Fundamental and practical studies of polymer separations by HPLC: I. Thermal gradients; II. Interparticle size exclusion; III. Ion-exchange separations of DNA oligomers

Reversed-phase liquid chromatography (RPLC) for polymer separations has gained much attention in the past decade but the actual retention mechanism is still a subject of debate. The first part of this dissertation studies the separation of polymers by RPLC. It concludes that the retention mechanism of polymers on RPLC can be explained by conventional, partition/adsorption chromatography theory. Linear Van't Hoff plots are obtained from isocratic, isothermal retention data of polystyrenes and poly(ethylene oxide)'s. Thermal gradients over the separation time and along the separation column's length were developed to enhance the separation of organic polymers. The advantages of these thermal gradients over conventional solvent gradients are discussed. A mathematical model is developed then to successfully predict the retention of polymers under thermal gradient conditions using isothermal retention data.; The second part of this dissertation focuses on one of the most basic and most important parameters in HPLC---the column void volume. Despite the fact that HPLC has been studied for over three decades, the column void volume remains a subject of debate as well. Most of the user-community assume that a column has a "fixed" void volume independent of the size of the analyte but leaders in the field argue that is not true for small solutes. It is concluded from the work reported here that a given column's void volume is unique for each different polymer analyte. The difference in the column void volume is attributed to exclusion effect caused by the different sizes of analytes. Void volumes of columns packed with various kinds of packing materials are examined. We report the first-ever observation that the void volume of a column packed with totally non-porous materials changes with the solute molar mass. A quantitative relationship between the column void volume and solute molar mass is established.; Finally, we investigate the retention behavior of synthetic oligonucleotides on a continuous-bed-matrix, strong anion-exchange column. The separation is shown to be based on the chain length of the oligonucleotide. The separation mechanism is an anion exchange process but hydrophobic interaction also plays a small role. Both organic mobile phase modifiers and column temperature significantly affect the retention of oligomers. A volatile buffer system, e.g., triethylamine acetate, is employed and no desalting procedure is required after the column separation step. The measured recoveries are 70% or higher. The loading capacity of an analytical column (UNOTM Q1 strong anion exchange column, 7 mm ID x 35 mm) is more than 366 micrograms.