TFA (trifluoroacetic acid, CF3COOH, CAS 76-05-1) is a fluorinated organic acid of molar mass 114.02 g/mol, pKa 0.23 (approximately 100000x more acidic than acetic acid), volatile liquid (bp 72°C) miscible with water and organic solvents. It is the most emblematic reagent of peptide chemistry, used both in SPPS synthesis, RP-HPLC purification and structural analysis (MS, Edman).
Final cleavage in Fmoc SPPS. In Fmoc SPPS synthesis, TFA is used as strong acid for simultaneous final cleavage of peptide from resin and side-chain protecting groups (tBu for Ser/Thr/Tyr, Trt for Cys/Asn/Gln/His, Pbf for Arg, Boc for Trp/Lys, OtBu for Asp/Glu). The standard cocktail is 95% TFA / 2.5% H2O / 2.5% TIS (triisopropylsilane as scavenger, 2-4 h at room temperature). Specialised formulations exist for Cys/Met-rich peptides (Reagent K: TFA/thioanisole/water/phenol/EDT 82.5:5:5:5:2.5) or Trp-rich.
Ion-pairing agent in RP-HPLC. Added at 0.05-0.1% in solvents A (water) and B (acetonitrile) of a peptide RP-HPLC gradient, TFA acts as ion-pairing agent: protonated TFA forms an ion pair with peptide basic groups (NH3+, guanidinium, imidazolium), masking charge and increasing hydrophobic retention on C18 stationary phase. This improves peak symmetry and resolution. Drawback: TFA suppresses signal in positive ESI-MS, motivating replacement by formic acid (0.1%) in analytical LC-MS.
Precautions and safety. TFA is corrosive (skin, eye, pulmonary burns upon inhalation), volatile and potentially ecotoxic (persistent degradation product). Storage is in borosilicate glass or PTFE bottle, under inert N2 atmosphere, shielded from light and heat. Elimination of TFA-containing peptide residues (TFA adducts) can be achieved by multiple lyophilisation from 10 mM HCl, C18 Sep-Pak desalting or anion exchange resin.
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.