Bonded Phases - Characteristics of Bonded Phases
Another bonding process developed by Grushka (8) involved the deposition of a polypeptide on the silica gel surface for specific use in the separation of substances of biological origin. The silica is first treated with 1-trimethoxysilyl-2(4-chloromethylphenyl)-ethane in benzene and subsequently shaken for about 6 hours to provide an anchoring group for the peptide. The reaction is shown follows:-
Grushka reported that the extent to which the second and third methoxy groups react with other surface silanol groups was uncertain, he assumed, however, that at least some double or more linkages occurred with the silica surface. The first protected amino acid, butoxycarbonyl glycine (or another selected protected peptide) is attached to the para phenylchloromethyl group by refluxing the material suspended in dry ethanol that contained triethylamine for a number of hours. The peptide is then synthesized, one amino acid at a time, employing the Merrifield procedure for the solid-state synthesis of peptides (9). The Merrifield procedure involves the following sequence of reactions for each amino acid addition.
1/ The bonded silica carrying the anchoring amino acid is washed with dioxane and the tertiary butoxy protecting group attached to the amino acid removed with 4N-HCl.
2/ The free acid is removed by washing with dioxane and the suspending solvent is changed to chloroform.
3/ The amino acid chloride that is now attached to the silica is treated with triethylamine in chloroform to release the base.
4/ The excess triethylamine is removed by washing with chloroform and the solvent changed to methylene dichloride.
5/ The next protected amino acid is coupled to the amino acid on the silica by a peptide bond using a solution of the amino acid in chloroform containing the coupling agent dicyclohexylcarbodiimide(DCC).
The bonded silica is then washed with methylene dichloride and finally methanol. At this stage the material has two amino acids attached to the surface coupled by a peptide bond. The product is now again suspended in dioxane and the sequence of operations repeated and the next amino acid is added to the chain. In this way (tedious though it may be) an octapeptide can be built onto the silica gel. Peptides having a greater number of amino acid residues than eight can be synthesized, but, unfortunately, as the number of residues increases so does the amount of peptide impurities that results from incomplete reaction in earlier stages of the synthesis. Nonetheless, although these types of bonded phase have not been available commercially to date, they offer interesting possibilities as stationary phases for the separation of biological macromolecules.
The reactions described are a meager sample of the many synthetic procedures that have been proposed and established for attaching a wide variety of organic groups to the silica gel surface. They also indicate the enormous variety of bonded phases that could be synthesized if required. There are a number of areas where unique bonded phases might be extremely useful, (e.g. in environmental chemistry and biotechnology) but so far, few of these alternatives have been exploited. Modifying the composition of the mobile phase is largely the preferred procedure to achieve any subtle retention adjustments that are necessary to achieve new and more difficult separations. Nevertheless, this approach will not necessarily achieve its goal but it is hoped, that as the need to separate essential materials produced by biotechnology arise, the use of more new bonded phases will be examined. It should again be emphasized, that the vast majority of bonded phases used today, are the simple reverse phases carrying C2, C8 or C18 hydrocarbon chains.