New monolith technology for automated anion-exchange purification of nucleic acids.

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.

Effect of ion-exchange site and eluent modifiers on the anion-exchange of carboxylic acids.

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.

New block-grafted anion exchanger for environmental water analysis by ion chromatography.

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.

Improved method for the determination of trace perchlorate in ground and drinking waters by ion chromatography.

Ammonium perchlorate, a key ingredient in solid rocket propellants, has been found in ground and surface waters in a number of U.S. states, and perchlorate contamination of public drinking water wells is now a serious problem in California. Perchlorate poses a health risk and preliminary data from the U.S. EPA reports that exposure to less than 4-18 microg/l provides adequate human health protection. An improved ion chromatographic method was developed for the determination of low microg/l levels of perchlorate in ground and drinking waters based on a Dionex IonPac AS16 column, an hydroxide eluent generated using an EG40 automated eluent generator, large loop (1000 microl) injection, and suppressed conductivity detection. The method is free of interferences from common inorganic anions, linear over the range of 2-100 microg/l perchlorate, and quantitative recoveries are obtained for low microg/l levels of perchlorate in spiked ground and drinking water samples. The MDL of 150 ng/l permits quantification of perchlorate below the levels that ensure adequate health protection.

Determination of inorganic anions in environmental waters with a hydroxide-selective column.

US Environmental Protection Agency Method 300.0 specifies the use of an IonPac AS4A anion-exchange column with a carbonate-hydrogencarbonate eluent and suppressed conductivity detection for the determination of inorganic anions in environmental waters by ion chromatography. Hydroxide eluents have not typically been used for the routine analysis of common inorganic anions due to the lack of an appropriate hydroxide selective column and the difficulty in preparing contaminant free hydroxide eluents. The use of ion chromatography with a hydroxide-selective IonPac AS17 column, automated eluent generation and potassium hydroxide gradient represents a new approach to the routine determination of inorganic anions in environmental waters. This new approach, which is a modification of Method 300.0, allows equivalent method performance with improved linearity, precision, and method detection limits. The AS17 column provides superior retention of fluoride from the column void volume and improved resolution from small organic acids, such as formate and acetate, compared to the AS4A column. Quantitative recoveries were obtained for all the common inorganic anions spiked into typical environmental waters using this new approach, and the Performance Based Measurement System Tier 1 method validation quality control acceptance criteria are well within the acceptable ranges defined by Method 300.0. In addition, the EG40 eluent generator eliminates the need to manually prepare eluents, increasing the level of automation and ease-of-use of the ion chromatography system.

Ion chromatographic determination of acidity.

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.

Practical approach to bacterial meningitis in childhood.

The classic triad of headache, fever and nuchal rigidity that occurs in adults with bacterial meningitis is often absent in children. Evaluation of the cerebrospinal fluid remains the gold standard for the diagnosis of bacterial meningitis. The choice of antibiotic therapy is dependent on the most likely age-specific pathogen and the drug's bactericidal activity in cerebrospinal fluid. Routine fluid restriction is no longer recommended in the initial management of critically ill patients. Dexamethasone has become an important adjunct to antimicrobial therapy for meningitis due to Haemophilus influenzae type b. Prevention, especially administration of H. influenzae type b vaccine at an early age, is probably the most effective way to reduce the significant mortality and morbidity associated with bacterial meningitis in children.