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.     

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.     

Computer simulation of ion chromatography separation: an algorithm enabling continuous monitoring of anion distribution on an ion-exchange chromatography column

A computer algorithm for the calculation of ion chromatography separation is presented. It is based on the calculation of equilibrium concentrations of present analyte in discrete column segments. The continuous column is treated as a number of discrete cells or segments where the equilibration process between the stationary phase and the eluent is simulated. The ion-exchange equilibration process is supposed to be instantaneous and quantitative. The continuous flow of the eluent is rendered by discrete transfers. The size of each transfer of the eluent corresponds to a portion of the volume contained in one column segment. The equilibrium calculations in all column segments are repeated for each transfer of the eluent, through all the stages of the chromatographic process. The distribution of the analytes between the stationary phase and the eluent can be monitored at any step and in any column segment which means that the described algorithm provides the spatial and time concentration profiles. The simulated chromatogram is acquired as a time-concentration profile in the last column segment. The obtained chromatograms are in good agreement with the experimental ones. The distribution of ions between the stationary phase and the eluent in the early stages of the ion chromatographic process can thus be studied with confidence.     

HPLC of nucleotides. II. General methods and their development for analysis and preparative separation. An approach to selectivity control

In order to develop efficient separation methods for nucleotides according to their size and heterocyclic composition, the application of ion-exchange, reverse-phase, and normal-phase adsorption HPLC has been studied. The comparative investigation of retention power and selectivity of various packings (non-polar bonded-phase and amino silicas) in relation to nucleotide length and composition yields data which enable suitable packings to be selected and a method of preparing the new packing for a particular separation problem to be formulated. Thus a new anion exchanger with high selectivity and dynamic mass transfer has been prepared for fractionation of large oligonucleotides. The effect of the eluent pH and composition (organic modifier, salt) on retention, selectivity, and resolution in ion-exchange and reverse-phase HPLC has been studied. The optimum separation conditions comprise elution with oppositely directed gradients of the salt and the modifier, use of a precolumn packing that provides the best protection for the main column without loss of its efficiency, and the optimum gradient program for the desired retention of the component of interest. The relation between loading and sample concentration has been studied and the system for gradient elution improved. Our work shows that two-dimensional separation is the most reliable and informative method for preparation of homogeneous oligonucleotides. The hydrophobic-pair ion-exchange mechanism is proposed for ion-pair chromatography. Protected and partially deblocked oligonucleotides, chemically synthesized for genetic engineering studies, have been separated with high selectivity by adsorption (normal-phase) HPLC which is efficient for gradient elution with isohydric eluents. The analysis of a monomeric composition of nano-(pico-) molar amounts of oligonucleotides has been developed; the procedure involves microcolumn digestion of the oligonucleotides with immobilized enzymes followed by microcolumn separation of the nucleoside-mononucleotide mixture. Also, a new slurry method for packing stable HPLC columns with a tightly consolidated, nonshrinkable bed of particles has been developed.     

吸附剂性能和操作参数对液相色谱流出峰不对称性的影响

色谱流出峰的拖尾现象普遍存在于制备和大型色谱分离过程中,它直接影响分离的产率和回收率．本文提出采用色谱流出峰形的不对称偏差度来表征色谱流出峰的拖尾程度;并基于液相制备色谱分离过程FAD－SMT模型及吸附速率理论,通过计算机模拟,定量分析了吸附剂性能和操作参数对色谱流出峰形不对称性的影响。结果表明：不仅是吸附剂的热力学和动力学性能（包括吸附相平衡关系、液固两相间的传质阻力）;而且柱的设计和吸附剂的装填状况（包括轴向扩散系数）,以及色谱分离的操作条件（进料时间、浓度和流速等）都直接影响色谱流出峰形的不对称性。随着吸附相平衡等温线的非线性程度增大,或者总传质系数的减小,色谱流出峰形的不对称偏差度明显增大;吸附剂吸附容量的减小也将引起色谱流出峰形的不对称偏差度的增加;色谱流出峰形的不对称偏差度与进科体积、浓度和流体线速,以及轴向扩散系数的增大成正比。     

Characterization of unstable ion-exchange chromatographic separation of proteins.

Very fine separation of proteins by stepwise elution ion-exchange chromatography is very often a unstable process. To characterize the unstability of such processes the elution volume variations were examined by the model equation which contained the ion-exchange capacity and the number of adsorption sites. The data needed for the model calculation were obtained from gradient elution experiments. As a model separation system stepwise elution of a model protein (beta-lactoglobulin) near the isoelectric point on a weak cation-exchange chromatography column was chosen. The elution volume varied significantly with a small change in the ion-exchange capacity. It was found that the ionic strength of the elution buffer must be adjusted in order to compensate a change in the elution volume due to the ion-exchange capacity variations. The ionic strength and the pH of the elution buffer were also found to be important variables affecting the elution volume. In this model separation system, it was indicated that the pH should be within +/-0.1 unit and the ionic strength within +/-0.002 mol/l in order to meet the criteria (+/-5% elution volume variation). It is recommended that gradient elution data be obtained for predicting elution volume variations in stepwise elution. By using the gradient elution data the process diagnosis can be performed, and the important information on the process stability can be obtained.     

Unique elution behavior of bromide and nitrate in anion-exchange chromatography using aqueous potassium chloride eluent containing cadmium or zinc ion

In anion-exchange chromatography using a high-concentration eluent and high-capacity ion-exchange resin, the effect of the countercation contained in the eluent was investigated. Cadmium(II) and zinc(II) ions were examined as additives in an aqueous potassium chloride eluent. The addition of these cations resulted in a reversed elution order of bromide and nitrate, as compared with conventional anion-exchange chromatography. The separation factor for these two anions increased as the cadmium concentration in the eluent was increased. Zinc(II) ion was also effective, but a relatively high concentration was necessary.     

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.     

离子色谱法测定氯膦酸二钠及其制剂的含量和有关物质

采用抑制型电导检测-离子色谱法同时测定氯膦酸二钠及其制剂的含量和有关物质.色谱条件为阴离子交换色谱柱(IonPac AS11-HC);检测方式为抑制电导检测;柱温30 ℃;流速1.2 mL/min;以KOH溶液为淋洗液,含量测定采用等度洗脱,有关物质检查采用梯度洗脱.氯膦酸二钠在0.0568～0.1895 g/L范围内线性关系良好(r＝0.9999); 注射液和胶囊的平均回收率分别为100.1%(RSD=0.7%)和98.9%(RSD=0.6%);检出限为0.3 ng.各杂质与主成分色谱峰能完全分离.     

Histidine as a dipolar eluent component in cation chromatography: II. Prediction of retention data for alkaline and alkaline-earth ions

The utility of cation chromatography has been developed by the application of -histidine as a multiprotic and dipolar (zwitterionic) eluent component. The method simplifies the cation analysis. The chromatographic characteristics of this system were studied in detail with a view to determining the selectivity and the mechanism by which the cations (Na⁺, K⁺, Mg²⁺, Ca²⁺) are retained. Complete separations were observed in the isocratic run over the eluent concentration range 3.0–6.0 mM at pH below 2.0. Sensitive detection was achieved using suppressed conductivity at the pH of isoelectric point of the histidine. Retention equations are derived for mono- and divalent cations eluted from ion-exchange separation column with multiple ionic eluents. The theory is based on the extension of ion-exchange equilibrium by protonation equilibria. The selectivity data for analyte and eluent species are determined using the model from the experimental retention data by computer-assisted iterative calculations. The model was utilized to predict retention data. The results in three-dimensional retention surfaces together with species distribution graphs are presented.     

Retention model for the separation of anionic metal-EDTA complexes in ion chromatography

A retention model is derived for complex anions eluted from an anion-exchange column with multiple ionic eluents containing hydrogencarbonate, carbonate, and hydroxyl species and the sample solution, containing transition metals, anions and complexing ligand. The theory is based on the generalized ion-exchange equilibrium, protonation and complex-formation equilibria. The unknown parameters of chromatographic ion-exchange equilibrium constants for sample and eluent species are determined from the experimental retention data by iterative minimization, using a non-linear regression algorithm. The model was utilized to predict the retention behaviour of CdEDTA²⁻, CoEDTA²⁻, MnEDTA²⁻ and NiEDTA²⁻ ions. The capacity factors of complex ions were determined for wide ranges of pH values and eluent concentrations. Good agreement was obtained between the observed and predicted retentions.     

Solubilization chromatography: Ethers,carboxylic acids,and hydrocarbons

Synthetic mixtures of aliphatic and aromatic ethers, satmated fatty acids, and substituted benzenes and naphthalenes were separated by elution through columns of ion-exchange resins with aqueous solutions of acetic acid as eluent An attempt was made to use a resin of low capacity for the separation of such mixtures, and the results of this attempt are used to help elucidate the theory of solubilization chromatography     

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.     

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.     

Radiochemical purification of microgram and sub-microgram amounts of beryllium

A method is presented for the separation and radiochemical purification of microgram and tracer amounts of beryllium from solutions. It is a four-stage ion-exchange procedure consisting of (1) selective adsorption of the beryllium onto NaDAP resin and its elution with ammonium fluoride, (2) adsorption of the fluoroberyllate complex onto an anion-exchange column and elution of the beryllium with hydrochloric acid, (3) adsorption and selective elution of the beryllium on a cation-exchange column and (4) a pass through an anion-exchange column in concentrated hydrochloric acid. The method is quantitative and requires no carrier. Decontamination factors from most radionuclides tested were greater than 10,000. The method can be used to determine beryllium-10 in environmental materials.     

Determination of monovalent inorganic anions in high-ionic-strength samples by electrostatic ion chromatography with suppressed conductometric detection

A new ion chromatographic (IC) system has been established by using micelles of 3-(N,N-dimethylmyristylammonio)propanesulfonate (Zwittergent 3-14) loaded onto a reversed-phase packed column as the separation column with an electronic rotary switching valve packed-bed suppressor for conductometric detection of inorganic anions. An aqueous H3BO3-Na2B4O7 solution has been demonstrated to be the most desirable eluent for this IC system. The relationship between retention time and the concentration of the borate eluent was determined for a series of model anionic analytes and this relationship was found to be opposite to that exhibited in a conventional anion-exchange IC system. The rapid elution and complete separation of monovalent inorganic anions were obtained by initially using a high-concentration borate solution as the eluent for a short-period, and then switching to a lower-concentration borate eluent to complete the separation. Detection limits for nitrite, bromide, nitrate, and chlorate were 0.85, 0.88, 0.95 and 4.8 microM, respectively, when a 7.0 mM Na2B4O7 eluent was used. Moreover, the ability to directly detect these monovalent anions in samples containing high concentrations of sulfate and/or chloride ions provided a major advantage of this approach.     

Simultaneous separation of inorganic anions and cations by using anion-exchange and cation-exchange columns connected in tandem in ion chromatography

Inorganic anions and cations in environmental waters were determined by ion chromatography. Stationary and mobile phases were examined for the simultaneous separation of both anions and cations. Cations detection by UV detection requires a mobile phase with a UV absorbing additive, which indirectly visualizes cations as negative peaks. Simultaneous separation of anions and cations were achieved when using an eluent that consists of inorganic acid with weak basic amino acid as additives. It was convenient to separate both anions and cations by coupling anion-exchange and cation-exchange columns in tandem. The order of the separation columns connected affected the elution profiles. When the eluent comprises of multiple anions and a single cation, the anion-exchange column should be connected in the upper stream, whereas when the eluent comprises multiple cations and a single anion, the cation-exchange column should be connected in the upper stream. Use of switching valves also allowed simultaneous separation of anions and cations in a single chromatographic run. In the present work, operating conditions were optimized for the simultaneous separation of anions and cations.     

Investigation of the adsorption behaviour of a chiral model compound on a tartardiamide-based network-polymeric chiral stationary phase

The adsorption behaviour of the enantiomers of 2-phenylbutyric acid on the chiral stationary phase (CSP) Kromasil CHI-TBB was studied using hexane/MTBE (90/10) as eluent. Adsorption isotherms were acquired at 40 different enantiomer concentrations in the interval between 7.6 microM and 305 mM, an approximately 40,000-fold dynamic range. The adsorption data fitted well to the bi-Langmuir model, indicating a heterogeneous surface with two different types of adsorption sites having different equilibrium constants and capacities; namely one chiral site and one non-chiral site. A comparison with earlier adsorption studies on modern CSPs revealed that the capacity value of the "true" chiral site of Kromasil CHI-TBB is the largest reported so far. The elution profiles simulated with these parameters show excellent agreement with the corresponding experimental profiles. Guidelines for comparisons of loading capacities of CSPs are presented.