Bonded Phases - The Stability of Oligomeric Phases

The Stability of Oligomeric Phases

Akapo et al (28), employed methyloctydichlorsilane as the silanizing reagent, and prepared a series of ten oligomeric phases each having 1, 2, 3, 4, 5, 6, 7 ,8, 9 and 10 methyloctyl chains attached. Unfortunately, it would appear that the synthesis was not very efficient and the products appeared to be a mixture of bonded phases that contained a range of oligomers of different sizes. Nevertheless, they could be used to test the stability of oligomeric phases in general. Each oligomer was packed into a column and a continuous flow of a mobile phase containing 70%v/v of water and 30%v/v of acetonitrile to which was added 0.1%v/v of trifluoroacetic acid (TFA) passed through it. Subsequent to 500 ml of mobile phase having passed through the column and after every following 500 ml had passed through, the retention volumes of benzene and analine were determined and the results obtained for aniline are shown in figure 10. Figure 10 demonstrates the effect of oligomer 'length' on reverse phase stability to mobile phases of low pH. It is clear that, on the column with only a single chain, surface erosion progressively and quickly takes place as more mobile phase is passed through the column, and, as a result the (k') of aniline rapidly increases due to the exposure of the acidic silanol groups. The acidic silanol groups interact with the basic aniline and its retention increases. As the layers of oligomeric phase become thicker, as more chains are boded bonded to the silica, the erosion of the phase becomes progressively and significantly less. This is clearly demonstrated by the small change in the retention volume of the aniline as progressively more mobile phase is passed through those column packed with oligomeric phases having more chains. Subsequent to five chains have been bonded to the silica, the bonded phase become quite stable and after ten oligomers have been attached, the bonded phase is very stable indeed.

Figure 10. Graph of (k') of Aniline against Volume of Mobile Phase Passed Through the Column

Such stable material could be very useful for the separation of peptides and polypeptides in those instances where TFA is used for pH adjustment in order to achieve adequate sample solubility. The oligomeric phase, despite solving the problem of reverse phase stability to low pH mobile phases, does not contend with protein denaturation. The data from the mobile phase stability tests for benzene are rather different and are shown in figure 11.

Figure 11. Graph of (k') of Benzene against Volume of Mobile Phase Passed Through the Column

The curves for benzene, that relates the (k') value of the solute to the volume of mobile phase through the column, shows similar effects as the curves for aniline, but in this case in a slightly different manner. As erosion occurs, some of the oligomeric phase is removed and simultaneously, more silanol groups are exposed. The dominant effect on the retention of aniline was an increase in ionic interactions with the exposed silanol groups and consequently, the retention volume of aniline increased. Benzene is, however, is neither ionic nor polar (although polarizable) and the major effect of phase erosion on benzene retention is the loss of reverse phase. Thus, the retention volume of benzene decreases as the erosion of the bonded phase surface progresses. Nonetheless, the same effect is shown, as the bonded phase layer thickness oligomer increases, so the reverse phase becomes more resistant to erosion.