Reversed-phase HPLC is probably the most widely used form of chromatography
mainly because of its flexibility and high resolution. It is widely used to analyse drugs
and their metabolites, insecticide and pesticide residues, and amino acids, peptides
and proteins. Octadecylsilane (ODS) phases bind proteins more tightly than do octyl-
or methylsilane phases and are therefore more likely to cause protein denaturation
because of the more extreme conditions required for the elution of the protein.11.5.4 Ion-pair reversed-phase liquid chromatography
Although the separation of some highly polar analytes, such as amino acids, peptides,
organic acids and the catecholamines, is not possible by reversed-phase chromato-
graphy, it is sometimes possible to achieve such separations by one of two approaches:- Ion suppression: The ionisation of the analytes is suppressed by using a mobile phase
with an appropriately high or low pH thus giving the molecules greater hydrophobic
character. For weak acid analytes, for example, an acidified mobile phase would be used. - Ion-pairing:Acounter ionthat has a charge opposite to that of the analytes to be
separated is added to the mobile phase so that the resulting ion-pair has sufficient
hydrophobic, lipophilic character to be retained by the non-polar stationary phase
of a reversed-phase system. Thus, to aid the separation of acidic analytes, which
would be present as their conjugate anions, a quaternary alkylamine ion such as
tetrabutylammonium would be used as the counter ion, whereas for the separation
Table 11.2Examples of silica bonded phases for reversed-phase HPLC
Product Particle size Pore size (A ̊)
mBondapak octadecyl 10 mm70
mBondapak phenyl 10 mm 125
mBondapak CN 10 mm 125
mBondapak NH 2 10 mm80
Zorbax octadecyl 6 mm70
Zorbax octyl 6 mm70
Zorbax NH 2 6 mm70
Discovery octyl 5 mm 180
Supelcosil LC-octadecyl 5 mm 120
Supelcosil LC-301 methyl 5 mm 300
Supelcosil LC-308 octyl 5 mm 300Note:1A ̊¼0.1 nm.457 11.5 Partition chromatography