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Ion chromatography-electrospray tandem mass spectrometry (IC-ESI-MS/MS) is used to determine nine haloacetic acids (HAAs), bromate, and dalapon in drinking water samples in U.S. EPA Method 557. In this method, all target analytes are separated and measured with good sensitivity without the need for sample preconcentration or derivatization. However, the separation time is relatively long. In order to reduce the sample analysis time in EPA Method 557, a new anion exchange column has been developed to perform fast separation of the target analytes. Using this new anion exchange column, nine HAAs, bromate, and dalapon can be resolved and separated from interfering matrix ions within 40 minutes, about 33% faster than the analysis time obtained using an earlier anion exchange column reported in EPA Method 557. The new anion exchange column has unique selectivity and high exchange capacity. Method optimization, simplification and improvements in robustness are demonstrated while validating the new column suitability for the determine of HAAs, bromate and dalapon according to EPA Method 557., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

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.

Characterization of glycans present on glycoproteins has become of increasing importance due to their biological implications, such as protein folding, immunogenicity, cell-cell adhesion, clearance, receptor interactions, etc. In this study, the resolving power of high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) was applied to glycan separations and coupled to mass spectrometry to characterize native glycans released from different glycoproteins. A new, rapid workflow generates glycans from 200 μg of glycoprotein supporting reliable and reproducible annotation by mass spectrometry (MS). With the relatively high flow rate of HPAE-PAD, post-column splitting diverted 60% of the flow to a novel desalter, then to the mass spectrometer. The delay between PAD and MS detectors is consistent, and salt removal after the column supports MS. HPAE resolves sialylated (charged) glycans and their linkage and positional isomers very well; separations of neutral glycans are sufficient for highly reproducible glycoprofiling. Data-dependent MS 2 in negative mode provides highly informative, mostly C- and Z-type glycosidic and cross-ring fragments, making software-assisted and manual annotation reliable. Fractionation of glycans followed by exoglycosidase digestion confirms MS-based annotations. Combining the isomer resolution of HPAE with MS 2 permitted thorough N-glycan annotation and led to characterization of 17 new structures from glycoproteins with challenging glycan profiles.

A microfluidic ion-suppression module for use in ion-exchange chromatography has been developed and evaluated. The device consists of an ion-exchange membrane clamped between two polymer chips featuring a 200×100μm (width×depth) eluent channel (l=60mm), and a 300×150μm regenerant channel (60mm), respectively. The suppression efficacy using a Nafion membrane was compared with that of a styrene-sulfonate grafted fluorinated ethylene propylene (FEP) membrane. The latter was found to outperform Nafion in terms of lowest attainable background signal (suppression efficacy) and dynamic suppression range. Increasing the suppressor temperature or the sulfuric acid regenerant concentration led to an extension of the operational suppression range, this however at the cost of an increased background signal due to enhanced diffusion, inducing sulfate bleed. Under optimized operating conditions, the microfluidic suppressor provided a dynamic capacity of 0.35μEq./min, being compatible with gradient separations applying up to 70mM KOH in combination with 400μm i.d. capillary columns operated at the optimal flow velocity. The applicability of the miniaturized suppressor is demonstrated for both isocratic and gradient separations of mixtures of inorganic anions. Band-broadening characteristics of the suppressor were optimized with respect to a commercial capillary hollow-fiber suppressor, yielding comparable overall system efficiency, e.g., 8500 plates for nitrate recorded on a 150mm long capillary column. A second chip device was also constructed, featuring suppression at both sides of the eluent flow path. This double-sided suppressor allowed to increase sample throughput and operate at eluent flow rates of 10μL/min, while maintaining efficient suppression characteristics., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This work describes chromatographic properties of reversed-phase/cation-exchange/anion-exchange trimodal stationary phases. These stationary phases were based on high-purity porous spherical silica particles coated with nano-polymer beads using an electrostatically driven self-assembly process. The inner-pore area of the material was modified covalently with an organic layer that provided both reversed-phase and anion-exchange properties while the outer surface was coated with nano-sized polymer beads with strong cation-exchange characteristics. This design ensured spatial separation of the anion-exchange and the cation-exchange regions, and allowed reversed-phase, anion-exchange and cation-exchange retention mechanisms to function simultaneously. Chromatographic evaluation of ions and small molecules suggested that retention of ionic analytes was influenced by the ionic strength, pH, and mobile phase organic solvent content, and governed by both ion-exchange and hydrophobic interactions. Meanwhile, neutral analytes were retained by hydrophobic interaction and was mainly affected by mobile phase organic solvent content. Depending on the specific application, selectivity could be optimized by adjusting the anion-exchange/cation-exchange capacity ratio (selectivity), which was achieved experimentally by using porous silica particles with different surface areas., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The effect of Fe³(+) treatment on weak cation exchange column chromatography was demonstrated for monoclonal antibody (MAb) analysis. Fe³(+)-exposed columns showed lowered relative peak areas of the parent MAb peak as well as both acidic and basic variant peaks that could lead to erroneous conclusions. Accurate measurement of relative amounts of variants to the parent MAb is essential for demonstrating the safety and efficacy of therapeutic molecules such as MAbs. Complete reversal of the compromised MAb analysis performance was observed after washing the column with chelating agents, confirming that metal contamination was responsible for the compromised column performance., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Synthetic nucleic acid analysis often employs pellicular anion-exchange (AE) chromatography because it supports very high efficiency separations while offering means to control secondary structure, retention and resolution by readily modifiable chromatographic conditions. However, these pellicular anion-exchange (pAE) phases do not offer capacity sufficient for lab-scale oligonucleotide (ON) purification. In contrast, monolithic phases produce fast separations at capacities exceeding their pellicular counterparts, but do not exhibit capacities typical of fully porous, bead-based, anion-exchangers. In order to further increase monolith capacity and obtain the selectivity and mass transfer characteristics of pellicular phases, a surface-functionalized monolith was coated with pAE nanobeads (latexes) usually employed on the pellicular DNAPac phase. The nanobead-coated monolith exhibited chromatographic behaviors typical of polymer AE phases. Based on this observation the monolithic substrate surface porosity and latex diameters were co-optimized to produce a hybrid monolith harboring capacity similar to that of fully porous bead-based phases and peak shape approaching that of the pAE phases. We tested the hybrid monolith on a variety of previously developed pAE capabilities including control of ON selectivity, resolution of derivatized ONs, the ability to resolve RNA ONs harboring aberrant linkages at different positions in a single sequence and separation of phosphorothioate diastereoisomers. We compared the yield and purity of an 8 mg ON sample purified on both the new hybrid monolith and a benchmark AE column based on fully porous monodisperse beads. This comparison included an assessment of the relative selectivities of both columns. Finally, we demonstrated the ability to couple AE ON separations with ESI-MS using an automated desalting protocol. This protocol is also useful for preparing ONs for other assays, such as enzyme treatments, that may be sensitive to high salt levels., (Copyright 2010 Elsevier B.V. All rights reserved.)

We have developed a number of new anion exchange stationary phases based on synthesis of hyperbranched condensation polymers prepared in situ on surface sulfonated wide pore, low surface area ethylvinylbenzene-divinylbenzene substrate beads. These new stationary phases are synthesized by exposing the substrate beads to a mixture containing one of several different diepoxides and methyl amine followed by exposure in an alternating fashion to diepoxide and then methyl amine in a repetitive manner. With each cycle of reaction with first diepoxide and then methyl amine, the capacity of the stationary phase doubles, allowing for the synthesis of high-capacity materials. These new phases are stable to hydrolysis in concentrated acid and base. They exhibit exceptionally low levels of hydrophobic interaction with anionic species commonly of interest in the analysis of environmental samples.

The United States Environmental Protection Agency (EPA) collected drinking water occurrence data for perchlorate in the Unregulated Contaminant Monitoring Regulation (UCMR 1; 2001-2005) using EPA Method 314.0. To address the interest in increasing sensitivity and selectivity for the analysis of perchlorate, three new methods, EPA Methods 314.1, 331.0 and 332.0, were subsequently published by EPA for the analysis of perchlorate in drinking water. In 2006, an automated two-dimensional ion chromatography (2D-IC) method for measuring perchlorate with suppressed conductivity detection was developed. Two-dimensional IC is essentially an automated "heart-cutting", column concentration and matrix elimination technique. In the first dimension, a large sample volume is injected onto a first separation column and the separated matrix ions are diverted to waste while the analyte(s) of interest are selectively cut, trapped and concentrated in a concentrator column. In the second dimension, the contents from the concentrator column are eluted onto a second analytical column for separation and quantitation of the analyte(s) of interest. Incorporation of two columns with different affinities for the analyte(s) in a single analysis can provide comparable selectivity and superior sensitivity to a method using second column confirmation in a second separate analysis step. Use of this approach led to the development of a new, highly sensitive and selective 2D-IC, suppressed conductivity method with a Lowest Concentration Minimum Reporting Level (LCMRL) of 55 ng/L for perchlorate in drinking water samples. This new method has comparable sensitivity and selectivity and is simpler and more economical than IC-mass spectrometric (MS) or IC-MS-MS techniques. The method is now being prepared for publication as EPA Method 314.2.

A capillary-scale high-pH anion-exchange chromatography (HPAEC) system for the analysis of carbohydrates was developed, in combination with two parallel on-line detection methods of sub-picomolar sensitivity: (1) pulsed amperometric detection (PAD); (2) capillary-scale desalting followed by electrospray ion-trap (IT) mass spectrometry (MS). The capillary chromatographic system combined the superb selectivity of HPAEC that allows routine separation of isomeric oligosaccharides with the information on monosaccharide sequence and linkage positions obtained by MS/MS fragmentation using the IT-MS. The applicability of the system in biomedical research was demonstrated by its use for the analysis of a urine sample of a GM1-gangliosidosis patient. Isomeric glycans in the sample could be resolved by HPAEC and assigned on the basis of the monosaccharide linkage information revealed by on-line IT-MS/MS.

Strong anion exchange columns are preferred for oligonucleotide analyses due to their ability to effectively control secondary structure and poly(G) interactions. Methacrylate-based anion exchange phases minimize hydrophobic interactions with oligonucleotides, but they also tend to hydrolyze under alkaline conditions. In this article, we report the use of an anion exchange column prepared from a new class of methacrylate monomers designed to improve hydrolytic stability. This column is used to show predictable adjustment of oligonucleotide retention by eluent pH and composition. Features of the new column include (i) large, predictable, pH-dependent retention shifts (varying with specific changes in 5' or 3' terminal bases with NaCl-based eluents); (ii) reduced retention when solvent is added to NaCl-based eluents; and (iii) suppression of much of the column's hydrophobic interactions when CH3CN is used with NaClO4-based eluents at a neutral pH (i.e., this eluent system separates oligonucleotides primarily in order of their length). These observations will aid the development of elution conditions for both size-dependent and base sequence-dependent (or base composition-dependent) separations.

Recent developments in new electrolytic devices that utilize the electrolysis of water and charge-selective electromigration of ions through ion-exchange media have significantly changed the routine operation of ion chromatographic methods. Examples of these new electrolytic devices include on-line eluent generators that produce high-purity electrolyte eluents using deionized water as the carrier stream, continuously regenerated trap columns that remove ionic contaminants in the eluents, and continuously regenerated suppressors that reduce eluent background conductance prior to conductivity detection. The combined use of these electrolytic devices has made it possible to perform various ion chromatographic separations using only deionized water as the mobile phase. This paper reviews the operation principles of these electrolytic devices and their applications in the ion chromatographic determination of anionic and cationic analytes.

This report documents the development of a new monosaccharide separator column (CarboPac PA20, 3x150 mm) that allows fast, efficient monosaccharide separations with good spacing. It is based on a new chemistry with a reduced resin particle size (from 10 to 6.5 microm). Faster, more efficient separations of glycoprotein monosaccharides with better spacing were achieved across a range of isocratic NaOH concentrations at lower flow rates. Detection sensitivity was improved, enabling routine low to sub pmol monosaccharide determinations. Glycoprotein monosaccharides eluted in less than 10 min at a flow rate of 0.5 ml/min. Furthermore, when used with an AminoTrap guard column, the protein matrix consisting of amino acids and peptides (released by acid hydrolysis of glycoprotein) did not interfere with monosaccharide analysis. Compared to previous CarboPac columns (CarboPac PA1 and CarboPac PA10), the CarboPac PA20 has improved selectivity with respect to glycoprotein monosaccharides. The improved selectivity results in better separation of glucosamine and galactose, enabling the accurate determination of monosaccharide ratios for undergalactosylated glycoproteins. Finally, disposable gold working electrodes that eliminate the possibility of working electrode recession affecting peak area response were used.

The term amines encompasses a wide variety of compounds: monovalent or polyvalent amines, hydrophobic or hydrophilic amines, and combinations of all of these. Due to their charge, polyvalent amines such as the biogenic amines have very strong cation-exchange interaction with the cation-exchange groups in the stationary phase. Very high acid concentrations are required to elute them effectively from a high-capacity, carboxylated cation-exchange column. The eluent must contain a divalent ion to elute them from a sulfonated cation-exchange column due to its selectivity. Chromatography for these amines with a new "tailored" amine column of moderate capacity using a simple acidic eluent is described. The hydrophobic nature of other amines, such as long-carbon-chained amines, results in partitioning into the polymeric substrate of previous carboxylated stationary phases, so that organic solvent is required to elute them effectively. The substrate resin of this new "tailored" amine column is first coated so that, when it is functionalized in a subsequent step, this type of interaction is minimized. Examples are given. Methods that require eluent gradients and/or step changes of eluent concentration are especially well suited to this column because the background conductivity remains almost unchanged under gradient conditions.

The chromatographic behavior of carboxylic acids has been investigated, on three different latex-based anion-exchange columns, in order to define the effect of the ion-exchange site structure on selectivity. The analytical columns produced are characterized by alkyl amines containing zero, one or two hydroxyl groups on the anion-exchange functional site. Divalent carboxylic acids, namely fumaric, maleic, trans-beta-hydromuconic, trans,trans-muconic, oxalic, malonic, succinic, glutaric, adipic, malic, tartaric and mucic acids, have been chosen as test solutes. The performance of the three stationary phases has been studied employing NaOH eluents and has been discussed with respect to the different hydrophilicity of the ion-exchange sites and analytes. Considering on previous results obtained using organic solvents (methanol and acetonitrile) with carbonate eluents on a highly hydrophilic column, the performance of the three exchangers has also been studied using acetonitrile, methanol and n-propanol. The chromatographic behavior was similar for the three columns studied, but the different organic solvents gave variations in selectivity. In order to characterize these differences, particle size measurements of the latices were performed both in pure water and in the presence of each organic solvent studied.

The IonPac AS14A is a recently developed stationary phase that was produced using a new block-grafting technique, which enables the preparation of high-water-content anion exchangers with excellent peak shape and good chromatographic efficiency. The performance of this column for the analysis of inorganic anions was compared to that obtained using an IonPac AS4A column, which is specified in US Environmental Protection Agency Method 300.0, in addition to another commonly used alternative: the AS14 column. The AS14A column is available in two different formats: 250x4 mm I.D. (7.0 microm diameter particle) and 150x3 mm I.D. (5.5 microm diameter particle). The IonPac AS14A (in 4 mm I.D. format) was found to provide similar performance to the AS14 column with increased peak efficiency and better pH stability and is a suitable alternative for the analysis of anions in moderate- to high-ionic-strength samples. The IonPac AS14A (in 3 mm I.D. format) provides comparable run times to the AS4A column with better overall peak selectivity and improved fluoride resolution, hence this column would be a suitable column to substitute in place of either the AS4A or AS14 columns for the analysis of inorganic anions in low- to moderate-ionic-strength environmental waters. The AS14A column used with an Atlas electrolytic suppressor provides equivalent method detection limits to those obtained when using a micromembrane suppressor but with the operational convenience of a self-regenerating suppressor.

The practice of determining acid concentrations by titrations has remained unchanged for more than a century. We introduce a new approach to the determination of acid concentrations based on cation exchange chromatography. We demonstrate the ability of sulfonated styrene-divinylbenzene based stationary phases to separate the hydrogen ion from other monovalent cations. The eluent is a dilute solution of a neutral salt, sometimes containing a small concentration of the corresponding acid, e.g., sodium ethanesulfonate, pH adjusted with ethanesulfonic acid. The high equivalent conductance (approximately 350 S.cm2/equiv) of H+ and relatively low eluent concentration allows sensitive conductometric detection of H+, down to the 50 microM level under favorable conditions. The conductometric response to H+ can be linear over a wide range of H+ concentrations, from sub-millimolar to several molar concentrations. The system allows the rapid quantitation of strong acids; weak acids can also be determined depending on pKa and injected concentration. The determinations of several strong and weak acids are presented along with factors that govern their chromatographic analysis.

In ion chromatography, samples of very different ammonium-to-sodium concentration ratios are difficult to quantify since these two cations have similar selectivities for stationary phases containing commonly used sulfonate or carboxylate cation-exchange functional groups. The IonPac CS15 cation-exchange column, with carboxylate and phosphonate functional groups as well as a crown ether group, was developed to address this limitation. Selectivity for the common inorganic cations on this column is different from that of conventional cation-exchange columns in that the separation between sodium and ammonium ions has been greatly increased, allowing for determinations of low levels of one in the presence of high levels of the other with an isocratic eluent. For larger than 4000:1 sodium-to-ammonium concentration ratios, an eluent step change or gradient elution is needed. For moderate ratios, combinations of this column with a carboxylate column, containing no crown ether group, can be used at room temperature with an isocratic eluent containing no organic solvent.

We developed a set of prototype cation-exchange column packings that are based on a hydrophilic coated, pellicular polymeric support with a grafted tentacular surface chemistry that is highly suited to resolving closely related protein variants. These column packings (1) afford minimal band spreading in conjunction with extremely high selectivity, (2) exhibit a very hydrophilic character and (3) have moderate loading capacity. Cytochrome c variants (bovine, horse, rabbit) were baseline-separated, as was native ribonuclease A and its two deamidation products, the Asp67 and isoAsp67 forms. Humanized monoclonal antibody variants differing in the presence of lysine at the C terminus of the heavy chains were baseline-resolved. Finally, the separation of hemoglobin variants found in a sample containing elevated levels of glycated hemoglobin was also demonstrated.

The monosaccharide content of a glycoprotein is often determined by acid hydrolysis at elevated temperature and subsequent high pH chromatography of the released, underivatized monosaccharides on pellicular anion-exchange resin (HPAE) using pulsed amperometric detection (PAD). We have found that for glycoproteins with low levels of glycosylation, monosaccharide quantitation can be compromised by amino acids fouling the working electrode surface. Specifically, lysine elutes on the CarboPac PA1 column just prior to galactosamine, whereas remaining amino acids and most peptides elute after the monosaccharides and do not interfere with monosaccharide quantification. A direct comparison of PAD vs Abs215 detection of lysine using the CarboPac PA1 column as the separator reveals that lysine does not cleanly come off the working electrode. The monosaccharide response inhibition caused by lysine could be corrected by the posthydrolysis addition of a rhamnose internal standard and the determination of "correction factors." We have developed a guard column with an altered selectivity for amino acids which, when used with a new separator, causes lysine to elute after the monosaccharides and also causes hydrophobic amino acids to elute further after the monosaccharides. Together the new separator and guard columns solve the lysine fouling problem, reduce sample-related baseline noise, and reduce the magnitude of correction factors.