Fractioncollectioninmicropreparativecapillary zoneelectrophoresisandcapillaryisoelectric 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 col- umn. 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 mm inner diameter (ID) were employed in CZE and 100 mm ID in CIEF for the micropre- parative mode. A sheath flow fraction collector was used to maintain permanent elec- tric 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, col- lection of several proteins from a mixture of standards is shown, followed by subse- quent analysis of each protein fraction by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Keywords: Fraction collection / Micorpreparative / Wide-bore / Mulitple-point detection / Optical fibers / Closed system / Capillary zone electrophoresis / Capillary isoelectric focusing / Matrix- assisted laser desorption/ionization EL 3706 MarekMinarik FrantisekForet BarryL.Karger Barnett Institute and Department of Chemistry, Northeastern University, Boston, MA, USA 1 Introduction CZE and CIEF are well-established high resolution sepa- ration techniques [1±4]. While both methods are generally performed on the analytical level, collection of separated fractions may be necessary for purification or further structural characterization [5±12). For example, DNA fragments can be separated, individually collected and then, after PCR, identified by DNA sequencing [6]. On- line structural characterization of proteins and peptides by MS, while powerful, can often be enhanced by enzymatic digestion of collected fractions, followed by MS of the digested products [7±11]. Moreover, CIEF can be used as a sample purification method for isolation of com- pounds from peptide and protein mixtures [13]. Most commercial instruments offer the possibility of frac- tion collection using an autosampler [14]; however, the accuracy for collection of several bands can be poor due to migration disturbances caused by repeated interrup- tions of the applied electric field when changing the col- lection vials. If the separation pattern includes adjacent migrating peaks, isolation of individual compounds can be difficult. A variety of laboratory-made designs have been developed to circumvent the problem of electric interrup- tions. For example, capillary fracture [15], on-column frit [16] or tee-junction [17, 18] have been employed to main- tain the permanent electric field during collection. Zones have been eluted from the capillary by electroosmotic [16, 19] or sheath flow [9, 20±22], with the eluting fractions being collected in vials [23] or directly deposited onto membranes [24±27] for (MALDI-TOF-MS) [7, 8, 26, 27]. Previously, our laboratory developed a sheath flow device with UV detection near the column exit for fraction collec- tion into glass capillaries [21]. The system was used for collection of DNA fragments [6, 21], as well as oligosac- charide analytes [28] without interrupting the applied elec- tric field. With fraction collection, an exact timing of the collector based on the calculated zone velocities is essential. With single point detection, the velocity determination is valid only if, following injection, the zones migrate with constant speed through the column. In reality, zone velocity is often not constant since the sample migration might be Correspondence: Professor B. L. Karger, Barnett Institute, Northeastern University, 341 Mugar Building, 360 Huntington Avenue, Boston MA 02115, USA E-mail: bakarger@lynx.dac.neu.edu Fax: +617-373-2855 Abbreviations: APTS, 1-aminopyrene-3,6,8-trisulfonate; PEEK, poly (ether-ether) ketone Electrophoresis 2000, 21, 247±254 247  WILEY-VCH Verlag GmbH, 69451 Weinheim, 2000 0173-0835/00/0101-0247 $17.50+.50/0 CEandCEC