Bonded Phases - Synthesis of Bonded Phases by Surface Cross-Linking

Eventually it was discovered that even the C4 phases could cause serious protein denaturation and accordingly a reverse phase with even shorter chains appeared desirable. Unfortunately, as the chain length is reduced, so is the retentive capacity of the reverse phase and in consequence, the resolution obtained from a column packed with short chain material is also impaired. An additional problem with short chain phases is that the loading capacity of the column (the maximum sample size) is correspondingly reduced with accompanying loss of peak capacity and a reduction in the practical concentration range available for analysis. In an attempt to provide a stationary phase that had adequate capacity and also maintained the effect of a very short chain length, Fung et al (22, 33) introduced a new form of bonded phase synthesis. The synthesis the authors have established is a very interesting synthetic procedure that may well have wider applications.

The basic concept of Fung et al was that, in the first instance, the surface of the silica should be covered with short chain interactive groups. Secondly, a long chain molecule should be chosen with repetitive interactive links along its length that could react with different surface groups. The result of this would be that, on reaction, the resulting product would comprise a series of long chains, lying flat on the silica surface, and anchored to it at various points along each chain. As far as the chromatographic process was concerned, the bonded phase would take the form of a surface covered with long hydrocarbon chains lying flat on the surface. However, the chains would be flat, only two or three carbon atoms from the surface but with the retentive capacity approaching that of a long chain reverse phase.

They selected the silanizing reagent dimethylchlorsilane as the material with which to cover the silica surface. This reagent linked a dimethylsilane residue to the silica surface that contained the highly reactive (silane) hydrogen on the attached silicon.

-Si-O-H + (CH3)2SiHCl = -Si-O-Si-(CH3)2H + HCl

The silica was then dispersed in a suitable solvent, squalene added, (squalene contains an number of double bonds distributed regularly along the length of the chain) and the mixture was heated in the presence of a catalyst. At those points of contact where, randomly, the double bonds of the squalene came in contact with the silane attached to the silica, the squalene chain was chemically linked to the surface.