Hydroxide Eluent Research Articles

On-column capillary suppressor for open tubular ion chromatography

BackgroundSuppressors with different dead volumes are required to match different suppressed ion chromatography systems. Especially for suppressed open tubular ion chromatography (SOTIC), the dead volume is a critical parameter. Both connection tubes between open tubular (OT) columns and suppressors and the dead volumes of the suppressors should be as short/small as possible to minimize peak dispersion. Suppressors with different dead volumes are required to match the various suppressed ion chromatography systems that operate at low flow rates 20–200 nL/min. ResultsWe describe three designs of on-column capillary suppressors for SOTIC: (A) on-column electrodialytic suppressor prepared by making small cracks on the cycloolefin polymer (COP) capillary at targeted locations, (B) on-column electrodialytic suppressor built on a polyether ether ketone (PEEK) capillary by removing the wall materials at target locations, (C) on-column chemical suppressor based on a single cut on a PEEK capillary at a targeted location a single cut on a PEEK capillary at a targeted location. The on-column electrodialytic suppressors work in two different modes with suppression voltage applied in co-current and counter-current direction to the eluent flow. Because of very narrow column inner diameter (i.d.), up to several hundred volts were required to suppress the hydroxide eluent, but it was found the there was a >90% loss of analytes in the suppressor accompanied with a high noise level after on-column electrodialytic suppression. Theoretical analysis reveals that high suppression voltage significantly affects the retention of specific analytes by electromigration. Further analysis indicated that the electrodialytic on-column suppressor in co-current mode would behave totally different from traditional suppressors. The on-column chemical suppression, with minimum dead volume of 0.27 nL, provides fairly well suppression of low hydroxide eluent without analyte loss in the suppressor. In design C, an efficiency of 47000 ± 1800 plates/m for Cl−, corresponding to a peak volume of 17.9 ± 0.7 nL, was obtained when separating five anion mixture (0.5 mM each) in the 25 μm i.d. AS18 latex coated PEEK OT column with an injection of 7.3 nL. Theoretical calculation revealed that a column efficiency loss of ≤3% would result in a cylindrical chemical suppression channel and thus it is taken as the acceptable dispersion contribution originating from the on-column chemical suppressor. SignificanceDifferent on-column suppressors have been designed on OT columns with i.d.s less than 30 μm. Two electrodialytic on-column suppressor designs with eluent flow parallel to the direction of electric field were proposed and tested. The eluent flow rate, analytes’ retention behavior, resistance of suppression channel, current-voltage relationship, and working principles in both co-current and counter-current were experimentally investigated and comprehensively discussed. It was found that although the on-column electrodialytic suppressions (Design A and B) are not feasible in practice, the electrodialytic on-column suppressor on co-current mode has a potential of being used as an enriching capillary column for analyte ions. Design C provides fairly well chemical suppression. Theoretical calculation indicates that the loss of column efficiency can be controlled within 3%.

Development of Analytical Methods for the Determination of N-Bromosuccinimide in Different Active Pharmaceutical Ingredients by High-Performance Ion Chromatography with Suppressed Conductivity Detection

Product safety is important for medicines. For drugs on the market, it must be demonstrated that the levels of toxic contaminants are below the permitted limits. These impurities are used as reagents or are generated during synthesis. N-bromosuccinimide is used as a brominating agent in the synthesis of some active pharmaceutical ingredients. The determination of N-bromosuccinimide is difficult due to its high reactivity. In this work, a high-performance ion chromatographic method was developed for the determination of N-bromosuccinimide. The ion chromatographic measurement can be performed in two ways, one involves the assay of the resulting bromide ion and the other is via the assay of the 3-carbamoyl propanoic acid ion produced from the succinimide. Both acid ions were analyzed on an anion exchange column by gradient elution with potassium hydroxide eluent and detection was performed by a suppressed conductivity detector. During the method development, the results showed that the measurement of bromide ion was more selective than the measurement of 3-carbamoyl propanoic acid ion. Two different types of active pharmaceutical ingredients (API), i.e., prasugrel and favipiravir, were chosen to test the developed method and sample preparation. For both APIs, sample preparation was performed in a vial and consists of liquid–liquid extraction with an alkaline reagent. Finally, the anion exchange ion chromatography method was validated at the limit value level, and harmonized with the guidelines. For prasugrel, the quantification limits and the accuracy at the limit level are 7.2 ppm and 96.4%, while for favipiravir these are 7.5 ppm and 114.7%, respectively.

Chromatographic analysis of oxidized cello-oligomers generated by lytic polysaccharide monooxygenases using dual electrolytic eluent generation

Research on oligosaccharides, including the complicated product mixtures generated by lytic polysaccharide monooxygenases (LPMOs), is growing at a rapid pace. LPMOs are gaining major interest, and the ability to efficiently and accurately separate and quantify their native and oxidized products chromatographically is essential in furthering our understanding of these oxidative enzymes. Here we present a novel set of methods based on dual electrolytic eluent generation, where the conventional sodium acetate/sodium hydroxide (NaOAc/NaOH) eluents in high-performance anion-exchange chromatography (HPAEC) are replaced by electrolytically-generated potassium methane sulfonate/potassium hydroxide (KMSA/KOH). The new methods separate all compounds of interest within 24–45 min and with high sensitivity; limits of detection and quantification were in the range of 0.0001–0.0032 mM and 0.0002–0.0096 mM, respectively. In addition, an average of 3.5 times improvement in analytical CV was obtained. This chromatographic platform overcomes drawbacks associated with manual preparation of eluents and offers simplified operation and rapid method optimization, with increased precision for less abundant LPMO-derived products.

Hyperbranched anion exchangers prepared from polyethylene polyamine modified polymeric substrates for ion chromatography

High hydrophilic anion stationary phases play a crucial role in the separation behavior of ion chromatography. Herein, we report novel polymeric anion exchangers grafted with polyethylene polyamines, including ethylenediamine, diethylenetriamine, triethylenetetramine and tetraethylene pentaamine, via a facile epoxy-amine polymerization method. The anion exchangers were characterized by scanning electron microscopy, thermogravimetry, Fourier transform infrared spectrometry and elemental analysis. The chromatographic performance of the stationary phases was evaluated with the separation of common inorganic anions, organic weak acids and highly polarizable anions. Seven common anions (F−, Cl−, NO2−, Br−, NO3−, SO42− and HPO42−) can be separated within 18 min by using hydroxide eluent in isocratic mode. By adopting different polyethylene polyamines as hyperbranched units, the four types of new stationary phases displayed high efficiencies and good reproducibility. The columns exhibit large exchange capacities at 76.5–184.8 μmol•column−1 (4.6 × 150 mm, i.d.) with efficiency up to 20293 plate m−1 (Cl−). The RSDs of the retention time were less than 0.27% and the RSDs of the efficiency were less than 1.95% by consecutive injections after working for two months. The self-fabricated column was successfully applied to determine the chloride content in exhaled breath condensate.

Hydrophobicity of polymer based anion-exchange columns for ion chromatography

The regularities of the retention of alkanoic and alkanesulfonic acids homologues were investigated for the set of 36 anion-exchange columns produced by various manufacturers. The role of hydrophobic and electrostatic interactions in the retention and separation of organic anions was studied. The methylene selectivity increments α(CH2) were measured for the studied columns with 10 mM sodium hydroxide eluent. The influence of matrix, surface area, polar group structure, ion-exchange capacity, the density of charged functional groups on the surface and other characteristics of anion-exchangers on resin hydrophobicity was considered.A unified approach for the measurements of hydrophobic properties of anion-exchange resins is proposed and the ratio of chloride retention factor (kCl) to α(CH2) was introduced as mixed-mode factor. The synergetic effect of electrostatic and hydrophobic interactions was observed.

Gradient nanopump based suppressed ion chromatography using PEEK open tubular columns

The progress of open tubular suppressed ion chromatography is reviewed from a broad perspective. Then a suppressed open tubular ion chromatography system, composed mostly of commercial components, is described: A splitless gradient-capable nanopump providing eluent delivery in the 10–1000 nL/min regime, a 7-nL injector integral to the pump, an anion exchange column based on a 25 µm i.d. poly(ether ether ketone) (PEEK) capillary, and an on-column admittance detector. The membrane suppressor was custom fabricated. Good gradient separation of seven common anions in 9 min is demonstrated using a manually prepared hydroxide eluent.

Manipulating selectivity of covalently-bonded hyperbranched anion exchangers toward organic acids. Part IV: General algorithm based on the variation of external part of the functional layer

Novel anion exchangers with mono- and dialkanolamines in the external part of covalently-bonded hyperbranched functional layer were synthesized. Comparison of the chromatographic properties of the prepared stationary phases in suppressed ion chromatography (IC) mode using hydroxide eluent allowed us to evaluate the effect of the number of substituents at nitrogen atom in alkanolamine on selectivity of anion exchangers toward organic acids. Obtained anion exchangers were also examined together with previously described hyperbranched stationary phases with different mono- and diamines in the external part of the layer for evaluating the influence of various parameters on their selectivity. Effects of hydrophilicity, functionality of amine, and the number of substituents at nitrogen atom of amine used in the last modification cycle were established independently from each other, which provided the possibility to tailor selectivity toward organic acids when preparing anion exchanger for solving particular analytical task. Predominance of hydrophilicity as a key factor affecting the separation of weakly retained organic acids over other studied parameters was demonstrated.

An electrodialytic potassium hydroxide eluent generator suited to small bore ion chromatography

An electrodialytic potassium hydroxide eluent generator (EDG) well-suited to small-bore (2 mm i.d.) ion chromatography (sIC) system is described. The basic configuration of EDG cartridge is much similar to that of conventional counterpart except lower void volume, which was measured to be ˜9 μL relative to ˜ 31 μL of conventional one. The corresponding dispersion volume was computed to be ˜58 μL and ˜106 μL, respectively. Lower void volume will improve the sharpness of a step gradient (or steeper rise curve), which is helpful to gradient elution. The device can bear the pressure up to at least 20.5 MPa and the effective KOH concentration that can be generated is ranging from 0.15 mM to 100 mM at the typical flow rate of sIC (0.4 mL/min). High purity of the produced KOH eluent and excellent running reproducibility of the device were observed, as indicated by the typical suppressed background (0.36 μS/cm) and <0.11% (or <0.23%) of the relative standard deviation of the retention times for common inorganic anions achieved under isocratic (or gradient) mode.

Manipulating selectivity of covalently-bonded hyperbranched anion exchangers toward organic acids. Part I: Influence of primary amine substituents in the internal part of the functional layer

Three covalently-bonded poly(styrene-divinylbenzene)-based (PS-DVB) hyperbranched anion exchangers prepared using primary amines with carboxylic, sulfonic or 2-hydroxyethyl substituents in the internal part of the functional layer were studied and compared for evaluating the effect of amines substituents on the chromatographic performance of the stationary phases. The hyperbranched coating was created on the surface of aminated PS-DVB substrate by repeating the modification cycles including alkylation with 1,4-butanediol diglycidyl ether (1,4-BDDGE) and amination with primary amine; glycine, taurine (2-aminoethanesulfonic acid) or ethanolamine were used in the first cycle, and 4 more cycles were conducted with methylamine (MA). The influence of the structure and type of primary amine used in the first cycle on the retention and selectivity toward inorganic anions, short-chain carboxylic acids, and polyphosphates was investigated using hydroxide eluent. The effect of temperature on the separation of organic acids was also studied for all stationary phases, which revealed the possibility to control and improve resolution for some pairs of analytes.

Dual Electrolytic Eluent Generation for Oligosaccharides Analysis Using High-Performance Anion-Exchange Chromatography.

The research on oligosaccharides is growing and gaining in importance at a rapid pace. The efforts to understand their bioactivity and to develop new products based on oligosaccharides in biotherapeutics and food industry require effective and reliable tools for analysis of oligosaccharides. Here we present a dual electrolytic eluent generation platform for the analysis of oligosaccharides by high-performance anion-exchange liquid chromatography (HPAE) in both analytical and capillary column formats. The system consists of one eluent generator producing methanesulfonic acid (MSA) connected in series with a second eluent generator producing potassium hydroxide (KOH). Through manipulating the concentration output of both eluent generators, chromatographic performance comparable to that obtained using the conventional sodium acetate/sodium hydroxide (NaOAc/NaOH) eluents is achieved using the electrolytically generated potassium methanesulfonate/potassium hydroxide (KMSA/KOH) eluent. This platform utilizes deionized water as the only carrier stream through a single isocratic pump, overcomes the various drawbacks associated with manually prepared NaOAc/NaOH eluents, and offers an easy to use, simplified operation solution for oligosaccharides profiling with increased precision and accuracy.

Assuring precision and calibration linearity in ultrasensitive suppressed ion chromatography by using deliberately contaminated hydroxide eluent

Experimental: and theoretical studies were conducted to investigate low and non-linear responses in sub-micro molar-level suppressed ion chromatography with a hydroxide eluent. A calculated response was derived using experimentally determined detector effluent ion composition data and compared with measured experimental responses. The calibration curve was non-linear, and its slope varied considerably with different instrumental setups and operating conditions. The non-linearity of the solution conductivity response was determined by two acid-base equilibria of water and carbonic acid, and fluoride ion. By using eluent contaminated with para-toluene sulfonate at micro molar level, the non-linear response was greatly alleviated.

Hyperbranched anion exchangers prepared from thiol-ene modified polymeric substrates for suppressed ion chromatography

The covalent modification of polymeric particles has been a challenge due to their chemical inertness. Herein we describe a facile and neat method via thiol-ene reaction to immobilize cysteamine/cysteine onto allylmethacrylate-divinylbenzene (AMA-DVB) and ethylvinylbenzene-divinylbenzene (EVB-DVB). The introduced functionalities were verified by elementary analysis and scanning electron microscope coupled with energy dispersive spectroscopy. This modification enabled the further grafting of hyperbranched condensation polymers (HBCPs), which converted the particles into anion exchangers. The anion exchangers demonstrated good separation with carbonate/bicarbonate eluents and hydroxide eluents for suppressed anion exchange chromatography. In the case of a 3-layered anion exchanger, simultaneous separation of nine model anions was achieved by isocratic elution in 17 min with efficiencies up to 16,100 plates m−1. High stability and dependable durability was exhibited in long-term tests.

Automated Programmable Preparation of Carbonate-Bicarbonate Eluents for Ion Chromatography with Pressurized Carbon Dioxide.

We introduce a novel carbonate-bicarbonate eluent generation system in which CO2 is introduced using programmable CO2 pressures across a membrane into a flowing solution of electrodialytically generated high purity KOH. Many different gradient types are possible, including situations where gradients are run both on the [KOH] and the CO2 pressure. The system is more versatile than current electrodialytic carbonate eluent generators and can easily generate significantly higher eluent concentrations (at least to 40 mM carbonate), paving the way for future higher capacity columns. Demonstrably purer carbonate-bicarbonate eluent systems are possible compared to manually prepared carbonate-bicarbonate eluents and with considerable savings in time. Performance in different modes is examined. The dissolved CO2 is removed by a carbon dioxide removal device prior to detection. Best case noise levels are within a factor of 2-3 of best case suppressed hydroxide eluent operation. The eluent system allows particular latitude in controlling elution order/time of polyprotic acid analytes. Although CO2 introduction is possible prior to hydroxide eluent generation, this configuration causes complications because of electroreduction of CO2 to formate.

Ion chromatographic determination of chlorite and chlorate in chlorinated food using a hydroxide eluent

This study was conducted to develop an analytical technique for determination of chlorite and chlorate concentrations in fresh-cut food and dried fish products by an ion chromatography/conductivity detection method using a hydroxide mobile phase. Deionized water was added to homogenized samples, which were then extracted by ultrasound extraction and centrifuged at high speed (8,500 rpm). Subsequently, a Sep-Pak tC18 cartridge was used to purify the supernatant. Chlorite and chlorate ions were separated using 20 mM KOH solution as the mobile phase and Dionex IonPac AS27 column as the stationary phase. Ethylenediamine was used as sample preservative and dibromoacetate was added to adjust for the disparity in extraction efficiencies between the food samples. The method detection limit) for chlorite and chlorate were estimated to be 0.2 mg/kg and 0.1 mg/kg, respectively, and the coefficient of determination (<TEX>$r^2$</TEX>) that denotes the linearity of their calibration curves were correspondingly measured to be 0.9973 and 0.9987. The recovery rate for each ion was 92.1 % and 96.3 %, with relative standard deviations of 7.47 % and 6.18 %, respectively. Although neither chlorite nor chlorate was detected in the food samples, the analytical technique developed in this study may potentially be used in the analysis of disinfected food products.

Anion exchangers with negatively charged functionalities in hyperbranched ion-exchange layers for ion chromatography

Novel pellicular poly(styrene-divinylbenzene)-based (PS-DVB) anion exchangers with covalently-bonded hyperbranched functional ion-exchange layers containing negatively charged functionalities are obtained and examined. The hyperbranched coating is created on the surface of aminated PS-DVB substrate by repeating the modification cycles including alkylation with 1,4-butanediol diglycidyl ether (1,4-BDDGE), and amination of the terminal epoxide rings with methylamine (MA) or glycine (Gly). The influence of the position and the number of the layers with glycine, as well as of the total number of the layers of amine in the coating on the chromatographic properties of the obtained stationary phases is investigated. Chromatographic performance of the obtained stationary phases is evaluated using the model mixtures of inorganic and organic anions with hydroxide eluent. It is shown that the best selectivity toward weakly retained organic acids and oxyhalides is possessed by the anion exchanger obtained after 5 modification cycles, with glycine being used in the first one. Such anion exchanger packed in 25-cm long column is capable of separating 22 anions in 58min including 7 standard anions, mono-, di- and trivalent organic acids, oxyhalides, and some other double- and triple-charged anions.

Functionalized Cycloolefin Polymer Capillaries for Open Tubular Ion Chromatography.

We describe novel cycloolefin polymer (COP)-based open tubular capillary ion exchange columns. COP capillaries (inner diameter of 19-28 μm) were successfully sulfonated at room temperature using a cocktail of ClSO3H (85-95% w/w) and HOAc or H2SO4. The cation exchange capacity is controlled by the sulfonation time and the sulfonation solution composition and can be as high as 300 pequiv/mm2. Following sulfonation, the capillaries were coated with 65-nm-diameter anion exchanger (AEX) latex nanoparticles that attach electrostatically. The typical anion exchange capacities were ∼20 pequiv/mm2. The chromatographic behavior of the AEX latex-coated COP capillaries are greatly dependent on the degree of sulfonation. When the base is heavily sulfonated, neutrals elute after the anions. The position of the water dip varies with the degree of sulfonation; the elution order is normal (water dip appear before anions) only with lightly sulfonated columns. On silica (-SiOH) or poly(methyl methacrylate) (-COOH) surfaces, AEX latex attachment is not stable over long periods in significant concentrations of strong base (e.g., ≥10 mM NaOH). Latex attachment on sulfonated COP surfaces are much stronger; several types show sufficient binding to be used over long periods at practical eluent concentrations, paving the way for suppressed hydroxide eluent ion chromatography (IC), which is discussed in a companion paper. Another interesting feature of COP capillaries lies in their flexibility. If softened at modestly elevated temperatures (e.g., boiling water), they can be coiled down to <1 mm coil radii, revealing, for the first time, the beneficial effects, albeit small, of centrifugal force on mass transfer in open tubular columns.

Low‐temperature Conductivity Detection for Ultrasensitive Ion Chromatography

The effects of conductivity detection temperature on calibration sensitivity and linearity in suppressed ion chromatography using hydronium or hydroxide eluent were investigated. Theoretical calibration curves for lithium and nitrate ions at 0–35 °C were calculated and compared with experimental data. As the detection temperature was lowered, both sensitivity and linearity of calibration at low concentrations were improved due to the reduced interference by water autoionization equilibrium; 4.3‐ and 1.3‐fold increases in linear regression slopes were observed in the 0–1 µmol/L range when the temperature was lowered from 35 to 5 °C for lithium and nitrate, respectively, along with significant increases in the correlation coefficient. Any remaining water autoionization effect was near completely removed by using eluents contaminated with rubidium or bromide ion at 0.1 µmol/L.

Anion exchangers with branched functional ion exchange layers of different hydrophilicity for ion chromatography

Novel polystyrene-divinylbenzene (PS-DVB) based anion exchangers differing from each other in the structure of the branched functional ion exchange layer are prepared to investigate the role of linker and functional site on ion exchange selectivity. The proposed method of synthesis includes the obtaining of aminated PS-DVB particles by means of their acylation with following reductive amination with methylamine. Further modification of the obtained secondary aminogroups is provided by the alkylation with either 1,4-butanediol diglycidyl ether (1,4-BDDGE) or resorcinol diglycidyl ether (RDGE), which form the linkers of different hydrophobicity, and amination of terminal epoxide rings with trimethylamine (TMA), dimethylethanolamine (DMEA), methyldiethanolamine (MDEA) or triethanolamine (TEA). The variation of the structure and hydrophobicity of the linker and terminal quaternary ammonium sites in the functional layer allows the alteration of selectivity and separation efficiency of the obtained adsorbents. The ion exchange selectivity and separation efficiency of the anion exchangers are evaluated using the model mixtures of anions (F−, HCOO−, Cl−, NO2−, Br−, NO3−, HPO42− and SO42−) in potassium hydroxide eluents. The adsorbents show the decrease of selectivity with increasing the hydrophilicity of the terminal functional site. The anion exchangers having more flexible and hydrophilic 1,4-BDDGE linker provide smaller separation factors for most of the analytes as compared with RDGE-containing adsorbents with the same terminal ion exchange sites, but are characterized with higher column efficiencies and better peak symmetry for polarizable anions. In case of 1,4-BDDGE-modified anion exchangers of the particle size of 3.3μm functionalized with DMEA and MDEA the calculated values of column efficiencies for polarizable NO3− and Br− are up to 49,000 and 53,000N/m, respectively, which is almost twice higher than the values obtained for the RDGE-containing analogues.

Enigmatic ion-exchange behavior of myo-inositol phosphates.

The separation of myo-inositol mono-, di-, tri-, tetra-, pentakis-, and hexakisphosphate (InsP1, InsP2, InsP3, InsP4, InsP5, InsP6) was carried out using hydroxide eluent ion chromatography. Acid hydrolysis of InsP6 (phytate) was used to prepare a distribution of InsPs, ranging from InsP1 to InsP5's and including unhydrolyzed InsP6. Counting all possible positional isomers (many of which have stereoisomers that will not be separable by conventional ion exchange), 40 chromatographically separable peaks are possible; up to 22 were separated and identified by mass spectrometry. InsPs show unusual ion-exchange behavior in two respects: (a) the retention order is not monotonically related with the charge on the ion and (b) at the same hydroxide eluent concentration, retention is greatly dependent on the eluent metal cation. The retention of InsP3-InsP6 was determined to be controlled by steric factors while elution was influenced by eluent cation complexation. These highly phosphorylated InsPs have a much greater affinity for alkali metals (Li(+) > Na(+) > K(+)) than quaternary ammonium ions. This difference in cation affinity was exploited to improve separation through the use of a tetramethylammonium hydroxide-sodium hydroxide gradient.

Novel Anion Exchangers with Spatially Distant Trimethylammonium Groups in Linear and Branched Hydrophilic Functional Layers

Novel polystyrene–divinylbenzene (PS-DVB) based anion exchangers having branched structure of ion exchange functional groups are prepared and characterised. Two proposed synthetic methods include acylation of PS-DVB microspherical particles followed by reductive amination with dimethylamine or methylamine, which results in obtaining tertiary or secondary amino groups on the surface, respectively. Further alkylation of amino groups was provided by reaction with either (3-chloro-2-hydroxypropyl)trimethylammonium chloride or glycidyltrimethylammonium chloride, which allowed the simultaneous insertion of trimethylammonium functional group and hydrophilic spacer into the structure. The other considered option is reaction with diglycidyl ethers, which requires further amination of terminal epoxide rings with trimethylamine. The proposed approaches resulted in preparation of the ion exchangers with extended linear or branched anion exchange structure with trimethylammonium groups located at a distance from the surface and connected via hydrophilic linkers. The ion exchange selectivity and separation efficiency of obtained anion exchangers are studied for the model mixture of inorganic anions (F−, HCOO−, Cl−, NO2−, Br−, NO3−, H\( {\text{PO}}_{ 4}^{2 - } \) and \( {\text{SO}}_{4}^{ 2- } \)) using hydroxide eluents. The adsorbents with branched ion exchange layer having three positively charged sites in the structure of attached groups demonstrate superior column efficiencies and better peak symmetry as compared with anion exchanger having linear structure with two positively charged sites in the chain. In case of branched anion exchangers the calculated values of column efficiencies for polarisable NO3− and Br− are 44,000 and 50,000 N/m, respectively, and significantly higher than 7,000 and 8,000 N/m obtained for anion exchanger with linear structure of the ion exchange layer.