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308 Heegaard et al.
Fig. 1. Images of capillaries cut by different methods. The capillary is a 375-m o.d.,
25-m i.d. polyimide-coated fused-silica capillary. The following cutting methods were
used: (1), standard cleave using a ceramic cleaving stone; (2) precision cleave using a
cleaving device (Polymicro); (3) saw cut and (4) laser cut using a programmable CO 2
laser station. Reproduced by permission from Polymicro Technologies, LCC AZ, USA.
3.2. Washing, Conditioning, Electrophoresis and Sample Buffers
At neutral pH in an uncoated capillary the wall charge is negative and
creates an electroendosmotic (EEO) flow towards the cathode, which in the
conventional set-up is situated at the detector end of the capillary. Actually, full
protonation of the siloxide groups (zero charge) first occurs at a pH as low as
2.0 (30). In addition, the magnitude of the EEO flow decreases with increasing
buffer ionic strength. All protein analytes/ligands that display positive charge
will be prone to attach to the fixed capillary wall charges by electrostatic
interactions. Therefore, proteins with low isoelectric points, i.e., negatively
charged at neutral pH, will be more likely to be recoverable than basic proteins.
However, even acidic proteins may – despite a low pI – contain patches of
positively charged side chains and display the hallmarks of disruptive wall
interactions: variable peak areas, tailing or other asymmetry or disappearance.