A 150-component, dynamic electrophoresis simulator was developed and applied to the description of capillary isoelectric focusing (CIEF) of amphoteric substances in quiescent solution. The simulator is shown to be capable of producing high-resolution pH 3-10 focusing data with 140 individual carrier ampholytes (20/pH unit) and at current densities that are used in CIEF, i.e., under conditions that were hitherto unaccessible by dynamic computer simulation. Having a focusing capillary of 5-cm length, the predicted focusing dynamics for amphoteric dyes obtained at a constant voltage of 1500 V (300 V/cm) are shown to qualitatively agree with data obtained by whole-column optical imaging. The simulation data provide detailed insight into the dynamics of the focusing process for the cases with the focusing column being sandwiched between 40 mM NaOH (catholyte) and 100 mM phosphoric acid (anolyte) or having the column ends only permeable for OH- and H+ at cathode and anode, respectively. Simulation data reveal that the number of sample boundaries migrating from the two ends of the column to the focusing positions is always equal to the number of sample components. The number of detectable migrating sample boundaries, however, can be lower. Whole-column optical imaging is demonstrated to be the method of choice for following the approach to equilibrium. With that detection format, transient sample peaks can be recognized and properly identified. This would also be possible with a scanning detector moving rapidly and repeatedly along the column but cannot be accomplished by a stationary detector placed at a specified location. The data presented demonstrate that the model together with imaging monitoring can be used to optimize the CIEF separation conditions.
The feasibility of isoelectric focusing (IEF) performed on-chip was demonstrated for the first time via absorption imaging detection. Microchannels on a quartz chip were fabricated using photolithography and a chemical etching process. The separation channels were 40 mm long, 100 mm wide and 10 mm deep, and were coated with linear polyacrylamide to reduce electroosmotic flow. A quartz chip cartridge for IEF was assembled in which two pieces of hollow fiber were glued to the two ends of the separation channel to isolate the electrolytes from the samples. Low molecular mass pI markers and myoglobin were selected as model samples which were mixed with 4% carrier ampholyte solution. Samples were injected into the channel via the connection capillary by pressure. A voltage of 3 kV was applied to perform IEF. The IEF current decreased from about 13.4 to 1.3 mA. The focused zones were monitored in real time by absorption imaging detection at 280 nm. The detection limit was about 0.3 mg ml 21 or 24 pg for pI marker 6.6, and 30 mg ml 21 or 2.4 ng for myoglobin with an optical pathlength of 10 mm. Good reproducibility and resolution were obtained for linear polyacrylamide coated channels. The total analysis time was less than 10 min. This imaged chip IEF provides a fast separation technique with quantitative ability and the potential for increasing throughput.
We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated fourcolor confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect. We present experimental evidence consistent with the predictions of the theory.
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