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.     

Fast, comprehensive two-dimensional liquid chromatography

The absolute need to improve the separating power of liquid chromatography, especially for multi-constituent biological samples, is becoming increasingly evident. In response, over the past few years, there has been a great deal of interest in the development of two-dimensional liquid chromatography (2DLC). Just as 1DLC is preferred to 1DGC based on its compatibility with biological materials we believe that ultimately 2DLC will be preferred to the much more highly developed 2DGC for such samples. The huge advantage of 2D chromatographic techniques over 1D methods is inherent in the tremendous potential increase in peak capacity (resolving power). This is especially true of comprehensive 2D chromatography wherein it is possible, under ideal conditions, to obtain a total peak capacity equal to the product of the peak capacities of the first and second dimension separations. However, the very long timescale (typically several hours to tens of hours) of comprehensive 2DLC is clearly its chief drawback. Recent advances in the use of higher temperatures to speed up isocratic and gradient elution liquid chromatography have been used to decrease the time needed to do the second dimension LC separation of 2DLC to about 20s for a full gradient elution run. Thus, fast, high temperature LC is becoming a very promising technique. Peak capacities of over 2000 and rates of peak capacity production of nearly 1 peak/s have been achieved. In consequence, many real samples showing more than 200 peaks with signal to noise ratios of better than 10:1 have been run in total times of under 30 min. This report is not intended to be a comprehensive review of 2DLC, but is deliberately focused on the issues involved in doing fast 2DLC by means of elevating the column temperature; however, many issues of broader applicability will be discussed.     

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.     

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.     

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.     

Statistical scanning method for the optimization of gradient elution high performance liquid chromatography

Summary A computer-assisted method is presented for the optimization of separation in gradient elution reversed-phase HPLC. The method is based on a polynomial estimation from nine preliminary experiments according to a two-factor (initial solvent composition C and gradient time T) rectangular design. This is followed by a two-dimension computer scanning technique. Resolution is used as the selection criterion. Good agreement was obtained between predicted data and experimental results.     

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.     

Optimization of stepwise gradient elution in reversed-phase chromatography

Summary Equations describing multi-step gradient elution with a mobile phase of constant composition in each step were derived. These equations useful for calculating the retention volumes in both gradient HPLC and TLC were derived on the basis of the relationship between the isocratic capacity factor and the volume fraction of the organic modifier. The validity of the equations was experimentally verified in a LiChrosorbRP-18-water/methanol system for 11 methyl- and chlorobenzenes and phenols. A satisfactory agreement between the theoretical and experimental k′ values was found.     

Preparative argentation reversed-phase high performance liquid chromatography

In this study, a preparative chromatography method named preparative argentation reversed-phase high performance liquid chromatography (Ag-RP-HPLC) was developed by adding silver ion to the mobile phase of preparative HPLC. Firstly, an analytical Ag-RP-HPLC method was developed, and the effects of silver content and acid content in the mobile phase on the column efficiency were studied. Based on the method of linear amplification, a preparative Ag-RP-HPLC technique with optimized separation conditions was developed. The new technique was applied successfully to the separation of the unsaturated aliphatic acid amide isomers contained in Asarum forbesii Maxim. Compared with the commonly used technique of argentation normal phase chromatography, this method with little solvent consumption is simple, fast, efficient, and flexible for the isolation and purification of the unsaturated compounds.     

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.     

Optimization of a comprehensive two-dimensional normal-phase and reversed-phase liquid chromatography system

The present investigation is based on the evaluation of the performance of a comprehensive two-dimensional liquid chromatography (LCxLC) system during method optimization. The LCxLC set-up, operated in normal phase (NP) mode (adsorption) in the first dimension (1D) and reversed-phase (RP) mode in the second dimension (2D), is equipped with a 1D microbore silica column and a 2D monolithic C(18) column with a 10-port two position valve as the interface. A photodiode array detector is used after the 2D separation. A possible cause of peak distorsion because of the immiscibility of the mobile phases employed in the two dimensions is resolved. The optimization of the analytical run time and flow rate for both dimensions and the initial gradient in the 2D is carried out with various standard compounds. The potential and versatility of this LCxLC approach is demonstrated through the separation of 11 standard components, most of them allergens. The latter, which are characterized by a scattered distribution on the 2D space plane, underwent separation on both a hydrophobicity and polarity basis.     

New approach to linear gradient elution used for optimisation in reversed-phase liquid chromatography

A new mathematical treatment concerning the gradient elution in reversed-phase liquid chromatography when the volume fraction psi of an organic modifier in the water-organic mobile phase varies linearly with time is presented. The experimental ln k versus psi curve, where k is the retention factor under isocratic conditions in a binary mobile phase, is subdivided into a finite number of linear portions and the solute gradient retention time tR is calculated by means of an analytical expression arising from the fundamental equation of gradient elution. The validity of the proposed analytical expression and the methodology followed for the calculation of tR was tested using eight catechol-related solutes with mobile phases modified by methanol or acetonitrile. It was found that in all cases the accuracy of the predicted gradient retention times is very satisfactory because it is the same with the accuracy of the retention times predicted under isocratic conditions. Finally, the above method for estimating gradient retention times was used in an optimisation algorithm, which determines the best variation pattern of psi that leads to the optimum separation of a mixture of solutes at different values of the total elution time.     

Effect of temperature on gradient reequilibration in reversed-phase liquid chromatography

The effect of mobile phase modifier and temperature on gradient reequilibration is examined using three different stationary phases. The stationary phases studied are a traditional C18 phase, a polar endcapped C18 phase, and an alkyl phase with a polar embedded group. It was observed that both temperature and choice of mobile phase organic modifier had an effect on gradient reequilibration volume on both the traditional C18 stationary phase and the polar endcapped phase. On both these phases, at any given temperature, the reequilibration volume was generally smaller when methanol was used as the mobile phase modifier as compared to acetonitrile. As the temperature is increased from 10 to 50 degrees C, significant reductions in reequilibration volume were observed with both mobile phase modifiers. In contrast, neither temperature nor choice of modifier appeared to have much effect on reequilibration volume when the polar embedded group stationary phase was considered.     

Influence of temperature in reverse-phase high-performance liquid chromatography with gradient elution

The influence of temperature on the retention of several species separated by reverse-phase liquid chromatography by gradient elution is shown to be of enough importance to warrant careful control of temperature if reproducible results are to be obtained. The smaller the particle size in the column, the greater the effect of temperature, and therefore the control should be greater. Likewise, it has been verified that for a given solvent gradient, independent of its complexity, there is a linear relation between ln k′ and 1/T, which also occurs in separations by isocratic elution. Dufek's equation can be adjusted perfectly to the experimental data obtained from gradient elutions, and may be used in the simulation and optimization of gradient chromatographic processes.     

Modeling of overloaded gradient elution of nociceptin/orphanin FQ in reversed-phase liquid chromatography

The Reversed-phase (RP) gradient elution chromatography of nociceptin/orphanin FQ (N/OFQ), a neuropeptide with many biological effects, has been modeled under linear and non-linear conditions. In order to do this, the chromatographic behavior has been studied under both linear and nonliner conditions under isocratic mode at different mobile phase compositions--ranging from 16 to 19% (v/v) acetonitrile (ACN) in aqueous trifluoracetic acid (TFA) 0.1% (v/v)-on a C-8 column. Although the range of mobile phase compositions investigated was quite narrow, the retention factor of this relatively small polypeptide (N/OFQ is a heptadecapeptide) has been found to change by more than 400%. In these conditions, gradient operation resulted thus to be the optimum approach for non-linear elution. As the available amount of N/OFQ was extremely reduced (only a few milligrams), the adsorption isotherms of the peptide, at the different mobile phase compositions examined, have been measured through the so-called inverse method (IM) on a 5 cm long column. The adsorption data at different mobile phase compositions have been fitted to several models of adsorption. The dependence of the isotherm parameters on the mobile phase composition was modeled by using the linear solvent strength (LSS) model and a generalized Langmuir isotherm that includes the mobile phase composition dependence. The overloaded gradient separation of N/OFQ has been modeled by numerically solving the equilibrium-dispersive (ED) model of chromatography under a selected gradient elution mode, on the basis of the previously determined generalized Langmuir isotherm. The agreement between theoretical calculations and experimental overloaded band profiles appeared reasonably accurate.     

梯度洗脱高效液相色谱法测定红花玉兰中4种植物激素

建立了梯度洗脱高效液相色谱法测定红花玉兰中赤霉素(GA3)、生长素(IAA)、脱落酸(ABA)和玉米素(ZT)等4种植物激素的方法。采用Agilent ZORBAX SB-C18柱和紫外检测器,以甲醇和0.1 mol/L乙酸作为流动相进行梯度洗脱,流速1 mL/min,进样量10μL。GA3,IAA和ABA的检测波长为254 nm,柱温35℃;ZT的检测波长为270 nm,柱温40℃。采用外标法进行定量测定,4种植物激素的相关系数均大于0.9990。4种激素的回收率为98.1%~125.2%,相对标准偏差为0.31%~0.92%,日内和日间精密度RSD均<10%。方法可适用于红花玉兰多种组织的植物激素测定,为红花玉兰生长发育特性的研究奠定了基础。     

Temperature selectivity in reversed-phase high performance liquid chromatography

Column temperature plays two important roles in reversed-phase high-performance liquid chromatography (RP-HPLC): control of retention (k) and control of selectivity (a). While changes in retention as a function of temperature are ubiquitous, selectivity changes for any given solute pair are more pronounced for ionized samples and samples with more polar substituents. With many samples, column temperature can be selected in a manner that optimizes resolution. The selectivity effects observed for temperature changes in RP-HPLC generally are complementary to those observed for mobile phase strength changes, so it is often possible to improve resolution by simultaneous optimization of temperature and mobile phase percent organic or gradient steepness. Computer simulation is a powerful tool for such optimization experiments. This paper reviews the influence of temperature on chromatographic selectivity for RP-HPLC.     

Simple algorithms for fitting and optimisation for multilinear gradient elution in reversed-phase liquid chromatography

The theory of the multilinear gradient elution in reversed-phase liquid chromatography (RP-HPLC) presented in [P. Nikitas, A. Pappa-Louisi, P. Agrafiotou, J. Chromatogr. A 1120 (2006) 299] is modified to increase its flexibility. In addition, it is embodied to simple algorithms for fitting gradient data and especially for resolution optimisation under multilinear gradient conditions. In particular, two new algorithms for fitting and one for optimisation are tested and compared with conventional algorithms. Their performance was examined using 13 o-phthalaldehyde (OPA) derivatives of amino acids with mobile phases modified by acetonitrile. It was found that the new proposed algorithms, a repeated application of the Levenberg-Marquardt (LM) method for fitting (R_LM) and a modified descent algorithm for optimisation (RND_D), in combination with the modified theory of the multilinear gradient elution can lead to high quality predictions of the retention times and optimisation results.