27
Gradient program:
Time Flow rate A (%) B (%) (min)
0 4
8.99 9
17
(mL/min) 0.4 0.4 0.4 0.4 0.4
Sample Preparation
The stock solutions containing 20 mg/mL each of Neomycin, Apramycin and Kanamycin were prepared by dissolving the appropriate amount of solid in 30/70 (v/v) acetonitrile/water solution. The standard solutions were prepared by mixing the stock solution with the same acetonitrile/water solution as described above.
50 5 5
50 50
50 95 95 50 50
Results and Discussions Separation with Zwitterionic HILIC Stationary Phase
The zwitterionic stationary phase used in this study is a silica base stationary phase bonded with sulfobetaine zwitterionic functionality, which has both a positively charged quaternary ammonium and a negatively charged sulfonic acid group in one single functional moiety. The unique zwitterionic structure and highly hydrophilic character makes it ideal for HILIC separation. The balance between the strong acidic and strong basic group makes this phase independent of pH, and the electrostatic interaction is significantly weaker compared to a conventional ion exchanger [16,17]
. Figure 2
shows the proposed retention mechanism for separating polar/hydrophilic compounds on such a zwitterionic HILIC phase. A crucial part in the separation mechanism is that a stagnant
water layer is built up when equilibrating the stationary phase with the mobile phase containing 3-50% aqueous buffer solution. A polar/hydrophilic analyte experiences hydrophilic partitioning between the water layer and the less polar mobile phase. In addition, a charged analyte may have weak electrostatic interactions (both attraction and repulsion) by the zwitterionic groups, which greatly contribute to the separation selectivity.
At the beginning of this work, we investigated a more neutral zwitterionic stationary phase to separate neomycin and apramycin with a similar gradient elution profile as described in the experimental section. However, the separation resolution between these two aminoglycosides was unfortunately unsatisfactory. It appeared that retention and selectivity based on hydrophilic partition are not sufficient in this case, especially when using a very fast gradient elution. We realized it was actually an advantage to have electrostatic interaction to improve the separation resolution due to the different charge states of neomycin and apramycin. This may be one of the reasons that zwitterionic HILIC stationary phases are the most used phases for the HILIC separation of aminoglycosides.
Figure 1: Chemical structures of the three aminoglycosides analysed in this study. The molecular weights and Log P values were obtained from PubChem (
http://pubchem.ncbi.nlm.nih.gov).
Separation of Aminoglycosides Aminoglycosides have multiple positive charges at pH conditions below their pKa values. This indicates that the electrostatic interaction between these analytes and the stationary phase will be much stronger than that for single charged molecules. The electrostatic interaction was examined by isocratic separation of aminoglycoside with a mobile phase containing 20-30% A and 80- 70% B, where the separation should be primarily dependent on the electrostatic interaction but not hydrophilic partitioning due to the very low organic solvent content in the mobile phase. It was found that retention factor (k’) for neomycin and apramycin decreased from 10.61 and 5.59 to 5.13 and 2.95, respectively, while the mobile phase B was increased from 70% to 80%. It was also found that the eluting peaks became broader. These observations indicate a separation mode based on electrostatic interaction.
Figure 2: Retention of polar/hydrophilic compounds on a zwitterionic HILIC stationary phase.
Ishii et al., Oertel et al., and Shen et al. [12-14] demonstrated the importance in using a mobile phase with high ionic strength and a fast gradient profile for aminoglycosides separation in HILIC. We used a standard HPLC-MS system to repeat the experiments with the same separation conditions and gradient profiles as those used by Ishii et al. yet neomycin exhibited strong peak tailing,
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
