Carrier-free zone electrophoresis, displacement electrophoresis and isoelectric focusing in a high-performance electrophoresis apparatus.
A characteristic feature of high-performance electrophoresis (HPE), the electrophoretic counterpart of high-performance liquid chromatography (HPLC), is that the separation chamber is a thin-walled, narrow-bore (0.05-0.3 mm) glass or fused-silica capillary tube for rapid dissipation of the Joule heat in order to minimize thermal zone deformation even at high field strengths. This paper is centered around the usefulness of HPE for separation in a carrier-free medium (i.e., in buffer alone) and deals with both zone electrophoresis, isoelectric focusing and displacement electrophoresis. Examples are given of analytical and micropreparative separations of inorganic and organic ions, proteins, viruses and bacteria. The run times are 5-30 min. Discontinuous buffer systems have up to now been used exclusively for the separation of proteins by electrophoresis in polyacrylamide gels ("disc electrophoresis"). However, the Ornstein and Davis discontinuous buffer system has been modified to adapt it to carrier-free zone electrophoresis in order to achieve automatic sharpening of the starting zone. Very high resolution of serum proteins was obtained when they were subjected to free high-performance disc electrophoresis in such a modified buffer system. To show that the HPE apparatus permits electrophoresis also in a gel medium, a polyacrylamide electrophoresis in SDS is presented. This experiment illustrates the difference between electropherograms obtained in free solution and in a molecular-sieving medium. Detection can be performed both on- and off-tube. The latter technique permits the rapid identification of the solutes by photodiode array spectrophotometry and the collection of fractions for further studies. The former detection method is simpler but mainly useful for analytical purposes. Non-UV-absorbing ions can be monitored with the aid of an on-tube UV detector if the run is performed in a UV-absorbing buffer.
D001798:Blood Proteins / Q000302:isolation & purification; D004586:Electrophoresis / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D007525:Isoelectric Focusing; D013056:Spectrophotometry, Ultraviolet; D014168:Transferrin / Q000302:isolation & purification; D005719:gamma-Globulins / Q000302:isolation & purification
D001798:Blood Proteins; D014168:Transferrin; D005719:gamma-Globulins