14
May/June 2013 Discussion
Characterisation tests The data sets for the separation factors generated in this study highlighted important retention characteristics and differences (Table 3-6). Stationary phase
characteristics have been visually illustrated by radar graphs [13, 16], which allow to express multi-
Figure 10. Effect of buffer concentration on retention of salicylic acid.
in terms of salicylic acid retention, is given in Figure 10. On Hypersil GOLD HILIC salicylic acid exhibits an opposite behaviour to what observed on Syncronis HILIC, with a dramatic decrease in retention, as the buffer concentration increases. The ion exchange interaction on this phase could have a major effect on the retention of acids. Salicylic acid is charged (mobile phase buffer pH= 6.7, pKa for salicylic acid= 2.9) and its retention drops as the buffer concentration increases (pH of mobile phase increases), which is typical of an ion exchange mechanism.
The effect of mobile phase pH on retention
Mobile phase pH can have a significant contribution on the variation of retention and selectivity in HILIC separations [6]. In this study the pH of stock salt solutions was changed before mixing with acetonitrile. Ammonium formate was used (because of its relatively low buffering range, pH 2.8-4.8); the pH of the 100mM ammonium formate stock solution (pH~ 6.4) was adjusted with formic acid to pH 3.3, 4.0 and 4.8. Table 7 summarises the retention data of the model compounds on the four columns. The retention is affected when the analyte ionisation state changes in the pH range considered for the study. This is the case for aspirin, where its retention gradually decreases with the buffer pH on the two silica materials, since aspirin is protonated and less hydrophilic. A similar behaviour is demonstrated by salicylic acid on Hypersil GOLD HILIC.
Aspirin retention is instead unchanged in the buffer range 6.4-4.8 on the zwitterionic material, but then the retention time drops when the pH is lowered to 3.3, probably due to ion-exchange effect no longer taking place. Cytidine and cytosine retentions only fluctuate slightly on the four columns in the pH range investigated.
dimensional data in a two- dimensional format and ultimately allow to visually
assess and compare columns. The separation factors obtained in the course of this assessment were therefore arranged in radar plots, which are shown in Figure 5. The following discussion will concern both tabulated data and their corresponding graphical representations.
From the radar plots illustrated in Figure 5 it is interesting to note that there is a positive correlation, for all of the materials, between
α (CH2) and α (OH)which matches the observations by Ikegami and his group [13].
A tentative interpretation for this observation is that the chemistry of the stationary phases does not have a substantial role on the selectivity of these two particular groups. Alternatively, the k uridine data demonstrate that the stationary phase chemistry has an effect on the absolute retention, probably due to the absolute volume of the water layer. It can be seen that the bare silica materials, the Trinity P1 and the mixed mode HILIC-1 exhibit lower values for k uridine. Syncronis HILIC demonstrated to be the most retentive material for uridine. The bare silica of Hypersil GOLD provided different
kuridine, α (OH) and α (CH2) values from the silica in Accucore HILIC and Syncronis Silica. These differences could be due to differences in pore volume, surface area and particle morphology for the three silica types. Syncronis Silica showed a higher retentivity than Hypersil GOLD Silica due to its higher nominal surface area. Accucore HILIC, in turn demonstrated higher k uridine,
α (OH) and α (CH2) values than the other bare silica columns.
From Table 4 it can be observed that Syncronis HILIC provided the best selectivity
for α (V/A) and α (2dG/3dG). Similar data were reported by Ikegami’s group for Nucleodur HILIC and ZIC-HILIC which have similar zwitterionic functionality [13]. Mixed Mode HILIC-1 cannot discriminate between
the two configurational isomers, as
demonstrated by the α (V/A) value of 1.0. This diol material showed a similar α (2dG/3dG) value to the 1.06 value reported by Ikegami et al. for Lichrosphere Diol [13].
The fact that α (2dG/3dG) values are about 1.1 for most materials (apart from HILIC-10) would indicate less specificity for positional isomers. From the radar plots it can be
observed some correlation between α (V/A) and α (2dG/3dG) for most phases, although the small variations for α (2dG/3dG) data are not sufficiently significant. These small
variations were also observed on the materials characterised by Ikegami and his group [13], suggesting that these probes are not selective enough. From Table 5 it can be observed that Hypersil GOLD HILIC and Acclaim Trinity P1 have the strongest anion interactions; these results are expected, considering that both materials possess amino groups, which work as AX functionalities at the pH experimental conditions of 4.7. The bare silica materials
exhibited the highest α (CX) values; bare silica phases are known to possess cation exchange ability due to their acidic silanols (SiOH) functionality.
For the mixed mode HILIC-1 the value for α (AX) was not reported, and the value for α (CX) was zero, since SPTS eluted faster than
t0 and TMPAC co-eluted with t0. It has been observed that some ligands exclude TMPAC
and SPTS from the pore volume, resulting in these compounds not being retained [13]. Pore exclusion could be advocated for the early elution of SPTS and TMPAC experienced on the mixed mode HILIC-1.
From the AX and CX characterisation study it can be concluded that cation exchange interactions have important effects in HILIC on bare silica phases. Syncronis HILIC showed considerable CX character, due to
the sulfo group in the phase; however, the α (CX) value for Syncronis HILIC was much lower than the values recorded by Ikegami’s group for Nucleodur HILIC and ZIC-HILIC (3.46 and 4.41 respectively) [13]. Experimental HILIC also demonstrated some CX character. The degree of ion exchange interactions has a major impact on the shape of the radar plots, as illustrated in Figure 5, with a distinct dichotomy between (i) the bare silica materials, which have strong cation exchange ability, and (ii) Trinity P1 and GOLD HILIC, which exhibit strong anion exchange activity. Very little ion exchange interactions were demonstrated by HILIC-10 and mixed mode HILIC-1.
In the study by Lämmerhofer et al. [10] it was
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