Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method. I. Injection procedure and column stability

We report a method for reliable routine polymer sample introduction with minimal bias, a separation method of the first six linear and cyclic oligomers by liquid chromatography, quantification using group equivalents and long term method performance. Injecting a polymer sample in a mobile phase containing an aqueous non-solvent often results in blocked systems as the polymer precipitates in the connecting capillaries. In this first part we focus on a new injection technique, in which the dissolved polyamide is placed between two zones of formic acid, preventing the polymer to precipitate before it reaches the column. Development of this sandwich injection method makes direct injection of the polymer into an aqueous acetonitrile gradient feasible. The oligomeric polyamide recovery of this technique, extraction, dissolution/precipitation and direct injection on a hexafluoro-isopropanol (HFIP) gradient were compared. With the sandwich injection method the polymer remains on the column, slowly changing the stationary phase. The influence of this on resolution and retention was studied. Column stability allows sixty injections before cleaning or replacing the column is necessary.     

On-column amperometric detection in capillary electrophoresis with an improved high-voltage electric field decoupler

The analysis of polyamide-6 oligomers and polymer is usually performed with expensive fluorinated alcohols like 2,2,2-trifluoroethanol (TFE) or 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). Formic acid is well known as a mobile phase additive to adjust pH in reversed-phase high-performance liquid chromatography. However, formic acid is seldom used as a modifier to perform gradient elution chromatography on octadecyl-modified silica-based columns. Here we demonstrate the determination of cyclic and linear polyamide-6 oligomers using formic acid as a modifier on an octadecyl-modified silica-based column. This column was shown to be stable for more than 5000 column volumes, even when a mobile phase of 65-95% formic acid in water at a flow of 1 ml/min is applied. With formic acid under the conditions used (65-95% formic acid in water) the oligomers are retained on the column, while the polymer does not precipitate. In comparison, during adsorption and separation with a HFIP gradient, precipitation of the polymer occurs. The implications of the different separation mechanisms, i.e., adsorption vs. precipitation chromatography are discussed. Loadability is shown to be much better with the formic acid system. However, with formic acid as a modifier UV detection below 250 nm is not feasible. The less sensitive evaporative light scattering detector is used to detect the polyamide oligomers in the formic acid phase. In addition it is shown that capillary zone electrophoresis (CZE) with UV-absorbance detection using HFIP is an attractive combination as HFIP is UV-transparent and CZE allows low modifier consumption.     

Methodology for porting retention prediction data from old to new columns and from conventional-scale to miniaturised ion chromatography systems

Several procedures are available for simulating and optimising separations in ion chromatography (IC), based on the application of retention models to an extensive database of analyte retention times on a wide range of columns. These procedures are subject to errors arising from batch-to-batch variability in the synthesis of stationary phases, or when using a column having a different diameter to that used when the database was acquired originally. Approaches are described in which the retention database can be recalibrated to accommodate changes in the stationary phase (ion-exchange selectivity coefficient and ion-exchange capacity) or in the column diameter which lead to changes in phase ratio. The entire database can be recalibrated for all analytes on a particular column by performing three isocratic separations with two analyte ions. The retention data so obtained are then used to derive a "porting" equation which is employed to generate the required simulated separation. Accurate prediction of retention times is demonstrated for both anions and cations on 2mm and 0.4mm diameter columns under elution conditions which consist of up to five sequential isocratic or linear gradient elution steps. The proposed approach gives average errors in retention time prediction of less than 3% and the correlation coefficient was 0.9849 between predicted and observed retention times for 344 data points comprising 33 anionic or cationic analytes, 5 column internal diameters and 8 complex elution profiles.     

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.     

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.     

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.     

Retention mechanism of poly(ethylene oxide) in reversed-phase and normal-phase liquid chromatography

The retention behavior of low- and high-molecular-mass poly(ethylene oxide) (PEO) in reversed-phase (RP) and normal-phase (NP) liquid chromatography was investigated. In RPLC using a C18 bonded silica stationary phase and an acetonitrile-water mixture mobile phase, the sorption process of PEO to the stationary phase showed deltaH(o) > 0 and deltaS(o) > 0. Therefore, PEO retention in RPLC separation is an energetically unfavorable, entropy-driven process, which results in an increase of PEO retention as the temperature increases. In addition, at the enthalpy-entropy compensation point the elution volume of PEO was very different from the column void volume. These observations are quite different from the RPLC retention behavior of many organic polymers. The peculiar retention behavior of PEO in RPLC separation can be understood in terms of the hydrophobic interaction of this class of typical amphiphilic compounds with the non-polar stationary phase, on the one hand, and with the aqueous mobile phase, on the other. The entropy gain due to the release of the solvated water molecules from the PEO chain and the stationary phase is believed to be responsible for the entropy-driven separation process. On the other hand, in NPLC using an amino-bonded silica stationary phase and an acetonitrile-water mixture mobile phase, PEO showed normal enthalpy-driven retention behavior: deltaH(o) < 0 and deltaS(o) < 0, with the retention decreasing with increasing temperature and PEO eluting near the column void volume at the enthalpy-entropy compensation point. Therefore, high-resolution temperature gradient NPLC separation of high-molecular-mass PEO samples can be achieved with relative ease. The molecular mass distribution of high-molecular-mass PEO was found to be much narrower than that measured by size-exclusion chromatography.     

Simple and Efficient Solution for Robustness Testing in Gradient Elution Liquid Chromatographic Methods

In this paper, an efficient way for robustness testing of gradient elution liquid chromatographic methods is proposed and tested on model mixtures comprising cilazapril, hydrochlorothiazide, and their degradation products, solutes that differ not only in polarities, but also in solubility and absorption characteristics. In general, the robustness could be tested with respect to various responses: resolution, retention factor, selectivity factor, change of detector response, etc. In chromatographic methods, the separation of the adjacent peaks is mandatory, and, consequently, the resolution is usually used as response. In isocratic elution methods, the resolution threshold depends on many factors, such as sizes of adjacent peaks, peak shapes, and asymmetry factor. At the same time, the situation is even more complex in gradient elution methods, because separation depends on a larger number of parameters, such as gradient profile, column geometry, mobile phase flow rate, column equilibration between gradient runs, etc. To ensure baseline separation, the authors propose application of separation criterion (s) as response and indirect modeling in the robustness evaluation. Examined response in this approach is represented by the difference between the retention time of the beginning of the peak and the retention time of the end of the previously eluting peak of the critical pair. Moreover, the proposed methodology included reusing experiments from the optimization phase to define a robust chromatographic region without increasing the number of experiments. It was shown that method robustness can be easily and efficiently evaluated by this methodology.     

The prediction of the peak width at half height in HPLC

The prediction of the peak width at half height is an important aspect in the optimization of the chromatographic operating conditions. In this paper, a linear relationship, between the peak widths at half height and the retention values with various isocratic elution is observed. In gradient elution, however, the relationship between the peak widths at half height and the so-called invented retention values that correspond to the mobile phase composition by eluting the solute from the column end is developed. We believe that there is almost the same band width at half height inside the column (in unit of length) for different solutes. The peak width at half height in the chromatogram (in unit of time) is mainly determined by the capacity factor of the solute when it is eluted from the column end. The larger the capacity factor of a solute eluted from the column end, the more slowly will be the solute eluted from the column end and the wider will be the peak width at half height. It is possible to predict the peak width at half height in various isocratic and gradient elutions by using this linear relationship.     

Separation of nucleotides by hydrophilic interaction chromatography using the FRULIC-N column

A stationary phase composed of silica-bonded cyclofructan 6 (FRULIC-N) was evaluated for the separation of four cyclic nucleotides, six nucleoside monophosphates, four nucleoside diphosphates, and five nucleoside triphosphates via hydrophilic interaction chromatography (HILIC) in both isocratic and gradient conditions. The gradient conditions gave significantly better separations by narrowing peak widths. Sixteen out of nineteen nucleotides were baseline separated on the FRULIC-N column in one run. Unlike other known HILIC stationary phases, there can be dual-retention mechanisms unique to this media. Traditional hydrogen bonding/dipolar interactions can be supplemented by dynamic ion interaction effects for anionic analytes. This occurs because the FRULIC-N stationary phase is able to bind certain buffer cations. The extent of the ion interaction is tunable, in comparison to stationary phases with embedded charged groups, where the inherent ionic properties are fixed. The best mobile phase conditions were determined by varying the organic modifier (acetonitrile) content, as well as salt type/concentration and electrolyte pH. The thermodynamic characteristic of the FRULIC-N column was investigated by evaluating the column temperature effect on retention and utilizing van’t Hoff plots. This study shows that there is a greater entropic contribution for the retention of nucleotide di and triphosphates, whereas there is a greater enthalphic contribution for the cyclic nucleotides with the FRULIC-N column.     

反相高效液相三元流动相台阶梯度分离条件的快速优化方法

 根据溶质在柱内的迁移规律 ,建立了一种利用线性梯度实验快速获得溶质保留值方程系数 ,然后以串行响应函数为优化指标进行多台阶梯度分离条件优化的方法。与利用等度实验获得保留值方程的方法相比 ,该法可以大大缩短优化时间。通过该方法对芳香胺和衍生化氨基酸样品进行了分离 ,获得了满意的分离度 ,表明该方法的预测精度很好。     

Reversed-phase high-performance liquid chromatographic separation of molecular species of alkyl ether, vinyl ether, and monoacyl lysophospholipids

An isocratic reversed-phase high-performance liquid chromatographic method was developed which resolved individual molecular species of choline and ethanolamine lysophospholipids utilizing a C13 bonded porous silica stationary phase with a mobile phase comprised of methanol--water--acetonitrile (57:23:20) containing 20 mM choline chloride. Solute retention was primarily determined by hydrophobic interactions with the stationary phase permitting separation of individual molecular species of lysophospholipids according to the composition of the aliphatic chain and the nature of its covalent attachment to the sn-1 hydroxyl group. The elution profile of unsaturated monoacyllysophospholipid or lysoplasmalogen molecular species was readily obtained by measuring UV absorbance at 203 nm. Identification of column eluates containing saturated monoacyl and alkyl ether lysophospholipids was possible utilizing relative retention factors that were obtained for the majority of molecular species present in animal tissues.     

Prediction of peptide retention times in normal-phase liquid chromatography with only a single gradient run.

Previous studies of peptide separation by normal-phase liquid chromatography have shown a linear relationship between the logarithm of the capacity factor and the logarithm of the volume fraction of modifier in the mobile phase. This permitted the use of a model to predict isocratic and gradient retention times based on data obtained by two initial gradient runs. In the present study, chromatographic behavior of 25 peptides in normal-phase liquid chromatography with isocratic elution have been studied and a linear relationship between the slope (S) and intercept [log k(0)] was obtained. This relationship was combined with the algorithm of prediction reported in the previous paper. The prediction of peptide retention times with only a single experimental gradient retention data was investigated.     

Stepwise gradient of buffer concentration for capillary electrochromatography of peptides on sulfonated naphthalimido-modified silyl silica gel

The advantage of using a stepwise gradient of buffer concentration in CEC was demonstrated with the mixed-mode stationary phase, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP). Before the application of a stepwise gradient, the effect of buffer concentration on the separations of six peptides and tryptic digests was investigated. Bubble formation caused by Joule heating at currents up to 95 microA was successfully suppressed by using SNAIP column even without pressurization, which contributed to a stepwise gradient of buffer concentration. Utilizing the stepwise gradient improved and shortened the separation of six peptides as compared to the separation under an isocratic elution.     

Development of an ion chromatographic gradient retention model from isocratic elution experiments

When facing separation problems in ion chromatography, chromatographers often lack guidelines to decide a priori if isocratic elution will give enough separation in a reasonable analysis time or a gradient elution will be required. This situation may be solved by the prediction of retention in gradient elution mode by using isocratic experimental data. This work describes the development of an ion chromatographic gradient elution retention model for fluoride, chloride, nitrite, bromide, nitrate, sulfate and phosphate by using isocratic experimental data. The isocratic elution retention model was developed by applying a polynomial relation between the logarithm of the retention factor and logarithm of the concentration of competing ions; the gradient elution retention model was based on the stepwise numerical integration of the corresponding differential equation. It was shown that the developed gradient elution retention model was not significantly affected by transferring data form isocratic experiment. The root mean squared prediction error for gradient elution retention model was between 0.0863 for fluoride and 0.7027 for bromide proving a very good predictive ability of developed gradient elution retention model.     

Relationship between isocratic and gradient retention times in the high-performance ion-exchange chromatography of proteins. Theory and experiment

Using isocratic retention parameters, the gradient elution retention time for several proteins has been calculated. The gradient retention time calculation is based on fitting the isocratic retention data to an equation of the form: log k' = m log (1/[Ca2+]) + log K and on applying well-established principles of gradient elution. A good correlation between the observed and calculated retention times for several test proteins was obtained at various total gradient times and column flow-rates. Conversely, isocratic retention parameters characterizing protein retention can be calculated from gradient elution retention data. However, even with retention data of high quality, small errors are amplified by the log-log nature of the ion-exchange isocratic retention model employed. Based on the close correlation between predicted and observed gradient retention times, no evidence for protein denaturation resulting from immobilization of the protein at high initial k' values at or near the column inlet was observed.     

Peak capacity in gradient reversed-phase liquid chromatography of biopolymers. Theoretical and practical implications for the separation of oligonucleotides

Reversed-phase ultra-performance liquid chromatography was used for biopolymer separations in isocratic and gradient mode. The gradient elution mode was employed to estimate the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.1 mm I.D. columns packed with a porous 1.7 microm C18 sorbent is below 0.2 mL/min for our analytes. However, the maximum peak capacity is achieved at flow rates between 0.15 and 1.0 mL/min, depending on the molecular weight of the analyte. The isocratic separation mode was utilized to measure the dependence of the retention factor on the mobile phase composition. Constants derived from isocratic experiments were utilized in a mathematical model based on gradient theory. Column peak capacity was predicted as a function of flow rate, gradient slope and column length. Predicted peak capacity trends were compared to experimental results.     

Controlling the retention in capillary LC with solvents, temperature, and electric fields

Once a suitable stationary phase and column dimensions have been selected, the retention in liquid chromatography (LC) is traditionally adjusted by controlling the mobile phase composition. Solvent gradients enable achievement of good separation selectivity while decreasing the separation time as compared to isocratic elution. Capillary columns allow use of other programming parameters, i.e. temperature and applied electric fields, in addition to solvent gradient elution. This paper presents a review of programmed separation techniques in miniaturized LC, including retention modeling and method transfer from the conventional to micro- and capillary scales. The impact of miniaturized instrumentation on retention and the limitations of capillary LC are discussed. Special attention is focused on the gradient dwell volume effects, which are more important in micro-LC techniques than in conventional analytical LC and may cause significant increase in the time of analysis, unless special instrumentation and (or) pre-column flow-splitting is used. The influence of temperature upon retention is also discussed, and applications where the temperature has been actively used for retention control in capillary LC are included together with the instrumentation utilized. Finally the possibilities of additional selectivity control by applying an electric field over a packed capillary LC column are discussed.     

Signal enhancement by on-column fluorimetric detection in gradient elution of dansyl amino acids in liquid chromatography

Summary Fluorimetric detection in the presence of a stationary phase has been applied to gradient elution of dansyl amino acids in liquid chromatography. A 1.5 mm ID quartz tube packed with the same materials as the separation column was employed for the flow cell. Conventional-size columns were employed. The peak height of analytes increased with increasing retention owing to focusing and environmental effects of the stationary phase, leading to improvements in sensitivity, which was pronounced for analytes eluting late. The lower the gradient, the larger the improvement in sensitivity achieved. Detection limits were improved by a factor of up to 5.1 by fluorimetric detection using the packed flow cell, compared with those achieved using a common empty flow cell.