LC/MS Analysis of Nucleotides on SeQuant® ZIC-HILIC. Effect of pH
CONDITIONS
column
SeQuant® ZIC-HILIC, 10 cm x 2.1 mm, 3.5 μm particles (1.50441.0001)
mobile phase
[A] acetonitrile; [B] 50 mM ammonium acetate, pH 4.5, 5.0, 5.3, or 5.5 with acetic acid in water, pH 6.9 not adjusted
gradient
26 to 27% B in 10 min; to 27 to 35% B in 10 min; to 26% B in 0.1 min; hold at 26% B for 4.9 min
flow rate
0.3 mL/min
pressure
1088 psi (75 bar)
column temp.
40 °C
detector
(Q-TOF (ESI-negative), XIC of the following m/z values: AMP-346, ADP-426, ATP-506, CMP-322, CDP-402, CTP-482, GMP-362, GDP-442, GTP-522, UMP-323, UDP-403, UTP-483)
injection
10 μL
sample
10-30 μg/mL of nucleotides (AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, UTP) (80:20, acetonitrile:50 mM ammonium acetate in water pH 5.3 with acetic acid)
Description
Analysis Note
ZIC-HILIC is a densely bonded zwitterionic stationary phase with sulfobetaine functional groups graft-polymerized to silica. The stationary phase has an overall 1:1 charge balance between the permanent positive quaternary ammonium groups close to the silica support and the distal negatively charged sulfonate groups. It has though a slight negative surface charge because of the spatial arrangement. A covalently bonded zwitterionic stationary phase, with permanent strong acidic and basic functionalities, provide hydrophilic partitioning combined with weak ionic interactions and makes the phase virtually pH independent as shown in this analysis.
Legal Information
SeQuant is a registered trademark of Merck KGaA, Darmstadt, Germany