James Grainger

Field-amplified sample stacking in micellar electrokinetic chromatography for on-column sample concentration of neutral molecules

On-column concentration of neutral molecules was achieved for the first time in micellar electrokinetic chromatography by means of field-amplified sample stacking. The stacking process was accomplished by dissolving the neutral analytes in a low-concentration micellar solution that was still above the critical micelle concentration. The lower total ionic strength in the sample buffer compared to the electrophoresis buffer allowed the negatively charged micelles to migrate rapidly into the boundary between the sample and the running buffer where they slow down. This field-amplified sample stacking was achieved by using normal or reversed electrode polarity and produced a 75-85-fold increase in sensitivity for 1,2,4,7- and 1,2,4,8-tetrachlorodibenzo-p-dioxins. The peak area counts obtained from the sample stacking process were proportional to the sample volume injected, and the stacking efficiency was dependent on the micellar concentration. The best stacking efficiency was obtained when the micelle concentration was slightly higher than the critical micelle concentration. When the injection volume was relatively small, the normal-polarity stacking procedure produced a higher stacking efficiency. However, when the injection volume was large, reversed polarity produced a higher stacking efficiency because the non-uniform distribution of the electrical field strength had been eliminated.

Application of comprehensive two-dimensional gas chromatography (GC×GC) to the qualitative analysis of essential oils.

This paper investigates the separation of moderately complex samples by comprehensive two-dimensional gas chromatography (GC x GC). The analysis of peppermint (Mentha piperita) and spearmint (Mentha spicata) essential oil components, including acetates, alcohols, furans, ketones, sesquiterpenes, and terpenes, was achieved by one-dimensional gas chromatography with quadrupole mass spectrometry detection (GC/MSD) and GC x GC with flame ionization detection. Peppermint essential oil was found to contain 89 identifiable peaks by GC x GC compared to 30 peaks in the GC/MSD chromatogram. Likewise, 68 peaks were found in the GC x GC chromatogram of spearmint (compared to 28 in GC/MSD). Plots of the first dimension versus second dimension retention times provided a fingerprint of the two essential oils, which revealed 52 similar compounds between the two essential oils as opposed to 18 matches by ID GC.

Determination of selected monohydroxy metabolites of 2-, 3- and 4-ring polycyclic aromatic hydrocarbons in urine by solid-phase microextraction and isotope dilution gas chromatography–mass spectrometry

Eighteen monohydroxy polycyclic aromatic hydrocarbon metabolites (OH-PAHs) representing polycyclic aromatic hydrocarbons (PAHs) containing up to four rings in human urine have been measured. The method includes the addition of carbon-13 labeled internal standards, enzymatic hydrolysis, and solid-phase microextraction followed by gas chromatography with high-resolution mass spectrometry. By using response factors calculated with the carbon-13 labeled standards, results are presented for calibration, relative standard deviations and analyte levels from an unspiked human urine pool. The method detection limits ranged from 0.78 ng/l for hydroxyphenanthrenes to 15.8 ng/l for 1-hydroxynaphthalene, and the recoveries ranged between 6% for hydroxychrysene and 47% for 1-hydroxypyrene. The relative standard deviation was lowest for 3-hydroxyphenanthrene at 2.4% and went up to 18.7% for 6-hydroxychrysene. The method was calibrated from 10 to 1200 ng/l. Eleven of the 18 metabolites were found in background pooled urine samples. This validated method is a convenient and reliable tool for determining urinary OH-PAHs as biomarkers of exposure to eight PAHs.

Stereoselective separation and detection of phenoxy acid herbicide enantiomers by cyclodextrin-modified capillary zone electrophoresis–electrospray ionization mass spectrometry

Abstract An application of on-line coupling of capillary electrophoresis and mass spectrometry (CE–MS) to the chiral separation of phenoxy acid herbicide enantiomers was investigated. As an ionization method, electrospray ionization (ESI) is used for a CE–MS interface. Generally, nonvolatile additives in separation solutions sometimes decrease the MS sensitivity and/or signal intensity. In this study, however, heptakis(2,3,6-tri- O -methyl)-β-cyclodextrin (TM-β-CD) was used as a chiral selector and it migrated directly into the ESI interface. By using the negative-ionization mode along with a methanol–water–formic acid solution as a sheath liquid and nitrogen as a sheath gas, stereoselective separation and detection of three phenoxy acid herbicide enantiomers were successfully achieved with a 20 m M TM-β-CD in a 50 m M ammonium acetate buffer (pH 4.6).

Separation of polycyclic aromatic hydrocarbon metabolites by γ-cyclodextrin-modified micellar electrokinetic chromatography with laser-induced fluorescence detection

Using a modified micellar buffer consisting of gamma-cyclodextrin (gamma-CD) and sodium dodecyl sulfate (SDS), we have obtained separations of hydroxy-polycyclic aromatic hydrocarbons (hydroxyPAHs). These compounds are oxidative products of mammalian PAH metabolism. The analytes were detected with a commercial laser-induced fluorescence (LIF) detector. A number of hydroxyPAH isomers could be separated by changes in gamma-CD concentration. Baseline resolution of 12 hydroxyPAHs was obtained using 30 mM borate, 60 mM SDS and 40 mM gamma-CD. The particular site substitution of the hydroxy group can produce changes in the hydroxyPAH fluorescence spectrum, and the effect of optical filter selection was studied for the LIF detection. The mass detection limits were in the (0.08-0.5) x 10(-15) mol range. To our knowledge, this is the first report of the separation of metabolic products of PAHs (and several positional isomers) using gamma-CD and micellar electrokinetic chromatography.

Molecular geometry approximations for chlorinated dibenzodioxins by Fourier transform infrared spectroscopy

Reference infrared vapor-phase spectra of 15 polychlorinated dibenzodioxin-p-dioxin (PCDD) isomers were recorded at low microgram concentrations. Ether linkage (COC) bond angles for these isomers and for the 22 tetrachlorodibenzodioxin (TCDD) isomers were calculated from infrared data, with the use of mass approximations for the terminal atom in a nonlinear XY2 model and by neglecting the valence force equation symmetric stretch bending term. Calculated bond angles show a good correlation with x-ray diffraction (XRD) and carbon-13 nuclear magnetic resonance (13C NMR) relaxation results. Molecular geometries in PCDD isomers, as defined by the COC bond angle and the COC stretching frequencies, were found to range from near planar, in laterally substituted isomers with high electron withdrawing capacity aromatic rings, to tetrahedral, for isomers with low electron withdrawing capacity rings. Non-bonded interactions were also found to influence molecular geometry. Molecular geometry was used to assign structures for the 1,2,3,6,7,8- and 1,2,3,7,8,9-hexachlorodibenzodioxin isomer mixture and was found to be an important factor in estimations of COC bond strength from empirical data. Correlations between infrared data and PCDD LD50 values suggest that molecular geometry, polarizability, and steric PCDD/receptor interactions are associated with isomer toxicity.

Chromatographic and spectroscopic properties of hemiacetals of aflatoxin and sterigmatocystin metabolites

Improved fluorescence detection of aflatoxin B1 by chromatographic analysis is accomplished by conversion to the corresponding hemiacetal, aflatoxin B2a. Because the metabolites aflatoxin M1, aflatoxin P1, aflatoxin Q1, sterigmatocystin, and O-methylsterigmatocystin have the same molecular conversion site, we investigated the chromatographic and spectroscopic properties of hemiacetals of these compounds to assist in confirming aflatoxins and sterigmatocystins in human urine. Nuclear magnetic resonance and infrared absorbance were used to confirm the hemiacetal structure for aflatoxin B1 and sterigmatocystin. The ultraviolet absorbance, fluorescence, and chromatographic properties of the metabolites were investigated. Using these data, we optimized the detection and solvent conditions for high-performance liquid chromatography. We determined that, of the conditions studied, maximum sensitivity and resolution for the native aflatoxins were achieved with a mobile phase of methanol, tetrahydrofuran, and water, a C8 column in series with a C18 column, and fluorescence detection with 365 nm excitation and 430 nm emission wavelengths for aflatoxins B1 and M1 and with 500 nm emission wavelength for aflatoxins P1 and Q1. For the analysis of the hemiacetals, a mobile phase of methanol, acetonitrile, and water provided better chromatography and fluorescence detection. Sterigmatocystin and O-methylsterigmatocystin were readily converted to the hemiacetal forms, which, like the aflatoxins, were more polar and, therefore, earlier eluting by reversed-phase HPLC (methanol, acetonitrile, and water, 236 nm absorbance). These data are important to maximize the sensitivity and confidence for detecting the mycotoxin metabolites in biological specimens.

Enhancement of the separation selectivity of a group of polycyclic aromatic hydrocarbons using mixed cyclodextrin-modified micellar electrokinetic chromatography

Abstract The separation selectivity of a group of twenty polycyclic aromatic hydrocarbons (PAHs) (including sixteen Environmental Protection Agency priority PAHs) was studied when mixtures of native β- and γ-cyclodextrins (CDs) are used as modifiers in the separation buffer in cyclodextrin-modified micellar electrokinetic chromatography. The ratio between the concentrations of β- and γ-CDs in the separation buffer as well as the total concentration was varied for various buffer concentrations. An enhancement in selectivity was obtained which enabled the separation of eighteen PAHs when using a 0.140-M borate buffer (pH 9) containing 0.100 M sodium dodecyl sulphate micelles, 0.042 M β-CD, 0.020 M γ-CD and 2.5 M urea. The correlation between the logarithm of the capacity factors of PAHs and the logarithm of their octanol–water distribution coefficients was also investigated.

HPLC Separation and FT-IR Isomer Differentiation of the 1,2,4,7/1,2,4,8-Tetrachlorodibenzodioxin Isomer Pair—A Theoretical/Empirical Approach to Dibenzodioxin Isomer Assignment

The 1,2,4,7- and 1,2,4,8-tetrachlorodibenzodioxin (TCDD) isomers were separated by reversed-phase high-performance liquid chromatography (HPLC) with the use of a pyrene column. Fourier transform infrared (FT-IR) matrix isolation and vapor-phase spectra of the individual isomers were recorded. The spectra of the HPLC-separated isomers correlate well with spectral subtraction results and were found to be distinct in three spectral regions—one of which allows for isomer structural assignment. Ambiguities and differences in published TCDD isomer FT-IR assignments are discussed in terms of a qualitative valence-bond approach and empirically derived estimates of ether linkage asymmetric stretching frequencies.

Isomer identification of chlorinated dibenzo-p-dioxins by orthogonal spectroscopic and chromatographic techniques

Isomer differentiation of chlorinated dibenzo-p-dioxin (CDD) isomer pair components was examined by three orthogonal chromatographic (gas chromatography, liquid chromatography, and micellar electrokinetic chromatography) techniques and three orthogonal spectroscopic (Fourier transform infrared and carbon-13 and proton nuclear magnetic resonance) techniques. Synthetic CDD isomer pair mixtures from the same sample set were separated by methods using independent partitioning dynamics and identified by spectroscopic methods using independent energy/structure transformations. This integrated approach using orthogonal methods minimizes isomer identification error probability and results in an excellent agreement among methods in isomer assignment.