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.     

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.     

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.     

Vitamin U-bonded stationary phase in capillary ion chromatography

A vitamin U-bonded stationary phase was prepared and the retention behavior of inorganic anions was examined using ion chromatography. Inorganic anions were retained on the vitamin U-bonded stationary phase under acidic as well as neutral eluent conditions in the ion-exchange mode. The elution order of the examined anions under neutral eluent conditions was nearly the same as that observed in common ion exchange mode, while the elution order observed under acidic eluent conditions was completely different from that observed in common ion exchange mode. The retention of the analyte anions under the neutral eluent conditions was due to the sulfonium groups of the vitamin U, while protonated primary amino groups caused retention of the analyte anions with different selectivity under acidic conditions. The retention factor of the analyte anions increased with decreasing eluent concentration under both eluent conditions. The present system was applied to the determination of bromide and nitrate contained in seawater.     

Separation of inorganic anions by liquid chromatography with crown ether as eluent additive

Inorganic anions were separated on a reversed-phase stationary phase dynamically modified with crown ether as a selector in capillary ion chromatography. The eluent contained crown ether, acetonitrile and a salt. Free and cation-trapped crown ether molecules in the eluent were adsorbed on a hydrophobic stationary phase such as triacontyl-functionalized silica (C30). The eluent cations trapped on crown ether worked as the ion-exchange sites, where the eluent anions and the analyte anions were competing for electrostatic interaction. The sizes of crown ether and the salt cation affected the retention of analyte anions. The concentrations of acetonitrile and crown ether as well as the eluent anion also affected the retention of analyte anions. An aqueous solution containing 18-crown-6-ether, potassium salt and acetonitrile achieved larger retention for analyte anions. Effects of the eluent conditions on the retention of analyte anions were examined in detail.     

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.     

Organic solvent and temperature-enhanced ion chromatography-high resolution mass spectrometry for the determination of low molecular weight organic and inorganic anions

There has recently been increased interest in coupling ion chromatography (IC) to high resolution mass spectrometry (HRMS) to enable highly sensitive and selective analysis. Herein, the first comprehensive study focusing on the direct coupling of suppressed IC to HRMS without the need for post-suppressor organic solvent modification is presented. Chromatographic selectivity and added HRMS sensitivity offered by organic solvent-modified IC eluents on a modern hyper-crosslinked polymeric anion-exchange resin (IonPac AS18) are shown using isocratic eluents containing 5–50 mM hydroxide with 0–80% methanol or acetonitrile for a range of low molecular weight anions (<165 Da). Comprehensive experiments on IC thermodynamics over a temperature range between 20–45 °C with the eluent containing up to 60% of acetonitrile or methanol revealed markedly different retention behaviour and selectivity for the selected analytes on the same polymer based ion-exchange resin. Optimised sensitivity with HRMS was achieved with as low as 30–40% organic eluent content. Analytical performance characteristics are presented and compared with other IC-MS based works. This study also presents the first application of IC-HRMS to forensic detection of trace low-order anionic explosive residues in latent human fingermarks.     

Determination of alkylphosphonic acids by microcolumn liquid chromatography with gradient elution coupled on-line with flame photometric detection

Microcolumn liquid chromatography with gradient elution and on-line flame photometric detection has been used for the selective and rapid determination of non-volatile alkylphosphonic acids, which are hydrolysis products of nerve agents, in aqueous samples. A make-up of water was used to maintain stable introduction of the eluent during a gradient run. The detection limits are strongly dependent on quenching, which is determined by the mass flow of methanol in the eluent. Large-volume injections of 100 microl of aqueous solutions of short- and long-chain alkylphosphonic acids resulted in detection limits in the range 6-800 ng/ml. The repeatability of the retention times and analyte response - peak area or peak height were 0.7-0.9% and 4-11%, respectively. The method was successfully applied to a local tap water sample and an aqueous soil extract.     

Interconversion of gradient and isocratic retention data in reversed-phase liquid chromatography: Effect of the uptake of eluent modifier on the retention of analytes

The experimental technique of mass spectrometric tracer pulse chromatography was used to study the effect of the sorption of eluent components by a C₁₈-bonded silica RPLC packing on the retention of a series of test analytes during isocratic and gradient elution experiments. The analytes of interest were a substituted phenol, a substituted nitroaniline, an anti-malaria drug, tetrahydrofuran, and methanol. The eluent used was a mixture of acetonitrile and water. The solutes and isotopically labeled eluent components were injected at fixed time intervals during each gradient run. The mass specific detector allowed the assignment of individual analyte peaks even when there was overlap in the chromatograms from successive injections. Thus, the retention time of each analyte could be determined as a function of gradient slope and initial eluent composition at the time of each injection. Experimental gradient retention time data were then compared with the calculated results from two theoretical models. The first model assumed the velocity of the mobile phase and eluent were equal. The second and most realistic model assumed the velocity of the eluent was less than the velocity of the mobile phase due to the uptake of eluent by the stationary phase. Gradient retention times predicted by the two models were reasonably accurate with the sorption model giving slightly more accurate values. Inverse calculations, i.e., calculation of isocratic retention factors from gradient elution data were also carried out with very similar results. That is, the model allowing for the uptake of eluent was slightly more accurate than the model assuming no eluent-stationary phase interaction.     

Polyoxyethylene as the stationary phase in ion chromatography

The present article reviews the use of polyethylene glycol (PEG) or polyoxyethylene (POE) as the stationary phase for the separation of inorganic anions in ion chromatography and discusses about the retention mechanisms involved in the separation of anions on the novel stationary phases. PEG permanently coated on a hydrophobic stationary phase retained anions in the partition mode and allowed us to use high-concentration eluents because the retention of anions increased with increasing eluent concentration for most of the eluents. This situation was convenient to determine trace anions contained in seawater samples without any disturbance due to matrices. Chemically bonded POE stationary phases retained not only anions but also cations. Anions were retained in the ion-exchange mode, although POE chains possess no ion exchange sites. The retention behavior suggested that eluent cations could be trapped among multiple POE chains via ion-dipole interaction, and that the trapped cations worked as the anion-exchange sites. Anions could be separated using crown ether, i.e., cyclic POE, as the eluent additive with a hydrophobic stationary phase, where analyte anions were retained via electrostatic interaction with the eluent cation trapped on the crown ether.     

Characteristics of a column suitable for capacity gradient chromatography with a borate eluent

In capacity gradient elution, the gradient separation of ionic species is achieved by decreasing the ion-exchange capacity of a column during the course of the separation. Diol-type hydroxy groups on the resin surface form anionic complexes with borate as an eluting reagent. Thus, a chemically bonded anion-exchange column enriched with residual hydroxy groups allows the creation of a capacity gradient. An increase in the amount of the complex formed gradually brings about a decrease in the ion-exchange capacity of the column, and strongly bound analyte ions are eluted. We investigated the characteristics of a column suitable for this eluent system. The concentration of borate eluent required to remove the ion-exchange capacity depended inversely on the ratio of the residual hydroxy groups to functional groups. On a column in which this ratio was approximately 100, the ion-exchange capacity could easily be adjusted by using a low concentration of mannitol as a competing reagent. Use of this column led to very small baseline shifts during the borate-mannitol gradients, and to the simultaneous determination of anions with widely varying retention times.     

Effect of eluent composition on retention behavior of anions in ion chromatography on anion-exchangers modified with heparin

Effects of eluent composition on retention behavior of inorganic anions have been investigated in ion chromatography using anion-exchangers modified with heparin. Both cation and anion of the eluent affected the retention of analyte anions and unusual retention behavior was observed on the modified stationary phase. The retention time of anions decreased with decreasing eluent concentration when sodium sulfate, magnesium sulfate and chlorides of alkali metals were used as the eluent, whereas it increased with decreasing eluent concentration when aluminum sulfate, copper sulfate and sulfuric acid were used as the eluent. The retention of nitrate increased in the order of Li⁺, Na⁺, K⁺, Rb⁺ and Cs⁺ when their chlorides were used as the eluent. When sodium perchlorate and chlorides of alkaline-earth metals were used as the eluent, the eluent should include heparin. Otherwise, the modifier was partially bled from the column.     

Fast ion chromatography using short anion exchange columns

A fast ion chromatographic system is described which uses shorter column lengths and compares various eluent profiles in order to maximise the performance without sacrificing the chromatographic resolution. Both isocratic and gradient elution profiles were considered to find the most efficient mode of separation. The separation and determination of seven target anions (chloride, chlorate, nitrate, chromate, sulfate, thiocyanate and perchlorate) was achieved using a short (4 mm ID, 50 mm long) column packed with Dionex AS20 high-capacity anion exchange material. A hydroxide eluent was used at an initial concentration of 25 mM (at a flow-rate of 1.0 mL/min) and two performance maxima were found. The maximum efficiency occurred at a normalised gradient ramp rate of 5 mM/t₀, resulting in a peak capacity of 16, while the fastest separation (<3 min) occurred at a normalised ramp rate of 30 mM/t₀. The retention time, peak width and resolution using the different eluent profiles on varying column lengths is also compared. Further investigations in this study determined that the highest peak capacity separation under gradient conditions could be approximated using an isocratic separation. The advantage of using this novel approach to approximate the maximum efficiency separation removes the need for column re-equilibration that is required for gradient elution resulting in faster analyses and enhanced sample throughput, with benefits in particular for multidimensional chromatography.     

Determination of pharmaceutically related compounds by suppressed ion chromatography: I. Effects of organic solvent on suppressor performance

This overall study aims to investigate gradient elution ion-exchange chromatography of pharmaceutically relevant compounds using universal nebulisation detectors, such as evaporative light scattering detection (ELSD). Addition of organic solvents to the eluent is necessary to minimise hydrophobic adsorption on the polymeric stationary phase and improve solubility of analytes. It is also necessary to de-salt the eluent prior to detection, and in this work, ion chromatography suppressors were used for this step. Such suppressors have been designed for aqueous eluents, so the purpose of the present study was to investigate the effects of methanol and acetonitrile on suppressor performance. Chemical and electrolytic suppressors were evaluated for baseline drift, noise and efficiency of suppression using aqueous/organic eluents containing up to 40% (v/v) methanol or acetonitrile. Chemical suppression of aqueous/organic eluents showed minimal noise levels, uniform low baseline and low gradient drift. Electrolytic suppression gave good performance, but with higher baseline conductivity levels and baseline drift than chemical suppression. The elevated baseline was found not to be caused by incomplete suppression of the eluent, but was attributed to chemical reactions involving the organic solvents and facilitated by high electric currents and heat generation. It was demonstrated that suppressed ion-exchange separation using a complex KOH elution profile could be coupled with ELSD, with the suppressor effectively de-salting the eluent, producing a stable baseline. Finally, complementary separation selectivity was demonstrated using a set of pharmaceutically related organic acids separated by reversed-phase and ion-exchange methods.     

Two-dimensional ion chromatography using tandem ion-exchange columns with gradient-pulse column switching

A two-dimensional ion chromatography (2D-IC) approach has been developed which provides greater resolution of complex samples than is possible currently using a single column. Two columns containing different stationary phases are connected via a tee-piece, which enables an additional eluent flow and independent control of eluent concentration on each column. The resultant mixed eluent flow at the tee-piece can be varied to produce a different eluent concentration on the second column. This allows analytes strongly retained on the first column to be separated rapidly on the second column, whilst maintaining a highly efficient, well resolved separation of analytes retained weakly on the first column. A group of 18 inorganic anions has been separated to demonstrate the utility of this approach and the proposed 2D-IC method provided separation of this mixture with resolution of all analytes greater than 1.3. Careful optimisation of the eluent profiles on both columns resulted in run times of less than 28 min, including re-equilibration. Separations were performed using isocratic or gradient elution on the first column, with an isocratic separation being used on the second column. Switching of the analytes onto the second column was performed using a gradient pulse of concentrated eluent to quickly elute strongly retained analytes from the first column onto the second column. The separations were highly repeatable (RSD of 0.01–0.12% for retention times and 0.08–2.9% for peak areas) and efficient (typically 8000–260,000 plates). Detection limits were 3–80 ppb.     

Tunable separation of anions and cations by column switching in ion chromatography

A convenient ion chromatography method has been proposed for the routine and simple determination of anions (Cl⁻, SO₄²⁻ and NO₃⁻) and/or cations (Na⁺, NH₄⁺, K⁺, Mg²⁺ and Ca²⁺) using a single pump, a single eluent and a single detector. The present system used cation-exchange and anion-exchange columns connected in series via two 6-port switching valves or a single 10-port valve. The connection order of the ion-exchange columns could be varied by switching the valve(s). The present system therefore allowed the separation of either cations or anions in a single chromatographic run. While one ion-exchange column is being operated, the other ion-exchange column is being conditioned, i.e., the columns are always ready for analysis at any time. When 2.4 mM 5-sulfosalicylic acid was used as the eluent, the three anions and the five cations could be separated on the anion-exchange column and cation-exchange column, respectively. In order to obtain the separations of the target ions, the injection valve was placed between the two columns. Complete separations of the above anions or cations were demonstrated within 10 min each. The detection limits at S/N = 3 were 19-50 ppb (μg/l) for cations and 10-14 ppb for anions. The relative standard deviations of the analyte ions were less than 1.1, 2.9 and 2.8% for retention time, peak area and peak height, respectively. This proposed technique was applied to the determination of common anions and cations in river water samples.     

Extended Thermodynamic Approach to Ion Interaction Chromatography: Influence of Organic Modifier Concentration

Summary The influence of organic modifier and ion interaction reagent (IIR) concentrations on analyte retention in ion interaction chromatography (IIC) was investigated via a bivariate approach. The system examined is as follows: ODS-Hypersils was used as stationary phase; the eluent was a variable mixture of a phosphate buffer pH 2.1 and methanol ranging 0%–40% (v/v). The organic modifier decreases both the analyte and the IIR free energy of adsorption hence their interaction is lower when the organic modifier concentration is higher. Retention equations describe how these two factors interact with each another. The estimated equilibrium constant for the IIR adsorption corresponds to G° = –17.3 KJ mol^–1. This value is reliable because it makes sense physically. The chi-squared (k _calc – k _exp)² is 1.11E + 00 and 2.85E-02, for octopamine and 2-naphthalenesulfonate, respectively. Retention equations were compared with those obtained from the outstanding retention models in IIC. For the modelled system, the extended thermodynamic approach is able to explain experimental evidence that cannot be rationalised by existing retention models: it proves superior for fitting experimental data and is also able to predict when the purely electrostatic approach may work properly. In the absence of IIR the present retention equation reduces, as expected, to the relationship that describes the influence of organic modifier on retention behaviour in reversed-phase liquid chromatography.     

Affinity partitioning for membrane purification exploiting the biotin-NeutrAvidin interaction. Model study of mixed liposomes and membranes

The linear-solvent strength (LSS) model of gradient elution has been applied to estimate parameters of lipophilicity and acidity of a series of drugs and model chemicals. Apparent pKa values and log kw values for individual analytes were determined in 2-3 gradient runs. The first experiment (or first two experiments) uses a wide-range organic modifier gradient with pH chosen for suppressed ionization of the analyte. The result of this experiment allows an estimate of contents of organic modifier of the mobile phase (%B) providing the required retention coefficient, k, for the non-ionized analyte. The following experiment is carried out with the latter %B and a pH-gradient of the aqueous component of the eluent that is sufficient to overlap the possible pKa-value of the analyte. The initial pH of the buffer used to make the mobile phase is selected to insure that the analyte is in non-ionized form. The resulting retention time allows an estimate of PKa in a solvent of the selected %B. At the same time, estimates of log kw can also be obtained. The log kw parameter obtained from gradient HPLC by the approach proposed correlated well with the corresponding value obtained by standard procedure of extrapolation of retention data determined in a series of isocratic measurements. Correlation between log kw and the reference parameter of lipophilicity, log P, was very good for a series of test analytes and satisfactory for a structurally diverse series of drugs. The approach supported with specific detection procedures can be recommended for fast screening of lipophilicity of individual components of complex mixtures like those produced by combinatorial chemistry. The values of pKa obtained in a study were found to correlate with the literature pKa data determined in water for a set of aniline derivatives studied. In case of a series of drugs the correlation was less than moderate if the general procedure of pKa determination was applied.     

Capacity gradient anion chromatography with a borate complex as eluent

Complex formation between borate compounds and vicinal diols is well recognized. Generally, in a chemically bonded anion-exchange resin, many hydroxyl groups are introduced on the surface of the resin in order to make the resin hydrophilic. The borate as an eluting reagent also reacts to these hydroxyl groups, and this complex formation decreases the apparent ion-exchange capacity of the column by being dissociated to the anion depending on the eluent pH. In the present work a method is described for the simultaneous determination of anions based on the capacity gradient for suppressed ion chromatography. A Tosoh IC-Anion-PW column and dihydroxyphenylborane–mannitol eluent system were used. To maintain baseline stability, it was helpful to keep the borate concentration constant during a gradient of 16 to 0 mM mannitol as a modifier to prevent the complex formation with the hydroxyl on the resin. The chemical composition of the eluents and gradient profiles are discussed and the application to the analysis of the condensed phosphates with widely varying retention times as food additives in a cheese sample is presented.     

Characterization of a microparticulate strong anion-exchanger in the HPLC of acidic drugs

The use of Waters Spherisorb S5SAX for the HPLC of acidic compounds, including a number of non-steroidal anti-inflammatory drugs (NSAIDs), has been investigated. Adequate retention, separation, and peak efficiency and symmetry were obtained for most analytes on a 250 x 4.6 mm i.d. column using methanol containing ammonium perchlorate (10 mmol L(-1), pH 6.7 or pH 8.3) as eluent. The results of changes in (i) eluent pH (constant ionic strength); (ii) eluent ionic strength (constant pH); and (iii) adding water to the eluent (constant pH) were consistent with a retention mechanism dominated by ion-exchange with the bonded strong anion-exchange (SAX) moieties. However, there were some unexpected observations, including (i) a general decrease in retention at eluent pH values above 7.7; (ii) a marked increase in retention on adding 1% (v/v) water to the eluent; (iii) a subsequent marked decrease in retention on adding 5% (v/v) or more water; and (iv) decreased column activity with time. These observations may be due to (i) interaction between the charged SAX moieties and ionised surface silanols (with ionization increasing at higher eluent pH values) and (ii) influence of the solvation of silanols, analytes, SAX moiety, and counter-ion varying with both pH and water content. Nevertheless, the factors influencing separation of individual NSAIDs remain unclear especially as no relation between log k and pKa exists for these compounds. Hydrophobic interactions are unlikely to be important since basic and neutral compounds were hardly retained. Ease of accessibility of the counter-ion to the SAX moiety for analyte displacement may be a factor.