Lane C. Sander

Selection of Column and Gradient Elution System for the Separation of Catechins in Green Tea Using High-Performance Liquid Chromatography

A study of a variety of stationary phases and elution conditions for the liquid chromatographic (LC) determination of six biologically active green tea catechins has resulted in the development of two well-defined, reproducible systems for such analyses which overcome limitations of previously described methods. Comparison of six reversed-phase columns indicates that deactivated stationary phases, which utilize ultrapure silica and maximize coverage of the silica support, provide significantly improved separation and chromatographic efficiencies for catechin analyses using LC, compared to conventional monomeric or polymeric C18 columns. Evaluation of elution conditions used for the separations reveals that the presence of acid in the mobile phase (0.05% trifluoroacetic acid) is essential for both the complete resolution of the catechins present in tea and the efficient chromatography of these compounds. The efficacy of one of the developed systems was demonstrated by the quantitative measurement of the six biologically active catechins in aqueous infusions of green tea (Camellia sinensis). Overall precision values for the analyses were within the range 0.3-1% (relative standard deviation).

Determination of polycyclic aromatic hydrocarbons by liquid chromatography

Abstract Reversed-phase liquid chromatography (LC) using fluorescence detection is a powerful analytical technique for the measurement of polycyclic aromatic hydrocarbons (PAHs) in environmental samples. The National Institute of Standards and Technology (NIST) has been involved in the development of LC methods for the measurement of PAHs since the mid- 1970s particularly for the development of standard reference materials (SRMs) for PAH measurements in environmental samples. The NIST experience in the use of LC for the determination of PAHs in environmental samples is summarized in this paper including: selection of the appropriate column, approaches to analyzing complex PAH mixtures, and the accurate quantitation of PAHs in environmental samples.

Capability of a polymeric C30 stationary phase to resolve cis-trans carotenoid isomers in reversed-phase liquid chromatography

Abstract A novel polymeric C30 stationary phase was tested for its ability to separate geometric isomers of six common carotenoids (lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene, and lycopene) prepared by photoisomerization of all-trans standards. Resolution and tentative identification of asymmetrical carotenoid isomers yielded the 13-cis, 13′-cis, all-trans, 9-cis, and 9′-cis isomers of both lutein and α-carotene, and the 15-cis, 13-cis/13′-cis, all-trans, 9-cis, and 9′-cis isomers of β-cryptoxanthin. Among symmetrical carotenoids, the 15-cis, 13-cis, all-trans, and 9-cis isomers of both zeaxanthin and β-carotene were resolved and tentatively identified, and nineteen geometric isomers of lycopene were separated. Separations were carried out using Vydac 201TP54 and Suplex pkb-100 stationary phases for comparison; in all cases, the C30 stationary phase gave superior resolution and produced unique separations.

Shape Selectivity for Constrained Solutes in Reversed-Phase Liquid Chromatography

In reversed-phase liquid chromatography (RPLC), the separation of compound mixtures of similar polarity can present a significant challenge for the analyst. Examples of such compounds include geometric isomers present in environmental samples (e.g., polycyclic aromatic hydrocarbons, polycyclic aromatic sulfur heterocycles, and polychlorinated biphenyl congeners) and compounds of biological significance (e.g., carotenoids and steroids). In general, compounds with rigid, well-defined molecular shape are best separated using a column with enhanced shape selectivity characteristics. This perspective presents an overview of column properties that influence shape selectivity for constrained solutes. Approaches to the characterization of stationary-phase structure are described, and the findings are correlated with chromatographic performance. Finally, retention models of shape discrimination are presented that are consistent with observed retention behavior. An appreciation for shape recognition effects in RPLC will facilitate method development for certain classes of difficult to resolve compounds.

C30 stationary phases for the analysis of food by liquid chromatography.

The introduction of a polymeric C30 liquid chromatographic column by Sander et al. [Anal. Chem., 66 (1994) 1667] designed for the separation of carotenoid isomers, has led to the development of improved analytical methods for these compounds. Subsequent commercial availability of polymerically bonded C30 columns has facilitated these advances, and applications to a wide variety of separation problems with biological samples have been described. This report provides a comprehensive review of applications of polymeric C30 columns, utilized in the determination of carotenoids, retinoids, and other nutrients and related compounds in complex, natural-matrix samples.

Shape selectivity in reversed-phase liquid chromatography for the separation of planar and non-planar solutes

Abstract Solute retention in reversed-phase liquid chromatography is the result of a variety of complex interactions between solute, mobile phase and stationary phase species. An understanding of the parameters that influence retention is useful in the development of separation methods with existing columns. Such knowledge is even more important for the design of new bonded stationary phases with engineered chromatographic properties. This review will examine some of the factors that affect retention and selectivity with alkyl-modified sorbents, particularly for the separation of solutes with well defined, rigid structure (e.g., polycyclic aromatic hydrocarbons). The chromatographic discrimination of compounds on the basis of molecular structure, namely “shape selectivity”, will be studied in terms of contributions from bonded phase morphology, and in terms of operational conditions. An emphasis is placed on practical choices that are available to control selectivity and optimize separations for isomers and related mixtures.

Influence of substrate parameters on column selectivity with alkyl bonded-phase sorbents

Abstract Differences in bonded-phase properties were studied for monomeric and polymeric C18 phases prepared on a variety of silica substrate materials. A total of 22 silicas with pore diameters ranging from 50-1000 A were used in syntheses. Phase loadings for the resulting bonded phases ranged from 1.3–5.4 μmol/m2. Physical properties of the substrates including surface area, pore volume, packing density, and background carbon were measured prior to bonding. Large differences were observed in the properties of the silica substrates and in the chromatographic behavior of the resulting phases. Differences in selectivity as well as absolute retention were observed as a function of pore size, with the greatest changes in selectivity occurring for the polymeric phases. The effect of silica pretreatment on phase synthesis and column selectivity was also examined for wide- and narrow-pore substrates. Phases prepared from silica pretreated with acid had greater polymeric character than those prepared from base-pretreated silica. Variation in phase loading and column selectivity is thought to be a function of both the reactivity of the silica surface and pore size. A model for polymeric phase synthesis is proposed where the extent of reaction is limited by a size-exclusion mechanism.

Combined use of temperature and solvent strength in reversed-phase gradient elution I. Predicting separation as a function of temperature and gradient conditions

It has been shown previously that computer simulation based on two initial experiments can predict separation in reversed-phase gradient elution as a function of gradient conditions (gradient steepness, gradient range and gradient shape) and column conditions (column length, flow-rate and particle size). The present study extends this capability for changes in temperature. Four initial experiments (two different gradient times, two different temperatures) provide input data that allow predictions of separation as a function of temperature as well as gradient and column conditions. A semi-empirical relationship, tR = a + bT, is able to relate gradient retention time tR to column temperature T (other conditions constant). The accuracy of this approach has been evaluated for 102 solutes and a variety of experimental conditions, including the use of five different HPLC instruments (four different models).

Evaluation of reversed-phase liquid chromatographic columns for recovery and selectivity of selected carotenoids

Sixty commercially available and five experimental liquid chromatography columns were evaluated for the separation and recovery of seven carotenoid compounds. Methanol- and acetonitrile-based solvents (either straight or modified with ethyl acetate or tetrahydrofuran) were compared to determine which solvent systems and which columns provided better selectivity and recovery. Methanol-based solvents typically provided higher recoveries than did acetonitrile-based solvents. Polymeric C18 phases generally provided better selectivity for the difficult separation of lutein and zeaxanthin than did monomeric C18 phases.

RECENT ADVANCES IN BONDED PHASES FOR LIQUID CHROMATOGRAPHY

I. INTRODUCTION Over the past 10 years high performance liquid chromatography (LC) has evolved into a mature analytical technique. Most LC separations are currently carried out in the reversed-phase mode using 25-cm columns filled with 5 or 10 μm nonpolar bonded substrates. A smaller fraction of separations are carried out in the normal-phase mode with polar bonded phases. Although major changes in column technology have become less commonplace in recent times, refinements continue to be made. New bonded phases have been developed for the separation of specific classes of compounds and new substrates have been introduced that extend column lifetimes and permit use over wide pH intervals. Even though bonded phase usage is now common, solute retention mechanisms with these materials are not fully understood (but considerable progress has been made). To aid the study of retention mechanisms, novel approaches have been taken in the physical and chemical characterization of bonded phase sorbents.