Types of Bonded Phases

1 Alkyl-Type Phases (C1-C18, C30). Probably 90% of all reversed-phase columns are alkyl-type bonded phases. An enormous amount of publications are devoted to the classiflcation, standardization, and comparison of these phases. In their book Practical HPLC Method Development, Snyder and Kirkland [57] indicate that reversed-phase retention for nonpolar and nonionic compounds generally follows the retention pattern Cl C4 C8 = Cl 8. At the same time, they refer to the comparison of Cl 8-type columns from different manufacturers and find dramatic variation in the retention of both polar and nonpolar compounds at the same conditions on different columns. 

HPLC retention expressed either in absolute values (T ) or in relative terms (retention factor) is a complex function of a multitude of parameters. Type of bonded phase is only one of them. Surface area of base material, pore size, bonding density, end-capping, and even the column history all can significantly affect analyte retention. 

2 Phenyl-Type Phases. Phenyl-type phases have been studied for a long time [58,59]. The presence of a phenyl ring on the surface of a bonded phase introduces so-called n-n interactions with some analytes that are capable of these types of interactions. This introduces an additional specificity for HPLC separations on these stationary phases. Compared to common alkyl-type phases, phenyl columns show lower methylene selectivity in other words, the separation of members of homologous series will be less selective on phenyl columns than on alkyl-modified phases. 

The second reason in the introduction of polar groups in the bonded ligands is that these groups interact with residual silanols, which make the silanols effectively inactive for the interaction with polar or basic analytes. Sometimes these phases are also end-capped with polar end-capping groups. These phases also show a significant difference in selectivity compared to conventional CIS-type phases. In some cases they show improvement in the peak shape for basic components. 

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FIGURE 2.3 Schematic presentation of the different types of bonded phases. 

The range of operating modes is much greater in LC than in GC because both phases, stationary and mobile, affect the separation and because a wide range of stationary phases can be used in LC. Stationary phases include many types of bonded phases with a wide range of polarities, and also materials with ion exchange and sieving (size exclusion) properties. 

Another type of bonded phase is intended to be used with biopolymers. In addition to the larger pore size that is required, these materials are intended to be used under milder conditions that will not denature the samples. When only water is used as the mobile phase, without any organic modifier, proteins will not be denatured and their hydrophobic char-... 

Several thousands of different columns are commercially available, and when selecting a column for a particular separation the chromatographer should be able to decide whether a packed, capillary, or monolithic column is needed and what the desired characteristics of the base material, bonded phase, and bonding density of selected column is needed. Commercial columns of the same general type (e.g., CIS) could differ widely in their separation power among different suppliers. Basic information regarding the specific column provided by the manufacturer, such as surface area, % carbon, and type of bonded phase, usually does not allow prediction of the separation or for the proper selection of columns with similar separation patterns. 

Figure 8-18. Effect of type of bonded phase on the separation selectivity.

Challenges for HPLC method development are for combination products where more than one active is present in the same formulation. Since degradation products are of big concern from a safety perspective, HPLC will continue to be utilized as a major separation technique during formulation development to develop the most safe and efficacious formulations to be used for human use. Many different types of bonded phases are currently available for routine HPLC analysis in addition, very selective and sensitive detection techniques can be integrated with HPLC to help an analytical chemist control the final quality of the drug product. Furthermore, the availability of this technique makes it the first and sometimes the only choice for the analysis of degradation products. 

The specific chemical interactions from the stationary phase have been approached in the same manner. Snyder (95) first proposed that the three types of bonded phases should provide maximum differences in selectivity. He assigned the amino-phase to group I, the cyano- phase to group VI, and the diol-phase to group IV. Cooper and Smith (98,99) have extensively studied the three common types of normal-phase, bonded-phase columns and, using extended solubility parameters, have experimentally located the three columns on a stationary-phase selectivity triangle, shown in Fig. 20. Both the amino-phase and the cyano-phase fall near the predictions however, the diol-phase shows significantly less... 

Monomeric versus Polymeric Bonding Types of Bonded Phases Reproducibility of Bonded Phases Alternative Bonding Technologies... 

Before bonding material to the surface, it is usual to heat the silica gel to at least 120°C and often to 200°C to remove all the adsorbed water. If the water is not removed, it will compete with the silanol groups for reaction with the derivatizing agent and may also cause a polymeric type of bonded phase to be formed. 

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