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May/June 2013
more retained than 2’-deoxyuridine; this reflects the fact that uridine is more hydrophilic than 2dU. The average α (CH2),
α (OH) and kuridine values are summarised in Table 3 and reported in Figure 5.
Structural and Configurational Isomers An important property that HILIC can offer is the ability to separate structural isomers and configurational isomers, typically found in saccharides and peptides. The selectivity of
configurational isomers, α (V/A) - obtained from a comparison of k vidarabine and
k adenosine, α (V/A) - and positional (regio) isomers, α (2dG/3dG) - calculated from the k 2dG/k 3dG ratio - were investigated. These
selectivity values also reflect shape selectivity [13]. Table 4 and Figure 5 summarise the
mean α (V/A) and α (2dG/3dG) values for the stationary phases tested.
Figure 4: Chromatograms for α (CX) test. Analyte: 1); 2) uracil; 3) N,N,N-trimethylphenylammoniumchloride, TMPAC.
Column Name Syncronis HILIC (5 µm)
Hypersil GOLD HILIC (5 µm) Hypersil GOLD Silica (5 µm) Hypersil GOLD Silica (1.9 µm) Syncronis Silica (5 µm) Accucore HILIC (2.6 µm)
Acclaim Mixed Mode HILIC-1 (5 µm) Acclaim HILIC-10 (3 µm) Acclaim Trinity P1 (3 µm) Experimental HILIC (3 µm)
α (V/A) 1.403 1.444 1.255 1.214 1.270 1.327 1.000 1.222 1.409 1.336
Hypersil GOLD HILIC
α (2dG/3dG) 1.129 1.082 1.092 1.092 1.100 1.114 1.102 0.963 1.023 1.111
Table 4. Separation factors for configurational isomers α (V/A) and positional isomers α (2dG/3dG) Syncronis HILIC
Hypersil GOLD Silica Ion Exchange Interactions
Ion-exchange interactions can be influential in HILIC, leading to drastic changes in selectivity, particularly when separating ionic species [15]. To evaluate the degree of ion exchange nature of the stationary phases a relatively hydrophobic organic anion, sodium p-toluenesulfonate (SPTS) and a relatively hydrophobic organic cation, N,N,N- trimethylphenylammoniumchloride (TMPAC) were chosen to see the effect of anion and cation interactions [13]. It is reasonable to postulate that these compounds would also be retained by hydrophilic interactions [13], so the retention factors k SPTS and k TMPAC were divided by k Uracil to account for the hydrophilic interaction contribution. The chromatography for both the anion and
Syncronis Silica Accucore HILIC
Acclaim Mixed-Mode HILIC-1
Acclaim HILIC-10
Acclaim Trinity P1
Experimental HILIC
Figure 5: Radar plots for HILIC stationary phases
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