Selection, stationary-phase chemical bonding

The PRISMA model was developed by Nyiredy for solvent optimization in TLC and HPLC [142,168-171]. The PRISMA model consists of three parts the selection of the chromatographic system, optimization of the selected mobile phases, and the selection of the development method. Since silica is the most widely used stationary phase in TLC, the optimization procedure always starts with this phase, although the method is equally applicable to all chemically bonded phases in the normal or reversed-phase mode. For the selection of suitable solvents the first experiments are carried out on TLC plates in unsaturated... 

Selection of columns and mobile phases is determined after consideration of the chemistry of the analytes. In HPLC, the mobile phase is a liquid, while the stationary phase can be a solid or a liquid immobilised on a solid. A stationary phase may have chemical functional groups or compounds physically or chemically bonded to its surface. Resolution and efficiency of HPLC are closely associated with the active surface area of the materials used as stationary phase. Generally, the efficiency of a column increases with decreasing particle size, but back-pressure and mobile phase viscosity increase simultaneously. Selection of the stationary phase material is generally not difficult when the retention mechanism of the intended separation is understood. The fundamental behaviour of stationary phase materials is related to their solubility-interaction... 

In gc there is only one phase (the stationary liquid or solid phase) that is available for interaction with the sample molecules. Because the mobile phase is a gas, all sample vapours are soluble in it in all proportions. In hplc both the stationary phase and the mobile phase can interact selectively with the sample. Interactions such as complexation or hydrogen bonding that are absent in the gc mobile phase may occur in the hplc mobile phase. The variety of these selective interactions can also be increased by suitable chemical modification of the silica surface, hence hplc is a more versatile technique than gc, and can often achieve more difficult separations. 

Locke, D. (1974) Selectivity in reversed-phase liquid chromatography using chemically bonded stationary phases. J. Chromatogr. Sci. 12, 433 137. 

Interestingly, in HPLC the stationary phase and the mobile-phase is able to interact with the sample selectively. Besides, such interactions as hydrogen bonding or complexation which are absolutely not possible in the GC-mobile phase may be accomplished with much ease in the HPLC-mobile phase. Furthermore, the spectrum of these selective interactions may also be enhanced by an appropriate chemical modification of the silica surface the stationary phase. Therefore, HPLC is regarded as a more versatile technique than GC and capable of achieving more difficult separations. 

Table 2 Selection of Commonly Used Ion-Pairing/Dynamic Liquid-Liquid Ion Exchange/ Mobile-Phase Additive Species, Which at pH <7.0 Modify the Retention Characteristics of Unprotected Peptides on Chemically Bonded Hydrocarbonaceous Stationary Phases3-11...

Many reviews have been written on the preparation, physico-chemical properties and application of silica in modem separation science [5-7]. RP LC with silica based bonded stationary phases is utilised for the majority of LC separations in laboratories world-wide. Their ubiquity derives from their versatility, in that generally a wide range of both ionic and non-ionic analyte species can be separated with these columns by careful selection of the stationary phase and mobile phase properties. 

For all these reasons, it will be understandable that LLC systems have been virtually replaced by chemically bonded phases (section 3.2.2) in current LC practice. Consequently, the various parameters of interest for the optimization of these systems will not be discussed extensively. With regard to the influence of temperature and mobile phase composition on retention and selectivity, it is suggested that the same relationships may be used for insoluble LLC stationary phases as are used for LBPC. LLC systems have been used extensively for the separation of ionic compounds by means of ion-pairing techniques. Such systems will be discussed in section 3.3.2. 

Besides the typical RPLC stationary phases with n-alkyl groups, various other functional groups may be chemically bonded to the surface in a similar way as described in section 3.2.2.1. A selection of possible bonded bases, arranged roughly in order of increasing polarity, is given in table 3.3. 

Eluent pH is limited to a maximum of 7 to 8 due to the reduced chemical stability of a chromatographic bed in an alkaline medium. The nucleophilic attack of Si-0 bonds by hydroxide ions leads to the erosion of the silica surface as shown by back pressure increases caused by the formation of Si(OH)4. With polystyrene-divinyl-benzene-based stationary phases, pH stability is not an issue and a very wide mobile phase pH range can be used, thereby providing additional selectivity [1]. Several silica-based and polymeric columns claimed to be stable in pH ranges from 1 to 13 are commercially available, however, they are not commonly used. 

In common with other application areas of chromatographic separation, a considerable amount of effort has been expended recently on the development of different elution conditions and types of stationary phases for peptide separations in attempts to maximize column selectivities without adversely affecting column efficiences. Peptide retention will invariably be mediated by the participation of electrostatic, hydrogen bonding, and hydrophobic interactions in the distribution phenomenon. The nature of the predominant distribution mechanism will be dependent on the physical and chemical characteristics of the stationary phase as well as the nature of the molecular forces which hold the solute molecules within the mobile and stationary zones. The retention of the solute in all HPLC modes can be described by the equation... 

---