Practical Protein Chemistry - A. Darbre 1989

Separation of protein and peptide mixtures by high-performance liquid chromatography
High-speed liquid chromatography of proteins

High-Performance Liquid Chromatography of Proteins (HPLCP) is widely used in biopolymer research; let us examine the main advantages of this method [20].

Pharmacia has developed a Column packed with 9.8 (±2%) µm particles that are stable across a 2<pH<12 range. The sorbent is available in cation-exchange and anion-exchange forms, as well as a support with a surface modified for chromatofocusing (Mono Q, Mono S, and Mono P columns). These sorbents feature high capacity and allow operation at high flow rates (up to 1 ml/min) under moderate pressures (10–20 bar) generated by reciprocating pumps. The low operating pressures make it possible to use Glass columns. The chemical Stability of the Supports permits The Use of a wide range of buffers, including detergents and chaotropic agents (e.g., Gu∙HCl). Pharmacia has developed a series of buffers that enable optimal Separation within specific pH ranges, avoiding the pH shifts characteristic of conventional Ion-exchange chromatography. Separation times are exceptionally short (1–5 min) because resolution depends much less on separation time than in other ion-exchange processes. Protein elution data obtained on conventional ion-exchangers can be directly translated to HPLCP experiments, usually resulting in improved resolution. When no information is available on The properties of the protein mixture to be fractionated, separation conditions are optimized by performing several HPLCP runs with different buffers, with the results represented as elution curves. Pharmacia's periodicals outline the extensive potential Applications of their columns designed for HPLCP. In principle, it is possible (though not recommended by the manufacturer) to use these columns with standard chromatographs equipped with appropriate fittings and pressure-relief Valves to prevent over-pressurization. The latter device is essential to protect the glass columns from Damage caused by high pressures.

When operating such columns in the presence of chloride ions at extreme pH values, pumps made of stainless steel should be avoided.

Chromatofocusing using Mono Q columns is a novel, high-resolution technique that could significantly impact the design of protein separation schemes. A major advantage of this method is The ability to exploit differences in protein net charge at pH values close to their respective pI. A detailed Description of the fundamental principles of separation by this method can be found in Pharmacia's technical literature.