RP-HPLC (Reverse-Phase High-Performance Liquid Chromatography) is the reference analytical technique for separation, purification and purity control of synthetic peptides. It relies on separation by hydrophobicity, using a non-polar stationary phase and polar mobile phase — inverse of normal-phase chromatography, hence the name.
Principle and stationary phase. The RP-HPLC column is typically functionalised silica with grafted alkyl chains: C18 (octadecyl, most universal for peptides <5 kDa), C8 (octyl, moderately hydrophobic peptides), C4 (butyl, peptides and small proteins up to 20 kDa), C3/C1 or phenyl (aromatic selective retention). Typical dimensions are 250 × 4.6 mm analytical, 250 × 21.2 mm semi-preparative, 250 × 50 mm preparative. Particle size (3, 5 or 10 μm) and porosity (100-300 Å) affect resolution and capacity.
Mobile phase and gradient. The standard gradient combines water + 0.1% TFA (solvent A, acidic aqueous phase) and acetonitrile + 0.1% TFA (solvent B, organic phase), TFA acting as ion-pairing agent neutralising peptide charges and improving peak symmetry. A typical 10 to 70% B gradient over 20-40 min allows separation of standard linear peptides. UV detection at 214 nm (peptide bond) and 280 nm (Tyr, Trp) is universal; PDA, ELSD and MS detectors (coupled LC-MS) provide additional information.
Peptide applications. Analytical RP-HPLC validates batch purity (≥98% on commercial RUO peptides), identifies impurities (deletion, deamidation, oxidation, racemisation) and their relative quantification. Preparative RP-HPLC is the critical final step of SPPS production to separate target peptide from truncated sequences, TFA-adduct fragments and synthesis impurities. The technique is documented in USP <621> and Ph. Eur. 2.2.46 monographs for pharmaceutical peptide products.
Décryptez la caractérisation complète d'un peptide : RP-HPLC préparative et analytique, LC-MS haute résolution Orbitrap/QTOF, RMN 2D (COSY/TOCSY/NOESY), dégradation d'Edman, cartographie peptidique MS/MS, dichroïsme circulaire. Les 10 techniques qui séparent un peptide research-grade d'un peptide douteux, avec seuils de détection et stratégie de QC.
Maitrisez les mecanismes qui detruisent vos peptides : oxydation Met, deamidation Asn, hydrolyse, aggregation. Protocoles de lyophilisation, conditions de stockage, reconstitution optimale et QC shelf-life. Le pilier technique que tout laboratoire doit integrer pour garantir l'integrite de ses peptides en recherche exploratoire.
Principe Merrifield, stratégies Fmoc et Boc, résines (Wang, Rink amide, 2-chlorotrityl), agents de couplage (HBTU, HATU, DIC/HOBt), cycle de synthèse, clivage TFA, purification RP-HPLC, contrôle qualité LC-MS et test Kaiser. Le pilier scientifique de référence sur la fabrication des peptides synthétiques.