Catherine A. Rimmer

The measurement and meaning of void volumes in reversed-phase liquid chromatography

The seemingly simple process of measuring the mobile phase volume, V0, in reversed-phase liquid chromatography has eluded unambiguous agreement for over 25 years. Examples exist in the literature where the reported volume is physically impossible, either equal to or larger than the empty column volume, or being so small that it would represent a total porosity of half the theoretical limit for well-packed columns. Here we review the many proposals for methods of measurement, and compare and critique them. At this time, there is still no consensus for the best method of measurement, and workers are urged to critically examine values they measure, to insure they are at least physically possible.

Development of a Standard Reference Material for Metabolomics Research

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research.

Gradient elution electrochromatography with a flow-injection analysis interface

A flow-injection analysis-capillary electrochromatography interface is used for gradient elution capillary electrochromatography giving purely electroosmotic flow through the analytical column. Solvent gradients were generated with a micro-LC system connected to the interface. Injections were carried out on-line using an inert rotary LC valve controlled by an electric actuator. Gradient shape was measured from acetonitrile (5% acetone)-water (50:50, v/v) to (100:0) in open tubular experiments. When compared to conventional instrumentation, peak tailing and peak width increased slightly using the interface. A test mixture of nine solutes was evaluated in isocratic and gradient elution modes. Using the interface, a gradient of MeCN-water (60:40) to (90:10) provided baseline separation of all nine solutes in under 18 min with good band spacing. Reproducibility of retention times in eight replicate injections was found to be better than 2% R.S.D. for all solutes. This interface also allows use of autoinjectors and dramatically lessens movement of the packed column, improving column lifetime.

Isotope Dilution Liquid Chromatography - Mass Spectrometry Methods for Fat- and Water-Soluble Vitamins in Nutritional Formulations

Vitamins are essential to human health, and dietary supplements containing vitamins are widely used by individuals hoping to ensure they have adequate intake of these important nutrients. Measurement of vitamins in nutritional formulations is necessary to monitor regulatory compliance and in studies examining the nutrient intake of specific populations. Liquid chromatographic methods, primarily with UV absorbance detection, are well established for both fat- and water-soluble measurements, but they do have limitations for certain analytes and may suffer from a lack of specificity in complex matrices. Liquid chromatography-mass spectrometry (LC-MS) provides both sensitivity and specificity for the determination of vitamins in these matrices, and simultaneous analysis of multiple vitamins in a single analysis is often possible. In this work, LC-MS methods were developed for both fat- and water-soluble vitamins and applied to the measurement of these analytes in two NIST Standard Reference Materials. When possible, stable isotope labeled internal standards were employed for quantification.

Gradient elution techniques for capillary electrochromatography.

Capillary electrochromatography (CEC) is a rapidly maturing technique, but still in need of further instrumental development and in need of unique applications that are not possible by traditional pressure-driven LC. We review the development of gradient elution schemes for CEC, beginning with pH gradients initially developed for capillary electrophoresis. Step gradients are the most easily instrumentally implemented, but provide less flexibility in separation than continuous gradients. Pressure-assisted CEC is easily adapted to gradient elution schemes, but does not offer the advantages of very high column efficiency provided by totally electro-driven mobile phases. The development of flow-injection interfaces allows a true solvent gradient to be generated by micro-LC pumps, with the mobile phase drawn into the separation capillary by pure electroosmotic flow. While requiring both a CEC instrument and a traditional pump or pumps capable of generating the gradient, this method offers advantages of greatly reduced column handling, prolonging column lifetimes, and allows simple autosampling. We also discuss voltage gradients, which provide a mobile phase velocity gradient.

Architecture and dynamics of C18 bonded interphases with small molecule spacers.

The relationship between alkyl phase structure and chromatographic performance is investigated for a series of octadecyl (C(18))-modified silica surfaces with defined spacing of the alkyl surface by a pre-end-capping technique. Stationary phases were prepared by a two step process with (1) reaction with less than stochiometric amounts of a small monofunctional silane, followed by (2) solution or surface polymerization with octadecyltrichlorosilane. The results of solid-state and suspension nuclear magnetic resonance (NMR) spectroscopy are correlated with the chromatographic behavior regarding shape selective separations. Two sets of six different stationary phases were prepared by solution and surface polymerization approaches, yielding materials with surface coverages from 2.7 to 5.6 micromol/m(2). (13)C cross-polarization magic angle spinning (CP/MAS) NMR spectra show a predominance of trans conformations for the set of surface polymerized phases with a C(18) coverage greater than 4.5 micromol/m(2). For the solution polymerized phases, no predominance for the trans conformation was observed, even for surface coverages greater than 5.1 micromol/m(2). Proton spectra in suspension indicate the trend that a higher coverage for the surface polymerized materials correlates with a more rigid alkyl chain conformation. The set of solution polymerized stationary phases confirms this tendency but minor deviations are observed for high coverages. These structural abnormalities are confirmed by differences in the (29)Si CP/MAS spectra. Furthermore, the (29)Si CP/MAS spectra indicate a lower amount of cross-linking for the materials with the highest amount of placeholder (spacer). The use of the different spectroscopic and chromatographic methods provides a wealth of information on the surface morphology of the systematically prepared C(18) materials and extends the understanding of surface morphology of alkyl modified silica and its influences of the molecular recognition process in liquid chromatography.

Preparation and Certification of Standard Reference Material 3278 Tocopherols in Edible Oils

Standard Reference Material (SRM) 3278 Tocopherols in Edible Oils has been issued for use as a quality assurance tool in the measurement of tocopherols. Like other natural-matrix SRMs, this material can be used in method validation or in assignment of tocopherol values to in-house quality control materials. Because most edible oils contain one predominant tocopherol isoform, the SRM is a blend of sunflower, soy, canola, and safflower oils to provide roughly comparable chromatographic peak heights of the two main tocopherols, γ and α, with smaller amounts of δ and β. The four tocopherol isoforms were determined by three independent liquid chromatography methods with absorbance and fluorescence detection. Various chromatographic and detection modes are used for assignment of certified values because biases inherent to one method should not be present in the other, and the existence of bias can therefore be identified.