Fast, reproducible size-exclusion chromatography of biological macromolecules

The size-dependent separation of biological macromolecules can be effectively carried out using size-exclusion chromatography (SEC) on silica-based HPLC columns. For this technique to be successful, appropriate methods should be chosen. This paper presents practical guidelines for the development of reproducible SEC methods based upon optimized sample volume, flow-rate, column length and use of mobile phase conditions that reduce non-ideal SEC behavior - parameters often ignored in SEC. Adjustment of these parameters often results in more accurate elution times for proper molecular-mass determination, sharper peaks for improved resolution and shorter run times for increased throughput. In general, sample volume and flow-rate should be kept to a minimum for optimal resolution in SEC. Increasing column length improves resolution and may be achieved by placing columns in tandem. In addition, adjustment of the mobile phase conditions can significantly enhance resolution. However, the results are difficult to predict because the sample plays a major role in this interaction, as does the column packing. When possible, mobile phase ionic strength and pH should be altered until the peak(s) of interest elute at the expected time and with good peak shape. Finally, use of smaller-diameter columns (i.e., 4.6 mm rather than 9.4 mm) and small-diameter packing (4.5 mu m) particles are also briefly discussed. The principles described here are demonstrated, using antibodies and a number of standard proteins under a variety of SEC conditions.