Retention time and peak width in the combined pH/organic modifier gradient high performance liquid chromatography

The purpose of this work was to test the applicability of the current theory to predict the peak retention time and the peak width in the combined pH/organic modifier gradient reversed phase high performance liquid chromatography (RP HPLC). A series of 38 isocratic measurements have been conducted for a wide range of pH and methanol contents for ketoprofen (weak acid) and papaverine (weak base). It served to find the model describing dependence of retention factor and the height equivalent of a theoretical plate (HETP) on pH and organic modifier content. The information gathered in the isocratic mode was used to simulate retention times and peak widths for 30 various methanol gradients, 25 pH gradients, and 3 combined pH/methanol gradients. The simulations were compared with the experimental data. We also proposed a simplified version of this model that was parameterized based on 12 initial organic modifier gradients carried out for different pHs and for the 20 min and 60 min gradient development times. The full and the simplified model described the experimental data very well. In conclusion, the proposed modeling approach allowed predicting analyte retention times and peak width for various pH and organic modifier changes. Its simplified version required only 12 initial experiments and seems to be very promising in the optimization RP HPLC separations for complex samples and for conditions providing peak compression.     

Exact peak compression factor in linear gradient elution. I. Theory

The only existing expression for the peak compression factor in linear gradient elution chromatography assumes that the linear-solvent-strength model (LSSM) applies to the retention of the compound studied, that the column efficiency is independent of the mobile phase composition, and that, during gradient elution, the relative retention factor of a compound inside its band varies linearly with the distance from the band center. Because the retention factors of many analytes in reversed-phase liquid chromatography do not rigorously follow the LSSM, we extend the theoretical approach of Poppe et al. to the prediction of peak compression factors in linear gradient elution chromatography for any retention model, when column efficiency varies with the mobile phase composition. Only the contribution of the chromatographic column to the peak compression was taken into account, the contribution of the dwell volume being neglected. A second restriction is the linearity of the relative retention factor as a function of the position along the band width inside the column. These constraints could be the sources for the difference observed between experimental and theoretical values of peak compression factors. When the retention factor varies steeply with the mobile phase composition, such as with proteins or large peptides in RP-HPLC, it is found that the thermodynamic compression term, which tends to sharpen the peak, is coupled with the column dispersion term, which tends to broaden the peak. This coupling term acts as an apparent dispersion term, contributing to broaden the peak. This result is consistent with the measurements of peak compression factors found in the literature.     

Reversed-phase chromatography of proteins with diphenyl-silica column and hydro-organic eluents containing two organic solvents

High-performance liquid chromatography of proteins with silica bonded diphenyl stationary phase and hydro-organic mobile phases with linear gradient elution has been carried out with binary organic modifiers. The use of a mixture of 2-propanol and 1-butanol facilitated elution at total organic modifier concentrations significantly lower than with the use of 2-propanol alone. Furthermore higher protein recovery and retention of biological activity was obtained with the binary organic modifier because the increase in eluent strength with binary organic modifier was greater than that of the denaturing strength of the eluent. The use of a short, 3 cm long, column, relatively high flow-rates and steep gradients was also advantageous in attenuating protein denaturation. The results were interpreted within the framework of a theoretical treatment of the combined effect of the retention process and denaturation reaction simultaneously occurring in the column.     

Improvement of an overloaded, multi-component, solvent gradient bioseparation through multiobjective optimization

Solvent gradient chromatography is quite often used in analytical studies for decreasing the analysis time of samples having components with widely different retention behaviour. Several studies, both theoretical and experimental, have been reported on the optimization of gradient profiles in improving analytical separation performance, suggesting various linear and non-linear gradients. In preparative chromatography, on the other hand, though solvent gradient is being increasingly used (especially in bioseparation) to improve the product yield and productivity, there is a dearth of literature and clearer understanding of the effect(s) of modifier gradients on the separation performance. For this, the gradients used in applications are of relatively simple profiles like step or linear gradients, obtained through hand optimization based on experience and intuition. Significant improvements, however, can be expected using the state-of-the art modelling of chromatographic processes and optimization routines running on widely available hi-speed desktop computers. In this work we are reporting such an optimization procedure to improve the purification of an industrial multi-component mixture, containing 65.8% of Calcitonin as the main product, in an overloaded reversed-phase column. The work comprises both theoretical simulations and their experimental validation using multilinear gradients as optimization variable. The study produced interesting insights for modifier gradient design, like using peak deformation of the target peptide to increase yield and productivity, and improved our understanding of the effect of modifier gradients in non-linear separations.     

电色谱中中性溶质柱内富集研究

中性溶质在毛细管电色谱中的富集可以通过调节有机调节剂在运行缓冲溶液和进样区段中的浓度及适当增加进样长度业实现,富集作用主要由两种过程控制,即进样过程中的自富集作用和运行过程中的一般输运富集作用,采用弛豫理论的研究方法得到了描述富集效果与操作条件关系的理论表达式,结果表明：随进样长度的增加,可以有效提高富集效果;进样长度对柱效的影响也与有机调节剂的浓度有关,当其在两区段中的浓度差别较大时,适当加长进样时间并不会对柱效产生太大的影响,对安息香和美芬妥因两种药物的实验研究达到了超过100倍的富集效果。     

Application of silica-based hyper-crosslinked sulfonate-modified reversed stationary phases for separating highly hydrophilic basic compounds

The separation and determination of hydrophilic basic compounds are of great importance in many fields including clinical and biological research, pharmaceutical development and forensic analysis. However, the most widely used analytical separation technique in these disciplines, reversed-phase liquid chromatography (RPLC), usually does not provide sufficient retention for several important classes of highly hydrophilic basic compounds including catecholamines, many drug metabolites and many drugs of abuse. Commonly eluents having little or no organic modifier and/or strong ion pairing agents must be used to achieve sufficient retention and separation. Use of highly aqueous eluents can lead to column failure by dewetting, resulting in poor retention, low selectivity and irreproducibility and slow recovery of performance. The use of a strong ion pairing agent to increase retention renders the separation incompatible with mass spectrometric detection and complicates preparative separations. This paper describes the successful applications of a novel type of silica-based, hyper-crosslinked, sulfonate-modified reversed stationary phase, denoted as (-)SO(3)-HC-C(8)-L, for the separation of highly hydrophilic cations and related compounds by a hydrophobically assisted cation-exchange mechanism. Compared to conventional reversed-phases, the (-)SO(3)-HC-C(8)-L phase showed significantly improved retention and separation selectivity for hydrophilic amines. Concurrently, due to the presence of both cation-exchange and reversed-phase retention mechanisms and the high acid stability of hyper-crosslinked phases, the separation can be optimized by changing the type or concentration of ionic additive or organic modifier, and by varying the column temperature. In addition, gradients generated by programming the concentration of either the ionic additive or the organic modifier can be applied to reduce the analysis time without compromising resolution. Furthermore, remarkably different chromatographic selectivities, especially toward cationic solutes, were observed upon comparing the (-)SO(3)-HC-C(8)-L phase with conventional reversed-phases. We believe that the combination of these two types of stationary phases will be very useful in two-dimensional liquid chromatography.     

Highly efficient monolithic silica capillary columns modified with poly(acrylic acid) for hydrophilic interaction chromatography

Monolithic silica capillary columns for hydrophilic interaction liquid chromatography (HILIC) were prepared by on-column polymerization of acrylic acid on monolithic silica in a fused silica capillary modified with anchor groups. The products maintained the high permeability (K=5 x 10(-14)m(2)) and provided a plate height (H) of less than 10 microm at optimum linear velocity (u) and H below 20 microm at u=6mm/s for polar solutes including nucleosides and carbohydrates. The HILIC mode monolithic silica capillary column was able to produce 10000 theoretical plates (N) with column dead time (t(0)) of 20s at a pressure drop of 20 MPa or lower. The total performance was much higher than conventional particle-packed HILIC columns currently available. The gradient separations of peptides by a capillary LC-electrospray mass spectrometry system resulted in very different retention selectivity between reversed-phase mode separations and the HILIC mode separations with a peak capacity of ca. 100 in a 10 min gradient time in either mode. The high performance observed with the monolithic silica capillary column modified with poly(acrylic acid) suggests that the HILIC mode can be an alternative to the reversed-phase mode for a wide range of compounds, especially for those of high polarity in isocratic as well as gradient elution.     

The bandwidth in gradient elution chromatography with a retained organic modifier

The variance of a chromatographic band is derived in the case of RPLC gradient elution when the organic modifier is significantly retained onto the stationary phase. This derivation is based on the extension of a model due to Poppe et al. [H. Poppe, J. Paanakker, M. Bronckhorst, J. Chromatogr., 204 (1981) 77] which assumes that the gradient front remains unchanged and propagates along the column at the same speed as the mobile phase, following piston flow. Theoretical and experimental results are compared in the case of caffeine on a C(1)-silica stationary phase eluted with an acetonitrile gradient. The actual retention behaviors of caffeine and acetonitrile were implemented in the theoretical calculations. The model predicts compression factors between 0.71 and 0.34 for relatively smooth gradient steepness, betat(0), between 0.009 and 0.054 while the corresponding experimental band compression factors vary between 1.01 and 0.43 for the very same gradient steepness. The model underestimation of these factors arises likely from the strong deviation of the actual retention behavior from the prediction of the Linear Solvent Strength Model (LSSM).     

Influence of mobile phase composition on electroosmotic flow velocity, solute retention and column efficiency in open-tubular reversed-phase capillary electrochromatography

The effects of some experimental parameters, such as the volume fraction and type of organic modifier in the mobile phase, and the concentration, type and pH of the buffer on the electroosmotic flow velocity, the retention behavior of test solutes, and the column efficiency have been investigated in capillary electrochromatography (CEC) using an open-tubular column of 9.60 microm I.D. with a porous silica layer chemically modified with C18 as stationary phase. The retention of a group of polycyclic aromatic hydrocarbons (PAHs) used as a test mixture varied significantly by changing the organic modifier content in the hydroorganic mobile phase according to the reversed-phase-like selectivity of the stationary phase. In addition, an increase in the percentage of organic modifier resulted in a slight increase in the linear velocity of the EOF. On the other hand, when the phosphate buffer concentration was increased over the range 1-50 mM, the electroosmotic mobility fell dramatically, the retention of the solutes decreased steadily, and the plate height showed a significant increase. The results obtained with phosphate, trishydroxymethylaminomethane or 2-morpholinoethanesulfonic acid as buffers were similar when pH remained constant. Optimization in CEC was essential to achieve further enhancement of separation performance, because the analysis time and separation resolution are essentially affected when varying operating parameters. Separations of seven PAHs with more than 100000 plates are presented within 4 min analysis time.     

Effects of pH in reversed-phase liquid chromatography

The effects of concomitant variations in pH and organic modifier concentration on retention, efficiency and peak symmetry are considered for reversed-phase liquid chromatography (RPLC) on octadecyl-modified silica (ODS) columns. A number of factors are discussed, which make the systematic exploitation of pH effects in RPLC more complicated than the optimization of solvent composition. If the pH is varied, a second factor (usually the concentration of organic modifier) will need to be varied simultaneously to maintain retention (capacity factors) in the optimum range. When pH is considered as a parameter in RPLC, not only its effects on retention, but also the variations in efficiency (plate count) and peak shape (asymmetry) need to be considered. These parameters turn out to vary drastically between individual solutes and between different experimental conditions. The results of a study involving a number of acidic, basic and neutral solutes, two different ODS columns and mixtures of either methanol or acetonitrile with aqueous buffers are reported. In the earlier part of the study, using methanol as the organic modifier, reproducible data for retention, peak width and peak symmetry were obtained and these data are reported. In the later part of the study, using acetonitrile, a gradual change in retention as a function of time was observed, this effect coinciding with a decrease in column efficiency. It is concluded that ODS columns are subject to considerable degradation during studies in which the pH is varied. Although this effect can be described mathematically, the preferred solution is thought to be the use of pH-stable columns.     

Application of retention modelling to the simulation of separation of organic anions in suppressed ion chromatography

The ion-exchange separation of organic anions of varying molecular mass has been demonstrated using ion chromatography with isocratic, gradient and multi-step eluent profiles on commercially available columns with UV detection. A retention model derived previously for inorganic ions and based solely on electrostatic interactions between the analytes and the stationary phase was applied. This model was found to accurately describe the observed elution of all the anions under isocratic, gradient and multi-step eluent conditions. Hydrophobic interactions, although likely to be present to varying degrees, did not limit the applicability of the ion-exchange retention model. Various instrumental configurations were investigated to overcome problems associated with the use of organic modifiers in the eluent which caused compatibility issues with the electrolytically derived, and subsequently suppressed, eluent. The preferred configuration allowed the organic modifier stream to bypass the eluent generator, followed by subsequent mixing before entering the injection valve and column. Accurate elution prediction was achieved even when using 5-step eluent profiles with errors in retention time generally being less than 1% relative standard deviation (RSD) and all being less than 5% RSD. Peak widths for linear gradient separations were also modelled and showed good agreement with experimentally determined values.     

Adsorption behavior of a teicoplanin aglycone bonded stationary phase under harsh overload conditions

Silica-bonded teicoplanin aglycone allows enantioseparation of amino acids by reversed-phase liquid chromatography with a low organic solvent content. However, a reversible change in the adsorption behavior leading to a retention time shift (RTS) was observed when a preparative scale column was treated with harsh preparative chromatography-like conditions between finite-injection HPLC runs conducted under exactly the same conditions. This behavior was observed for all five investigated aliphatic and aromatic amino acids. In all cases, the retention times were prolonged after the overload conditions and the RTS was more pronounced for the later eluting d-enantiomer. We defined a standardized method for measuring the RTS and performed a systematic investigation on the influence of experimental conditions (type and concentration of pH modifier and organic modifier, temperature, pH) on the RTS. In this way a solvent composition--90/10 50 mM NH4Ac pH 5.8/MeOH--was identified that yielded no observable shift in retention time after overload conditions for both enantiomers. In order to treat the observed phenomenon on a mechanistic level, we applied band profile analysis based on the stochastic theory of chromatography and identified two different enantioselective sites. When the band profile analysis was performed on elution profiles obtained from runs with prolonged retention time after harsh overload conditions, the retention time shift could be attributed to both differentiable types of adsorption sites. One site was found to make both, enantioselective and non-selective contributions.     

Void volume determination and experimental probes in reversed phase chromatography

Summary In reversed-phase liquid chromatography with n-alkyl bonded silica, the dead volume (V₀) of the column is theoretically indeterminate owing to adsorption of organic modifier on n-alkyl chains and of water on silanol groups. With binary mobile phases, retention volumes of the mobile phase components and of their deuterated species are relaeed to the adsorption isotherms and V₀ by equations which can be solved with some assumptions on the adsorbed layer composition. Methanol-water and acetonitrile-water systems are studied. As the experimental excess isotherm shows a linear part in the concentration range 50–80% in organic modifier, the hypothesis of an adsorbed layer of constant composition in this range is possible. When increasing the water content of the mobile phase, adsorption of water occurs up to saturation of silanol groups. Then the assumption of a constant water content for a mobile phase having more than 50% of water is applied. With the hypothesis of a constant adsorbed content of organic modifier when the eluent has more than 80% of organic modifier, V₀ and the absolute isotherms are calculated over the entire range of mobile phase composition. Experimental retention behavior of the mobile phase components are totally explained by these V₀ determinations. The retention times of commonly used V₀ markers are compared with V₀ values. It is shown that, when buffering the eluent, no visible effect on the distribution equilibrium is observed, so that injection of concentrated potassium nitrate is a convenient method to measure V₀. With a few solutes with are UV detectable it is possible to measure V₀ whatever the mobile phase composition in methanol-water and acetonitrile-water systems.     

Influence of the mobile phase on salmon calcitonin analysis by reversed-phase liquid chromatography

The retention properties of calcitonins on a reversed-phase column are examined using salmon calcitonin as the model compound. The effect of the concentration of organic modifier, buffer strength, pH of the mobile phase, and ion-pair reagent are studied. In the absence of an ionic modifier in the eluent the calcitonin peak shapes are not symmetrical. The addition of 0.1% trifluoroacetic acid (TFA), however, results in good peak characteristics without the need to add nonvolatile salts. The retention of the calcitonins was found to be very sensitive to the concentration of the organic modifier (acetonitrile) present in the mobile phase. A change of pH between 2 and 5 has only a slight effect of the k' of salmon calcitonin, but the k' increases significantly at higher pH values. The addition of a phosphate buffer to the mobile phase and an increase in the buffer concentration (0-0.2 M) causes a decrease in the retention of salmon calcitonin. Evidence shows that reproducible, quantitatively measurable data can be obtained using reversed-phase chromatography if the ion-pairing reagent and organic modifier concentrations are carefully controlled. The system also shows a good selectivity for the calcitonin series. Therefore, both highly selective methods (qualitative) as well as quantitative methods for analytical, pharmaceutical, and manufacturing use can be developed by adjusting the high-performance liquid chromatography (HPLC) conditions as discussed.     

Development of dual gradient column in liquid chromatography

The dual gradient column, in which both the chemical property of the stationary phase and the flow velocity in the mobile phase are heterogeneous longitudinally along the column, is developed to obtain the mobile phase gradient-like elution in an isocratic condition. Here, the step-wise dual gradient columns were prepared by connecting an inlet column (I.D. 50 microm, packed with ODS) serially to an outlet column (I.D. 100-200 microm, packed with the mixture of ODS and C1 [9:1]). The retention behavior of alkylbenzenes was able to be controlled in the dual gradient column depending on the variation in the flow velocity. Moreover, the change in retention behavior induced by the flow velocity variation for the dual gradient columns was quite different from that by the variation in organic modifier content of the mobile phase in isocratic elution for a single gradient column and can induce the similar effect with an ordinary gradient elution in a mobile phase composition.     

Packed column supercritical fluid chromatographic separation and estimation of acetaminophen, diclofenac sodium and methocarbamol in pharmaceutical dosage forms

A reproducible and fast method has been developed for the assay of acetaminophen, methocarbamol, and diclofenac sodium in bulk and drug forms using packed column supercritical fluid chromatography employing internal standard method. The analytes were resolved by elution with supercritical fluid carbon dioxide doped with 11.1% (v/v) methanol on a Shendon-Phenyl (250x4.6 mm) 5 mum column with detection monitored spectrophotometrically at 225 nm. The densities and polarities of the mobile phase were optimised from the effects of pressure, temperature and modifier concentration on chromatograhic figures like retention time (t(R), min), retention factor (k(')) etc. Modifier concentration proved to be the most effective means for changing both retention and selectivity. Calibration data and recovery of the drug from spiked concentrations were determined to assess the viability of the method. The supercritical fluid chromatography (SFC) method was directly compared to an HPLC assay, developed in the laboratory, of the same analytes. With respect to speed and use of organic solvents SFC was found to be superior, while in all other aspects the results were similar to HPLC. The method has been successfully used for the assay of two formulations containing a combination of (A) acetaminophen and methocarbamol and (B) acetaminophen and diclofenac sodium. There was no interference from excipients. The present work validates the recent proposition that supercritical fluid chromatography using CO(2) and modifiers is a viable, faster alternative to reverse phase HPLC.     

Effect of Column Dimensions on HPLC Separations Using Constant Volume Columns

Abstract

The effect of column dimension on resolution, sample capacity, retention time, efficiency and mobile phase composition were studied, using both constant flow rate and constant linear velocity. The four columns selected (A = 238 × 3.2 mm, B = 153 × 4.0 mm, C = 116 × 4.6 mm and D = 50 × 7 mm) had the same volume. K¹ values were found to be constant, within experimental error, for all columns. At constant linear velocity, the retention time was found to be a linear function of column length, while at constant flow rate retention time was constant for all columns. The longest column (A) generated the largest N values while columns 3 and C gave the lowest H values, for dilute solutions, while they decreased with decreasing column length. On the other hand, it was observed that as the sample size increased, N generated by column A decreased more rapidly and eventually fell below the values generated by columns B and C. These two columns (B & C) can tolerate a larger sample size with less reduction in N value than the longest column. It is important to note that although there were minor differences in performance between columns B and C, there were significant differences between them (B and C) and the other two columns (A and D). Column A offered the highest sensitivity (narrower peaks) for dilute solutions, while columns B and C offered higher loadability. The volume of organic modifier in the mobile phase affected the retention equally in the four columns. It was also found that equal separation (a) was obtained for each column at constant flow rate and constant linear velocity, except with the latter the retention times were longer.     

Fast Separation of Nucleosides by Capillary Electrochromatography on Non‐Endcapped Phenyl‐Bonded Silica Phase Using Short‐End Injection Method

The influence of several experimental parameters (pH, ionic strength, organic modifier content of hydro‐organic buffer) upon EOF, migration time, and retention factor has been studied in CEC with a phenyl‐bonded silica column on a model mixture of five nucleosides. This paper illustrates the current interest in CEC as a method of resolving complex mixtures of neutral and ionic solutes and demonstrates the potential of the short‐end injection method as a means of reducing analysis time.     

Packed column supercritical fluid chromatography with light-scattering detection. II. Retention behaviour of squalane and glucose with mixed mobile phases

Summary Evaporative light scattering detectors can be used to detect organic substances without chromophoric groups in packed column supercritical fluid chromatography (SFC). A detector of this type has been used to detect squalane and glucose after SFC with various packed columns and binary mobile phases. In this study, the amount of organic modifier in carbon dioxide/modifier mixtures was varied. The results give further insight into the mechanisms that influence retention behaviour in packed column separations with super- and subcritical mobile phases. Squalane is an ideal non-polar test solute which shows long retention times on non-polar columns while its elution can be accelerated by non-polar modifiers in carbon dioxide. Glucose is an extremely polar solute containing hydroxyl groups. Elution of this sugar can be improved with polar modifiers. Column packings with polar end groups lead to high capacity ratios and long retention times for glucose. Most columns used in this study contained silica-based packing materials. For purposes of comparison, a polymeric packing (HEMA RP-18) was also employed.