Wide Pore RP Phases (Part 1 of 2)
Wide Pore Reversed Phase Stationary Phases for Biochromatography
In biochromatography, mostly macromolecules are analyzed or purified. Due to their large molecular size and properties these macromolecules require special stationary phases with pores larger than the more standard 100 Å pores that stationary phases typically have. Thus, many manufacturers offer columns with wide pore phases.
Properties and advantages of the new wide pore RP phases NUCLEODUR® 300 are presented below.
HPLC phases for separation of biological macromolecules
Biological macromolecules – proteins, peptides, and nucleic acids – have a higher molecular weight and a greater hydrodynamic radius than low molecular weight analytes such as amino acids, catecholamines, steroids, and nucleotides. HPLC stationary phases with a narrow pore size of mostly 100 Å show insufficient retention of biological macromoledules. This is due to a possible size exclusion of macromolecules by their molecular size. Thus, for the separation of biological macromolecules “wide pore phases” with a pore size of 300 Å to 4000 Å (30nm to 400nm) are used. Wide pore phases based on both polymers and silicas are commercially available. According to the separation mechanisms required, determined by the polarity and hydrophobicity of the analytes, stationary phases with a surface chemistry of both ion exchange and RP (reversed phase) modified phases can be used. An overview of MACHEREY-NAGEL biochromatography phases is found under this link.
The figure shows the possibilities of a column selection of RP phases by molecular weight and analyte hydrophobicity.
Column selection by analyte characteristics
Wide pore RP phases based on high purity silicas
The influence of pore size is shown by the comparison of narrow pore NUCLEODUR® C18 Gravity, 5 µm (110 Å pore size) and wide pore NUCLEODUR® 300-5 C18 ec (300 Å pore size) for the separation of a protein standard. The macromolecular proteins can better penetrate into the larger pores of the wide pore phase, resulting in stronger retention and peak resolution despite lower C18 coverage.
The pictured Tanaka plots are useful for a characterization of RP properties of the two NUCLEODUR® 300 RP phases. Even with its short C4 chains, NUCLEODUR® 300-5 C4 shows a relative distinct hydrophobicity, approximating the more hydrophobic C18 phase. Due to the longer C18 chains, NUCLEODUR® 300-5 C18 features a stronger steric selectivity. The C4 phase shows a higher ion exchange capacity at pH 2.7, caused by the more distinct remaining hydrophilic character of the underlying silica skeleton.
The below pictured column test with characteristic polar, hydrophobic and sterically different analytes shows the specific properties of both phases. As expected, polar analytes (e.g., ethyl benzoate) show high retention on the C4 phase. Hydrophobic analytes (e.g., naphthalene) also have a good retention. Even if the organic part in the eluent is reduced, the low steric selectivity does not allow separation of the sterically different analytes biphenyl and acenaphthene on the C4 phase. Depending on the properties and the partial structures of the macromolecules the behavior of these low molecular analytes can be transferred. That is, the C4 phase is preferred for the separation of proteins with predominantly polar partial structures.
Due to a homogeneous coverage of the silica with alkyl chains and an exhaustive endcapping, a high pH stability of the NUCLEODUR® 300 RP phases is achieved, shown with application 126770 (protein test mixture) and 126760 (PNA test mixture) at pH 1, and with application 126750 (PNA test mixture) at pH 9.