11
Figure 6a
Figure 6b
Figure 6a and 6b. Effect of acetonitrile content on the retention of salicylic acid (a) and cytosine (b) Column temperature: 30ºC. Flow rate: 1mL/min. UV detection at 228nm for salicylic acid and 248nm for cytosine.
cation exchange interactions on the selected columns is shown in Figure 3 and Figure 4, respectively. The resulting mean separation
factors, α (AX) and α (CX) for the stationary phases tested are reported in Table 5; the values are also plotted in Figure 5.
Figure 3 and Table 5 highlight the variability in elution orders obtained for the different columns. The majority of columns show that
the elution order is toluene (t0 marker), uracil, and SPTS, although Hypersil GOLD HILIC and Acclaim Trinity P1 have a reversal of the two test analytes. Anion exchange selectivity also varied for Acclaim Mixed Mode HILIC-1, where SPTS was not retained and it eluted before toluene and for Acclaim Mixed Mode HILIC-10, where SPTS co-eluted with uracil.
From Figure 4 and Table 5 a similar set of results demonstrating a variability in elution order can be seen when performing the cation test. Thus, it can be observed that for some materials TMPAC elutes after uracil, apart from Hypersil GOLD HILIC, where it elutes before uracil. Different cation exchange selelctivity was also exhibited by Acclaim Mixed Mode HILIC-1 and Acclaim Trinity P1, with TMPAC co-eluting with uracil.
Acidic-Basic nature of the stationary phase surface
Since many compounds analysed in HILIC have ionisable functional groups, knowing the acid-base properties of the stationary phase is important for controlling the separation [13]. Xanthine derivatives have been used as test samples in HILIC; the pKa values for theophylline and theobromine have been reported as pKa= 8.6 and pKa= 10 respectively, so theobromine is more basic
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)
Table 5: Separation factors for α (AX) and α (CX) Column Name Syncronis HILIC (5 µm)
Hypersil GOLD HILIC (5 µm) Acclaim HILIC-10 (3 µm)
Acclaim Mixed Mode HILIC-1 (5 µm) Acclaim Trinity P1 (3 µm) Syncronis Silica (5 µm)
Hypersil GOLD Silica (1.9 µm) Hypersil GOLD Silica (5 µm) Accucore HILIC (2.6 µm)
Experimental HILIC (3 µm) Table 6: Separation factors for α (Tb/Tp)
than theophylline. If an HILIC mode separation applies, an elution order of theophylline < theobromine would be expected, based on their log D values.
It follows that the selectivity values, k Tb/k Tp
should be > 1 [10]. From the selectivity data obtained during our investigation (reported in Table 6 and Figure 5) it can be seen that this is the case for the acidic bare silica materials. This elution order is the opposite
α (Tb/Tp)
1.000 1.000 1.000 0.860 0.671 1.151 1.102 1.091 1.189
1.269
pH conditions of stationary phase
Neutral
α (AX) 0.723 1.878 0.609 0.549 0.581 0.521 -
1.000 9.241 0.454
α (CX) 1.115 0.554 4.832 5.951 5.614 3.992 0.000 1.919 1.000 1.660
Basic
Acidic
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