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APPLICATION NOTE

AN040608-54
 

STYROS™ Amino HILIC Simulated Monolith Polymeric: Separation of Angiotensins I, II and III

 

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

The following chromatogram shows the separation of 3 closely related peptides on a STYROS™ Amino-HILIC Simulated Monolith column at 30°C.

 

Chromatogram 1

Separation of  3 Angiotensins at 360 cm/hr on STYROS™ Amino HILIC
(Flow Rate: 2 ml/min on normal bore column at 30˚ C)

 

Table 1. Operating parameters.

HPLC System.

Agilent 1100 with thermostatted column compartment.

Columns

STYROS™ Amino HILIC 4.6 X 100 mm (V=1.66 ml)

Mobile phase.

A: ACN
B: 50 mM CH3COONH4, pH=6.8

Flow rate

1 ml/min (360 cm/hr of linear flow rate )

Gradient

Isocratic: 90 % A, 10 % B

Temperature

30°C

Detection

230 nm

Injection volume

2 ul of each

Sample:

1 mg/ml of each Angiotensins in DI H2O

 

Angiotensins I, II and III are oligopeptides found in the blood.

Angiotensin I is derived from the precursor molecule angiotensinogen, a serum globulin produced in the liver. It is a decapeptide made of the following amino acids sequence:

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu

Angiotensin II is an octapeptide with the following amino acid composition:

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe where the His-Leu terminal of Angiotensin I have been cleaved.

Angiotensin III is a heptapeptide that derives from Angiotensin II it is made of the following sequence of amino acids:

Arg-Val-Tyr-Ile-His-Pro-P

These peptides are usually found together in the blood stream.

The present Amino HILIC separation procedure provides the proper LC method in hyphenation with mass spectroscopy to analyze these compounds.

 

Chromatogram 2

Separation of  3 Angiotensins at 180 cm/hr linear flow rate on STYROS™ Amino HILIC
 

STYROS™ Amino-HILIC Simulated Monolith columns are stable in the full pH range and high temperatures.

Unlike Monolith, STYROS™ Simulated Monolith columns are available in many sizes for additional resolving capabilities.

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