How to verify the structure and purity of peptides?
The purity requirements for synthetic peptides depend on the specific needs. For certain experiments or applications, peptide purity may not be critical. However, in other cases, high purity peptides, free from racemization, deletions, or side-chain modifications, may be required. Historically, thin-layer chromatography (TLC) and analytical reverse-phase high-performance liquid chromatography (HPLC) have been used to assess purity and the likelihood of successful purification strategies. For example, if all peaks are well-separated in an analytical HPLC chromatogram, low-pressure reverse-phase HPLC may work effectively. However, in high-throughput settings using automated solid-phase peptide synthesis (SPPS), high-pressure preparative HPLC is commonly used. Nowadays, methods based on MALDI-TOF-MS and LC-ESI-MS(MS) are more routinely employed. Preferred techniques for verifying the structure and purity of synthetic peptides include amino acid analysis and mass spectrometry. Nevertheless, no single biomolecular characterization technique or procedure is infallible, and it is recommended to use multiple techniques to analyze the final product.
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