Preparation of Solutions and Reagents

INTRODUCTION Trifluoroacetic acid (TFA) is commonly used in the manufacturing process to release synthesized peptides from solid-phase resins. TFA or acetate is also used during reversed-phase HPLC purification of peptides. TFA is manufactured using acetate and fluoride as precursors, and residual levels of these compounds may be present whenever TFA is used. Residual TFA, fluoride, and, to a much lesser extent, acetate are toxic and undesirable in peptides intended for preclinical and clinical studies. A method for the determination of TFA, acetate, and fluoride must be suitable for peptide formulations and be capable of verifying the removal of these anions during the production process. TFA has been assayed by gas chromatography, GC mass spectroscopy, reversed-phase HPLC, isotachophoresis, infrared spectrometry, titration, spectrophotometry, and ion-exchange chromatography. Ion chromatography (IC) is advantageous because it is sensitive, simple, and can be automated. The separation mechanism of IC is based on an anionexchange displacement process occurring between the sample ions and eluent ions with the anion-exchange functional groups bonded to the stationary phase. A typical stationary phase consists of a grafted, solvent-compatible, alkyl-based ion-exchange resin. The separation of TFA, acetate, and fluoride illustrated in this application note uses a stationary phase functionalized with alkyl quaternary ammonium groups. Effluent from the analytical column is passed through a suppressor that reduces the total background conductance of the eluent and increases the electrical conductance of the analyte ions. With suppressed conductivity, signal-to-noise ratios are improved approximately 50-fold compared to nonsuppressed conductivity. This application note describes the analysis of commercially prepared, water-soluble peptides using ion chromatography. This method requires minimal sample preparation, and the analytes, fluoride, acetate, and TFA, are easily separated without significant peptide interference.