1. Instrumentation for Medium-Throughput Two-Dimensional Capillary Electrophoresis with Laser-Induced Fluorescence Detection.
- Author
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Cuiru Zhu, Xinya He, Kraly, James R., Jones, Megan R., Whitmore, Cohn D., Gomez, David G., Eggertson, Michael, Quigley, Wes, Boardman, Anna, and Dovichi, Norman J.
- Subjects
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CAPILLARY electrophoresis , *RATIONAL numbers , *DISTRIBUTION (Probability theory) , *ANALYSIS of variance , *STATISTICS , *DATA transmission systems , *FLUORESCENCE , *FLUORIMETRY , *STANDARD deviations - Abstract
In two-dimensional capillary electrophoresis, a sample undergoes separation in the first dimension capillary by sieving electrophoresis. Fractions are periodically transferred across an interface into a second dimension capillary, where components are further resolved by micellar electrokinetic capillary electrophoresis. Previous instruments employed one pair of capillaries to analyze a single sample. We now report a multiplexed system that allows separation of five samples in parallel. Samples are injected into five first-dimension capillaries, fractions are transferred across an interface to 5 second-dimension capillaries, and analyte is detected by laser-induced fluorescence in a five-capillary sheath-flow cuvette. The instrument produces detection limits of 940 ± 350 yoctomoles for 3-(2-furoyl)quinoline-2-carboxaldehyde labeled trypsin inhibitor in one-dimensional separation; detection limits degrade by a factor of 3.8 for two-dimensional separations. Two-dimensional capillary electrophoresis expression fingerprints were obtained from homogenates pre- pared from a lung cancer (A549) cell line, on the basis of capillary sieving electrophoresis (CSE) and micellar electrophoresis capillary chromatography (MECC). An average of 131 spots is resolved with signal-to-noise greater than 10. A Gaussian surface was fit to a set of 20 spots in each electropherogram. The mean spot width, expressed as standard deviation of the Gaussian function, was 2.3 ± 0.7 transfers in the CSE dimension and 0.46 ± 0.25 s in the MECC dimension. The standard deviation in spot position was 1.8 ± 1.2 transfers in the CSE dimension and 0.88 ± 0.55 s in the MECC dimension. Spot capacity was 300. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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