New approaches to concentration, desalting and separation of biopolymers in capillary electrophoresis

Sammanfattning: In the first part of the thesis five new on-tube methods for concentration and desalting ofampholytes, such as peptides and proteins, are described in Papers I and II. The methods arebased on the fact that electrophoretic migration velocities decrease upon a decrease in theabsolute value of the zeta potential of a solute and the pore size of the electrophoresis mediumand upon an increase in the cross section of the electrophoresis chamber, the viscosity and theelectrical conductivity of the electrophoresis medium. A combination of displacementelectrophoresis and a hydrodynamic counter flow is also utilized to create a stationary zone inwhich the sample solute can be concentrated continuously. Paper III describes an on-tubedesalting technique for IEF, which is based on an automatic substitution of the salts in thesample with an ampholyte solution in a short focusing pre-step. A simple off-tubeconcentration and desalting method using a hollow fiber is described in Paper IV, which isbased on the transport of water by evaporation or the Donnan effect out of the fiber throughthe pores in its wall and has the advantage that solute adsorption is negligible. The method canbe used not only for macromolecules but also for low-molecular-weight compounds.The second part of thesis consists of two publications about HPCE in the presence ofadditives. In Paper V liposomes were used as a pseudostationary phase in CZE. The decreasein the mobility of an analyte owing to the presence of liposomes reflected interaction betweenanalytes and liposomes. Paper VI deals with the shifts in mobilities of peptides and proteinswhen buffers are supplemented with β-cyclodextrin sulfate and 6-amino β-cyclodextrin. A newdefinition of resolution of two very adjacent peaks, without knowing peak widths, is suggested.Studies of crystals of lysozyme and MBP by HPCE were presented in the last part of thethesis (Paper VII), which show that HPCE can be used to shed some light on the question whysome protein crystals afford low resolution upon X-ray diffraction.