Michael Merion

On-line sample preconcentration on a packed-inlet capillary for improving the sensitivity of capillary electrophoretic analysis of pharmaceuticals

Abstract An on-line preconcentration method is reported that improves the sensitivity of capillary electrophoretic analyses by at least two orders of magnitude. The method uses a concentrator capillary with a 75 μm I.D. and a 1-mm length packed bed at the inlet end that can concentrate samples using the principles of liquid chromatography. Using low-molecular-mass pharmaceutical standards as examples, the parameters used in developing a preconcentration sensitivity enhancement method were optimized. The optimized method was then used to evaluate the quantitative aspects of capillaries of this type, including run to run and capillary to capillary reproducibility, linearity, and efficiency and resolution. In addition, the analysis of a urine sample spiked with doxepin at the 500 ppb level is reported.

High-resolution chromatography of nucleic acids on the gen-pak fax column

High-performance liquid chromatography (HPLC) on a Gen-Pak FAX column has been used to separate and purify microgram amounts of single- and double-stranded DNA and RNA molecules. HPLC of mixtures of DNA restriction fragments showed that fragments within the size range 0.125-23.1 kilobase were easily resolved. Supercoiled (form I) plasmid DNA molecules were readily separated from single-stranded circular DNA of the same length and from various DNA conformational isomers including nicked (form II) and linear (form III) species. Topological isomers generated from supercoiled plasmid DNA molecules by DNA topoisomerase I exhibited different retention times than supercoiled molecules. Supercoiled (form I) DNA molecules were resolved from fully relaxed (form IV) molecules. Synthetic oligonucleotides of 74 and 128 nucleotides in length were separated from failure sequences, as well as from other contaminating synthesis products. Single-stranded circular M13mp18 DNA molecules sufficiently pure for use in automated DNA sequencing systems were prepared by HPLC on a Gen-Pak FAX column. HPLC was also used to fractionate linear double-stranded porcine rotavirus genomic RNA fragments into size classes between 0.3 and 3 kilobase. Finally, HPLC of unfractionated Escherichia coli tRNA molecules resolved multiple species. In all cases, HPLC on Gen-Pak FAX was carried out in phosphate or Tris buffers at neutral pH in the presence of sodium chloride. Columns were not damaged by repeated exposure to impure samples, provided they were cleaned frequently with sodium hydroxide and acetic acid. Although procedures for resolution of the various size ranges for each class of DNA and RNA molecules require further optimization, our preliminary data on the separations obtained, the moderate salt concentrations employed, and the durability of the matrix suggest that this column merits further study.