How to choose reversed-phase HPLC Columns?
Different bonded phases for reversed-phase separations exhibit different combinations of possible solute-stationary phase interactions. For bonded phase, the variety of separation mechanisms will influence the overall selectivity. This predominance depend on the properties of the analyte, and the applied chromatographic conditions. The most significant interactions between solute and stationary phases that contribute to column selectivity are as below.
- Hydrophobic interaction is a dominant retention mechanism for all reversed-phase columns and the most significant interaction of alkyl phases. For a given phase, retention time is proportional to the hydrophobicity of the molecule.
- Hydrogen-bonding capacity of a phase generally involves the interaction of a basic solute group with an acidic group within the stationary phase, possibly from unbonded silanol groups.
- π-π interactions are observed between an aromatic or unsaturated solute and an aromatic stationary phase.
- Steric selectivity is a measure of the accessibility of solutes to the stationary phase. Larger solute molecules may be excluded from the stationary phase.
- Dipole-dipole interactions, between a dipolar solute group and a dipolar group in the stationary phase are most important in the case of cyano and PFP bonded columns.
- Cation-exchange interactions may occur between a cationic solute and an ionised silanol within the stationary phase.
A general summary of these interactions for typical reversed-phase bonded phases is given below. Different interactions may be dominant for different analytes and interaction strengths will vary amongst different manufacturers’ bonded phases. However, this table gives a useful indication of likely interaction strengths.
C18 Reversed-phase Column
Hydrophobicity is the primary mechanism of analyte interaction with C18 and other alkyl-bonded stationary phases. In addition, the polarity of the phase will also contribute to the overall selectivity observed.
The strength of hydrophobic interaction can be measured by the retention of neutral (non-polar) molecules. The k values (retention factors) for a neutral species, for a given C18 phase, will give an indication of the surface area and surface coverage (ligand density) of the silica.
The percentage of carbon in the material is a simplistic but useful guide to the hydrophobic retention characteristics of a column. In Figure 1 this loose correlation is demonstrated by the increase in retention observed when alkyl chain length (i.e. carbon load) is increased. This increase results from an increase in hydrophobicity of the stationary phase. Similarly, an increase in retention would be expected in going from a C18 phase with a low carbon load to one of high carbon load.
Hydrophobic selectivity can be determined from the retention factor ratio between two neutral species. This is a better measure of surface coverage than carbon content, as surface area and porosity may vary from silica to silica.
The second key property of C18 materials is their silanol activity, often discussed in terms of polarity. This can be determined by measuring the retention factor ratio between a basic and an acidic compound. At pH >7 the total ion-exchange capacity will correspond to a measure of the total silanol activity. At acidic pH (e.g. pH 2.7) an indication of the acidic activity of the silanol groups can be obtained. The presence of metal ions in the base silica increases the level of silanol activity. Older generation silicas have higher and less tightly controlled levels of metal ions, and hence higher
silanol activity compared to newer generation alkyl bonded phases. For this
and other reasons, it is strongly recommended that new method development
should be approached using newer generation higher purity silicas.
High Purity Base Deactivated Phases
Modern alkyl bonded phases have very low cumulative metal ion levels within the base silica (<10ppm), resulting in the number of isolated silanol groups, and hence the polarity of the silica surface, also being reduced. Combined with more effective and reproducible bonding processes, these newer generation reversed-phase materials lead to significantly improved chromatography for the more basic polar solute molecules. The use of bonded alkyl groups containing hydrophilic substituents (i.e. polar embedded) can either enhance this effect and/or offer alternative selectivity.
Figure 2 illustrates the relationship between the change in polarity and hydrophobicity for typical C18, C8, and C4 materials, showing a decrease in hydrophobicity on reducing alkyl chain length. Greater ligand density, and hence
lower polarity, is also seen as the length of the alkyl chain is reduced. However, changing the alkyl chain length may reduce analysis time but will not significantly affect selectivity. Changing the chemistry to an alternative bonded phase is a more powerful tool to achieve this.
Older Generation ‘Traditional’ Phases
The older ‘traditional’ C18 phases are hydrophobic and have a high polarity due to the lower purity silica containing a higher level of acidic silanol groups on which they are based. The use of the newer high purity silicas reduces the resultant phases’ silanol activity and improves reproducibility. Employing a polar embedded functionality may also result in a reduced polarity material. For basic solutes that will interact strongly with surface silanols, lower polarity phases are generally recommended. However, for certain analyses, the additional interactions provided by the surface silanols of a ‘traditional’ C18 material may be beneficial to the overall separation.
Phenyl Reversed-phase Column
Phenyl bonded silica phases offer an alternative reversed-phase selectivity to alkyl bonded phases. They show lower hydrophobic retention than their C18 counterparts, with similar retention characteristics to C8-bonded phases. Phenyl stationary phases interact with compounds containing aromatic groups or unsaturated bonds through the involvement of π-π interactions. For aromatic solutes containing an electronegative atom or group (e.g. F, NO2), the degree of π-π interactions with the phenyl phase will increase.
Due to the rigid nature of the phenyl ring, the solute shape can also influence selectivity.
Traditional phenyl phases tend to be less stable than the corresponding C8 or C18 reversed-phases. Additionally, the larger steric size of the phenyl group reduces surface coverage, leaving a greater number of exposed silanol sites. More recently introduced phenyl phases show greater stability. The use of a purer silica base, more effective and reproducible bonding procedures and the availability of a sterically protected phenyl silane all contribute to greater phase robustness and reduced column bleed.
Conventional phenyl phases are bonded to the silica through a propyl spacer. The incorporation of the longer chain hexyl spacer results in increased hydrophobic retention and aromatic selectivity.
Polar Bonded Silica Phases
Polar bonded silica phases offer an alternative selectivity to alkyl bonded materials. In general, they have a lower hydrophobicity but higher polarity. Cyano, amino and diol bonded phases can be used in both normal- and reversed-phase modes. In normal-phase, they equilibrate more rapidly with the eluent than silica itself and are not deactivated by traces of water.
Cyano bonded phases show unique selectivity for polar compounds and are more suitable than bare silica for normal-phase gradient separations. The cyano functional group is a strong dipole that can interact with other dipoles or induce dipoles on solutes. These phases also exhibit moderate hydrophobicity due to the alkyl linker.
Amino bonded phases show alternative normal-phase selectivity to unbonded silica, especially for aromatics. Amino columns are also used in the HILIC mode for carbohydrate analysis and for other polar compounds. Their weak anion-exchange properties can be used in the analysis of anions and organic acids.
Diol bonded phases are a versatile alternative to unbonded silica for normal-phase separations. The hydroxyl groups provide good selectivity without excessive retention since H-bonding with the diol layer is weaker than with silanols. Some diol bonded phases have been developed specifically for HILIC applications. Differing pore size materials are used in size-exclusion separations.
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