The formation of 3.5% T, 3.3% C cross-linked polyacrylamide is monitored in 75-microns-i.d. electrophoresis capillaries by Raman microprobe spectroscopy. The disappearance of the acrylamide 1292-cm-1 band is followed with 60-s time resolution for 30 min, and 2-4 min resolution for up to 10 h. Polymerization is 98% complete in 1.5 h and greater than 99% complete after 2 h. In the 900-1700-cm-1 region no bands attributable to cross-linking are observable. Reaction in the capillary follows second-order kinetics. The reaction is faster in the bulk system because heat dissipation is not sufficient to maintain a constant temperature.
Variable frequency pulsed field capillary gel electrophoresis of nucleic acid restriction fragment mobility and resolution is reported. The frequency of the sine wave superimposed on the dc driving field was randomly varied throughout the duration of the electrophoresis in order to enhance the fragment separations. By frequent changes of frequency, fragments of various sizes experienced optimum conditions for separation sometime during the electrophoresis. The mobilities of phi X 174 HaeIII fragments in 9% T linear polyacrylamide were primarily dependent on power dissipation in the capillary, independent of whether the driving field was dc only or modulated. Resolution of long fragments exhibited a strong modulation frequency dependence. Modulated driving fields provided better resolution of the 603-1353 base pair fragments than dc only fields. Modulation protocols with center frequencies near 100 Hz gave the highest resolution.
An aluminum heat sink and radiator are used with forced air cooling of an electrophoresis capillary. Theoretical analyses of the operating limits and heat dissipation characteristics are presented. A system designed for power dissipation as high as 5 W is shown to dissipate heat efficiently and to operate without arcing at voltages higher than 30 kV.
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