Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing.
A new fraction collection system for capillary zone electrophoresis (CZE) and capillary isolelectric focusing (CIEF) is described. Exact timing of the collector steps was based on determining the velocity of each individual zone measured between two detection points close to the end of the capillary. Determination of the zone velocity shortly before collection overcame the need for constant analyte velocity throughout the column. Consequently, sample stacking in CZE with large injection volumes as well as zone focusing in CIEF could be utilized with high collection accuracy. Capillaries of 200 microm inner diameter (ID) were employed in CZE and 100 microm ID in CIEF for the micropreparative mode. A sheath flow fraction collector was used to maintain permanent electric current during the collection. The bulk liquid flow due to siphoning, as well as the backflow arising from the sheath flow droplet pressure, were suppressed by closing the separation system at the inlet with a semipermeable membrane. In the CZE mode, the performance of the fraction collector is demonstrated by isolation of individual peaks from a fluorescently derivatized oligosaccharide ladder. In the CIEF mode, collection of several proteins from a mixture of standards is shown, followed by subsequent analysis of each protein fraction by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).
Minarik
M
M
Barnett Institute and Department of Chemistry, Northeastern University, Boston, MA 02115, USA.
Foret
F
F
Karger
B L
BL
D000818:Animals; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D007525:Isoelectric Focusing / Q000295:instrumentation* / Q000379:methods; D011506:Proteins / Q000032:analysis / Q000737:chemistry
D011506:Proteins