Julia Khandurina

Multidimensional separations in the pharmaceutical arena

The introduction of novel, powerful and rapid multidimensional separation and characterization methods has produced revolutionary global changes at the genome, proteome and metabolome level, bringing about a radical transition in our views of living systems, at the molecular level. The age of proteomics and metabolomics demands high-resolution multidimensional separation techniques. Multidimensional gas and liquid chromatography techniques, in addition to capillary and microchip electrophoresis methods, offer increased resolution and sensitivity, while also affording adequate throughput and reproducibility to meet the demands of the modern pharmaceutical industry. Coupled with MS, these techniques provide not only separation but also reliable identification of the sample components. The resolving power of these methods has proved to be superior over individual one-dimensional approaches, enabling the comprehensive separation of complex biological mixtures, with excellent resolution and reproducibility. High capacity computer systems that are capable of rigorous qualitative and quantitative analysis of the separation profiles allow the establishment and mining of large databases. Examples of various modern multidimensional separation techniques, and their integration with MS, are reviewed, here, with respect to pharmaceutical analysis.

Micromachined capillary cross-connector for high-precision fraction collection

A new approach for high-precision fraction collection of double-stranded DNA fragments by capillary electrophoresis coupled to a micromachined plastic capillary cross-connector is presented. The system design integrates four fused-silica capillaries with an acrylic cross-channel connector. The cross-channel structure was introduced to enhance the efficiency of the fraction collection process by electrokinetic manipulations. Following the detection of the sample zone of interest at or slightly upstream of the cross during the separation mode, the potentials were reconfigured to collection mode to direct the selected analyte zone into the corresponding collection vial, while keeping the rest of the sample components virtually stopped within the separation capillary. In this way the spacing between consecutive bands of interest can be physically increased, allowing precise isolation of spatially close sample zones. After collection of the target fraction the separation mode is resumed, and the separation/collection cycle is repeated until all desired sample zones are separated and captured. The capillary cross-connector was fabricated of a transparent acrylic substrate by microdrilling flat end and through channels, matching precisely the O.D. and I.D. of the connected capillary tubing, respectively. This design provided a close to zero dead volume connection assembly for the separation and collection capillaries causing minimal extra band broadening during high-precision micropreparative DNA fractionation.

Differential gene expression analysis by micro-preparative capillary gel electrophoresis

Differential display analysis by cDNA fractionation, collection of differentially expressed fractions of interests and their downstream characterization is demonstrated. cDNA pools from two strains of Cochliobolus heterostrophus fungus were generated by specific restriction digestion and selective ligation. Micropreparative separation and isolation of differentially expressed transcript representatives were accomplished by high-performance capillary gel electrophoresis. The collected individual DNA molecules were polymerase chain reaction amplified and sequenced to create expressed sequence tags for the genes of interests. High resolving power and sensitivity of capillary gel electrophoresis enabled fast and automated processing of minute amounts of cDNA samples with high precision.

Chapter 11 Microfabricated analytical devices

Publisher Summary Microfabricated analytical devices are referred to as “lab-on-a-chip systems” and include microseparation units, miniaturized reactors, and microarrays. This chapter reviews recent developments and trends in microfluidic analyses of biological interest, including DNA, proteins, and complex carbohydrate analysis and high-throughput screening. The microfabrication methods comprise photolithography in rigid materials and fabrication in plastics and elastomers. Glass substrates are common because of their good optical properties and well-developed microfabrication technology and surface chemistry. For miniaturized analysis systems, sample handling and manipulation are of great importance. Specific problems resulting from the shrinking of macroscopic systems include the failure of samples to be representative and of manipulations to be reproducible. This makes it necessary to incorporate specific microscale techniques and components in such devices. The analytical performances of microfluidic devices are drastically affected by dead volumes in the system and also by the surface properties of the fluidic channels.

High Precision Micropreparative Separation System Based on Plastc Microfluidic Module — Capillary Coupling

We report on a new approach for high precision fraction collection of electrophoretically separated DNA molecules. The fraction collection system is based on a microfluidic cross-connector module coupled to four fused silica capillaries. The cross-connector was fabricated of an acrylic substrate by microdrilling flat end and through channels to ensure close to zero dead volume connections with capillary tubing. DNA fragments were size separated and physically isolated by appropriate electrokinetic manipulations. Selected portions of analyte were redirected to the corresponding collection reservoirs, while maintaining the rest of the sample virtually stopped or slowly reversed within the separation capillary. In this way, the spacing between consecutive bands of interest can be increased enabling high precision isolation of spatially close analyte zones. The separation/collection cycle was repeated until all desired sample zones are captured. The amounts of the collected DNA fractions were enough for further downstream sample processing, such as PCR amplification and analysis.