12
May/June 2013
Figure 7a
Figure 7b Figure 7a and 7b. Effect of column temperature on retention of salicylic acid (a) and cytosine (b). Mobile phase: acetonitrile/water (90/10, v/v), containing 10mM ammonium acetate.
on the basic Acclaim Mixed Mode HILIC-1, which produced a negative selectivity value; the stronger retention of theophylline on this material would indicate an additional ionic interaction between the more acidic theophylline and the column surface as compared to theobromine. On the remaining columns the two xanthines co-elute, resulting in selectivity values = 1.
Chromatographic parameters investigation: their effect on HILIC retention
The effect of acetonitrile content on retention
In HILIC, the level of organic solvent in the mobile phase can have a large influence on retention. Separation of the two test mixtures was carried out on the four stationary phases (marked with an asterisk in Table 1). The percentage of acetonitrile in the mobile phase was varied whilst keeping ammonium
acetate concentration constant at 10mM. The logarithmic retention factors (ln k) for two model compounds (salicylic acid and cytosine) were plotted against the acetonitrile content (Figure 6a and 6b respectively).
Salicylic acid shows HILIC behaviour of decreasing retention with decreasing acetonitrile content on Syncronis HILIC and on Hypersil GOLD Silica. On Hypersil GOLD HILIC and on Accucore HILIC the retention of salicylic acid decreases initially as the acetonitrile content decreases, but as this reaches 80% the retention gradually increases. This behaviour could be due to ion-exchange interactions. Cytosine shows HILIC behavior of decreasing retention with decreasing acetonitrile content on the four stationary phases. This is also true for the other components of the basic test mixture.
The effect of column temperature on retention
In this study the temperature effect on the retention of salicylic acid and cytosine was investigated for the four stationary phases; the column temperature was varied from 20 to 70°C. The logarithmic retention factors for the two model compounds were used to generate van’t Hoff diagrams. Figure 7a shows van’t Hoff plots for salicylic acid and Figure 7b for cytosine, on the four selected columns. From Figure 7a it can be seen that a decrease in retention, indicating a negative heat of enthalpy and hence an exothermic reaction, is observed as the column temperature is increased on Accucore HILIC, Hypersil GOLD Silica and to a lesser extent on Syncronis HILIC. The retention increases with the temperature on Hypersil GOLD HILIC. Positive enthalpy was obtained for Hypersil GOLD HILIC, indicating an endothermic process of transferring salicylic acid from the mobile phase to the stationary
Figure 8a
Figure 8b Figure 8a and 8b. Effect of buffer concentration on retention of basic and acidic mixtures, for Syncronis HILIC.
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