| This work is presented in two parts. In Part I, Chapter 1, the relative importance of three purported retention processes (hydrophobic, silanophilic and metallophilic) in reversed-phase high performance liquid chromatography (RP-HPLC) of small molecules is studied. Also, the chromatographic effects of stainless steel frits are critically evaluated.; Part I, Chapter 2 is concerned with the general chromatographic properties observed for fluorinated bonded phases. These results are compared to the properties of a hydrocarbonaceous support. Test solutes containing a wide range of functional groups are studied in order to determine if any specificity of functional group/stationary phase exists.; Part II, Chapter 3 focuses on the extremely complex area of the RP-HPLC of proteins. Almost since the inception of chemically modified silica, these bonded phases have been used to separate peptides and proteins. Although some of the original problems have essentially been eliminated (long analysis time, poor resolution, large sample size requirement), others still plague the chromatographer (irreversible protein adsorption, protein denaturation, loss of enzyme activity). The contribution of stainless steel frits to the irreversible loss of proteins is studied. Replacement of the frits with meshes allows for the study of those chromatographic parameters which play important roles in protein adsorption and denaturation on the packing material surface. These studies also help define the mechanism of protein adsorption onto RP supports. Finally, a quantitative evaluation is done for a selected group of packing materials as to their applicability for use as chromatographic supports in protein separations.; Part II, Chapter 4 deals with the modification of the base silica through the use of mobile phase modifers (MPMs) and the permanent alteration of the silica surface through the polymerization of previously adsorbed cyclic tetraaza compounds. These cyclic tetraaza compounds are also used as MPMs. |
