UBC Theses and Dissertations
Optimization of capillary isoelectric focusing for rapid analysis of biological molecules Lee, Alexis Kyungmi
Currently in the field of bioanalysis, a large amount of information must be obtained and processed in a timely manner. Despite its exceptional separation power, progress using capillary isoelectric focusing (cIEF) as an analytical tool has been stagnating in this rapidly advancing field without significant means to shorten analysis time. To address this limitation, 30-μm internal diameter capillaries were used to achieve full pH range (pH 3-10) cIEF within 15 minutes. The benefits of using narrow diameter capillaries are demonstrated by comparing with results obtained by using 50-μm internal diameter capillaries. Optimization of additional experimental parameters such as presence of cathodic spacer, concentration of anticonvective media, and focusing time helped to achieve much shorter analysis time. While sensitivity is sacrificed in the process of reducing analysis time with the change of some parameters, such loss can be reasonably accommodated given the exceptional sensitivity of cIEF. Prior to achieving rapid cIEF with narrow diameter capillary, reproducibility of cIEF is examined by 60 consecutive experiments on a single capillary. Despite measures taken to ensure minimal analyte adsorption and protein precipitation, migration times of analytes increased in subsequent experiments until the capillary was conditioned by extended rinsing. Unfortunately, conditioning of the capillary degrades the internal coating and shortens its lifespan to approximately 50 consecutive experiments when conditioned every 10 experiments. Notwithstanding substantial variation in migration times, calculated pI values of the sample protein, myoglobin, are accurately and precisely determined throughout repeated experiments. Capillary isoelectric focusing can therefore be relied upon to separate and identify biological analytes such as proteins.
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