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APPLICATION NOTE

AN011703-30

SIMULATED MONOLITH™: Comparison With Non Covalently Coated Polymeric Hard Gel.

Polymeric hard gel media with large throughpores are commonly produced as hydrophobic particles that are subsequently coated to generate hydrophilic activated surfaces to be functionalized.

It is crucial for the coating process not to result in the obstruction of the pores or the alteration of their size.

STYROSä media are covalently coated with a single hydrophilic layer to fully preserve the pore structure.

A typical separation of some standard proteins on similar phases (HIC-Phenyl), shows the outcome of the two coating processes.

STYROSä HIC-Phenyl/XH is compared to its commercial counterpart in which the throughpores are for the most part obstructed.

Chromatogram 1
Protein Separation on Commercial Porous HIC-Phenyl Column.

 

Table 1. Operating parameters.

HPLC System

Agilent 1100, Standard Cell

Columns

4.6 x 100 mm. Commercial HIC-Phenyl, hard gel porous polymeric column.

Eluent A

0.1M Phosphate, pH = 7

Eluent B:

A + 3 M SO4(NH4)2, pH = 7

Detection:

280 nm

Flow rate:

5 ml/min  (1,800 cm/hr)

Temperature

20 °C

Gradient:

100 to 0%B in 5 minutes ( 15 C V ) as suggested by the manufacturer

Sample

1. Myoglobin, 2. Ribonuclease A, 3. Lysozyme, 4. a-Chymotrypsinogen.

 


Chromatogram 2
Protein Separation on STYROSä HIC-Phenyl

 

Table 2. Operating parameters.

HPLC System

Agilent 1100, Standard Cell

Columns

4.6 x 100 mm. STYROSä HIC-Phenyl/XH.

Eluent A

0.1M Phosphate, pH = 7

Eluent B:

A + 2.1 M SO4(NH4)2, pH = 7

Detection:

280 nm

Flow rate:

3 ml/min  (1,100 cm/hr)

Temperature

20 °C

Gradient:

100 to 0%B in 6 minutes ( 11 C V )

Sample

1. Cytochrome c, 2. Myoglobin, 3. Ribonuclease A, 4. Lysozyme, 5. a-Chymotrypsinogen.

 

The optimum capacity chosen for the STYROSä HIC media allows the use of lower salt to retain the proteins. As a result, a shallower gradient can be chosen for the baseline separation in terms of absolute salt concentration.

In addition, the pore structure of the Simulated Monolithä bed provides unobstructed throughpores for a uniform flow path.

It is evident from the second chromatogram, that there are no long obstructed or diffusive pores involved in the separation process.

The present set of polymeric columns are not only a considerable improvement over soft gel columns with pressure and flow restrictions, they also far exceed the characteristics of other non covalently coated hard gel media with either obstructed throughpores or a wide range of throughpore distribution.

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