Michael W. Dong, HPLC and UHPLC For Practicing Scientists, 2nd Edition #
RPC Theory and Fundamentals #
Under RPC conditions, proteins and peptides are separated based on their hydrophobicity and their interaction with the hydrophobic ligands in the stationary phases (see Section 1.2.2). The stationary phase ligands are typically composed of carbon chains of varying lengths, from 4 carbons (C4 phase) to 18 carbons (C18 bonded phase), with the longer carbon chains providing a stationary phase with higher hydrophobicity. Other ligands such as diphenyl are also commonly utilized. RPC of proteins and peptides typically uses higher temperatures and organic solvents, which denature the proteins and expose the hydrophobic cores. The use of shallow gradients is common since the variants tend to elute in a very narrow gradient segment. The pore size of the support material is also important, with large proteins such as antibodies using 300$\mathring{A}$ pore materials, while small proteins or peptides are better resolved in columns packed with smaller pore supports (e.g. 120 $\mathring{A}$ ). The phenomenon of “entangled diffusion” of large proteins in small-pore support materials leading to poor mass transfer and band broadening is discussed in Section 13.6.1.
Method Conditions #
Higher column temperatures (up to 110^0^C) are often used to reduce carryover, while the column temperature could be lower for small protein and peptide separation.
In addition to commonly used acidic mobile phases, alkaline buffers have been used to separate small proteins and peptides due to the unique selectivity at high pHs. However, columns packed with bonded phases to hybrid particles designed to tolerate high-pH mobile phases, such as bridged ethylene hybrid (BEH) columns or other similar columns, must be used (see Section 3.5.2).
Recently, superficially porous particles (SPPs) have been developed for RPC applications. These particles consist of a solid core, which is impervious to the solutes, and a porous outer layer, which provides shorter diffusion pathways in the particle, thus yielding significantly higher column efficiency (20–40%) vs. those packed with totally porous particles (see Section 3.5.7). SPP columns can have lower backpressures than other particle types – a reason for the popularity of small-particle SPP (<3 μm) [57].
Purolite reversed-phase chromatography #
Reversed-phase chromatography uses a polar (aqueous) mobile phase where hydrophobic molecules will adsorb to the hydrophobic stationary phase, and hydrophilic molecules will pass through uninterrupted. Hydrophobic molecules are eluted from the resin by decreasing the polarity of the mobile phase via use of non-polar solvent such as alcohol, which reduces hydrophobic interactions.
The more hydrophobic the molecule, the higher the concentration of solvent needed to elute the molecule.
Reverse Phase Chromatography is a frequently used analytical method to quantify and separate various molecules such as betalactam antibiotics, flavors, polyphenols, vitamins, peptides, oligonucleotides and many more. Some applications where additional separations are needed such as ion exchange in a mixed mode type of separation (such as a reverse phase chromatography resin with ion exchange capabilities). Purolite offer such resin for the separations of peptides and oligonucleotides such as 10AD2S (cation exchanger) and 10AD2Q (anion exchanger).