The HPLC Resolution of Nucleic‐Acid Bases and An a Log Hypoxanthine on R,S‐2‐Hydroxypropyl Derivatized β‐Cyclodextrin Bonded Chiral Stationary Phase Using a Water‐Based Mobile Phase

A HPLC approach using R,S‐2‐hydroxypropyl derivatized β‐cyclodextrin packed column as the stationary phase was developed to resolve five nucleic‐acid bases and an a log hypoxanthine in the reversed‐phase mode. These bases are not only similar in structure but also very close in basicity. However, the resolution can be completed in less than ten minutes and is considered to be better carried out on the R,S‐2‐hydroxypropyl derivatized β‐cyclodextrin phase than that obtained on the native β‐cyclodextrin phase under the same chromatographic conditions. The mechanism involved in the resolution is believed to be inclusion complexation between the analyte and the cavity of cyclodextrin in the reversed‐phase mode. The retention time was found relevant to the size of the analyte. The number of groups on analyte that is available to form hydrogen bonding with hydroxyl groups on CDs also affects the retention scale. Factors of introducing organic acid and base or organic modifier such as methanol to the water‐based mobile phase or increasing their percent ages in the mobile phase decreases the retention time without de grading the resolution significantly.     

Evaluation of the analytical method performance for incurred samples

A methodology for the evaluation of the performance of an analytical method for incurred samples is presented. Since this methodology is based on intra-laboratory information, it is suitable for analytical fields that lack reference materials with incurred analytes and it can be used to evaluate the analytical steps prior to the analytical portion, which are usually excluded in proficiency tests or at the certification of reference materials. This methodology can be based on tests performed on routine samples allowing the collection of information on the more relevant combinations analyte/matrix; therefore, this approach is particularly useful for analytical fields that involve a high number of analyte/matrix combinations, which are difficult to cover even considering the frequent participation in expensive proficiency tests.This approach is based on the development of a model of the performance of the analytical method based on the differential approach for the quantification of measurement uncertainty and on the comparison of recovery associated with each one of the analytical steps whose performance can vary with the analyte origin, for spiked and incurred samples.This approach was applied to the determination of pesticide residues in apples. For the analytes covered, no evidence was found that the studied sample processing and extraction steps performance for this matrix varies with the analyte origins.     

Predicting the chromatographic retention of polymers: poly(methyl methacrylate)s and polyacryate blends

The suitability of a retention model especially designed for polymers is investigated to describe and predict the chromatographic retention behavior of poly(methyl methacrylate)s as a function of mobile phase composition and gradient steepness. It is found that three simple yet rationally chosen chromatographic experiments suffice to extract the analyte specific model parameters necessary to calculate the retention volumes. This allows predicting accurate retention volumes based on a minimum number of initial experiments. Therefore, methods for polymer separations can be developed in relatively short time. The suitability of the virtual chromatography approach to predict the separation of polymer blend is demonstrated for the first time using a blend of different polyacrylates.     

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.     

Reversed-phase high-performance liquid chromatography of ionogenic compounds: comparison of retention models

Two kinds of retention models describing a behaviour of ionogenic substances in reversed-phase chromatographic systems were compared. Model A utilises a concept of limiting retention factors and is especially suitable for the prediction of retention of compounds co-existing in several forms in mobile phase. An effect of the concentration of organic modifier (e.g., methanol) on the magnitudes of the limiting retention factors and equilibrium constants (dissociation constants of the separated substances) can be expressed with the aid of various, more or less sophisticated, relationships. A stoichiometric displacement model (model B) in its original form simply relates the analyte retention to the content of organic modifier in the mobile phase. In this work, it was modified to also express an effect of the mobile phase pH introducing side equilibria (acid-base) into the model. Both models predict a sigmoidal dependence of the analyte retention factor on the mobile phase pH in accordance with experimental data, and allow, among others, to estimate dissociation constants from those data. Experimental dependencies between the analyte retention and the concentration of methanol in the mobile phase comply well with model A, whereas the stoichiometric displacement model could be used only in a limited range of the methanol concentrations.     

A physical approach to the interpretation of the mechanisms and kinetics of analyte release in electrothermal atomic absorption spectrometry

The first part of this review is devoted to a substantiation of the physical approach to the interpretation of the exponential dependence of the rate of heterogeneous reactions on temperature proposed by Hertz and Langmuir, and developed by the author as an alternative to the traditional chemical approach based on the Arrhenius concept of the activation effect. The second part of this work is devoted to the application of this approach to the identification of the mechanisms of analyte release in electrothermal atomic absorption spectrometry. The most important results of this application, which support advantages of the physical approach over the traditional chemical approach, are the following: a method of theoretical calculation of the absolute rates for solid-state decomposition reactions has been developed; some unusual peculiarities in kinetics of analyte atomization (double values of the E-parameter and high values of the pre-exponential factor in the Arrhenius equation) were interpreted; the mechanism of low-temperature migration of analyte in graphite tubes at pyrolysis temperatures and, in particular, analyte migration onto a palladium modifier was explained; and two simple criteria have been proposed for the identification of the true mechanism of analyte release as either bulk evaporation/desorption alternative and on this basis the mechanism of analyte retention on a Pd modifier has been interpreted. It has been concluded that the application of the chemical approach, based on the Arrhenius concept of the activation effect to the kinetics of heterogeneous reactions, was wrong, and has resulted in a stagnation of theory over many years.     

Retention profiles and mechanism of anion separation on latex-based pellicular ion exchanger in ion chromatography

A retention model based on stoichiometric approach has been developed in order to describe analyte retention of anions on latex-based pellicular ion exchanger. The chromatographic process entails two stepwise and complex equilibria, first is ion-pair forming of analyte or eluent ion with ion-exchange sites under the effect of electrostatic forces due to the sulfonic layer behind the aminated functional groups of stationary phase. Second component is the ion-exchange between the analyte and eluent ions. As a new parameter of the fractional electrostatic coefficient of the ion exchange capacity was introduced to develop retention profiles of anions. Analysis of the dependence of the capacity factors on the eluent concentrations at different values of fractional coefficient shed light on the possible complex mechanism. Extensive experimental retention data were obtained for 14 anions (formate, acetate, propionate, pyruvate, lactate, chloride, nitrate, oxalate, malonate, succinate, tartarate, fumarate, maleate, sulphate) using hydroxide eluents of varying concentration. The ion-pair formation and ion-exchange selectivity constants for analyte and eluent species are determined using derived retention equation from experimental data by nonlinear iterative calculation. The model was utilized to predict retention data under elution conditions of practical importance. The predicted and obtained retention factors are in good agreement, which confirms the predictive power of the model.     

Evaluation of the interrelationship between mass resolving power and mass error tolerances for targeted bioanalysis using liquid chromatography coupled to high‐resolution mass spectrometry

The determination of acceptable mass error tolerances for high‐resolution mass spectrometry based signals has been evaluated in a comprehensive way. This was achieved by using a technical approach which is based on the post‐column infusion of an analyte containing solution. This well‐known experimental setup was not used to spot signal suppression regions of a particular analyte, but to spot regions of the chromatogram where a systematic mass drift of the analyte ion can be observed (isobaric interference plot). Not the changing signal intensity but the stability of the measured analyte mass was observed. A wide range of different analytes in combinations with potentially interfering matrices has been evaluated. Furthermore, different mass resolving power settings were evaluated. Isobaric interferences between matrix compounds and analytes were common at mass resolving powers <50 000 full width at half maximum. The proposed post‐column infusion technique is a useful tool for the determination of the assay and matrix‐specific mass error tolerances. It aims to ensure the highest possible selectivity, at the same time preventing the encounter of detrimental mass error related peak deformations as well as false negative findings. Unlike conventional matrix spiking approaches, isobaric interference plots provide information of potential interferences across the whole chromatographic time range. This becomes relevant when there is a relative retention time shift between the analyte and potential interfering matrix compounds. Furthermore, the described setup can be used to study how the mass accuracy of any mass spectrometer is affected by a widely varying total ion current. Copyright © 2012 John Wiley & Sons, Ltd.     

Hydrophilic interaction ultra performance liquid chromatography retention prediction under gradient elution

The development and application of new separation mechanisms such as hydrophilic interaction chromatography (HILIC) is of high importance for the simultaneous analysis of polar molecules such as primary metabolites. However the retention mechanism in HILIC is not fully understood and as a result retention prediction tools are not at hand for this chromatographic approach. In the present report we study the utility of a simple algorithm, based on a simple linear and/or a simple logarithmic retention model, for retention prediction in HILIC gradient separation of a mixture of 23 selected compounds including (poly)amines, amino acids, saccharides, and other molecules. Utilizing two types of gradient elution programs with or without an isocratic part, retention data were collected in order to build prediction models. Starting from at least three gradient runs the prediction of analyte retention was very satisfactory for all gradient programs tested, providing useful evidence of the value of such retention time prediction methodologies.     

Prediction of retention indexes. III. Silylated derivatives of polar compounds.

Polar compounds containing hydroxyl, amino and carboxyl groups, singly or in combination, can be chromatographed after the polar functional groups are silylated. The silylated derivatives of acids, alcohols, amines, diols, amino alcohols, amino acids are shown to behave chromatographically as hydrocarbons, and their retention indexes can be readily predicted from their base values. The column difference, namely, the difference between the retention indexes of the analyte on polar and non-polar columns is minimal for the silylated derivatives in comparison to that observed for the underivatized analytes. This minimal column difference is attributed to the hydrocarbon-like chromatographic characteristics of the silylated derivatives. The retention indexes of the silyl derivatives appear to correlate with the atom number Z of the analyte.     

Low-energy interactions in high-performance liquid chromatography

Liquid chromatographic systems with very weak excessive analyte-adsorbent interactions have been studied. These systems consisted of a homologous series of n-alkanes as both analytes and mobile phases with a C18 reversed-phase adsorbent. A linear decrease of the analyte retention volume with an increase of the number of analyte carbon atoms was found. Corresponding increases of analyte retention with an increase in the number of eluent carbon atoms was also discovered. An explanation of these two effects on the basis of adsorption theory is proposed. A good correlation of column hold-up volume calculated by interpolation of the retention dependencies for above mentioned systems with that measured by the minor disturbance method has been shown. A study of the temperature dependencies of these alkane systems has shown entropy-governed retention dependencies.     

Influence of inorganic mobile phase additives on the retention, efficiency and peak symmetry of protonated basic compounds in reversed-phase liquid chromatography

Inorganic eluent additives affect the retention of protonated basic analytes in reversed-phase HPLC. This influence is attributed to the disruption of the analyte solvation-desolvation equilibria in the mobile phase, also known as "chaotropic effect". With an increase of counteranion concentration analyte retention increases with concomitant decrease in the tailing factor. Different inorganic counteranions at equimolar concentrations affect protonated basic analyte retention and peak symmetry to varying degrees. The effect of the concentrations of four different inorganic mobile phase additives (KPF6, NaClO4, NaBF4, NaH2PO4) on the analyte retention, peak symmetry, and efficiency on a C8-bonded silica column has been studied. The analytes used in this study included phenols, toluene, benzyl amines, beta-blockers and ophthalmic drugs. The following trend in increase of basic analyte retention factor and decrease of tailing factor was found: PF6- > ClO4- approximately BF4- > H2PO4-. With the increase of the counteranion concentration greater analyte loading could be achieved and consequently an increase in the apparent efficiency was observed until the maximum plate number for the column was achieved. At the highest concentration of counteranions, the peak efficiency for most of the basic compounds studied was similar to that of the neutral markers. In contrast, the neutral markers, such as phenols, showed no significant changes in retention, efficiency or loading capacity as counteranion concentration was increased.     

Impact of methanol and acetonitrile on separations based on pi-pi interactions with a reversed-phase phenyl column

Studies were performed to investigate the roles of methanol and acetonitrile on the retention mechanism of an active pharmaceutical ingredient (API) and related compounds with a reversed phase phenyl column. Different retention orders were observed depending upon whether acetonitrile or methanol was used as the organic modifier. We propose that acetonitrile impedes the selective pi-pi interactions between the analyte molecules and the phenyl groups in the stationary phase. Further study with 1-naphthoic acid and 1-naphthol as test compounds in the HPLC separation provides additional support for the influence of acetonitrile on pi-pi interactions between analyte molecules and a phenyl stationary phase. This study suggests that methanol be used as the preferred organic modifier with phenyl columns to achieve selectivity based upon pi-pi interactions.     

Electrochemically controlled solid-phase microextraction and preconcentration using polypyrrole coated microarray electrodes in a flow system

Polypyrrole coated microarray electrodes have been used for electrochemically controlled solid-phase microextraction and preconcentration on individually addressable gold microband electrodes. In this study, a flow of analyte solution was maintained over the band electrodes by positioning a capillary in a vertical position over the electrode array during both the extraction and the detection of the desorbed compounds. This experimental set-up was used to evaluate the possibilities of using electrochemically controlled solid-phase microextraction with conducting polymers as a preconcentration step in miniaturised flow systems. The performance of the polymer, which was prepared by electrochemical polymerisation using a solution of 0.05 M pyrrole and 0.1 M LiClO4, was investigated using chloride as a model analyte employing different extraction times and analyte concentrations. It was found that significant preconcentration was possible using extraction times of only a few minutes and that a good linearity between the extraction time and detection response was present both for mM and microM chloride concentrations. Compared to a recent study (Liljegren et al., Analyst, 2002, 127, 591-597), using a more traditional solid-phase microextraction technique under electrochemical control, the preconcentration factor could be increased by a factor of about 210 by using the present flow system based approach. This increase in the preconcentration factor can be explained by the significant decrease in the desorption volume (i.e. reduced dilution of the desorbed analyte) associated with the use of the present flow system. With the present approach, the detection limit for the model analyte chloride could be decreased from 10 microM to 625 nM employing an extraction time of 180 s.     

A solvation-based screening approach for metabolite arrays

This paper explores a new method for screening metabolites in an array format based on relative polarity using selective solvent dissolution. A synthetic cocktail of metabolites was spotted onto a hydrophobic silicon surface, and solubilised with solvents of varying polarity. The metabolites retained on the silicon surface after the solvent treatments were detected using time-of-flight static secondary ion mass spectrometry (ToF-sSIMS). Solvent-specific metabolite retention was clearly evident on multivariate analysis of the dataset, using principal component analysis. Selective removal of metabolites was observed when solvents with different polarity were used, with the metabolite retention or removal in most cases correlating to the polarity of the solvent used, although consideration of other forces in operation may be needed to arrive at fully predictable behaviours. This approach provides the basis for development of a technique to separate complex metabolites into simpler constituents in a metabolite array prior to identification and quantification using mass spectrometry. It is an analytical approach that is intermediate between the more rapid but less informative direct analysis methods (such as DIMS) that do not involve any analyte separations and the more comprehensive but time consuming methods (such as GC- and LC-MS) that involve chromatographic or electrophoretic separations. The approach has the potential to be successfully developed for rapid, yet informative screening of metabolomes.     

Isomeric Distinction of Small Oligosaccharides: A Bottom-Up Approach Using the Kinetic Method

Isomeric distinction of di- and tri-saccharides could be efficiently achieved by using data previously obtained while performing experiments aimed at discriminating monosaccharides using trimeric ion dissociation with data analysis by the kinetic method. This study shows that effects observed for lower homologues when one of the partners is changed in the metal/reference system (typically a transition metal divalent cation associated to amino acids) can be extrapolated to upper homologues, at least for the tested analyte series. Systems allowing galactose, glucose, and fructose distinction were used as starting conditions to resolve cellobiose, lactose, maltose, and saccharose disaccharides. When a unique dissociation reaction was observed from the trimeric clusters, a new reference was selected based on its propensity to favor the analyte or the reference release, as revealed from monosaccharide experiments, depending on the desired effect. The same approach could be implemented from data obtained for disaccharides to select efficient metal/reference systems to distinguish cellotriose, isomaltotriose, maltotriose, and panose trisaccharides. As a result, method optimization is greatly improved due to an enhanced rationalization of the search for discriminant systems. While 40 systems had to be tested for monosaccharides, by screening five transition metals and eight amino acids, the proposed approach allowed efficient metal/reference systems to be found for disaccharides after testing 18 combinations; then, only four systems had to be scrutinized to achieve trisaccharide distinction. Accurate quantitative analyses could be performed in binary mixtures using three-point calibration curves to correct for competition effects between analytes for the formation of the trimeric clusters.     

A general strategy for performing temperature-programming in high performance liquid chromatography--prediction of segmented temperature gradients

In the present work it is shown that the linear elution strength (LES) model which was adapted from temperature-programming gas chromatography (GC) can also be employed to predict retention times for segmented-temperature gradients based on temperature-gradient input data in liquid chromatography (LC) with high accuracy. The LES model assumes that retention times for isothermal separations can be predicted based on two temperature gradients and is employed to calculate the retention factor of an analyte when changing the start temperature of the temperature gradient. In this study it was investigated whether this approach can also be employed in LC. It was shown that this approximation cannot be transferred to temperature-programmed LC where a temperature range from 60°C up to 180°C is investigated. Major relative errors up to 169.6% were observed for isothermal retention factor predictions. In order to predict retention times for temperature gradients with different start temperatures in LC, another relationship is required to describe the influence of temperature on retention. Therefore, retention times for isothermal separations based on isothermal input runs were predicted using a plot of the natural logarithm of the retention factor vs. the inverse temperature and a plot of the natural logarithm of the retention factor vs. temperature. It could be shown that a plot of lnk vs. T yields more reliable isothermal/isocratic retention time predictions than a plot of lnk vs. 1/T which is usually employed. Hence, in order to predict retention times for temperature-gradients with different start temperatures in LC, two temperature gradient and two isothermal measurements have been employed. In this case, retention times can be predicted with a maximal relative error of 5.5% (average relative error: 2.9%). In comparison, if the start temperature of the simulated temperature gradient is equal to the start temperature of the input data, only two temperature-gradient measurements are required. Under these conditions, retention times can be predicted with a maximal relative error of 4.3% (average relative error: 2.2%). As an example, the systematic method development for an isothermal as well as a temperature gradient separation of selected sulfonamides by means of the adapted LES model is demonstrated using a pure water mobile phase. Both methods are compared and it is shown that the temperature-gradient separation provides some advantages over the isothermal separation in terms of limits of detection and analysis time.     

Retention of ionisable compounds on high-performance liquid chromatography XVI. Estimation of retention with acetonitrile/water mobile phases from aqueous buffer pH and analyte pKa

In agreement with our previous studies and those of other authors, it is shown that much better fits of retention time as a function of pH are obtained for acid-base analytes when pH is measured in the mobile phase, than when pH is measured in the aqueous buffer when buffers of different nature are used. However, in some instances it may be more practical to measure the pH in the aqueous buffer before addition of the organic modifier. Thus, an open methodology is presented that allows prediction of chromatographic retention of acid-base analytes from the pH measured in the aqueous buffer. The model presented estimates the pH of the buffer and the pKa of the analyte in a particular acetonitrile/water mobile phase from the pH and pKa values in water. The retention of the analyte can be easily estimated, at a buffer pH close to the solute pKa, from these values and from the retentions of the pure acidic and basic forms of the analyte. Since in many instances, the analyte pKa values in water are not known, the methodology has been also tested by using Internet software, at reach of many chemists, which calculates analyte pKa values from chemical structure. The approach is successfully tested for some pharmaceutical drugs.