Bonded phase Chemically modification

Reversed-phase PLC precoated plates are based on silica gel matrices with chemical modifications in such a manner that the accessible polar, hydrophilic silanol groups at the silica gel surface are replaced by nonpolar, hydrophobic alkyl chains via silicon-carbon bonds. For preparative purposes, up to now only PLC precoated RP plates with C-18 modification are available. This abbreviation is often also designated as RP-18, meaning that an octadecyl alkyl chain is chemically bonded to the silica gel surface. 

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. 

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. 

It is very instructive to compare the kinetics and plausible mechanisms of reactions catalyzed by the same or related catalyst(s) in aqueous and non-aqueous systems. A catalyst which is sufficiently soluble both in aqueous and in organic solvents (a rather rare situation) can be used in both environments without chemical modifications which could alter its catalytic properties. Even then there may be important differences in the rate and selectivity of a catalytic reaction on going from an organic to an aqueous phase. TTie most important characteristics of water in this context are the following polarity, capability of hydrogen bonding, and self-ionization (amphoteric acid-base nature). 

It is generally accepted that the only important polar adsoiption sites on the silica surface are the silanol functions, i.e., hydroxyl groups, that are attached to silicon atoms (2). They can interact with the sample molecules by hydrogen bonding and various physical observations ctAi be used to prove this statement. Complete dehydration of silica by beating, i.e., removal of all surface hydroxyl groups, yields a hydrophobia silica which no longer shows adsorption for unsaturated and polar molecules and is no more wetted by water (15). Chemical modification of the surface hydroxyls such as used in the preparation of chemically bonded phases also eliminates the selective adsorption properties of the silica. ... 

Besides the above differentiation, restricted-access media can be further subdivided on the basis of the topochemistry of the bonded phase. Packings with a uniform surface topochemistry show a homogenous ligand coverage, whereas packings with a dual topochemistry show a different chemical modification of the pore internal surface and the particle external surface (114). Restricted-access media of the former type are divided into mixed-mode and mixed-function phases, bonded-micellar phases, biomatrix, binary-layered phases, shielded hydrophobic phases, and polymer-coated mixed-function phases. Restricted-access media of the latter type include the Pinkerton s internal surface reversed-phase, Haginaka s internal surface reversed-phase diol, alkyl-diol silica, Kimata s restricted-access media, dual-zone phase, tris-modified Styrosorb, Svec s restricted-access media, diphil sorbents, Ultrabiosep phases. Bio Trap phases, and semipermeable surface phases. 

Reversed phase ion-pair systems (RP-IPC) could be of the LLC type, but the use of chemically bonded (alkyl) phases has become increasingly popular, because of the increased stability and flexibility of the system. Even if an LLC system is used for RP-IPC, then a chemical modification of the surface is still required to coat an organic liquid on the particles of (for example) silica. 

Types Modification Reactions. Chemical modification of hydroxy-lated surface of base material in more than 90% of commercially available HPLC stationary phase is performed by its reaction with alkylchlorosilane-type modifiers, reaction (3-9). HCl is generated as a byproduct, and typically a scavenger for HCl is employed in the synthesis. For most common bonded phases the two side groups are methyl however, other side groups such as isopropyl and fert-butyl have been used. 

Fig. 1.8. Chemical modification of silica gel in the preparation of (A) a monomeric and (B) a polymeric non polar alkyl bonded stationary phase for RPC by reaction with mono-, di- and tri-funclional alkylchlorosilanes.

For steric reasons bifunctional or trifunctional silanes can react with either one only or, at most, two silanol groups on the silica gel surface (second reaction in Fig. 1.8A). Thus, some of the functional (Cl or alkoxy) groups remain unreacted and easily hydrolyse to form new silanol groups. If the reaction mixture contains even traces of water, the hydrolysis occurs during chemical modification of silica and the new silanol groups react with excess molecules of reagents to form a polymerised surface layer (Fig. 1.8B). These bonded phases may be more stable and usually show stronger retention than monomeric phases at low pH. However, the reaction is difficult to reproduce and various batches of the same material may have different properties, so that the reproducibility of separation is poorer than with monomeric phases. Polymeric phases are more resistant to penetration of analytes and may show increased mass-transfer resistance and decreased efficiency (plate number) of separation [- 91. 

In a later study, Pesek et al. reported the separation of other proteins using a diol stationary phase [61-64]. The use of a diol stationary phase should result in a surface that is more hydrophilic than a typical alkyl-bonded moiety, like Cis or Cg. The overall results showed significant variations in retention times due to differences in solute-bonded phase interactions. Other factors, such as pH, could also influence this interaction, due to its influence on charge and protein conformations. Combining all these factors in the separation of peptides and proteins provides an experimentalist with many decisions to be made in the optimized experimental conditions to be used. Other chemical modifications of etched fused silica need to be studied in order to provide a better understanding of their interactions with proteins and peptides, as well as other classes of biopolymers. 

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