resolution was achieved with an optimal trilinear gradient. Excellent agreement between experimental and predicted optimal chromatograms was found. With the proposed approach, a degree of completion of the separation capability of the chromatographic system of 21.2, 49.7, 81.5 and 88.5% was accomplished with optimal gradients with one, two, three and four segments, respectively. More complex gradients did not enhance the latter figure significantly. Also, multi-linear gradients gave rise to more benefits than complementary gradients.     

Effect of temperature on the adsorption behavior of tryptophan in reversed-phase liquid chromatography

Single-component adsorption isotherm data of l-tryptophan on a C(18)-bonded silica column were acquired by frontal analysis (FA), with aqueous mobile phases containing 5% of acetonitrile at five different temperatures between 23 and 62 degrees C. The non-linear fitting of these data provided the bi-Moreau model for all temperatures as the best isotherm model. The inverse method (IM) was used to derive the parameters at these temperatures from the parameters of the 25 degrees C isotherm. The adsorption constants and the saturation capacities of the low and high-energy sites decreases by increasing the temperature, while the adsorbate-adsorbate parameters of both sites increase. An excellent agreement was found between the experimental and calculated overloaded band profiles at all the temperatures used. The breakthrough curves obtained and the overloaded band profiles obtained were found to have different shapes according to the range of concentration studied and the temperatures. At low concentration 0.05-0.5 g/L the breakthrough curves and the overloaded band profiles have a front shock and diffuse rear, which indicates langmuirian behavior, but at intermediate 1-2 g/L and high concentration 8 g/L they start to have diffuse fronts and shocks at the rear or more than one shock at the rear which indicates non-langmuirian behavior. At 23 degrees C the isotherm has another langmuirian part, which appears at high concentration. The behavior of the breakthrough curves is explained by the shape of the isotherm in which all of the isotherms have a langmuirian part (the isotherm is concave upward) and an antilangmuirian part (the isotherm is concave downward). The temperature affected the breakthrough curves by decreasing the time of the appearance of the fronts for all concentration ranges studied, and by decreasing the time difference between the highest concentration and lowest concentration of the fronts, especially the low concentration range at 0.5 g/L. The fronts of the breakthrough curves at high concentration seems to be the most affected by temperature.     

Control of column temperature in reversed-phase liquid chromatography

When separations by reversed-phase liquid chromatography (RP-LC) are carried out at temperatures other than ambient, resulting retention times and bandwidths can depend on the equipment used. As a result, an RP-LC separation that is adequate when carried out on one LC system may prove inadequate when the separation is repeated on a second system. In the present study, various temperature-related problems which can result in a failure of method transfer for non-ambient RP-LC methods were examined. Means for correcting for such effects and thereby ensuring method transferability are described.     

Column equilibration effects in gradient elution in reversed-phase liquid chromatography

The fundamental equations and conditions for linear and stepwise gradient elution in reversed-phase liquid chromatography are applied to a mixture of amino acids in their underivatized form in aqueous mobile phases modified by 2-propanol, acetonitrile or methanol for examining column equilibration effects. It was found in all cases systematic deviations between experimental and calculated retention times, which are prominent in 2-propanol, reduced in acetonitrile and practically negligible in methanol. These deviations appear within a chromatogram just after the first change in the composition of the mobile phase reaches the detector and last ca. 5 min, where the magnitude of errors reduces exponentially with time. Based on these observations we propose a simple way to correct the calculated from the gradient elution theory retention times of sample solutes. The origin of the discrepancies between theory and experiment as well as their impact on the resolution is also discussed.     

Effect of the temperature on the isotherm parameters of phenol in reversed-phase liquid chromatography

Adsorption isotherm data of phenol from an aqueous solution of methanol onto a C18-bonded silica (Symmetry-C18) were acquired by frontal analysis (FA) at six different temperatures, in a wide concentration range. The non-linear fitting of these data provided the bi-Langmuir model as best isotherm model, a conclusion further supported by the results of the calculation of the affinity energy distribution (AED). The isotherm parameters were obtained using several methods, the fitting of FA isotherm data, the calculation of the AED, and the inverse method, that uses overloaded elution band profiles. The different values obtained are in close agreement. They allow a quantitative investigation of the separate properties of the low- and the high-energy sites on the adsorbent surface. Increasing the temperature decreases the saturation capacity of the low-energy adsorption sites and the adsorption constant of the high-energy sites. In contrast, increasing the temperature does not cause any significant changes in either the saturation capacity of the high-energy sites or the adsorption constant of the low-energy sites.     

Optimisation technique for stepwise gradient elution in reversed-phase liquid chromatography

An optimisation technique of reversed-phase liquid chromatographic separations based on gradient elution with a stepwise variation pattern of the volume fraction phi of the organic modifier in the water-organic mobile phase is presented. It uses a non-linear least-squares programme with a Monte-Carlo search for initial estimates in order to determine the best variation pattern that leads to the optimum separation of a mixture of solutes. The validity of the above methodology was tested by separating eight catechol-related solutes with mobile phases modified by methanol or acetonitrile and variation patterns of two, three or four steps in the psi values. It was found in all cases a very satisfactory accuracy of the predicted gradient elution times, which is of the same order with the accuracy of the retention times predicted under isocratic or linear gradient conditions. In addition, it was shown that the proposed optimisation technique is both effective and flexible but well-shaped chromatograms are obtained under electrochemical detection only if steps with increasing psi are used and the change in psi is programmed to occur at the intermediate of the predicted peaks.     

The temperature convergence of biopolymers in reversed-phase liquid chromatography

Summary Based on a stoichiometric displacement model for retention (SDM-R) in liquid chromatography, the two linear plots, log/(a constant relating to the affinity of one mole of solute to the stationary phase) andZ (the total moles of the displacing agent released at the interface between stationary phase and solute molecules as one mole of the solute is absorbed by the stationary phase) of small solutes and biopolymers versus the reciprocal of absolute temperature, l/T, in reversed-phase liquid chromatography were theoretically derived and experimentally proved to be linear and to have a common point called the temperature convergent point. The two linear plots could be used instead of the plot of logk to l/T which is normally only valid for small solutes but not for biopolymers for investigations of chromatographic thermodynamics and temperature convergence. The average convergence temperature (T _conv) of biopolymers was theoretically derived to equal the ratio between the slope and the intercept from either one of the two linear plots and to be 130.8±9.3°C for five proteins which are very close to those of biopolymers by calorimetry. A new methodology to investigate chromatographic thermodynamics and possibly for investigating the temperature convergence of biopolymers in the process of protein folding is also presented.     

Fast, comprehensive online two-dimensional high performance liquid chromatography through the use of high temperature ultra-fast gradient elution reversed-phase liquid chromatography

A new approach to high speed, comprehensive online dual gradient elution 2DLC (LCxLC) based on the use of ultra-fast, high temperature gradient elution reversed phase chromatography is described. Entirely conventional gradient elution instrumentation and columns are assembled in a system which develops a total peak capacity of about 900 in 25 min; this is equivalent to roughly one peak/2 s. Each second dimension gradient is done in a cycle time of 21 s and the peak retention times measured for a set of twenty six indole-3-acetic acid (IAA) derivatives are reproducible to 0.2 s. Each peak eluting from the first dimension column is sampled at least twice across its width, as the corresponding peaks on the second dimension column appear in two or three consecutive second dimension chromatograms, clearly indicating that there is little loss in the resolution gained in the first dimension separation. Application to the separation of the low molecular weight components of wild-type and mutant maize seedlings indicates the presence of about 100 peaks on a timescale of 25 min. Compelling illustrations of the analytical potential of fast, high temperature 2DLC are evident in the clear presence of nine distinct peaks in a single second dimension chromatogram from a single quite narrow first dimension peak, and the great power of 2DLC to solve the "analytic dynamic range" problem inherent in the measurement of small peaks that are neighbors to a gigantic peak.     

Multilinear gradient elution optimisation in reversed-phase liquid chromatography using genetic algorithms

The treatment presented in a recent paper [P. Nikitas, A. Pappa-Louisi, J. Chromatogr. A, 1068 (2005) 279] is extended to multilinear gradients, i.e. continuous gradients consisting of a certain number of linear portions. Thus, the experimental lnk versus phi curve, where k is the retention factor of a sample solute under isocratic conditions and phi is the volume fraction of the organic modifier in the water-organic mobile phase, is subdivided into a finite number of linear portions resulting in simple analytical expressions for the solute gradient retention time. These expressions of the retention time are directly used in an optimisation technique based on genetic algorithms. This technique involves first the determination of the theoretical dependence of k upon phi by means of gradient measurements, which in turn is used by the genetic algorithm for the prediction of the best gradient profile. The validity of the analytical expressions and the effectiveness of the optimisation technique were tested using fifteen underivatized amino acids and related compounds with mobile phases modified by acetonitrile. It was found that the adopted methodology exhibits significant advantages and it can lead to high quality predictions of the gradient retention times and optimisation results.     

Retention models for isocratic and gradient elution in reversed-phase liquid chromatography

One- and multi-variable retention models proposed for isocratic and/or gradient elution in reversed-phase liquid chromatography are critically reviewed. The thermodynamic, exo-thermodynamic or empirical arguments adopted for their derivation are presented and discussed. Their connection to the retention mechanism is also indicated and the assumptions and approximations involved in their derivation are stressed. Special attention is devoted to the fitting performance of the various models and its impact on the final predicted error between experimental and calculated retention times. The possibility of using exo-thermodynamic retention models for prediction under gradient elution is considered from a practical point of view. Finally, the use of statistical weights in the fitting procedure of a retention model and its effect on the calculated elution times as well as the transferability of retention data among isocratic and gradient elution modes are also examined and discussed.     

Microbore reversed-phase chromatography of proteins with conventional gradient equipment for high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography with microbore columns (50 X 1.0 mm) was used effectively for the separation and analysis of proteins down to 1 ng at flow-rates of 0.1-0.2 ml/min. With the use of standard low-pressure gradient HPLC equipment, the peak volumes were five times smaller when compared with a conventional column at equal chromatographic efficiencies and analysis time. The sensitivity of detection was further increased by a reduction in solvent peaks, resulting in a 20-fold overall increase.     

An abnormal temperature dependence of alkylpyrazines' retention in reversed-phase liquid chromatography

Retention behaviors of pyrazine and alkylpyrazines on various stationary phases in reversed-phase liquid chromatography were examined. An abnormal temperature effect on the retention of alkylpyrazines with a mobile phase consisting of acetonitrile and water was observed when changing the column temperature. On the other hand, no similar trend was found with a methanol-water mobile phase. For all the columns investigated in this work, the above tendency to the temperature-dependence was consistently observed, suggesting that the abnormal temperature effect on the retention of alkylpyrazines could be mainly induced by an acetonitrile-based mobile phase.     

Optimisation of multilinear gradient elutions in reversed-phase liquid chromatography using ternary solvent mixtures

The multilinear gradient elution theory for binary mobile phases in reversed-phase liquid chromatography presented in [P. Nikitas, A. Pappa-Louisi, A. Papageorgiou, J. Chromatogr. A 1157 (2007) 178] is extended to ternary gradients. For the evaluation of this theory and the performance of the various fitting and optimisation algorithms we used 13 o-phthalaldehyde (OPA) derivatives of amino acids with mobile phases modified by acetonitrile and methanol. It is shown that the theory can lead to high quality predictions of the retention times under gradients elutions and optimisation of ternary gradients provided that we use a six-parameter expression for the logarithm of the retention factor, lnk, and the adjustable parameters of this expression are determined from ternary isocratic data.     

Effect of temperature on competitive adsorption of the solute and the organic solvent in reversed-phase liquid chromatography

In analysis of the temperature effect on chromatographic separations the influence of the adsorption of organic solvent on the retention properties of solute is generally not taken into account. In fact, adsorption behavior of solutes is strongly affected by competitive adsorption of organic solvents, which is temperature dependent. In this work changes of adsorption equilibrium of an organic solvent as well as a solute with temperature have been analyzed. Data of the excess adsorption of methanol from aqueous solutions on octadecyl-bonded silica have been acquired at different temperature. Experiments have been performed over a relatively narrow temperature range corresponding to typical chromatographic conditions, i.e., 10-50 degrees C. The competitive adsorption equilibria of model solutes (i.e., two homologous compounds: cyclopentanone and cyclohexanone) have been measured at different temperature and composition of the mobile phase. Temperature alterations to the retention properties were found to result from combined effects of changes in adsorption behavior of the organic solvent and of the solute. The influence of temperature on the separation selectivity has been considered.     

Microfabricated refractive index gradient based detector for reversed-phase liquid chromatography with mobile phase gradient elution

Typical refractive index (RI) detectors for liquid chromatography (LC) are not well suited to application with mobile phase gradient elution, due to the difficulty in correcting for the detected baseline shift during the gradient. We report a sensitive, highly reproducible, microfabricated refractive index gradient (micro-RIG) detector that performs well with mobile phase gradient elution LC. Since the micro-RIG signal remains on-scale throughout the mobile phase gradient, one can apply a baseline correction procedure. We demonstrate that by collecting two mobile phase gradient blanks and subtracting one of them from the other, a reproducible, flat baseline is achieved. Therefore, subtracting a blank from a separation provides a baseline corrected chromatogram with reasonably high signal-to-noise ratio for eluting analytes. The micro-RIG detector uses a collimated diode laser beam to optically probe a RIG formed perpendicular to the laminar flow direction within a microfabricated borosilicate glass chip. The chip-based design of the detector is suitable for either traditional bench-top or LC-on-a-chip technologies. We report reversed phase high performance liquid chromatography (RP-HPLC) separations of proteins and polymers, over mobile phase gradient conditions of 67% A:33% B to 3% A:97% B by volume, where A is 96% methanol:3.9% water:0.1% trifluoroacetic acid (TFA), and B is 3.9% methanol:96% water:0.1% TFA. The separations were performed on a Jupiter 5 mu C4 300 A 150 mm x 1.0 mm Phenomenex column at a flow rate of 20 microl/min. Viscosity changes during the mobile phase gradient separation are found to shift the on-chip merge position of the detected concentration gradient (i.e., RIG), in a reproducible fashion. However, this viscosity effect makes detection sensitivity vary throughout the mobile phase gradient, due to moving the optimized position of the probe beam in relation to the analyte concentration gradient being probed. None-the-less, consistent limits of detection (LODs) were achieved. The 3-sigma deflection angle LOD was 16 microrad for micro-RIG detection, corresponding to an injected concentration LOD of 7 ppm (mass/mass) for cytochrome c.     

Axial temperature gradient and mobile phase gradient in microcolumn high-performance liquid chromatography

The effect of axial temperature gradient (ATG) along a microcolumn on the separation performance at both isocratic and gradient elution mode was investigated. A thermostat system was designed to form an ATG along the packed column. Polycyclic aromatic hydrocarbons (PAHs) were separated on a 0.53 mm  × 150 mm i.d. 5 μm C₁₈ microcolumn, with water and acetonitrile as mobile phase. The separation results obtained at mobile phase gradient (MPG) and ATG in microcolumn HPLC were compared with the results performed at ambient conditions. Extrapolated curves of peak width at half height (wh)versus lnk showed that wh is narrower at the same retention time when ATG was applied in addition to MPG. The column efficiency was enhanced 20-30% and the resolution was slightly reduced because of reduction of selectivity at elevated temperature at ATG condition. The RSD of retention time in ATG mode was less than 2.5%.