OraChrom, Inc.

  
       

 

 

Home
Up

APPLICATION NOTE

AN080406-43

Hydrophobic Interaction Chromatography: Comparison of STYROS™ HIC-Phenyl with TSKgel Phenyl-5PW from TOSOH.

 

A mixture of 7 proteins were separated on a STYROS™ HIC-Phenyl 4.6 x 100 mm column (volume 1.7 ml) at linear flow rates of 720 cm/hr (2 ml/min volumetric flow) and compared with the performance of a TOSOH TSKgel Phenyl-5PW 7.5 mm x 7.5 cm, 10 µm particles column (volume 3.3 ml) run by the manufacturer at a linear flow rate of 136 cm/hr (1 ml/min volumetric flow) using 5 proteins.

Both resins are porous; STYROS™ is a Simulated Monolith™ column made of hard gel polymeric that can withstand high back pressures and therefore can be run at high flow rates, whereas TSKgel is a soft gel resin with pressure and flow rates limitations.

 

Chromatogram 1

Separation of 7 proteins on STYROS™ HIC-Phenyl/XH

(Linear Flow Rate: 720 cm/hr)

 

Table 1. Operating parameters.

HPLC System.

Agilent 1100 with thermostatted column compartment.

Columns

STYROS™ HIC-Phenyl/XH 4.6 X 100 mm

Mobile phase.

A: 0.1 M Phosphate, pH=7
B: A + 2.1 M SO4(NH4)2, pH=7

Flow rate

2 ml/min (720 cm/hr )

Gradient

100 to 50 % B in 5 min, to 45% B in 7 min, to 0% B in 10 min (12 cv).

Temperature

30°C

Detection

280 nm

Injection volume

10 ml

Sample:

1- Cytochrome c, 0.1 mg/ml, 2- Myoglobin 2.5 mg/ml, 3-Ribonuclease A, 5 mg/ml, 4- Lysozyme 2 mg/ml, 5- Ovalbumin 5 mg/ml, 6- -Chymotrypsin 2.5 mg/ml, 7- α-Chymotrypsinogen A 0.5 mg/ml in buffer A.

 

Chromatogram 2

Separation of 5 proteins on TSKgel Phenyl-5PW

(Linear Flow Rate: 136 cm/hr)

 

Table 2. Operating parameters.

Columns

TSKgel Phenyl-5PW, (7.5 mm x 7.5 cm)

Mobile phase.

A: 0.1 M Phosphate, pH=7
B: A + 1.8 M SO4(NH4)2, pH=7

Flow rates

1 ml/min (136 cm/hr)

Gradient

100 to 0 % B in 60 min

Temperature

25°C

Detection

280 nm

Injection volume

100 µl (50-100 µg)

Sample:

1- Myoglobin, 2- Ribonuclease, 3- Lysozyme, 4- α-Chymotrypsin,
5- α-Chymotrypsinogen.

 

The clear advantage of hard gel polymeric which STYROS™ media are made of is obvious from these chromatograms.

The porosity in the case of STYROS™ enhances the resolving power whereas it is a detriment in the case of TSKgel.

The chemical and mechanical stability of hard gel are also advantages soft gels polymeric do not have.

[ Home ] [ Up ]

Copyright © 1997-2009 OraChrom, Inc.