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HPLC Mass Spec Testing Peptide Purity Reports: Methodology Comparison
HPLC-UV (214 nm) Relative purity. Percentage of target peptide vs peptide impurities 0.1% impurity detection Quantifying synthesis byproducts and peptide-related substances Cannot confirm molecular identity or detect non-peptide contaminants ESI-MS (Electrospr
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- HPLC-UV (214 nm)
- Relative purity. Percentage of target peptide vs peptide impurities
- 0.1% impurity detection
- Quantifying synthesis byproducts and peptide-related substances
- Cannot confirm molecular identity or detect non-peptide contaminants
- ESI-MS (Electrospray Ionization Mass Spec)
- Exact molecular weight and charge state distribution
- ±0.5 Da accuracy for peptides under 3000 Da
- Verifying correct amino acid sequence and detecting truncations
- Does not quantify purity percentage
- MALDI-TOF MS (Matrix-Assisted Laser Desorption)
- Molecular weight confirmation for larger peptides
- ±1–2 Da accuracy depending on size
- Rapid screening of peptide identity in high-throughput synthesis
- Lower resolution than ESI. Less suitable for complex mixtures
- LAL Endotoxin Assay (Limulus Amebocyte Lysate)
- Bacterial endotoxin contamination in EU/mg
- 0.01 EU/mg detection threshold
- Ensuring biological safety for cell culture and in vivo use
- Does not detect non-endotoxin contaminants like DNA or host proteins
- Karl Fischer Titration
- Water content as percentage of total sample mass
- 0.01% water detection
- Calculating net peptide content for accurate dosing
- Only measures water. Does not account for salts or residual solvents