OraChrom, Inc.

  
       

 

 

Home
Up

APPLICATION NOTE

AN080806-44

Hydrophobic Interaction Chromatography: Comparison of STYROS™ HIC-Butyl with TSKgel Butyl-NPR from TOSOH.

 

A mixture of 7 proteins were separated on a STYROS™ HIC-Butyl 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 Butyl-NPR 4.6 mm x 3.5 cm, 2.5 µm particles column (volume 0.6 ml) run by the manufacturer at a linear flow rate of 360 cm/hr (1 ml/min volumetric flow) using 5 proteins.

STYROS™ is a Simulated Monolith™ fully porous resin whereas the TSKgel NPR is a non porous resin made of 2.5 µm particles operating at 1000 to 2000 psi at flow rates of 1 ml/min with a 4.6 mm ID and a column length of 3.5 cm.

The following 2 chromatograms highlight the difference between the two stationary phases.

 

Chromatogram 1

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

(Linear Flow Rate: 720 cm/hr)

 

Table 1. Operating parameters.

HPLC System.

Agilent 1100 with thermostatted column compartment.

Columns

STYROS™ HIC-Butyl/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 30 % B in 10 min (12.5 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- α-Chymotrypsin 2.5 mg/ml, 7- α-Chymotrypsinogen A 0.5 mg/ml in buffer A.

 

 

Chromatogram 2

Separation of 5 proteins on TSKgel Butyl NPR

(Linear Flow Rate: 360 cm/hr)

 

Table 2. Operating parameters.

Columns

TSKgel Butyl-NPR, (4.6 mm x 3.5 cm)

Mobile phase.

A: 0.1 M Phosphate, pH=7
B: A + 2.3 M SO4(NH4)2, pH=7

Flow rates

1 ml/min (360 cm/hr)

Gradient

100 to 0 % B in 12 min (21cv)

Temperature

25°C

Detection

280 nm

Sample: 20 µl (1.5-4.0 µg)

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

 

Although the TSKgel Butyl-NPR has a higher pressure tolerance than the soft gel series, the size of the particles (2.5µm) generates high back pressure that limits the flow and therefore slows dawn the chromatographic run as well as the re-equilibration and reconditioning of the column.

In comparison, the STYROS™ column with 3 times the length can be run at twice the flow rate with less than half the back pressure and provide substantially higher resolution. The components of a commercial sample of Ovalbumin have also been separated in the corresponding chromatogram.

Neither the decrease in size, nor the absence of pores has provided the TSKgel Butyl-NPR any advantage that would justify the high back pressure as well as the restrictions in column length.

[ Home ] [ Up ]

Copyright © 1997-2009 OraChrom, Inc.