Sarath R. Sirimanne

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 cloud-point extraction-reversed-phase high-performance liquid chromatography. A preliminary study of the extraction and quantification of vitamins A and E in human serum and whole blood.

Methods available for quantification of vitamins A and E in serum or blood requires preconcentration and clean-up by liquid-liquid extraction, evaporation of the extract, and reconstitution of the extract in a solvent of choice before analysis. This process not only involves the use of toxic organic solvents but also requires a long sample preparation time. The lipids and other non-polar coextractants often require additional steps for sample clean-up and evaporation, which may cause sample losses. The use of cloud-point extraction eliminates most of these sample clean-up problems. We recently demonstrated that cloud-point extraction (CPE) can be used for extraction and quantification of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated dibenzo-p-dioxins (PCDDs) from human serum. We now demonstrate how CPE can be used with human serum and blood, at volumes as low as 50 microl, and report a methodology for extracting and quantifying two clinically important vitamins, (A and E) from human serum and blood. Vitamins A and E were extracted from human serum and blood by using Genapol X-80 as the cloud-point extractant under salting out conditions. Serum and blood samples were diluted in organic-free water to get sufficiently large sample volumes for CPE. The surfactant-rich phases were separated by centrifugation, and the samples were analyzed by HPLC-UV after deleterious coextractants were removed by precipitating them with acetonitrile. The recoveries of spiked vitamins A and E were found to be 85.6+/-0.4% and 82.6+/-5.2%, respectively. The average concentration of vitamins A and E in a serum pool after correction for recoveries were found to be 43.4+/-1.8 microg/dl (1.5+/-0.1 micromol/l) and 564.3+/-65.3 microg/dl (13.1+/-1.5 micromol/l), respectively. Vitamin A and E concentrations in whole blood were found to be 26.3+/-0.4 microg/dl (0.92+/-0.01 micromol/l) and 457.5+/-15.6 microg/dl (10.6+/-0.4 micromol/l), respectively. These values are comparable with those obtained by the reference method used at the Centers for Disease Control and Prevention. The success of the preliminary study will lead to a comprehensive validation of this method for vitamins A and E in serum and blood.

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.