40 MORAVCOVÁ AND PLANETA Figure 1: HILIC separation of four purine bases in a bare silica monolithic capillary column. Mobile phase: 95% (v/v) ACN, 5% (v/v) 100 mM ammonium acetate pH=4.5; flow rate 500 nL/min; UV detection at 210 nm. Peak iden- tification: 1 – toluene (unretained compound), 2 – xan- thine, 3 – caffeine, 4 – theobromine, and 5 – hypoxanthine. silica capillary 0.025 mm × 150 mm in length). The inlet section of the monolithic column was inserted through the body of the splitter into the injection valve to mini- mize extra-column dispersion. The outlet of the monolithic column was connected to a Spectra 100 UV/Vis detector (Thermo Separation Products, Waltham, Massachusetts, USA) via a 0.035 mm × 120 mm-long fused silica capillary (with an optical bubble cell window inner diameter (ID) 0.110 mm) us- ing a polytetrafluoroethylene (PTFE) sleeve. UV detec- tion was performed at 210 nm. The detector signal was processed by the chromatography station software Clar- ity (DataApex, Prague, Czech Republic). 3. Results and Discussion 3.1 Chromatographic properties of the bare silica monolith The prepared bare silica monolithic capillary columns were evaluated under HILIC conditions by employing a mixture containing toluene as an unretained compound and four purine bases, namely xanthine, caffeine, theo- bromine and hypoxanthine. The obtained chromatogram Table 1: The retention factor (k), the height equivalent to the theoretical plate (H), the number of theoretical plates per meter (N), and peak asymmetry (As) obtained on the silica monolithic column. See Fig. 1 for separation condi- tions. Compound k H/µm N As Toluene – 7.4 135,185 0.67 Xanthine 0.03 4.5 222,547 0.88 Caffeine 0.13 5.8 172,795 1.00 Theobromine 0.19 5.2 192,528 1.13 Hypoxanthine 0.87 4.8 209,601 1.33 Table 2: The retention factor (k), number of theoretical plates per meter (N), and peak asymmetry (As) obtained on the MEPC column. See Fig. 2 for the separation condi- tions. Compound k N As Toluene – 138,955 1.0 Adenosine 0.16 201,048 1.0 Uridine 0.25 289,537 1.1 Cytidine 0.41 244,314 0.9 AMP 1.24 206,697 1.3 UMP 1.66 183,465 1.5 CMP 2.50 157,760 1.4 ADP 3.98 122,169 2.0 UDP 5.34 112,389 2.4 CDP 7.58 81,476 2.8 ATP 9.73 61,462 2.1 where all purine bases are baseline separated and have narrow symmetrical peaks is presented in Fig. 1. The prepared column exhibits a high degree of sepa- ration efficiency reaching values of between 135,000 and 220,000 theoretical plates/m which corresponds to the minimum plate height within the range of 4.5–7.4 µm, as outlined in Table 1. 3.2 HILIC separation of nucleosides and nu- cleotides The commercially available methacrylate-based monomers MEDSA and MEPC were selected for the preparation of zwitterionic stationary phases. The bare silica monolith prepared in a 0.1 mm ID fused silica capillary was modified by 3-(trimethoxysilyl)propyl methacrylate and a polymer layer, with the appro- priate zwitterion grafted onto it. The suitability of these columns for the isocratic HILIC separation of nucleosides and nucleotides was studied. Chromatograms for selected compounds obtained on MEDSA (Fig. 2A) and MEPC (Fig. 2B) capillary columns are presented in Fig. 2. An enhanced degree of separation was obtained on the MEPC column (Fig. 2B) where all 11 compounds present in a sample mixture were well separated within 25 mins. The value of peak asym- metry (As) reflects the change in the molecular shape of the analyte and increases slightly as the number of phos- phate groups increases in the relevant nucleotide, as pre- sented in Table 2. On the other hand, the separation efficiency de- creases along with the number of phosphate groups in the molecule of nucleotide exhibit lower values of N com- pared to nucleosides. A similar trend was also observed on the MEDSA-modified monolithic capillary column. The MEDSA-modified monolithic column exhibits a poorer separation efficiency than the MEPC column for all nucleosides and nucleotides, even the number of the- oretical plates per meter for toluene is comparable, as il- lustrated in Table 3. The MEDSA column also exhibits Hungarian Journal of Industry and Chemistry SILICA MONOLITHIC CAPILLARY COLUMNS FOR HILIC SEPARATIONS 41 Figure 2: Separation of nucleosides and nucleotides on prepared MEDSA (A), MEPC (B) and bare silica (C) monolithic columns. Mobile phase: 70% (v/v) ACN/30% (v/v) 50 mM ammonium acetate pH = 4.5; flow rate 500 nl/min; UV detection at 210 nm. a lower retention factor for selected compounds, espe- cially for di- and triphosphates of the relevant nucleo- sides. This can be explained by the fact that monomers used for column modification differ in terms of their ter- minal groups, which affects the selectivity of the prepared stationary phases. The MEDSA monolithic capillary col- umn contains negatively charged terminal sulfo groups which decrease the retention factor of negatively charged nucleotides. On the other hand, the MEPC monomer pro- vides the stationary phase with a slightly positive charge originating from the phosphorylcholine functional group which improves the retention factor of negatively charged Table 3: The retention factor (k), number of theoretical plates per meter (N), and peak asymmetry (As) obtained on the MEDSA column. See Fig. 2 for the separation con- ditions. Compound k N As Toluene – 131,000 1.2 Adenosine 0.12 135,641 1.1 Uridine 0.19 129,895 1.2 Cytidine 0.28 130,754 1.2 AMP 0.46 101,991 1.5 UMP 0.66 89,032 1.0 CMP 0.91 90,838 1.1 ADP 0.97 71,440 1.7 UDP 1.40 48,371 5.1 CDP 1.78 49,250 3.1 ATP 1.78 59,378 3.5 nucleotides under the HILIC separation conditions, e.g. the retention factor k for ATP is approximately 5 times higher on the MEPC column compared to the MEDSA column. The separation of nucleosides and nucleotides on a bare silica monolithic capillary column is shown in Fig. 2C. The bare silica monolith does not exhibit a sufficient degree of selectivity to compounds of the target. All com- pounds are eluted from the column within 1 min and ac- companied with the coelution of most compounds. 4. Conclusion A silica-based monolith in a capillary format coated with zwitterionic polymer layers is a good alternative for the analysis of complex mixtures containing highly po- lar compounds such as nucleosides and nucleotides. In particular, the MEPC stationary phase containing posi- tively charged terminal groups enables the highly selec- tive HILIC separation of nucleosides and nucleotides to be achieved. The utilization of such a stationary phase with regard to the analysis of real samples, where usually other highly polar-interfering species originating from complex matrices are present and complicate the analy- sis, should be evaluated in the future. Acknowledgement This research was supported by the Czech Science Foun- dation (Project No. 16-03749S), the Ministry of the Inte- rior of the Czech Republic (Project No. VI20172020069), and by the Czech Academy of Sciences (Institutional Support RVO:68081715). REFERENCES [1] Nakanishi, K., Soga, N.: Phase separation in silica sol-gel system containing polyacrylic acid I. Gel formation behavior and effect of solvent compo- sition, J. Non-Cryst. Solids, 1992 139 1–13 DOI: 10.1016/S0022-3093(05)80800-2 46(1) pp. 39-42 (2018) 42 MORAVCOVÁ AND PLANETA [2] Minakuchi, H., Nakanishi, K., Soga, N., Ishizuka, N., Tanaka, N.: Octadecylsilylated Porous Silica Rods as Separation Media for Reversed-Phase Liq- uid Chromatography, Anal. Chem., 1996 68(19) 3498–3501 DOI: 10.1021/ac960281m [3] Ikegami, T., Tanaka, N.: Recent Progress in Mono- lithic Silica Columns for High-Speed and High- Selectivity Separations, Annu. Rev. Anal. 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