DEVELOPMENT OF SUPPORT MATERIALS FOR HIGH - PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC, SILICA, POLYETHYLENEIMINE)
Abstract
Large pore ((GREATERTHEQ) 300(ANGSTROM)), spherical microparticulate silicas possessing n-alkyl hydrocarbon ligands were found to be optimal for the purification of large denatured peptides. Various macroporous silicas were tested and it was found that some silicas offered better selectivity than others for reversed-phase peptide and protein separations. Additional variables found to affect protein resolution were thermal pretreatment of silica, silylation reaction conditions, and n-alkyl chain length. A test mixture for monitoring protein resolution potential was established because theoretical plate values were poor indicators of column performance. Peptides and proteins were nicely resolved on columns only 5 cm in length. The interaction between protein and stationary phase was based on an adsorption rather than partition mechanism. A method for fractionating transfer ribonucleic acids by descending salt gradient elution on reversed-phase columns was developed. Stationary phase variables such as n-alkyl chain length and silica type were investigated and it was concluded that ethyl or n-butyl groups covalently attached to macroporous silica worked the best. Mobile phase parameters including salt concentration, organic solvent, pH and temperature were found to affect resolution. Polyethyleneimine anion-exchangers were developed for both protein and oligonucleotide fractionations. The process consisted of adsorbing polyethyleneimine onto silica and then covalently immobilizing the polyamine layer with a bifunctional crosslinker. The type of crosslinker employed was found to contribute to stationary phase selectivity for both proteins and mononucleotides. Changes in ionic strength could be used to alter retention times of various proteins on anion-exchangers depending upon the relative lipophilicity of the crosslinking agent. Development of oligonucleotide anion-exchangers based on polyethyleneimine was also achieved and a general method for converting pre-packed silica columns into anion-exchangers was outlined. Short, 5 cm anion-exchange columns were capable of fractionating oligonucleotide homologs of up to at least 35 bases in chain length.
Degree
Ph.D.
Subject Area
Biochemistry|Analytical chemistry
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