Introduction

In order for a sample molecule to freely access the interior of the pores of the packing material, its diameter must be smaller than the average pore diameter. For high molecular weight solutes, the use of lower pore size materials of 60-120Å may result in frictional drag within the pore leading to restricted diffusion and reduced column efficiency.
The use of larger pore silica-based bonded phases leads to improvements in resolution, capacity and recovery of proteins and other biomolecules, due to a reduction in size-exclusion mechanism and enhanced molecular diffusion rates. A pore size of 300Å has become the accepted standard for wide pore silicas, and has been found to be suitable for a broad range of molecular weight proteins, peptides and oligonucleotides. In general, peptides exceeding about 50 amino acids and oligonucleotides greater than 25 residues are preferentially analysed on 300Å materials.

Separations of very large biomolecules (MW >100,000 Da) may require larger pore size packings (500 to 4000Å). 

Bonded phases

Alkyl bonded silica phases are the most commonly used materials for the reversed-phase separation of biomolecules. The shorter C4 matrices are generally recommended for large hydrophobic peptides and most proteins. Peptide maps, natural and synthetic peptides and small hydrophilic proteins are best chromatographed on C8 columns. C18 columns are often chosen for the analysis of small peptides. Other bonded wide pore phases, including cyano and phenyl, are available in some brands.

The Table below summarises a range of wide pore alkyl-bonded reversed-phase silica materials. Ion-exchange and size exclusion packings are also available as wider pore materials. 

Column Dimensions

Wide pore silica phases are available in a range of column dimensions from rapid analysis to preparative and process scale. Increased column capacity favours these wide pore materials for preparative separations of samples with molecular weight >5000 Da.
 

300Å Reversed-Phase Bonded Silica Phases