Stationary phases
Conventional TLC plates can be prepared in the laboratory by standardised methods, but reproducible layer preparation is easier to achieve in a manufacturing setting. Few laboratories pre pare their own plates today, finding it more efficient to purchase TLC plates from commercial suppliers. Precoated plates for high performance, conventional and preparative TLC are available in a range of sizes and different layer thickness, supported on glass, aluminium or plastic backing sheets. To impart the desired mechanical stability and abrasion resistance to the layer, a binder such as poly (vinyl alcohol), poly (vinyl pyrrolidone), gypsum or starch in amounts from 0.1% to 10% (w/w) is incorporated into the layer. An ultraviolet (UV) indicator, such as manganese-activated zinc silicate of a similar particle size to the sorbent, may be added to the layer for visualisation of separated samples by fluorescence quenching. TLC plates with a narrow preadsorbent zone located along one edge of the layer are available to aid manual sample application.
Silica gel is the most important stationary phase for TLC, with other inorganic oxide ad sorb ents, such as alumina, kieselguhr (a silica gel of low surface area) and Florisil (a synthetic magnesium silicate), of minor importance. Most silica gel sorbents have an average pore size of 6nm and are designed for the separation of small molecules (relative molecular mass 700). The chromatographic properties of the inorganic oxide adsorbents depend on their surface chemistry and specific surface area. For silica gel, silanol groups (1Si1OH) are the dominant adsorption sites (Fig. 1). The complementary sample properties that govern retention are the number and type of functional groups and their spatial location (see Fig. 2). The influence of functional group properties on selectivity is illustrated in Fig. 3 for the separation of ethynyl steroids. The steroids with phenolic groups are the most strongly retained, followed by hydroxyl groups, and then ketone and ester groups. Subtle separation differences through steric hindrance at a functional group and differences in ring conformations are also seen, which allow the separation of steroids with very similar chemical properties.
Figure 1 Structure of silica gel.
Figure 2 General adsorption scale for separations by silica gel TLC.
Figure 3 Separation of ethynyl steroids (birth-control pill components) by high-performance TLC. Two 15 min developments with the mobile phase hexane–chloroform–carbon tetrachloride–ethanol (7:18:22:1) on a silica gel 60 high-performance TLC plate. Chromatogram was recorded by scanning densitometry at 220 nm.
Separations on silica gel are considered as ‘normal’ phase separations (see pp. 514 and 515), where the stationary phase is polar relative to the mobile phase. TLC separations can be carried out in reversed-phase mode. In this mode, the mobile phase is more polar than the stationary phase. Chemically bonded stationary phase layers are prepared from silica gel by reaction with various organ silane reagents to form siloxane bonds. The degree of chemical bonding can be varied to form stationary phases with a high coverage of bonded phase and few residual silanol groups on the silica surface to phases with a lower bonded phase coverage and more (higher concentration or proportion of) residual silanol groups (Table 13.5). Reversed-phase alkyl siloxane-bonded layers with a high level of surface bonding cannot be used with mobile phases that contain a significant amount of water (>30% v/v) because of the inadequate mobile-phase velocity generated by capillary forces. Water compatibility for alkyl siloxane bonded layers is achieved by increasing the particle size of the silica support material, using a reproducible although lower degree of silanisation, and by using modified binders. These layers are referred to as water wettable and are used for all types of reversed-phase separations, while layers with a high degree of silanisation are used predominantly with non-aqueous mobile phases. Alkyl siloxane bonded phases are used primarily (but not exclusively) for the separation of water-soluble polar drugs and weak acids and bases after ion suppression (buffered mobile phase) or ion-pair formation. Water compatibility is not a problem for chemically bonded phases where the bonded group is polar in nature e.g. aminopropyl siloxane or spacer bonded propanediol (Table 13.5). These phases can be used for both normal- and reversed-phase separations. For separations that cannot be achieved on silica gel, the polar chemically bonded phases are the most widely used stationary phases. The 3-aminopropylsiloxane bonded layers can function as a weak anion exchanger for the separation of polyanions with a buffered mobile phase. Cellulose layers provide only weak retention of common drug substances and are used primarily to separate very polar compounds in biochemistry.
TLC has found limited use for the separation of enantiomers. The most widely used approach employs ligand-exchange chromatography on reversed-phase layers impregnated with a solution of copper acetate and (2S,4R,2RS)-N-(2 hydroxy dodecyl)-4-hydroxyproline. Separations result from stability differences in diastereomeric complexes formed between the drug, copper and the proline selector. Suitable drugs for this application require an amino acid or a-hydroxycarboxylic acid group for complex formation. A more versatile approach to the separation of enantiomeric drug substances by reversed-phase TLC is the use of chiral selectors, such as cyclodextrins or bovine serum albumin, as mobile-phase additives.
