Joseph J. Kirkland

Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples. I. Optimizing the separation of a growth hormone tryptic digest.

Peptide and protein samples are often complex mixtures that contain a number of individual compounds. The initial HPLC separation of such samples typically results in the poor resolution of one or more band pairs. Various means have been suggested for varying separation selectivity so as to minimize this problem. In this study of a tryptic digest of recombinant human growth hormone, the simultaneous variation of temperature and gradient steepness was found to be a convenient and effective means of varying selectivity and optimizing the separation. The use of computer simulation greatly facilitated this investigation.

Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples: II. Selectivity effects observed in the separation of several peptide and protein mixtures

Changes in band spacing as a function of temperature and/or gradient steepness were investigated for four peptide or protein samples. Reversed-phase HPLC in a gradient mode was used to separate tryptic digests of tissue plasminogen activator and calmodulin. Additionally, a synthetic peptide mixture and a storage protein sample from wheat were studied. Simultaneous changes in gradient steepness and temperature were found to provide considerable control over band spacing and sample resolution. The effects of temperature and gradient steepness on selectivity in these systems appear to be complementary. Simultaneous optimization of both temperature and gradient steepness thus represents a powerful and convenient means of controlling band spacing and separation. Because of the complexity of these sample chromatograms, computer simulation proved to be a useful tool in both interpreting these experiments and in optimizing final separations.

Functionalized Porous Silica Microspheres as Scavengers in Parallel Synthesis

The use of solid scavengers in parallel solution-phase organic synthesis is an effective method for work-up and purification. Functionalized macroreticular or gel-form polystyrene particles are generally used for scavenging applications, how ever these materials have some limitations. We have developed new scavenging reagents based on ultrapure silica microspheres displaying a variety of functional groups useful for sequestering impurities from reaction products. These materials are easy to handle, have excellent mass-transfer properties, and are efficient scavengers in both polar and nonpolar organic solvents. The properties of these materials were tailored specifically to fit the needs of a medicinal chemist employing parallel synthesis techniques in current commercial equipment. Results are presented from head-to-head comparisons with conventional scavengers in tests designed to demonstrate the versatility of these new materials.