There are primarily two types of RPC media, one based on silica beads covered with a bonded non-polar phase of carbon chains and one based on a naked non-polar polymer matrix (Fig 5.1).
Fig 5.1. Properties of RPC media.
There is no big difference in selectivity between polymer-based and silica-based RPC in the commonly used pH range (2 - 7.5), at least not with ACN as the organic modifier. Above pH 7.5 , however, silica dissolves leaving polymer based RPC as the only alternative.
It is interesting to note that the amino acids cysteine, tyrosine, and lysine all have pKa values in the pH range
9.1 - 10.4. RPC in this pH interval would consequently provide new selectivities for peptides and proteins containing these amino acids.
Polymer-based media can be cleaned by alkali, a great advantage when running crude protein and peptide samples.
Small organic molecules behave as if they were dissolved in the hydrocarbon phase, while peptides and proteins behave as if they were adsorbed to it. As a consequence, organic molecules are sensitive to the chain length (the depth) of the bonded phase.
Proteins and peptides, on the other hand, are much less sensitive in this respect (Fig 5.2).
Proteins and many peptides carry both hydrophilic and hydrophobic areas on their surfaces and the net hydrophobicity varies depending on the ratio between the two.
Fig 5.2. "Classical" hydrophobic organic molecules are sensitive to the carbon chain length, while more or less identical results are obtained for proteins and larger peptides, regardless of the carbon chain length.
Generally, most components interact with RPC media in a way characterised partly by dissolution and partly by adsorption. |
