Reversed phase HPLC loading capacity

The scale of preparative HPLC is normally larger than that of conventional HPLC. Therefore, a practical starting point is to develop an analytical separation that optimizes the isolation conditions. Optimization of the analytical method implies seeking conditions that combine maximum resolution of the peak of interest and minimum elution time, under the restriction of a limited pressure drop. The optimized conditions determine the column, mobile phase, flow rate, and sample loading capacity for the particular column. The conditions may be either normal phase or reverse phase. The mobile phase should be chosen carefully to avoid salt complexation with the compound to be isolated. Volatile acid salts such as trifluoroacetic acid, formic acid, and acetic acid are acceptable mobile phase additives, and the ammonium counterion is preferred for pH adjustment to any of these acids. 

Regardless of the procedure employed preparative HPLC has most commonly been used in the adsorption mode with silica packings due to their sample versatility and loading capacity however large bore preparative columns are now commercially available, packed with any of the proprietary reverse-phase and ion exchange media. 

Figure 5.3 Protein loading capacity of RP-HPLC materials of different particle sizes. Source Protein and Peptide Analysis and Purification, Vydac Reversed Phase Handbook, 5th edition, W.R. Grace, 2013.

Short (3-10 cm) HPLC columns can also be used for rapid sample concentration. Microbore reversed-phase columns have been used for preconcentration of proteins prior to microsequence analysis, after collection and dilution of milliliter fractions from conventional HPLC columns [36]. Gradient elution at low flow rates (0.1-0.2 mL/min for 2-mm-ID or 0.02-0.04 mL/min for 1-mm-ID columns) permits recovery of proteins in volumes as small as 25 pL, with concentration factors up to 80-foId. The high capacities of porous microparticulate packings enable 50-100 pg to be loaded onto microbore columns as long as the sample is introduced in a weak solvent. 

---