13
phase. For Hypersil GOLD HILIC, the positive enthalpy of salicylic acid could also be the evidence of both ion exchange and partitioning processes taking place.
Figure 7b shows a decrease in retention on the four stationary phases as the column temperature is increased. Linear van’t Hoff plots resulted from this study. Negative retention enthalpy values were obtained, indicating an exothermic process. The enthalpy values derived were very different, which could indicate the existence of strong specific (secondary) interactions between cytosine and the functional groups of the four stationary phases [6].
The effect of buffer concentration on retention
In this study the effect of salt concentration on the retention was investigated. Separation of the two test mixtures was carried out on Syncronis HILIC and Hypersil GOLD HILIC. The content of acetonitrile in the mobile phase was kept constant, whilst varying the ammonium acetate concentration from 2.5 to 20 mM. The capacity factors for the two model compound mixtures were plotted against the buffer molar concentrations for the two columns. Figure 8a and 8b show the data relative to the basic and acid test mixtures respectively obtained on Syncronis HILIC. The five bases are fully resolved on Syncronis HILIC when low levels of ammonium acetate are used (2.5, 5 and 10 mM). Further increase in the salt content leads to an increase in retention, with co-elution of adenosine and uridine. Apart from uracil, the other 4 bases show an increase in retention as the ammonium acetate concentration is
Table 7. The effect of buffer pH on retention of model compounds
increased above 5mM, the increase being more pronounced for cytidine. Interestingly, the retention decreases when the buffer concentration is increased from 2.5 to 5 mM. The general trend of increased retention as the buffer concentration increases has been related to a hydrophilic partitioning process [6]. The partitioning model for HILIC assumes the presence of a water-rich liquid layer on the stationary phase. Salt is more soluble in the aqueous rich sub-layer, so increasing the organic will effectively increase the concentration in this layer. The four acids are not fully resolved on Syncronis HILIC, as shown in Figure 8b, with salicylic acid co- eluting with salicylamide when low levels of ammonium acetate are used (2.5, 5 and 10 mM). Further increase in the salt concentration to 15mM leads to an increase
in retention for salicylic acid and salicylamide. It is possible that electrostatic repulsions from the negatively charged sulphonate groups prevented the acid molecules from reaching the quaternary amine groups (located closer to the silica surface), resulting in lower retentions. Higher salt concentration could have weakened these repulsions, leading to salicylic acid and salicylamide being more retained. Aspirin elutes as a split peak and dhpa is very broad when the salt content was increased to 15mM and above.
Figure 9a and 9b show the data relative to the basic and acid test mixtures respectively, obtained on Hypersil GOLD HILIC.
To elucidate the different behaviours exhibited by the two columns, a comparison,
Figure 9a
Figure 9b Figure 9a and 9b. Effect of buffer concentration on retention of basic and acidic mixtures, for Hypersil GOLD HILIC.
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60
