APPLICATION NOTE

AN040410-57

STYROS™ HILIC Simulated Monolith™ Polymeric Normal Phase: Separations of Nucleotides.

HILIC or Hydrophilic Interaction Chromatography is a variation of normal phase chromatography. It provides complementary selectivity compared to reversed phase chromatography.

The following chromatograms show the separation of Adenosine and Guanosine Phosphates on a STYROS™ HILIC Simulated Monolith™ column at 30˚C.

Chromatogram 1

Separation of 3 Adenosine derivatives on STYROS™ HILIC
(Flow Rate: 1 ml/min)

Table 1. Operating parameters.

	HPLC System	Agilent1100, Standard Cell with Thermostatted Column Compartment
	Detector	254 nm
	Column	STYROS™ HILIC 4.6 X 100 mm (V=1.66 ml)
	Mobile Phase	A: DI H2O B: ACN B: 100 mM CO3(NH4)2, pH=9.6
	Separation	Isocratic: 15% A, 75% B, 10% C (total ionic strength 10 mM)
	Flow rate	1ml/min (360 cm/hr of linear flow rate )
	Temperature	30 °C
	Injection volume	5 µl
	Sample:	Adenosine-5’-monophosphate, Adenosine-5’-diphosphate, Adenosine-5’-triphosphate (330 ug/ml each in B:C 80:20)

Adenosine-5’-monophosphate and the similar structures di- and triphosphate derivatives are baseline separated.

Chromatogram 2

Separation of 3 Guanosine derivatives on STYROS™ HILIC
(Flow Rate: 1 ml/min)

Table 1. Operating parameters.

	HPLC System	Agilent1100, Standard Cell with Thermostatted Column Compartment
	Detector	254 nm
	Column	STYROS™ HILIC 4.6 X 100 mm (V=1.66 ml)
	Mobile Phase	A: DI H2O B: ACN B: 100 mM CO3(NH4)2, pH=9.6
	Separation	Isocratic: 15% A, 75% B, 10% C (total ionic strength 10 mM)
	Flow rate	1ml/min (360 cm/hr of linear flow rate )
	Temperature	30 °C
	Injection volume	5 µl
	Sample:	Guanosine-5’-monophosphate, Guanosine-5’-diphosphate, Guanosine-5’-triphosphate (167 ug/ml each in B:C 70:30)

To be noted is the total ionic strength of 10 mM of Ammonium Carbonate required for the elution.

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