QUANTITATIVE-ANALYSIS OF PEPTIDES WITH NMR-SPECTROSCOPY

The determination of peptide concentration with H-1 nuclear magnetic r esonance (NMR) spectroscopy using an internal standard or an external standard in a sealed glass capillary was investigated for three tyrosi ne-containing tripeptides. Trimethylsilylpropionic acid (TSP) and male ic acid were tested as external standards for quantitation by proton N R IR. Although comparable results were obtained for either standard, t he performance of maleic acid was found to be superior because of its better long-term stability in the sealed capillary. Loss of TSP from s olution occurred over time due to adsorption onto the walls of the cap illary, necessitating frequent recalibration against the primary stand ard, potassium acid phthalate (KHP). The peptide contents of solid pep tides determined with H-1 NMR are compared with those obtained from ul traviolet (UV) absorbance measurements of the tyrosine chromophore. Th e versatility of NMR for the quantitative analysis of peptides that do not contain an appropriate UV chromophore make it well-suited for the determination of peptide concentration in aggregation studies or for the preparation of solutions for high-throughput screening of biologic al activity.