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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. |
Partial Structure
 |
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) |
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 |
|
|
| Support
Matrix |
Crosslinked Cellulose |
| Particle
Size |
53 – 125 µm |
| Particle
Shape |
Spherical beads |
|
Exclusion Limit |
4,000 kD |
| Functional
Group |
Butyl, Phenyl, Octyl |
| 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 < Octyl. In general hydrophobic proteins will be more
strongly adsorbed to Cellufine Octyl 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 either the
Butyl or Octyl matrices. Consequently it is difficult to generalize
and each application needs to be evaluated separately to select
the optimal media functionality.
 |
| Column : |
8.2 x 150 mm |
| Column Vol. : |
8 ml |
| Buffer : |
2.0 – 0.0M
Ammonium Sulfate
in 0.01M phosphate, pH 7.0 |
| Flow Rate : |
1.32 ml/min |
| Sample : |
5 mg/3 ml– 100 µl |
|
| Figure 1. The retention
increases with an increase in the carbon chain and aromatic
structure of the functional group as a result of stronger
hydrophobic interaction. |
|
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.
 |
| 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 |
Cellufine
Octyl |
| |
Adsorption
mg/ml |
Recovery
% |
Adsorption
mg/mL |
Recovery
% |
Adsorption
mg/ml |
Recovery
% |
| BSA |
25 |
87 |
30 |
92 |
23 |
87 |
| Catalase |
42 |
62 |
35 |
71 |
25 |
62 |
| Myoglobin |
19 |
62 |
11 |
63 |
9 |
61 |
| Glucose Oxidose |
38 |
97 |
37 |
99 |
15 |
100 |
| Ovalbumin |
31 |
87 |
31 |
89 |
19 |
88 |
| 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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| Description |
Quantity* |
Catalogue
No. |
| Cellufine
Butyl |
100
ml
500 ml
5 liters
10 liters |
19905
19906
19879
6769 733 35 |
| Cellufine Phenyl |
100 ml
500 ml
5 liters
10 liters |
19900
19901
19881
6769 753 35 |
| Cellufine
Octyl |
100
ml
500 ml
5 liters
10 liters |
19895
19896
19880
6769 743 35 |
|
|
| *Larger quantities are
available on request. |
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