Analytical Techniques
Laboratory methods for peptide characterization including HPLC, mass spectrometry, and amino acid analysis.
High-Performance Liquid Chromatography (HPLC)
HPLC is the primary analytical tool for peptide purity assessment. Reverse-phase HPLC separates peptides based on hydrophobicity, with more hydrophobic species eluting later under increasing organic solvent gradients. UV detection at 214 nm monitors peptide bond absorbance, while 280 nm detects aromatic residues.
Purity is expressed as the percentage of total peak area attributed to the primary peptide peak. Research-grade peptides are typically held to ≥95% purity, with some applications requiring ≥99%.
Mass Spectrometry
Mass spectrometry provides definitive identity confirmation by measuring the mass-to-charge ratio (m/z) of ionized peptide molecules. The observed molecular mass is compared to the theoretical mass calculated from the amino acid sequence.
Electrospray ionization (ESI) is commonly used for peptides in solution, producing multiply charged ions that extend detection to larger peptides. MALDI-TOF MS is applied for rapid screening and mixture analysis. Tandem MS (MS/MS) enables sequence confirmation through fragmentation pattern analysis.
Amino Acid Analysis
Amino acid analysis (AAA) determines the molar composition of amino acids in a peptide following acid hydrolysis. The hydrolyzed amino acids are derivatized and quantified by HPLC, providing compositional data that confirms the expected amino acid ratios.
AAA serves as an orthogonal method to mass spectrometry, detecting compositional errors that may not alter the overall molecular mass. It is particularly valuable for verifying the content of amino acids with identical masses.
UV-Vis Spectrophotometry
Peptide concentration is estimated spectrophotometrically using the Beer-Lambert law. Peptides containing tryptophan or tyrosine residues are quantified at 280 nm using calculated molar extinction coefficients. For peptides lacking aromatic residues, the BCA or Bradford protein assay provides concentration estimates.
Capillary Electrophoresis
Capillary electrophoresis (CE) separates peptides based on charge-to-size ratios under an applied electric field. CE complements HPLC by resolving charged variants and isoforms that may co-elute under reverse-phase conditions. Capillary zone electrophoresis (CZE) is the most commonly applied CE mode for peptide analysis.
Certificate of Analysis Interpretation
A Certificate of Analysis (COA) documents the analytical results for a specific peptide lot. Key parameters reported include HPLC purity, molecular mass confirmation by MS, water content, and net peptide content. Researchers should verify that COA values meet their specific application requirements before use.