DEVELOPMENT AND APPLICATION OF COUNTERFLOW METHODS: GEITP, GEITP-CZE, TGF, and TGDF

Abstract

Extensive research on amino acids, and even other biochemical assays usually present in low concentration and volume face challenges using known analytical techniques for analysis of traces amounts. Some limiting factors are the achievable efficiency, sensitivity (resulting from instrument limit of detection and/or experimental methods), volume requirement, and total analysis time. Counterflow electrofocusing techniques combining forces of electrophoresis and bulk flow (pressure driven flow and/or electroosmotic flow) provides a basis for the development of alternative detection techniques geared towards improving peak efficiency, sensitivity and time. The work presented gives a vivid description of recently developed capillary counterflow techniques: gradient elution isotachophoresis (GEITP) using UV detection, GEITP coupled to Capillary Zonal Electrophoresis (GEITP-CZE), temperature gradient focusing (TGF), and temperature gradient denaturing focusing (TGDF). A first demonstration of GEITP using UV detection was applied to enrichment and separation of tyrosine and tryptophan under optimized conditions. Primarily, separation is achieved as the result of the difference in electrophoretic velocity of analytes in a discontinuous buffer system. First, a plug of sample is allowed to preconcentrate (or enrich) between high mobility leading electrolyte (LE) and low mobility trailing electrolyte (TE) under controlled hydrodynamic pressure and continuous injection. This preconcentration is initiated outside the capillary in a conductivity bubble. Although analyte focus according to their electrophoretic velocity, the inclusion of spacer molecule in sample matrix was instrumental in achieving separation with tradeoff between analyte resolution and enrichment. Gradient produced results from reduction in pressure as sample is loaded on column. Separation using this technique is a one step process. A hybrid method marking the first successful coupling of GEITP to CZE with laser induced fluorescence detection was used for separation of six fluorescently labeled amino acids (which formulates the Mars-7). An eleven minute separation was achieved under optimized conditions. A proof-of-concept demonstration of TGF with LIF detection showed focusing and separation of fluorescein and carboxyfluorescein dye molecules, and carboxyfluorescein-labeled glutamate and aspartate. The generation of null focusing points along the thermal separation column (set between 80-20oC) was produced in collaboration with continuous sample injection, discontinuous buffer system and balancing of counterflows (electrophoresis and bulk flow). Preliminary results showed stability in instrument. The TGDF method carried out on a TGF apparatus is a modification to the temperature gradient gel electrophoresis and denaturing gradient gel electrophoresis methods. In principle, TGDF primarily achieves focusing and separation on a thermal separation column (set between 20 to 80 oC) as a result of conformational changes. It is currently being developed for the detection and simultaneous separation of single and double stranded DNA. Preliminary results show enrichment of wildtype and mutant synthetic DNA strands (containing twenty-four base pairs in sequence) in different buffer matrices.