Practical Protein Chemistry - A. Darbre 1989

Separation of protein and peptide mixtures by high-performance liquid chromatography
Reversed-phase separation of peptides
Selection of mobile phase composition

The COMPOSITION OF THE Mobile phase must meet the requirements for the isolation conditions and the state of the purified preparation.

1. The SPECTRAL PROPERTIES OF the eluent should be taken into account. The peptide bond absorbs at 206–215 nm, which allows for the Quantitative evaluation of the elution of all Peptides. This circumstance must be considered when selecting the mobile phase composition. Organic acids are relatively transparent at 206–215 nm, although at high concentrations, a baseline drift may occur due to Light absorption by these compounds. A specific solvent can be chosen using reference tables published in manufacturer brochures or textbooks. Acetonitrile, methanol, n-propanol, and ethanol are most commonly used. Solvent purity affects the baseline shift that occurs during gradient elution. Solvents are commercially available that allow operation at a wavelength of 200–215 нм.

2. The size, charge, and Structure/106.html">Hydrophobicity of the peptides must be taken into account. Until recently, the Separation of large peptides (containing more than 30–40 residues) was carried out according to molecular size. On RP Supports, peptides are separated due to hydrophobic interactions; therefore, an increase in peptide hydrophobicity leads to an increase in retention time. Since the pH range is limited to 2.0–7.5 (due to the hydrolytic instability of the support), changes in buffer pH mainly affect the carboxyl and a-amino groups of the peptides. Consequently, at a pH<pI of the Asp and Glu side chains, the hydrophobicity of the peptide will increase because the side-chain carboxyls will be largely uncharged. Peptide hydrophobicity can be altered using ion-pairing interactions of charged side chains. For example, at pH 4, ammonium salts form an ion pair, which helps suppress the ionic interactions of carboxyl groups. Ion-pairing interactions with side-chain anions or cations are used both to increase and decrease peptide polarity. The retention time of a peptide on the Column after entering into an ion-pairing interaction depends on the ability of the complex to participate in partition Chromatography or on the adsorption of the ion-pairing reagent by the stationary hydrophobic phase, which results in ion-exchange interactions.

For most separations, it is sufficient to use phases containing TFA, phosphoric acid, or ammonium acetate. In special cases (e.g., the separation of peptide mixtures with an elevated content of acidic groups), more complex ion-pairing systems are employed [4].

3. If it is necessary to separate a peptide from all accompanying solution components, lyophilization should be performed instead of Desalting, which is accompanied by significant sample losses. In this case, preference is usually given to easily vacuum-volatile ammonium acetate and bicarbonate, triethylammonium formate, TFA, HFBA, formic acid, and acetic acid.

4. Sample isolation conditions must not interfere with the subsequent determination of its properties. For many types of bioassays, organic solvents must be removed. This is typically achieved by evaporation under a stream of nitrogen. Care should be taken to ensure that the salts present and the pH of the solution do not affect the assay results.