Adsorption Chromatography of Small C-Terminal Peptide Amides on Dihydroxyalkyl Bonded Silica High Performance Liquid Chromatography Columns and Application to Purification of Insect Neuropeptides
The chromatography of small neuropeptides (< 1400 mw) was studied on a high performance liquid chromatography (HPLC) sized exclusion column designed with an aqueous-compatible dihydroxyalkyl bonded silica support. The major mechanism of retention for small peptide amides on this column was clearly not size exclusion but adsorption. Peptide amides were nearly always retained beyond the calculated salt volume of these columns. This retention was nearly five column volumes for selected peptides like substance P. Under the same conditions, peptide C-terminal acid analogs always eluted much closer to retention times predicted by their molecular weight. Changes in the composition of the mobile phase indicated that the selective retention of the peptide amides was primarily the result of selective yet weak hydrophobic interactions. However, most peptide amides will elute from these supports without the aid of organic solvents, an ideal characteristic for direct coupling of this method to reversed-phase HPLC. A pilot two-dimensional (dihydroxyalkyl HPLC/reversed-phase HPLC) HPLC purification of a diuretic peptide from mosquito head extracts showed that the coupling of these two forms of chromatography could be used to quickly isolate a minor component of a complex mixture.
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