APPLICATION NOTE

 Separation of Angiotensin Variants at Basic pH.

It is well established that the separations of Angiotensins II and III is not possible on reversed-phase stationary phases at acidic pH.

In order to achieve such separation, which also holds true for numerous other peptides of similar structure, it is necessary to operate at relatively high pH (>10) where the use of silica based media is not possible.

The present application note provides an example where the use of STYROSä becomes a convenient tool to operate at high pH and resolve the separation.

The optimized pore structure of the polymeric matrix that makes up the core of STYROSä 1 and STYROSä 2 can not only operate in full pH range, it also makes it possible to maintain high resolution even at high speed. The following chromatograms highlight such characteristic.

Chromatogram 2. Separation of Angiotensin variants at pH=11.2, on STYROSä 1 R/XH at 900 cm/hr.

Chromatogram 2. Separation of Angiotensin variants at pH=11.2, on STYROSä 1 R/XH at1,800 cm/hr.

Table 1. Operating Parameters for Chromatograms 1 and 2.

	HPLC System	Hewlett Packard 1050
	Detector	214 nm
	Column	STYROSä 1 R/XH 100x4.6mm
	Mobile Phase	A: 10 mM Phosphate in water, pH=11.2 B: Acetonitrile/water
	Gradient	0-40% B in 25 ml
	Flow rate	2.5 and 5ml/min
	Temperature	Ambient
	Injection volume	10 ml
	Sample: 1 mg/ml each	1: Angiotensin II, 2: Angiotensin III, 3: Angiotensin I

STYROSä 2 provides a different surface chemistry within the same optimized pore structure. As a result, while maintaining a similar high performance at low and high flow rates, it is possible to get different selectivity and retentivity by using STYROSä 2.


Chromatogram 3. Separation of Angiotensin variants at pH=11.2, on STYROSä 2 R/XH at900 cm/hr.




Chromatogram 4. Separation of Angiotensin variants at pH=11.2, on STYROSä 2 R/XH at 1,800 cm/hr.

Table 2. Operating Parameters for Chromatograms 3 and 4.

	HPLC System	Hewlett Packard 1050
	Detector	214 nm
	Column	STYROSä 2 R/XH 100x4.6mm
	Mobile Phase	A: 10 mM Phosphate in water, pH=11.2 B: Acetonitrile/water
	Gradient	0-40% B in 25 ml
	Flow rate	2.5 and 5ml/min
	Temperature	Ambient
	Injection volume	10 ml
	Sample: 1 mg/ml each	1: Angiotensin II, 2: Angiotensin III, 3: Angiotensin I

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