APPLICATION NOTE

Comparison of STYROSä 1R and 2R with Silica C4 Stationary Phase.

Most of the reversed-phase analytical separation methods have been developed on Silica C4 stationary phases. In the last several years, numerous companies have introduced macroporous poly (styrene-divynilbenzene) (PS-DVB) media to replace silica. The polymeric stationary phases provided the advantage of high chemical stability, but they had several shortcomings, namely low pressure tolerance (<3000 psi) and low capacity (5-10 mg/ml Lysozyme).

We have addressed these deficiencies during the development of the first two generations of STYROSÔ chromatographic media family. The retentivity of the two new media is compared, in Figure 1, to that of PLRP-S 4000 [1] (PS-DVB) and Vydac[2] C4 ( silica based aliphatic ligand) using Angiotensin III. The retentivity of STYROSÔ 1R closely matches that of C4 while the retentivity of STYROSÔ 2R is in between the C4 and PLRP-S. Both STYROSÔ stationary phases can tolerate pressure up to 5,000 to 10,000 psi without irreversibly collapsing

Figure 1. Retention map of Angiotensin III on different stationary phases

Standard protein and peptide separations on STYROSä 1 R and on VYDAC C4 have been compared in chromatograms 1-4.
Chromatograms 1 and 2 show the selectivity of the two columns to be practically the same for proteins.

STANDARD PROTEIN SEPARATIONS

Table 1. HPLC Operating Parameters for Chromatograms 1 and 2.

	HPLC System	Hewlett Packard 1100
	Detector	214 nm
	Column	STYROSä 1 R/XH 50x4.6mm (Chromatogram 1).VYDAC C4 50x4.6 mm (Chromatogram 2).
	Mobile Phase	A: 0.1 % TFA in water B: 0.1 % TFA in Acetonitrile/water (95/5)
	Gradient	15-80% B in 2 minutes
	Flow rate	2.5 ml/min
	Temperature	Ambient
	Injection volume	10 ml
	Sample: 1 mg/ml each	1: Cytochrome C, 2: Lysozyme 3: b-Lactoglobulin, 4: Ovalbumin

 

Chromatogram 1: Standard protein separation on STYROSä 1 R/XH

Chromatogram 2: Standard protein separation on VYDAC C4

SEPARATION OF ANGIOTENSIN VARIANTS

 

Table 2. HPLC Operating Parameters for Chromatograms 3 and 4.

	HPLC System	Hewlett Packard 1050
	Detector	214 nm
	Column	STYROSä 1 R/XH 50x4.6mm (Chromatogram 3). VYDAC C4 50x4.6 mm (Chromatogram 4).
	Mobile Phase	A: 0.1 % TFA in water B: 0.1 % TFA in Acetonitrile/water (95/5)
	Gradient	5-40% B in 10 minutes
	Flow rate	1 ml/min
	Temperature	Ambient
	Injection volume	10 ml
	Sample: 1 mg/ml each	1: Angiotensin II, 2: Angiotensin III, 3: Angiotensin I

Chromatogram 3: Separation of Angiotensin Variants on STYROSä 1 R/XH

Chromatogram 4: Separation of Angiotensin variants on VYDAC C4

The selectivity of the two columns is slightly different towards the Angiotensin variants (chromatograms 3 and 4). In general, the peaks are less tailing on STYROSÔ than on VYDAC C4, most probably due to the absence of silanol groups. Similarly to numerous other peptides, Angiotensin II and III can be separated only under basic conditions as depicted in chromatogram 6. However, silica based media can not be used above pH 8.

 

SEPARATION OF ANGIOTENSIN VARIANTS AT BASIC pH.

Table 3. HPLC Operating Parameters for Chromatogram 5.

	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
	Gradient	0-40% B in 25 ml
	Flow rate	2.5 ml/min
	Temperature	Ambient
	Injection volume	10 ml
	Sample: 1 mg/ml each	1: Angiotensin II, 2: Angiotensin III, 3: Angiotensin I

Chromatogram 5. Separation of Angiotensin Variants on STYROSÔ 1 R/XH at 2.5 ml/min, pH 11.2

 

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