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APPLICATION
NOTE
AN080497-04
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.
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Chromatogram 2.
Separation of Angiotensin variants at pH=11.2, on
STYROSä
1 R/XH at 900 cm/hr.
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Chromatogram 2.
Separation of Angiotensin variants at pH=11.2, on
STYROSä
1 R/XH at1,800 cm/hr.
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Table 1. Operating Parameters for Chromatograms 1 and 2.
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HPLC System |
Hewlett Packard 1050 |
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Detector |
214 nm |
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Column |
STYROSÔ
1 R/XH 100x4.6mm |
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Mobile Phase |
A: 10 mM Phosphate in
water, pH 11.2
B: Acetonitrile |
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Gradient |
0-40% B in 25 ml |
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Flow rate |
2.5 and 5 ml/min |
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Temperature |
Ambient |
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Injection volume |
10
ml |
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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.
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Chromatogram 3.
Separation of Angiotensin variants at pH=11.2, on
STYROSä
2 R/XH at900 cm/hr.
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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 |
STYROSÔ
2 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 and 5 ml/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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