Analytical HPLC of Peptides (C18, C8 & C4 Columns)
Analytical Reverse-Phase HPLC (RP-HPLC) is the primary method used to determine peptide purity, impurity profile, and chromatographic behavior. Method selection—including stationary phase choice (C18, C8, or C4)—is optimized based on peptide length, hydrophobicity, and analytical objective.
Why Analytical HPLC Is Critical
- Purity determination: Calculates % purity based on integrated peak area.
- Impurity profiling: Detects deletion sequences, truncations, oxidation, and synthesis-related by-products.
- Method comparability: Enables lot-to-lot and stability trending.
- Process verification: Confirms effectiveness of purification and salt-exchange procedures.
Stationary Phase Selection
C18 Columns
C18 columns provide strong hydrophobic interaction and are widely used for general peptide purity analysis. Suitable for most short- to mid-length peptides.
C8 Columns
C8 columns offer moderate hydrophobic retention and may improve peak shape or resolution for peptides that are overly retained on C18 phases.
C4 Columns
C4 columns provide reduced hydrophobic interaction and are often preferred for longer or more hydrophobic peptides where stronger phases may cause excessive retention or peak broadening.
Column selection is determined by peptide properties and analytical objectives.
Mobile Phase & Gradient Strategy
Separation is achieved using gradient elution with aqueous and organic mobile phases. Gradient slope, run time, and temperature are optimized to improve resolution between the main peptide peak and related impurities.
- Controlled gradient ramp for reproducible retention
- Optimized resolution between closely related species
- UV detection at peptide-relevant wavelengths
What Analytical HPLC Detects
- Main full-length peptide
- Deletion or truncated sequences
- Oxidized or modified species
- Aggregation or secondary peak formation
- Process-related impurities
System Suitability & Data Quality
Analytical runs are evaluated for retention consistency, peak shape, resolution, and baseline stability under defined conditions. Method performance is reviewed to ensure reliable purity interpretation.
Purity Reporting
Purity is typically reported as percent peak area of the principal peak relative to total integrated peaks under defined method conditions. Chromatograms with retention times and method parameters are included in the data package.
Chromatographic purity is method-dependent. Orthogonal confirmation by LC-MS may be recommended when identity clarification is required.
Applications
- Release testing for research and regulated non-GMP programs
- Stability and time-point trending
- Comparability studies after process modification
- Salt exchange and counterion evaluation support
Bio-Synthesis, Inc. provides analytical HPLC testing using C18, C8, and C4 methodologies aligned with GLP / ISO 13485 quality systems for regulatedprograms.