Hydrophobic Interaction Chromatography Media
Cellufine™ Butyl, Phenyl

For purification of proteins and macromolecules

Hydrophobic Interaction Chromatography (HIC) is a method which separates proteins on the basis of their differential interactions with a mildly hydrophobic surface.

 

HIC media are porous chromatography particles, manufactured from crosslinked cellulose to which either a butyl, phenyl or octyl functionality has been covalently bonded via a short spacer.

 

Factors which affect hydrophobic interactions include: salt concentrations, temperature, pH, surfactant and organic solvents. Usually the higher the ionic strength (salt concentration) the stronger the hydrophobic bond. Consequently the interaction is enhanced by conditions inverse to that of ion exchange chromatography. HIC is, therefore, an effective complementary tool for separating and purifying substances which are difficult or cannot be separated by ion exchange.


Cellufine Butyl > Reference > MSDS / Technical Information > Catalog No.


Cellufine Phenyl > Reference > MSDS / Technical Information > Catalog No.



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Partial Structure
Butyl, Phenyl & Octyl


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Features

 

► Spherical particles exhibiting high mechanical strength

► Butyl, Phenyl, Octyl functionality

► Pre-swollen

► Virtually no shrinkage or swelling

► Stable in organic solvents and surfactants

► Stable coupling chemistry

► Resistant to 0.2 M NaOH

► Autoclavable (121 °C, 20 min)



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Benefits

 

► High flow rates allowing rapid chromatography and direct scale-up

► Enables optimum selectivity to be obtained

► Easy packing

► Easy large scale operation. No shrinkage at high salt concentrations

► Enables range of solvent systems to be utilized

► Resistant to cleaning and elution conditions

► Sterilizable

► Regulatory support


Characteristics
Support Matrix Crosslinked Cellulose
Particle Size ca. 40-130 μm
Particle Shape Spherical beads
Exclusion Limit 4,000 kD
Functional Group Butyl, Phenyl
Shrinkage/Swelling Negligible
pH Stability pH 1 – 13
Environmental Resistance Resistant to 0.2M NaOH
Operating Pressure Up to 1 bar (15 psi)
Solvent Resistance Resistant to detergents, organic solvents, salts
Supplied Suspension in 20 % Ethanol
Density 1.3 ml/g wet gel
Autoclavable 121 °C, 20 min


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Hydrophobicity of Matrix

 

The degree of hydrophobicity increases in the order of Butyl < Phenyl. In general hydrophobic proteins will be more strongly adsorbed to Cellufine Phenyl than Cellufine Butyl. However, if the protein is too strongly adsorbed, difficulty may be experienced in elution. The aromatic nature of the Cellufine Phenyl may, in certain cases, give improved selectivity compared to Cellufine Butyl. Consequently it is difficult to generalize and each application needs to be evaluated separately to select the optimal media functionality.


Columm : φ 6.6 mm × 5 cmL
Protein : Ribonuclease A, Lysozyme,
α-Chymotripsinogen A (2mg/ml)
Injection : 100 μL
Elution : 10 mM PB pH 7.0
1.5 → 0 M (NH4)2SO4 gradient (30CV)
Figure 1. Separation of proteins by Cellufine Butyl and Cellufine Phenyl


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Flow Properties

 

The semi-rigid structure of Cellufine HIC, combined with the spherical bead shape, gives excellent flow rates with higher operating pressures. Flow rates in excess of 100 cm/hr are achieved at pressure drops of 1 bar, even in large diameter process columns.


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Buffer : 0.01M Phosphate buffer (pH 7.0)
Temperature : 23 °C
Columns :
(a) 22 x 300 mm
(Vc = 0.11 liters)
(b) 90 x 200 mm
(Vc = 1.27 liters)
(c) 250 x 250 mm
(Vc = 12.26 liters)


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Adsorption Capacity and Recovery Ratio

  Cellufine
Butyl
Cellufine
Phenyl
  Adsorption
mg/ml
Recovery
%
Adsorption
mg/mL
Recovery
%
BSA 25 87 30 92
Catalase 42 62 35 71
Myoglobin 19 62 11 63
Glucose Oxidose 38 97 37 99
Ovalbumin 31 87 31 89

Sample concentration : 0.1 %
Adsorption buffer : 0.1M phosphate buffer, pH 7.0 + 2M (NH4)2SO4
Recovery buffer : 0.01M phosphate buffer, pH 7.0


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