De Smet, Seppe

Lynen, Frederic

English

Ghent University Academic Bibliography

Hydrophilic interaction electrochromatography applied in packed capillaries  S. De Smet1 , F. Lynen1  1 Separation Sciences Group, Department of Organic Chemistry, Universiteit Gent, Krijgslaan 281 S4-bis, B-9000 Gent INTRODUCTION EXPERIMENTAL CONCLUSION      Capillary packing procedure:  • The inlet frit is formed by dipping the tip of the capillary in  mixture of 5 µm silica particles wetted with a silicate/water (1/1) solution followed by sintering • 50 mg of the packing material (3 µm silica particles) is suspended  in 2 ml of an acetone /H2O (60/40) solution and introduced in the packing reservoir • The packing reservoir is  connected to the  LC pump and pressure is immediately raised to 600 bar for 2 hours • The capillary is  flushed for two hours with the packing solvent and  subsequently flushed for at least 12 hours with H2O • The outlet frit is sintered with a heating element (370°C), while the capillary is pressurized at 600 bar with water • The excess material at the outlet frit is removed by flushing the column with the mobile phase buffer during 2 hours • The detection window is 1.5 cm away from the outlet frit formed by thermolytical removal of the polyimide                       RESULTS & DISCUSSION • Hydrophilic interaction chromatography is a strong tool for analysis of polar solutes  • HICEC has been described on organic and inorganic monoliths and open-tubular coated capillaries, as the incorporation of polar functional groups can be achieved most easily in this way  • In HICEC the efficiency and retention can be the result of both electrophoresis and chromatographic phenomena in the case of ionized components. To disconnect both phenomena only polar neutral solutes should be allowed for the in depth study of the hydrophilic interaction mechanism in CEC  • Narrow fused silica capillaries are there fore packed with native silica particles and evaluated in HICEC    RESULTS & DISCUSSION Retaining frit formation Removal of excess material H2O  600 bar + -     Rinsing  1) NaOH (1 M)  2) H2O 3) MeOH Formation of detection window - + Inlet frit sintering 1) Acetone/H2O (with packing reservoir), 600 bar    2) Acetone/H2O (without packing reservoir), 600 bar  Packing process H2O  600 bar Flushing LC-pump 600 bar  Acetone/ H2O (60/40)  ultrasonic bath capillary outlet  frit pressure  release valve waste Packing reservoir Analytical conditions  • Instrument:  CE 7100 system (Agilent Technologies, Waldbronn, Germany) • Buffer 1: 100 mM ammonium acetate pH 5.5 with 97.5 % ACN • Buffer 2: 100 mM ammonium acetate pH 5.5 with 90 % ACN • Voltage conditioning: Stepwise (5 kV) increase to 30 kV in 60 minutes + 60 minutes at 30 kV • Sample: 125 µg of each standard was dissolved in 1 ml of the analysis buffer prior injection  • Injection: electrokinetically  (8 kV x 4 sec.) • Operating voltage: 30 kV  • Detection: UV detection at 214 or 254 nm • Pressurization: All analyses were done while applying 12 bar N2 pressure on both the inlet and the outlet vial                                                         + - while conditioning with buffer Fig 1: Schematic representation of the packing procedure of HICEC capillaries Fig 2: Schematic representation of the packing set-up of capillaries                         • Standards were always selected in such a manner that they don’t exhibit any electrophoretic behavior at the applied mobile phase conditions       • The proof of principle of HICEC  with packed capillaries is done by the separation of aromatic nitro-derivates          • Future prospects of HICEC in packed capillaries is the analyses of oligonucleotides and oligosaccharides. The packed capillaries and the applied mobile phase conditions are compatible with MS. To illustrate the power of packed capillary HICEC a high efficient analysis of nucleotides was performed on a 60 cm packed bed capillary         00.511.522.533.544.550 5 10 15 20 25 30 35 40 45 50Intesnity (mAu) Time (min) 00.511.522.533.544.550 2 4 6 8 10 12 14 16 18 20Intesnity (mAu) Time (min) 1 u 2 3 4 5 1 2 3 4 5 HILIC-HPLC HICEC 60 cm packed bed (3 µm, 75 µm i.d.) Figure 4: The analysis of 4 nucleotides and toluene in HPLC and CEC. Sample: 1) thiourea, 2) thymine, 3) adenine, 4) guanine, 5) cytosine, u) uracil. Background electrolyte: 100 mM ammonium acetate pH 5.5 with 90% ACN.  Component name Retention time k’ Plates(60 cm) Plates/ m Toluene 18.235 / / / Thymine 24.093 0,32 41509 69182 Cytosine 42.081 1,30 66178 110296 Guanine 45.719 1,50 72195 120325 Adenine 47.323 1,60 77685 129475 Table 1: The chromatographic characteristics of the eluting components 0510152025Intesity (mAu) Time (min) 1,2,3,4,5 Figure 5: Identical same analysis as in figure 4  but in CZE mode  (without stationary phase) 6 7 8 • The packing conditions for bare silica were optimized and columns with a packed bed of 30 cm and 60 cm were successful fabricated  • A successful separation of the nucleotides was obtained with a maximum efficiency of 130000 plates/meter  • The elution order between  HILIC-HPLC and HICEC of neutral components remains  the same  01020304050600 5 10 15 20 25 30Intensity (mAu) Time (min) Seppe De Smet gratefully acknowledges the financial support of the Agency for Innovation by Science and Technology in Flanders (IWT) Any retention is caused by chromatographic interaction of the  solutes with stationary phase. This is attested with confirmatory CZE analyses (where no separation is observed)    1 2 3 4 Figure 4: The analysis of thiourea (1), 3-nitro-phenol (2), 4-nitro-phenol (3) and 2,4-dinitro-toluene. Background electrolyte: 100 mM ammonium acetate pH 5.5 with 97.5% ACN.  30 cm packed bed (3 µm, 75 µm i.d.)  

Hydrophilic interaction electrochromatography applied in packed capillaries

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Hydrophilic interaction electrochromatography applied in packed capillaries

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