Preparative HPLC column overloading

Low loading on to preparative columns results in poor yields due to nonspecific losses. On the other hand, overloading could cause peak broadening due to solubility problems on elution from the column. RP-HPLC columns have high capacities for protein retention and a loading of about 50% of total capacity of the column is best. A guide is 2 g for 25 x 250 mm column, 100 mg for (10 x 250 mm column) and 5 mg for (4.6 x 250 mm column). 

Preparative HPLC may involve either scale-up operations where analytical chromatographic conditions are used but with slightly larger sample loads, or large scale operations where the column is used under overloaded conditions. 

The injector is an interface that achieves reproducible and accurate transfer of a sample volume between the system operator and the chromatographic column in a front (band) as narrow as possible. Usually, for analysis by high-performance liquid chromatography (HPLC), samples are prepared in a liquid state (as a solution in an appropriate solvent or mixture of solvents, obtained as a result of sample preparation of solid or liquid samples). For practical reasons (avoiding column overloading), even for analytes in a liquid state under ambient conditions, dilution in appropriate solvents is more often used. 

Another important issue that must be considered in the development of CSPs for preparative separations is the solubility of enantiomers in the mobile phase. For example, the mixtures of hexane and polar solvents such as tetrahydrofuran, ethyl acetate, and 2-propanol typically used for normal-phase HPLC may not dissolve enough compound to overload the column. Since the selectivity of chiral recognition is strongly mobile phase-dependent, the development and optimization of the selector must be carried out in such a solvent that is well suited for the analytes. In contrast to analytical separations, separations on process scale do not require selectivity for a broad variety of racemates, since the unit often separates only a unique mixture of enantiomers. Therefore, a very high key-and-lock type selectivity, well known in the recognition of biosystems, would be most advantageous for the separation of a specific pair of enantiomers in large-scale production. 

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