Christopher A. Pohl

Determination of carbohydrates by anion exchange chromatography with pulsed amperometric detection

Abstract Carbohydrates such as sugar alcohols, monosaccharides, disaccharides, and other oligosaccharides are separated as anions by ion exchange chromatography with a sodium hydroxide eluent. Retention time and selectivity are controlled by varying eluent strength and column temperature. The carbohydrates are detected by oxidation at a gold electrode. A repeating sequence of three potentials electrochemically cleans the electrode surface of oxidation products and other interfering species. Detection limits are as low as 30 ppb for sugar alcohols and monosaccharides, and about 100 ppb for oligosaccharides. Other species containing CHOH groups can also be detected, such as alcohols and glycols.

Gradient elution in ion chromatography

Abstract Both theoretical and practical considerations for the use of gradient elution in ion chromatography are discussed. The theory for the dependence of an analytes retention on eluent concentration for both isocratic and gradient elution is presented and shown to agree with experimental results. Practical solutions to problems not encountered in isocratic ion chromatography are presented. Background conductivity changes caused by increasing eluent concentration are greatly minimized by the use of eluents containing salts of weak acids with p K a values greater than 7, which are converted to low-conductivity free acids in the suppressor. Sodium hydroxide based eluents produce a minimal baseline shift. Small baseline shifts produced by other eluents can be compensated by chemical means. Using a gradient, as many as 36 ions can be separated and eluted in one run.

Factors controlling ion-exchange selectivity in suppressed ion chromatography

The control of ion-exchange selectivity is the most important means of moderating separations in suppressed ion chromatography. Selectivity variations are primarily achieved through the use of different stationary phases, hence the construction of the stationary phase plays a key role. The major factors which determine the selectivity of the ion-exchange phase are: the polymer composition of the stationary phase, the type of ion-exchange site and the structure of the ion-exchange site. The eluent can also play a significant role in determining the overall selectivity of an ion-exchange separation. The mobile phase parameters which affect the separation selectivity are: the choice of eluent ion, the concentration (and pH) of the eluent, the presence of non-ionic eluent modifiers and the eluent temperature.

HILIC behavior of a reversed‐phase/cation‐exchange/anion‐exchange trimode column

The hydrophilic interaction liquid chromatography behavior of a novel trimodal stationary phase with reversed-phase/cation-exchange/anion-exchange characteristics was studied. This material is based on nanopolymer silica hybrid technology and provides reversed-phase, strong cation-exchange and weak anion-exchange properties at the same time. It is constructed with high-purity porous spherical silica particles coated with charged organic polymer nano-beads (d(p) approximately 100 nm). The inner-pore area of the silica gel is covalently modified with an organic layer that provides both reversed-phase and weak anion-exchange properties. The outer surface is modified with strong cation-exchange functionality. This chemistry ensures distinctive spatial separation of the anion-exchange and cation-exchange regions, which allows both retention mechanisms to function simultaneously and provides more flexibility in selectivity control. The new phase exhibits hydrophilic interaction liquid chromatography characteristics: at high acetonitrile levels, the retention for charged analytes is governed by an ion-exchange process, accompanied by hydrophilic interaction (partitioning mechanism). This feature provides good flexibility in pharmaceutical method development such as simultaneous separation of hydrophilic drug molecule and its counterions (e.g. penicillin G potassium salt).

Protein variant separations by cation-exchange chromatography on tentacle-type polymeric stationary phases

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.

New hydrophilic interaction/reversed-phase mixed-mode stationary phase and its application for analysis of nonionic ethoxylated surfactants.

We have developed a new stationary phase that combines both hydrophilic interaction and reversed-phase characteristics. The new phase is based on high-purity, porous and spherical silica gel functionalized with a silyl ligand consisting of both hydrophilic and hydrophobic functionalities. This phase can be operated in both HILIC mode (high organic solvent) and RPLC mode (low organic solvent). An optimal balance of hydrophilic and hydrophobic moieties on the silica surface provides unique chromatographic properties that make it useful for determination of alkyl chain distribution and degree of ethoxylation (EO number) of nonionic ethoxylated surfactants.

Capillary ion chromatography

This review summarizes progress in capillary ion chromatography. Theoretical aspects and practical limitations of packed and open tubular capillary columns are considered. Applications of packed and open tubular capillary IC are described. Emerging technologies such chip-scale IC and the use of monolithic columns are discussed.

Anion-exchange selectivity in latex-based columns for ion chromatography

Abstract The anion-exchange columns employed in suppressed ion chromatography contain packings which are agglomerations of anion-exchange latexes and functionalized polymeric substrate. Differences in anion-exchange selectivity studied here are achieved through variations of the properties of the ion-exchange sites and charge density. Column capacity is influenced by the particle sizes of the latexes and substrate. Efficiency is most strongly effected by size of the substrate resin. This paper discusses selectivity variations that are associated with differences in the nature of the quaternary ammonium ion-exchange sites of the latexes and their relationships with various eluents employed in suppressed ion chromatography. Hypotheses are proposed to explain retention behavior of model analyte anions.

Novel cation-exchange stationary phase for the separation of amines and of six common inorganic cations

Abstract Currently available polymeric cation-exchange phases tend to be best suited to the separation of either amines as a class or the inorganic cation as a class. In this paper we will describe a new cation-exchange phase based on a polymeric, highly crosslinked macroporous substrate to which two different monomers, one with carboxylate and the other with phosphonate functional groups, have been covalently grafted. Due to the type of monomers used and the conditions under which the resin is grafted, it offers good selectivity for both inorganic cations and amines, and is more hydrophilic than its predecessor, IonPac CS12 (Dionex). It is possible to chromatograph the more hydrophilic amines without the use of solvent, and for the more hydrophobic amines solvent can be added to the eluent, as the resin is solvent-compatible. Applications in which temperature is used on the column to affect chromatography will be shown.

A new approach to dealing with high-to-low concentration ratios of sodium and ammonium ions in ion chromatography

Abstract In ion chromatography, samples of very dissimilar concentration ratios of ammonium-to-sodium are difficult to quantify since these two cations have similar selectivities for stationary phases containing commonly used sulfonic acid or carboxylic acid cation-exchange functional groups. A new column with carboxylic acid and phosphonate functional groups as well as a crown ether group has been developed to address this limitation. Selectivity of the common inorganic cations is different from conventional columns in that the separation between sodium and ammonium has been greatly increased, and potassium ion elutes after magnesium and calcium. Applications involving very dissimilar concentration ratios of cations can now be done isocratically, with a single column. Other applications of this new column will also be discussed.