High-Performance Liquid Chromatography
HPLC serves as the primary analytical technique for peptide purity assessment and purification. Reversed-phase HPLC (RP-HPLC) separates peptides based on hydrophobic interactions with a nonpolar stationary phase, typically C18 or C8 bonded silica.
Analytical HPLC employs UV detection at 214-220 nm, where peptide bonds absorb strongly. Peak integration provides quantitative purity data expressed as percentage of total peak area.
Typical RP-HPLC Parameters
- Column: C18, 4.6 x 150 mm, 5 μm particle size
- Mobile Phase A: 0.1% TFA in water
- Mobile Phase B: 0.1% TFA in acetonitrile
- Gradient: 5-65% B over 30 minutes
- Flow Rate: 1.0 mL/min
- Detection: UV at 214 nm
Mass Spectrometry
Mass spectrometry confirms peptide identity by measuring molecular mass. Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are the primary ionization methods for peptide analysis.
ESI-MS generates multiply charged ions from peptides in solution, useful for LC-MS applications. MALDI-TOF produces singly charged ions and provides rapid analysis of purified samples. Mass accuracy within 0.1% of theoretical confirms peptide identity.
Amino Acid Analysis
Amino acid analysis (AAA) determines peptide composition through hydrolysis and chromatographic quantitation of individual amino acids. The technique confirms the presence and ratio of expected amino acids in the peptide sequence.
Acid hydrolysis (6N HCl, 110°C, 24 hours) cleaves peptide bonds, releasing free amino acids for derivatization and chromatographic separation. Some amino acids (tryptophan, cysteine) are destroyed or modified during hydrolysis, requiring alternative methods for quantitation.
Peptide Content Determination
Peptide content represents the mass fraction of actual peptide in a lyophilized sample. The remainder consists of counterions (acetate, trifluoroacetate), water, and residual solvents. Typical peptide content ranges from 70-90%.
Peptide content is determined through amino acid analysis or UV spectroscopy using calculated extinction coefficients. This value enables accurate mass-based preparation of peptide solutions for research applications.
Water Content Analysis
Karl Fischer titration quantifies water content in lyophilized peptides. Water absorption occurs during handling and storage, affecting both weight-based calculations and long-term stability.
Typical specifications limit water content to less than 8-10% of total mass. Proper storage conditions (desiccated, frozen) minimize water uptake between analysis and use.
Endotoxin Testing
Limulus amebocyte lysate (LAL) assays detect bacterial endotoxin contamination. Endotoxin limits are specified when peptides are used in applications where bacterial contamination could affect experimental results.
Standard specifications for research peptides typically require less than 1 EU/mg (endotoxin units per milligram). Lower limits may be specified for specialized applications.
Educational Notice
This material is provided for educational purposes related to laboratory research methodology. It does not constitute comprehensive analytical method documentation. Practitioners should consult validated protocols and instrument manufacturer guidelines for their specific applications.