The purity determined by HPLC (%) corresponds to the percentage of requested peptide in relation to the total amount of material absorbing at 210 – 220 nm i.e. the desired product, peptidic by-products and other impurities.
The net peptide content (NPC) is the fraction of peptidic material (i.e. the requested peptide and the peptidic impurities) relative to counter-ions and residual water. The latter do not contain nitrogen, allowing the determination of the net peptide content by elemental analysis.Not all impurities can be removed by a single HPLC run. Usually a few peptidic contaminants remain, mostly deletion sequences (peptides lacking one or more amino acids of the target sequence). Such by-products differ only slightly in polarity, hence their removal may fail. Truncation sequences (which may be generated deliberately by capping steps to avoid the formation of deletion peptides), incompletely deprotected peptides and by-products generated during the synthesis or during the final cleavage may also be found among the impurities.
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