I. High Pressure Liquid Chromatography of Fatty Acids, α-Keto Acids and Nucleotides, Nucleosides and Thier Bases on Reversed Phase Columns II. Solute Interactions with Carrier Solutions and Stationary Reversed Phases in High Pressure Liquid Chromatography

Part I: High pressure liquid chromatographic methods for the determinations of fatty acids, α-keto acids and nucleotides, nucleosides and their bases were developed. A. Fatty Acids. Saturated and unsatureated fatty acids were quantitatively converted to p-methoxyanilides to enhance their uv detectability. Rapid methods for the synthesis of the anilides with triarylphosphine reagents were developed. The p-methoxyanilides were separated by reversed phase chromatography using a 10 μm stationary phase in a 4.2 mm i.d. X 30 cm long column. Water-acetonitrile and water-methanol were used in hyperbolic gradient elution. B. α-Keto Acids. Pyruvic and α-Ketoglutaric acids were quantitatively and rapidly converted to quinoxalones by a new method to enhance their uv detectability. The quinoxalones were separated by reversed phase chromatography. Aqueous ammonium acetate solutin and methanol were used in linear gradient elution. The method was applied to the determination of these acids in the urine of normal, and diabetic patients and patients with renal dysfuntin. C. Nucleotides, nucleosides and their bases. Uracil, cytosine, adenine, and guanine and the ribonucleosides of these bases were separted isocratically on a reversed phase column, 4.6 mm i.d. X 25cm length, in the presence of 10-camphorsulfonate ion. The groups UMP, CMP, AMP, and GMP; UDP, CDP, ADP, and GDP; UTP, CTP, ATP, and GTP; and AMP, ADP, ATP were separated isocratically in the presence of tetra-n-butylammonium ion. The twelve nucleoside phosphates and cyclic AMP could not be separated isocratically but could be separted by gradient elution with increasing ionic strength and methanol concentration. Part II. The interactions of homologous series of organic ions and neutral organic molecules with stationary reversed phases and carrier solvents in high pressure liquid chroatography were studied. A. Organic Ions. Two solvent systems, aqueous ammonium acetate and ammonium chloride-phosphoric acid solution with varying amounts of organic solvent, were used. The retention behavior of charged species in the presence of salts was related to the species hydrophobic size, concentration of aqueous salt solution, organic composition of the mobile phase, and the charge character of the stationary phase surface. A practical result of this study was the demonstration of isocratic separations of carboxylic acids and amines in their base and acid forms respectively. B. Organic molecules. The retention of six homologous series of organic compounds was found to be a function of the water content of the organic mobile phase in reversed phase chromatography. Methanol and acetonitrile were found to be the most selective mobile phases. The sensitivity of the homologous series\u27 retentions to the water content of methanol increased with higher octadecyl content of the stationary phase. The sensitivity of retentions of n-alkanes, ethers and esters to water content of acetonitrile increased with lower octadecyl content of the stationary phase. n-Alcohols, amides and ketones interacted with negative sites on the silica surface of the stationary phase when acetonitrile was used