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instrument	Agilent^® 6530 Q-TOF LC/MS
column	SeQuant ZIC-cHILIC, 10 cm x 2.1 mm, 3.0 µm
mobile phase	[A] acetonitrile; [B] 50 mM ammonium acetate, pH 5.0 with acetic acid in water
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 9.9 min
flow rate	0.3 mL/min
pressure	1059 psi
column temp.	50 °C
detector	Q-TOF (ESI-negative) \r\n
detector other	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

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Analysis Note	HILIC separation is an alternative that permits sensitive MS detection and without the use of ion-pair reagents. Nucleotides are the building blocks of nucleic acids. They comprise a nitrogenous base, a sugar, and a phosphate group making them highly polar and ionic. This causes a challenge for typical reversed phase chromatography (RPC). These charged polar analytes are poorly retained by RPC, and thus typically chromatographed by either ion-exchange chromatography or RPC in conjunction with an ion-pairing reagent. Nucleotide analysis using ion-pair reagents or high concentration of phosphate buffers cause high background and ion source pollution for MS detection and is undesirable compared to HILIC.
Featured Industry	Life Science and Biopharma
Legal Information	SeQuant is a trademark of Merck KGaA, Darmstadt, Germany \r\nLiChrosolv is a trademark of Merck KGaA, Darmstadt, Germany \r\n
Application No.	ASC140218E

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